Atlas of Preimplantation Genetic Diagnosis [2 ed.] 1842142453

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Atlas of

PREIMPLANTATION GENETIC DIAGNOSIS Second Edition

Atlas of PREIMPLANTATION GENETIC DIAGNOSIS Second Edition

Yury Verlinsky PhD and

Anver Kuliev MD PhD Reproductive Genetics Institute Chicago, Illinois

informa healthcare New York London

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2004 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20130415 International Standard Book Number-13: 978-1-4822-1065-1 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified. The reader is strongly urged to consult the drug companies’ printed instructions, and their websites, before administering any of the drugs recommended in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents

List o f c o n trib u to rs

vii

A ckno w ledg m en t Preface

viii ix

F ore w o rd

xiii

Professor Robert G Edwards

Introd uction

SECTION I

1

I

Review of Current Methods and Experience in Preimplantation Genetic Diagnosis

5

N o rm a l and abnormal human preim plantation developm ent in relation to preim plantation

7

genetic diagnosis and establishm ent o f em bryonic stem cells Introd uction

7

O ocytes at d iffe re n t stages o f meiosis I

7

O ocytes at metaphase II and variations in first polar body form atio n

8

First polar body m orp ho log y in relation to chrom osom al abnorm ality in oocytes

8

O ocytes fo llow ing fertilizatio n and second polar body extrusion N o rm a lly and abnorm ally cleaving em bryos

II

N o rm a l and abnorm al blastocyst fo rm a tio n

11

Establishment o f human em bryonic stem cells

11

References 2

10

12

M icrom anipulation and biopsy o f polar bodies and blastomeres

15

First polar body rem oval

18

Intracytoplasm ic sperm injection

19

First and second polar body rem oval

20

Blastom ere rem oval

21

Blastocyst biopsy References 3

N uclear transfer techniques fo r preim plantation diagnosis and prospect fo r artificial

22 22 23

gam ete form atio n

v

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

4

Visualization o f polar body and blastomere chrom osom es

23

Sperm duplication

26

D evelopm ent o f artificial human gametes in vitro

26

References

27

Preimplantation diagnosis fo r aneuploidies

29

Introduction

29

Preparation o f polar bodies and blastomeres fo r fluorescence in situ hybridization

3I

analysis Pretreatm ent, probe application, hybridization and washing

34

Fluorescent signal evaluation

36

Chrom osom al abnormalities in polar bodies

37

Chrom osom al abnormalities in cleaving embryos

39

References 5

40

Preimplantation diagnosis fo r translocations

43

References 6

47

Preimplantation diagnosis fo r single-gene disorders

49

Updated procedure o f single-cell D N A analysis fo r PGD o f single-gene disorders Diagnostic accuracy PGD fo r specific genetic disorders

7

49 53 55

Conclusion

60

References

61

Future perspectives fo r preim plantation diagnosis

63

Im proving accuracy o f PGD fo r single-gene disorders

63

Preconception diagnosis fo r paternally derived genetic disorders

64

Developments in sampling procedures

64

Towards PCR-based karyotyping

64

Production o f male and female gametes fro m human somatic cells

65

Stem cell transplantation and availability o f human em bryonic stem cells

65

Introduction o f PGD as the future IVF standard

66

References

67

S E C T IO N II Preimplantation Genetic Diagnosis Illustrated

71

1

73

N orm al and abnormal human preim plantation developm ent in relation to preim plantation genetic diagnosis and establishment o f em bryonic stem cells

2 3

M icrom anipulation and biopsy o f polar bodies and blastomeres

103

Nuclear transfer techniques fo r preim plantation diagnosis and prospect fo r artificial

I 17

gamete form ation 4

Preimplantation diagnosis fo r aneuploidies

141

5

Preimplantation diagnosis fo r translocations

179

6

Preimplantation diagnosis fo r single-gene disorders

2 13

Index

vi

281

List of contributors

All the contributors are affiliated with the Reproductive Genetics Institute, 2825 N. Halsted Street, Chicago, IL 60657, USA

Jeanine Cieslak-Janzen MLT Embryology and chromosomal disorders Vasily Galat PhD Artificial gametes and visualization of blastomere chromosomes Veleriy Kuznyetsov PhD Sperm duplication and visualization of blastomere chromosomes Svetlana Rechitsky PhD Single-gene disorders Nick Strelchenko PhD Embryonic stem cells Oleg Verlinsky MS Single-gene disorders

Acknowledgment

We are indebted to our colleagues in the DNA, FISH and embryology laboratories: T Sharapova, S Ozen, K Lazyuk, G Wolf Y Illkevitch, V Koukharenko, Z Zlatapolsky and I Kirillova, and also to our genetic counselors, C Lavin, R Beck, R Genoveze and D Pauling for their participation in acquisition of the data and technical assistance.

Preface

Preimplantation genetic diagnosis [PGD], intro­ duced in 1990 as an experimental procedure1'2, has now been developed into a practical tool for avoid­ ing the birth o f affected children. It represents a valuable complement to traditional prenatal diagno­ sis and a clinical option in reproductive medicine. The number of unaffected children born following PGD is already over 10003, a testimony to the accu­ racy and safety of PGD, which is now being used to establish a potential donor progeny for the stem cell treatment of siblings4. PGD has allowed hundreds of ‘at risk’ couples, not only to avoid producing offspring with genetic disorders, but also, more importantly, to have unaffected healthy babies of their own, without facing the risk of pregnancy termination after traditional prenatal diagnosis3-6'7. The technique was first applied to pre-existing Mendelian diseases, such as cystic fibrosis and Xlinked disorders, but initially did not seem to have any practical value. However, after the introduction of fluorescence in situ hybridization (FISH) analysis in 1993-1994 for PGD of chromosomal disorders, the number of PGD cycles began to double annually, yielding more than 100 unaffected children by 1996. Although the accuracy of PGD for aneuploidies has improved considerably, there is still the risk of misdi­ agnosis due to a high prevalence o f mosaicism at the cleavage stage7,8. A sequential sampling of both oocytes and the resulting embryos may improve the accuracy of aneuploidy testing, and may also allow the detection of embryos with uniparental disomies (Chapter 4). Application of PGD increased further when the ability to detect translocations became possible in 1996, first using locus-specific FISH probes and then the more widely available subtelomeriC probes8.

Because many carriers of balanced translocations have a poor chance of having an unaffected preg­ nancy, PGD has a clear advantage over the tradi­ tional prenatal diagnosis in assisting these couples to establish an unaffected pregnancy and deliver a child free from the disorder. Current developments and application of nuclear transfer techniques (Chapter 3), and microarray technology may further contribute to improvements of PGD for transloca­ tions (Chapter 5]. The natural extension of PG D ’s ability to allow transfer o f euploid embryos is its positive impact on the liveborn pregnancy outcome910. This is espe­ cially applicable to poor-prognosis in vitro fertiliza­ tion (IVFj patients, including prior IVF failures and those with a maternal age over 37. The introduction of commercially available five-color probes in 1998-1999 led to the present accumulated experi­ ence of approximately 5000 clinical cycles for aneu­ ploidy testing. This has resulted in the birth of close to 1000 aneuploidy-free children. The overall preg­ nancy rate per transfer is much higher than in IVF patients o f comparable age. Widespread confirma­ tion of these results would indicate that the current IVF practice o f transferring embryos based solely on morphological criteria is inefficient and in need of revision, given that half of these embryos are chromosomally abnormal and would compromise outcome (Chapter 4). A high prevalence of aneu­ ploidies in oocytes and embryos may also affect the accuracy of PGD for single-gene disorders7, making aneuploidy testing an important part of PGD for mendelian diseases and preimplantation testing for human leukocyte antigen (HLA) typing (Chapter 6). Further expansion of PGD occurred in 1999, when the technique was applied to late-onset

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

diseases with a genetic predisposition, a novel indi­ cation that had not been previously considered for the traditional prenatal diagnosis11,,z. For patients with an inherited pathological predisposition this option provided realistic grounds for undertaking pregnancy, with a reasonable chance of unaffected offspring. Prospective parents at such risk should be made aware o f this emerging option (Chapter 6). Another unique option that can now be consid­ ered concerns H LA typing during PGD which was introduced in 2000 (Chapter 6). This option has not been considered during traditional prenatal diagno­ sis, but with PGD it offers, not only preventative technology to avoid affected offspring, but also a new method for treating (older) siblings with congenital or acquired bone marrow diseases. This approach was first applied to souples desiring an unaffected (younger) child free from the disorder in the older sibling4. In addition to diagnosis to ensure a genetically normal embryo, HLA-matched, unaf­ fected embryos were replaced. At delivery, cord blood (otherwise to be discarded) was gathered for stem cell transplantation, resulting in complete cure in the case o f a sibling with Fanconi anemia. This approach was then also used without testing of the causative gene, with the sole purpose of finding matching H LA progeny for a source o f stem cell transplantation for affected siblings with congenital or acquired bone marrow disease or cancer13. Together with progress in the establishment of embryonic stem cells, this may contribute to the development and application o f stem cell therapy, so a specific discussion in the second edition of this Atlas (Chapter 7) will be devoted to this issue. As in the previous edition, Chapter 1 details normal and abnormal preimplantation development, including oocyte maturation and fertilization, cleav­ age stage and blastocyst formation. In addition to observations on the morphological parameters of polar bodies and pronuclear formation, recent data on the relationship between morphological parame­ ters and chromosomal status are presented, offering the future possibility of predicting the developmen­ tal potential of embryos without chromosomal analysis. This chapter is o f particular relevance for PGD, because appropriate selection of the material for genetic analysis at each stage of preimplantation development will affect the test results and their interpretation, avoiding misdiagnosis and achieving the highest accuracy and reliability of PGD.

X

Since the first edition of this Atlas, PGD has also been improved, involving macromanipulation proce­ dures, including the originally introduced microma­ nipulation techniques to remove the first and second polar body and biopsy cleaving embryos, together with other related methods, such as intracytoplasmic sperm injection, which has become an integral part of PGD for single-gene disorders (Chapter 2). The techniques have been reproduced in more than 50 PGD centers all over the world. These have performed more than 6000 clinical cycles to date, resulting in the birth of more than 1000 apparently unaffected children, with an overall congenital malformation rate no different from that o f the general population14. More than two-thirds of these clinical cycles were performed in the US, from which the largest number (over 2000 cycles) has been contributed by our center. With the greatly improved accuracy o f genetic analysis and indications expanding well beyond those for prenatal diagnosis, more than 1000 PGD cycles are now performed annually. Experience during the past 2 years has resulted in the birth o f nearly the same number of children as during the entire preced­ ing decade. The list o f conditions for which PGD is performed is being extended rapidly, and presently includes single-gene disorders presented at birth, latc-onset disorders, H LA genotyping and chromoso­ mal abnormalities. PGD offers particular advantages not attainable with traditional prenatal diagnosis including avoidance of clinical pregnancy termina­ tion, which is especially attractive for couples carry­ ing translocations, couples at risk for producing offspring with common diseases of autosomal domi­ nant or recessive etiology, and, finally, for couples wishing not only to have an unaffected child, but also an HLA-compatible cord blood donor for treatment o f an older sibling with a congenital disorder. Yet the greatest numerical impact of PGD is in standard assisted reproduction, where improved IVF effi­ ciency through aneuploidy testing is evolving to become standard practice. Finally, the second edition of this Atlas presents for the first time progress in the use of PGD as a source o f embryonic stem cell (ESC) lines and future developments in the use of nuclear transfer tech­ niques for improving PGD, and producing human gametes.

PREFACE

REFERENCES 1.

Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplan­ tation embryos sexed by Y-specific D NA amplifica­ tion. Nature 1990;344: 768-70

2.

Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990;5:826-9

3.

Verlinsky Y, Cohen J, Munne S, et al. Over a decade of preimplantation genetic diagnosis experience - a multi-center report. Fertil Steril 2004;82:292-4

4.

5.

6.

Verlinsky Y, Rechitsky S, Schoolcraft W, et al. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. J Am Med Assoc 2001;285:3130-3 International Working Group on Preimplantation Genetics. Preimplantation Genetic Diagnosis. Experience of three thousand clinical cycles. Report of the 11th Annual Meeting International Working Group on Preimplantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 15, 2001. Reprod BioMed Online 2001;3:49-53 European society of Human Reproduction and Embryology. Preimplantation Genetic Diagnosis Consortium. Data Collection III, May 2002. Hum Reprod 2002; 17:233—46

7.

KuLev A, Verlinsky Y. Thirteen years experience of preimplantation genetic diagnosis. Reprod BioMed Online 2004;8:229-35

8.

Munn£ S. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities, Reprod BioMed Online 2002;4:183

9.

Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999;72:837^14

10.

Munne S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod BioMed Online 2003; 7: 91-7

11.

Rechitsky S, Verlinsky O, Chistokina A, et al. Preimplantation genetic diagnosis for cancer piedisposition. Reproductive BioMed Online 2002;5:148-55

12.

Verlinsky Y, Rechitsky S. Verlinsky O, et al. Preimplantation diagnosis for early onset Alzheimer disease Caused by V 7 17L mutation. J Am Med Assoc 2002;287:1018-21

13.

Verlinsky Y, Rechitsky S, Sharapova T, et al. Preimplantation HLA typing. J Am Med Assoc 2004;291:2079-85

14.

Kuliev A, Verlinsky Y. Current features of preimplan­ tation genetic diagnosis. Reprod BioMed Online 2002;5:296-30

Foreword

Preimplantation genetic diagnosis (PGD) is spread­ ing worldwide, as it is introduced into more and more IVF clinics. Yet it has been almost 50 years since the concept of diagnosing inherited diseases in preimplantation embryos began with a study by Richard Gardner and myself, in which small pieces of trophectoderm were excised from rabbit blastocysts and scored for sex using the presence or absence of sex chromatin. We were delighted when all offspring proved to be correctly sexed at full term. So, with this in mind, imagine the absolute joy afforded to a couple attending for PGD who would know that their child was safe from a debilitating family disease gene or that their embryos did not carry lethal chro­ mosomal disorders. Scientists who are newcomers or those who are already deeply versed in its methods will gain immensely from the details presented in this book. Nevertheless, current methods to achieve PGD remain a worrying prospect for many couples. The greatest concern arises through the difficulty in attaining high rates of implantation, which both reduces the success of the treatment and the number of cycles with a successful diagnosis. IVF remains expensive, tedious and sometimes uncertain. Every new advance in human preimplantation embryology helps to remit such apprehensions as outlined in this book. Molecular aspects of PGD are under finer control and new techniques are introduced yearly to improve their scope and accuracy. It is difficult even for professionals in the field to predict where the future lies, although let us hope that new genetic studies will identify embryos with a high implanta­ tion potential. Advances in computers to further improve or replace current techniques would seem to be inevitable. Computer diagnoses may be applied

on a wider scale than hitherto and will be essential in the development o f new approaches to alleviate genetic disease or to identify couples needing PGD. New approaches using gene therapy may even become available long term, as is the case in current studies that use this approach to alleviate various forms of hemopoietic failure. Will such approaches challenge PGD? I doubt this will be likely in the near future since PGD is so accurate and is steeped in modern technologies. Nor does it suffer from the uncertainties that accompany current forms of gene insertion into human beings. Even though well versed in molecular genetics, I stand impressed by the analytical methods described in this textbook. Standard and novel concepts are demonstrated in the text and in wonderful color images. Elegant methods for polar body or blas­ tomere excision and analysis are described, with nuclear transfer serving to enhance the diagnosis of inherited disease by converting nuclei to mitotic chromosomes in recipient enucleated cells. I have long admired the works of various investigators into translocation inheritance, surely the most complex of chromosomal disasters and which demands immense diagnostic skills. Future developments are inevitable. Stem cell lines are being established to open future research on specific disease genes. Artificial gametes are not too far away; a major departure from current clinical techniques aimed at gathering the desper­ ately few gametes surviving in a testis or an ovary in hypogonadal individuals. Such novelties could well demand PGD to ensure that artificially derived gametes or embryos derived from them are safe. Advanced molecular techniques might replace FISH by direct scoring of the numbers and quality of

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

genes. Will PGD replace prenatal diagnosis? Perhaps to some extent since it does not rely on abortion. A stunning array of photographs and diagrams throughout the book portray an astonishing degree of variability associated with embryos in the initial early stages o f human life. Micromanipulation looks so smooth and simple, as depicted in the figures displaying polar body removal or ICSI. The incredi­ ble level o f care devoted to ensuring a normal birth is also revealed in these images, together with impressive amounts o f information and insight into chromosomal and genetic factors influencing early development. Turning these pages raises the question yet again why so many human embryos are abnor­ mal. Furthermore, why does a woman ovulate 80% of eggs incapable of implantation? Why do men have so many anomalous spermatozoa after investing so much biological capital in spermatogenesis? And are the frequencies of these anomalies associated with

PGD sufficient to explain why only one-fifth 01 human embryos can implant? So much is known that it has become increasingly difficult to handle or even comprehend the detailed data available on the web and elsewhere. Trophectoderm is revealed as an astonishing tissue in need of further study. PGD could help by assessing the nature o f its genes regulating implantation. This studv could be done today using methods such as real-time PCR, new forms o f detecting allele drop­ out, the identification of tumor suppressor genes and combinations of HLA matching. Such methods have already greatly added to the diagnosis of gene muta­ tions, and make one wonder where we will be in 5 or 10 years, given current rates of progress. Will we be analyzing quantitative genetic inheritance, or perhaps measuring chromosomal sites of genes inserted into embryonic genomes? I feel certain we will. Robert G Edwards Chief Editor Reproductive BioMedicine Online

xiv

Introduction

Preimplantation genetic diagnosis (PGD) has become an essential part of preventive measures for genetic disorders, enabling couples to avoid termina­ tion of pregnancy after routine prenatal diagnosis1-3. It allows the detection of errors in early preimplan­ tation development and prediction of the genotype of the resulting embryo prior to implantation, so that only normal embryos and those with optimal devel­ opmental potential are preselected for transfer to the uterus, ensuring an unaffected pregnancy and the birth of a healthy baby. In this way, couples at high risk o f having offspring with genetic disease have an option to control the outcome of their pregnancy from the outset. Although the procedure involves ovarian hypf'rstimulation and in vitro fertilization (IVF), 13 years of PGD experience shows that it is an acceptable procedure in many ethnic groups all around the world. The application of PGD is o f particular interest for assisted reproduction practices, because of the extremely high prevalence o f chromosomal aneu­ ploidies in preimplantation development, which are known to be the major contributor to spontaneous abortions and implantation failures. This provides obvious potential for improving the efficiency of IVF through preselection of euploid embryos for transfer, making PGD an integral component o f assisted reproduction technologies (ART). As will be described below, the accumulated data provide strong evidence of a PGD contribution to the improvement in the effectiveness of IVF, particularly in patients of advanced reproductive age4'5. PGD is currently performed by two main approaches, one involving testing of female gametes, and the other testing of single blastomeres removed from the preimplantation embryo. Both of these

methods have been used extensively in clinical prac­ tice and have proved to be accurate and reliable in predicting the genotype ot the resulting pregnancy. These methods will be described based on the origi­ nal experience o f more than 2000 PGD cycles, representing approximately one-third of the world experience. As in the first edition of this Atlas, the major emphasis will be on illustrative material, which has been considerably updated especially in Chapters 4, 5 and 6, and may provide a working manual for the establishment and performance of PGD within the framework of IVF and genetic services. Although PGD was initially offered to couples at high risk for having children with single-gene disor­ ders, more than two-thirds of the procedures to date have been performed for age-related aneuploidies. The list of conditions for which PGD has been performed is being rapidly extended, with the avail­ ability of sequence information for many diseases, and with the application of PGD for late-onset disor­ ders with genetic predisposition6-7. Thus the second edition presents the experience of PGD for almost 100 different conditions. Because of the obvious usefulness of PGD for ART, many centers still concentrate on PGD for chromosomal aneuploidies using the fluorescent in situ hybridization (FISH) technique. However, an increasing number o f centers presently also include in their activities single-cell polymerase chain reaction (PCR) analysis for Mendelian disorders, with some focusing on PGD for the most prevalent conditions, such as PGD for cystic fibrosis and thalassemias. To date, over 6000 PGD cycles have been performed, resulting in more than 1000 healthy chil­ dren being born8. As already mentioned, over 2000

I

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

of these cycles were performed in our center, of which more than 1500 were done for chromosomal aneuploidy, using mainly the first polar body fPB l] and second polar body (PB2) sampling, and recently also in combination with blastomere analysis for IVF patients of advanced reproductive age, demonstrat­ ing the clinical significance o f this procedure for improving the chances of IVF patients to become pregnant. The data demonstrate an extremely high prevalence of aneuploidies in oocytes from IVF patients o f advanced maternal age, making obvious the requirement for genetic testing o f oocytes in assisted reproduction practices for older women9" 11, To date, PGD provides the best solution for couples carrying translocations, as their reproductive outcome is very poor even with the use of routine prenatal diagnosis12-14. As described in the previous edition of this Atlas, PGD for maternal translocations was initially performed by PB1 removal and FISH analysis using whole-chromosome painting probes, with further addition of PB2 and blastomere testing1415. In the absence o f commercial probes for the detection of complex rearrangements, casespecific probes for detection o f normal and deriva­ tive chromosomes in interphase cells were also prepared to facilitate PGD for any translocation16; however, this was extremely labor intensive, required custom-made preparations and could not be applied as a method of choice in routine practice. In the meantime, FISH analysis using chromosome-specific probes has been proven to be extremely efficient and reliable, despite its limitations for testing some translocations and for complete karyotyping. As will be described in the present edition of this Atlas, these limitations have recently been overcome by different approaches for visualization and cyto­ genetic analysis of single blastomeres and PB2, which allow the accuracy of PGD for both maternally and paternally derived translocations to be improved14,15'1715. Another possible approach for PGD o f translocations is comparative genomic hybridization (C G H ), which was applied experi­ mentally to investigate the feasibility o f accurate prediction of balanced and normal meiotic outcome; however, its accuracy has still to be demon­ strated19,20. Whatever method is used for PGD of translocations, a significant impact of PGD on the pregnancy outcome is obvious owing to a consider­ able reduction in the numbers of spontaneous abor­ tions after PGD, compared to those before under­ taking PGD for translocations13’14. Because of

2

considerable impact, a separate chapter has been devoted to PGD for chromosomal rearrangements in the second edition of this Atlas (Chapter 5], As will also be described, considerable progress has been achieved in the detection and exclusion of sources for misdiagnosis in PGD for single-gene disorders, based on overcoming of the major limita­ tions of single-cell PCR. A description of the current progress in PGD, with presentation of the original data from the largest PGD world experience, will make it obvious that PGD has become an established procedure in assisted reproduction practices and genetic services. There is no doubt that PGD improves the standard of IVF, providing the possibil­ ity to better the outcome of pregnancy for patients with advanced reproductive age and those with poor prognosis. For genetically disadvantaged couples, PGD makes it possible to plan for healthy progeny without facing the need for prenatal diagnosis and termination o f wanted pregnancies. The technique has great potential even for those who cannot accept any manipulations at the embryo stage, through the methods o f preconception or pre-embryonic diagno­ sis.

REFERENCES

1

International Working Group on Preimplantation Genetics. Preimplantation Genetic Diagnosis Experience of Three Thousand Clinical Cycles. Report of the 11th Annual Meeting International Working Group on Preimlantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 15, 2001. Reprod BioMed Online 2001; 3:49-53

2.

European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium. Data Collection III, May 2002. Hum Reprod 2002;17:233-46

3.

Kuliev A, Verlinsky Y. Thirteen-years experience of preimplantation genetic diagnosis. Reprod BioMed Online 2004;8:229-35

4.

Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999;72:837-44

5.

Munne S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod BioMed Online 2003;7:91-7

IN T R O D U C T IO N

6.

Rechitsky S, Verlinsky O, Chistokina A, et al. Preimplantation genetic diagnosis for cancer predis­ position. Reprod BioMed Online 2002;5:148-55

j^

Verlinsky Y, Kuliev A. Current status of preimplanta­ tion diagnosis for single gene disorders. Reprod BioMed Online 2003;7:23-8

,„

8.

Verlinsky Y, Cohen J, Munne S, et al. Over a decade of preimplantation genetic diagnosis experience - a multi-center report. Fertil Steril 2004;82:292-4

9.

Munne S. Preimplantation genetic diagnosis o f numerical and structural chromosome abnormalities. Reprod BioMed Online 2002;4:183-96

10.

Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Nuclear abnormlities in series of 6733 human oocytes. Reprod BioMed Online 2003;6:54-9

11.

Kuliev A, Verlinskv V. The role of preimplantation genetic diagnosis in women of advanced reproduc­ tive age. C urr Opin Obstet Gynecol 2003;15:233-8

12.

Munne S, Morrison L, Fung J, et al. Spontaneous abortions are significantly reduced after preconcep­ tion genetic diagnosis of translocations. J Assist Reprod Genet 1998;15:290-6

13.

Munn£ S, Sandalinas M, Escudero T, et al. Outcome of premplantation genetic diagnosis of translocations. Fertil Steril 2000;73:1209-18

14.

Verlinskv Y, Cieslak J, Evsikov S, Galat V, Kuliev A. Nuclear transfer for full karyotyping and preimplan­

tation diagnosis of translocations. Reprod BioMed Online 2002;5:302-7 Verlinsky Y, Evsikov S. Karyotyping of human oocytes by chromosomal analysis of the second polar body. Mol Hum Reprod 1999;5:89-95

lb

Weier HUG, Munne S, Fung JL. Patient-specific probes for preimplantation genetic diagnosis of structural and numerical aberrations in interphase cells. J Assist Reprod Genet 1999;16:182-91

17.

Verlinsky Y, Evsikov S. A simplified and efficient method for obtaining metaphase chromosomes from individual human blastomeres. Fertil Steril 1999;72:1-6

18.

Willadsen S, Levron J, Munne S, et al. Rapid visual­ ization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs, llum Reprod 1999;14:470-5

19.

Malmgrcn H, Sahlcn S, Inzunza J. Single cell CGH analysis of human preimplantation embryos from PGD patients with balanced structural chromosome aberrations. Reprod BioMed Online 2002;4 (Suppl 2):14-15 Kuliev A, Verlinsky Y. Current features of preimplan­ tation genetic diagnosis. Reprod BioMed Online 2002;5:296-301

3

Section I

R eview o f C u rre n t M eth od s and Experience in P reim p lan tatio n G en etic Diagnosis

I

Normal and abnormal human preimplantation development in relation to preimplantation genetic diagnosis and establishment of embryonic stem cells

IN T R O D U C T IO N

Preselection of oocytes and embryos with the highest developmental potential, currently based on morphological criteria, is of obvious interest for improving the efficiency of assisted reproductive technologies. To date, there are insufficient data on the morphological parameters that may be of value to exclude aneuploid embryos from transfer. Currently this requires chromosomal studies of oocytes and (or) embryos and is limited to only a small proportion of IVF centers. Different approaches have been tested for possi­ ble prediction of the developmental potential of oocytes, such as pronuclear morphology scoring1'2, microtubule and microfilament organization assess­ ment3, and the first polar body (PB1) grading4-6. It is currently known that approximately 42% of oocytes obtained from women of advanced reproductive age have PB1 aneuploidies7, but because chromosomal studies are not readily available and require special­ ized expertise, the possibility of PB1 morphological grading as a potential means for preselection of viable oocytes and embryos for transfer is very attrac­ tive, particularly since it could be easily adopted in IVF practice. However, preliminary reports on the utility of PB1 morphology have been controversial, suggesting a positive correlation between well shaped, round PB1 within a cohort and fertilization rates, embryo quality and implantation rates4,5, or no correlation with fertilization but a positive correla­ tion between implantation rates and the presence of fragmented PB1 within a cohort6. However, these were retrospective studies in which the chromoso­ mal status of the oocytes and embryos was not inves­ tigated. Thus, the observations on the prospective

study of PB1 morphology in relation to fertilization, chromosomal status, developmental potential of the resulting embryos and outcome of embryo transfer may represent practical interest and will be described below.

O O C Y TE S A T D IFFER EN T STAGES OF M EIO SIS I

Current superovulation regimes create a population of follicles that are not totally synchronous in devel­ opment8. Although the preoperative injection of human chorionic gonadotropin (hCG) may induce germinal vesicle breakdown (GVBD), the cytoplas­ mic maturation of the oocyte may not be complete and appropriate for the initiation of normal embry­ onic development. A number of ovarian markers have been demonstrated to be useful for predicting the developmental potential of oocytes9'10. Immature oocytes are usually retrieved from small follicles and may not have undergone GVBD. Accordingly the surrounding cumulus cells are well compacted around the oocyte and difficult to remove. Although maturation in vitro for a propor­ tion of these immature oocytes in culture and full development to the blastocyst stage can be achieved, the fertilization rate of the in vitro matured oocytes and the competence of embryos derived from such oocytes to establish a pregnancy is reduced11,12. The first set of photographs demonstrates the heterogeneity of the oocyte-cumulus complex, which may represent the degree of maturity of the oocytes, their competence for fertilization, as well as the viability of the resulting embryos (Figures 1.1-1.4). As seen from these oocyte-cumulus complexes, obtained in the course of a standard

7

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

ovarian hyperstimulation procedure, not all of the oocytes may be selected for testing, since detailed examination of the preovulatory oocyte is obscured by the large cumulus mass. Although the stage of maturation can be deduced from the state of expan­ sion of the cumulus mass, the exact stage of matura­ tion cannot be assessed without removal of the cumulus cells. Removal of the cumulus cells prior to insemination is not detrimental to fertilization and is performed using hyaluronidase. Information about the exact stage of maturation is obligatory for PGD, since only oocytes with a mature nucleus can be the source of PB1 and subsequently PB2 for genetic testing. The accurate assessment of oocyte maturity is based on the absence of a germinal vesicle and the presence of PB1. The germinal vesicle in prophase I oocytes is more or less spherical in shape and usually contains a single eccentrically placed nucleolus (Figures 1.5 and 1.6). The germinal vesicle is centrally located within the cytoplasm in early immature oocytes and becomes more eccentrically located prior to GVBD (Figure 1.6). The typical metaphase I (intermediate) oocyte displays neither germinal vesicle nor PB1 (Figure 1.7). The cytoplasm of these oocytes is round and even, slightly colored or granular. The dynamics of the oocyte’s transition from prophase I to metaphase I, revealed by immunochemical staining, is presented in Figure 1.8. These examples illustrate variations of oocyte matu­ ration and possible abnormalities at each of these stages that may influence the selection and availabil­ ity of material for PGD.

O O C Y T E S A T M E T A P H A S E II A N D V A R IA T IO N S IN FIR ST P O LA R B O D Y F O R M A T IO N

Despite a small proportion of immature oocytes, the majority of retrieved oocytes have a mature nucleus and are suitable for PGD. The typical metaphase II (mature, preovulatory) oocyte displays PB1, extruded within 36-40 h of hC G injection, which is usually round and non-fragmented, with its morphology differing from oocyte to oocyte. PB1 is the by-product of the first meiotic divi­ sion, providing the material for evaluation of the resulting genotype of the corresponding oocyte following maturation. Therefore, observation of the transition of oocytes from prophase I to metaphase II, as well as the outcome of the second meiotic divi­

8

sion, is of special relevance for PGD. Together with analysis of PB2, which is the by-product of the second meiotic division, the majority of maternally derived genetic defects may be detected, to avoid the transfer of the embryos resulting from the corre­ sponding abnormal oocytes. Neither polar body has any biological significance in preimplantation and post-implantation development, and may be removed and analyzed to investigate the genetic normality of oocytes. It is, of course, possible to obtain the genotype of an oocyte by direct analysis, but this will destroy the oocyte. Therefore, removal and analysis of PB1 and PB2 is the only way for eval­ uating the genetic quality of oocytes. Figures 1.9-1.15 show mature oocytes, based on PB1 extrusion, but clear abnormalities are seen. PB1 formation in some of them (Figures 1.12-1.14) suggests possible errors in the corresponding oocytes and their derivatives13. Although most of these abnormalities are rare (Figures 1.16-1.19), such oocytes have to be recognized and removed from genetic testing. Avoidance of inclusions of cumulus cells (Figures 1.20-1.26) in the process of PB1 removal is of great practical importance for prevent­ ing D N A contamination, which might lead to mis­ diagnosis in PGD of single-gene disorders.

FIR ST PO LA R B O D Y M O R P H O L O G Y IN R E L A T IO N T O C H R O M O S O M A L A B N O R M A L IT Y IN O O C Y T E S

Comparison of PB1 morphology with its chromoso­ mal contents was performed on oocytes obtained from 90 patients in 91 IVF cycles for different indi­ cations, using a standard IVF protocol14. PB1 morphology was assessed in 831 mature oocytes prior to intracytoplasmic sperm injection (ICSI) (day 0) and at fertilization assessment (day 1), and placed into three main categories: grade 1 representing a round or oval shape which may sometimes be flat­ tened; grade 2 representing a non-fragmented PB 1 of irregular shape; and grade 3 representing a partially or totally fragmented PB1 (Figure 1.27). PB1 obser­ vation was performed using an inverted microscope (Diaphot, Nikon, Garden City, NY, USA) with Hoffman modulation contrast optics and a magnifi­ cation of x200. Prior to ICSI, oocytes were rotated so that both the side and top views of the PB1 were observed. Oocytes were then followed up after ICSI from fertilization throughout preimplantation development and embryo transfer. Data on PB1

NORMAL AND ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

grading for each oocyte were correlated to fertiliza­ tion rate, day-3 embryo quality as to cell number and degree of fragmentation present, development to the blastocyst stage and embryo transfer outcome. Due to differing patient responses to hormonal stimulation, patients were divided into two groups, consisting of 42 poor responders (50 cycles; group I) in which the number of retrieved oocytes did not exceed ten, and 48 good responders (50 cycles; group II] with more than ten available oocytes. Chromosomal status of the resulting embryos was available for 49 patients in 50 cycles in which 395 oocytes and embryos were tested on day 1 (polar body analysis), day 3 (blastomere analysis) or both, using micromanipulation techniques and fluorescent in situ hybridization (FISH) analysis as described in Chapter 3. The overall distribution of oocytes according to PB1 grades on day 0 and day 1 in group I and group II was as follows. The number of oocytes with PB 1 of different grades on day 0 was similar and distributed equally in both patient groups. PB1 grading on day 1 revealed the changes of PB1 morphology in both patient groups for each grade category except grade 3. Overall, the grade changes were observed in 331 (36.2%) of 831 PB1, and were similar in both groups I and II. Significant differences for such changes were observed only for the oocytes with grade 2 PB1, 79.1% and 66.3% o f which, in group I and II respec­ tively, became grade 3 on day 1. Lower fertilization rates were observed for oocytes with grade 1 PB1 (69.5% in group I and 73.6% in group II), compared to grade 2 (85.5 %) in group I, and to grade 3 (83.6%) in group II. The cleavage rates of embryos deriving from oocytes with different PB1 grades were similar in each PB1 cate­ gory for each patient group. The morphological quality of the embryos, with respect to the degree of fragmentation observed on day 3 in relation to the oocytes with different PB1 grading, showed no difference in the three PB1 grades or patient groups, as demonstrated by the proportion of grade 1-1.5 embryos (little or no fragmentation present), result­ ing from oocytes with PB 1 grades 1, 2 and 3. Similar results were observed in the potential of embryos to reach the blastocyst stage on days 5 and 6. O f 109 group I embryos, 35 (32%) reached the blastocyst stage of development with 24 (22%) at a gradable expanded stage according to Gardner's criteria15, with no significant difference in any of the PB1 grade groups. The same was true for 555 group

II embryos, of which 249 (44.9%) reached the blas­ tocyst stage with 148 (26.7%) at a gradable expanded stage, again with no significant difference in any of the PB 1 grade groups. Implantation rates of 317 embryos, grouped according to the oocytes’ corresponding PB1 grade from which they were derived, showed no differ­ ence, also taking into consideration the embryo transfers consisting of a combination of embryos in which there were two different PB1 grades. The overall implantation rate for group I, group II and the group of PGD patients was 20.2%, 32.8% and 22.5%, respectively. Results of aneuploidy testing in 395 embryos, resulting from oocytes with different PB1 grades obtained from 49 patients (50 cycles) of mean age of 37 years, showed no differences in aneuploidy rates in any PB1 grade category, which were 75.9%, 65.3% and 66.4% for PB1, grade 1, 2 and 3, respectively. The data also failed to reveal any difference in the frequency of error for each chromosome tested in relation to the PB1 grade, as seen from examples of normal PB1 morphology and abnormal chromosome pattern, as well as abnormal PB1 morphology and normal FISH results (Figures 1.28-1.31). A higher prevalence of aneuploidy for chromosomes 21 and 22 for all PB1 grades was observed, which is in agree­ ment with the previous findings for women of advanced reproductive age7. The data show that PB1 morphology may not be a useful predictor of developmental potential or chromosomal normalcy of the resulting embryos, in either good or poor responders. Because PB1 morphology was graded in sequence before and after fertilization, it was possible to detect the grading changes we observed in more than one-third of the oocytes in both patient groups. These changes were significant for the grade 2, intact, irregular-shaped PB1, in which the majority on day 0 became grade 3 by day 1. Although this may be representative of postextrusion changes, it appears to have no practical relevance, sincc all the clinical parameters studied showed no correlation with any of the PB 1 grades. The relevance of the higher fertilization rate observed in oocytes with the irregular PB1 shape, compared to those with the regular shape PB1 in poor responders, as well as to those with the frag­ mented PB1 in good responders is not clear, since these findings conflict with other data. Previous retrospective studies suggested no correlation6, or even the possibility of a positive relationship of PB1

9

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

morphology with higher fertilization rate4,5. These conflicting data may be attributed to the timing of the ICSI procedure in relation to the cytoplasmic maturation of the oocytes. On the other hand, in contrast to previous reports, there was no relationship of PB I morphol­ ogy to thp embryo quality and cleavage rate, which makes PB1 grading questionable for reliable prese­ lection o f cleavage stage embryos for transfer. Furthermore, in contrast to the earlier findings of a positive relationship of PB1 morphology with blasto­ cyst formation potential4, the prospective data showed no relationship in either good or poor responders. In addition, no relationship was observed between PB1 grading and the outcome of embryo transfer, which was similar in different patient groups, based on PB1 morphology. This is not in agreement with previous data, some of which suggested higher implantation and pregnancy rates for embryos derived from oocytes with intact, round versus fragmented PB14S, nor does it agree with another report which describes an association between higher implantation and pregnancy rates and cohorts of oocytes in which a greater percentage of fragmented PB1 are present6. Finally, aneuploidy testing of embryos resulting from oocytes with different PB1 grading, failed to reveal any relationship between PB1 morphology and karyotype, suggesting that PB1 morphology is not useful for diagnosing or screening for chromoso­ mal aneuplodies in preimplantation development. In conclusion, the data provide no evidence for any relationship o f PB1 morphology with chromoso­ mal normalcy, embryo quality and developmental potential, and outcome o f embryo transfer, suggest­ ing that PB1 grading is of no prognostic value for the developmental potential o f embryos to be used in preselection of embryos for transfer.

OOCYTES FOLLOWING FERTILIZATION A N D SECOND POLAR BODY EXTRUSION The occurrence of normal fertilization is usually assumed from the presence of two pronuclei within the ooplasm observed approximately 12-18 h after insemination and PB2 in the perivitelline space (Figure 1.32). PB2 formation is an important sign of the fertilization process following exposure of mature oocytes to sperm by conventional insemina­ tion or intracytoplasmic sperm injection (ICSI). However, the fertilization process can be delayed,

10

interrupted at any point or proceed in some peculiar way, as shown in Figure 1.33. In some instances, the penetration of sperm leads to premature condensa­ tion of the oocyte, eventually leading to premature condensation of the sperm chromatin; while the PB2 may or may not have been extruded, the oocyte enters metaphase III stage (Figure 1.34). In other eases, one (Figure 1.35), three (Figure 1.36) or four pronuclei (Figure 1.37) are formed. Also, the number and morphology of polar bodies can vary from zygote to zygote. The time of the appearance (usually 6-8 h after ICSI) or disappearance (usually 20-24 h after insemination) o f pronuclei, their size and location in the ooplasm can also differ from oocyte to oocyte8. As mentioned, without testing the PB2, the geno­ type of the oocyte cannot be adequately assessed. PB2 is extruded within the first two hours after insemination or ICSI. Pronuclei may be formed as early as 4 h post-insemination, although the zygote is not yet formed at this stage, and is defined as prezygotic (pronuclear stage). Usually male and female pronuclei are still present 20 h post-insemination, so genetic testing of PB1 and PB2 may be completed before syngamy16. Technically, this may represent preconception, or pre-embryonic diagnosis, prevent­ ing the discarding of embryos, which is not accept­ able in some populations and ethnic groups. Pronuclear morphology has been demonstrated to be an important predictive indicator for embryo potential and also may be correlated with chromoso­ mal content of the oocyte and embryo1-2. This is in agreement with the fact that the nuclear and Cyto­ plasmic maturation should be synchronized to ensure normal meiosis resumption, fertilization and cleavage. Thus, any abnormality in formation of the paternal or maternal pronucleus, and asynchrony of nuclear and cytoplasmic maturation processes may lead to fertilization failure and irregular cleavage. For example, poorer pronuclear morphology was observed in couples with severe infertility undergo­ ing ICSI, which was also associated with higher chro­ mosomal abnormalities in the resulting preimplanta­ tion embryos1. Analysis of pronuclear morphology in relation to the resulting nuclear morphology, polar body alignment, chromosomal status and the outcome of the transfer of the resulting embryos, revealed the relationship between chromosomal abnormalities and embryo quality with only a few pronuclear types2. Thus, a possible correlation between pronuclear zygote morphology, chromosomal

NORMAL AND ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

content and embryo development may, in the future, help to predict the presence of chromosomal abnor­ malities and preselect the embryos with higher developmental potential based on morphological characteristics.

NORMALLY A N D ABNORMALLY CLEAVING EMBRYOS Since testing o f PB1 and PB2 may provide PGD for only maternally derived single-gene and chromoso­ mal disorders, embryo biopsy with blastomere analy­ sis is one of the basic approaches for PGD of pater­ nally derived conditions, as well as for X-linked disorders by gender determination17. As mentioned, the first cleavage o f the zygote is observed no earlier than 20 h post-insemination, and cleavage continues every 12-18 h (Figures 1.38-1.45]. However, asym­ metric or asynchronous divisions can occur, resulting in uneven distribution o f zygote cytoplasm, anucleate blastomeres and fragmentation, as well as slowly developing embryos (Figures 1.46-1.52], One or two blastomeres are usually removed for PGD at the six- or eight-cell stage: (Figures 1.53a-d], i.e. not earlier than 2 days after insemination. For slowly developing embryos, biopsy is delayed or in some cases their development prohibits testing entirely. As will be shown below, there are limitations to PGD by blastomere biopsy due to the high rate of mosaicism18-20 and allele-specific amplification failure in cleaving embryos 1. Complete failure of amplification may be also observed, when anucleate blastomeres are removed for testing. As in polar body sampling, embryo biopsy has been suggested to have no deleterious effects on the viability o f the embryo22, although this cannot be completely excluded until more data are collected on the clini­ cal outcomes of PGD at the cleavage stage. Nevertheless, blastomere biopsy remains the major approach for PGD in many centers around the world.

NORMAL A N D ABNORMAL BLASTOCYST FORMATION With the present tendency o f in vitro fertilization centers to shift from cleavage stage to blastocyst transfer, blastocyst biopsy will probably be of great potential value for PGD of single-gene and chromo­ somal disorders23. Until recently, only a few clinical

Cycles have been performed using blastocyst biopsy, which did not result in pregnancy or the birth o f a healthy baby^4. However, most recently, the first cases of PGD by blastocyst biopsy were reported to result in clinical pregnancies25. Different stages of blastocyst formation arc presented in Figures 1.54-1.65, including possible abnormalities deter­ mined by morphological appearance and special staining (Figures 1.58, 1.62, 1.65). The advantages of PGD using blastocyst biopsy are based on the premise that trophectoderm cells rather than the inner cell mass could be biopsied to avoid reducing the number of cells forming the fetus26. In contrast to cleavage-stage embryo sampling, not one or two, but a dozen cells may be biopsied from the trophec­ toderm, providing more accurate analysis.

ESTABLISHMENT OF HUM AN EMBRYONIC STEM CELLS PGD provides a novel source for the establishment o f embryonic stem (ES) cells27. Although ES cells are usually derived from culture of the inner cell mass (ICM) of the preimplantation blastocyst (Figure 1 66d-f), a highly efficient and original technique was developed for the establishment of ES cell lines from human embryos at the morula stage. To establish human ES cells from a morula stage embryo, zona pellucida is removed and the morula placed under a middle density feeder layer (Figure 1,66a). Within the next few days, cells outgrow and spread into the feeder layer (Figure 1.66b). The passage 0 or primary cell disaggregation is performed with ethylenediamine tetraacetic acid (EDTA) or ethylene glycol bis-2-aminoethyl ether-N, N ', N ", N'-tetraacetic acid (EGTA), and the loose cells are transferred back to thp feeder layer to proliferate. Rapidly proliferating colonies are isolated and prop­ agated further. The typical human morula-derived stem cells are shown in Figure 1.66c. In contrast to morula, the establishment of human ES cells from a blastocyst involves immunosurgery, requiring the isolation and placement of the ICM on a feeder layer (Figure 1.66d). The typical outgrowth of ICM is shown in Figure 1.66e. The passage 0 is performed with EDTA and the rounded cells are transferred back to a fresh feeder. As seen from Figures 1.66d and 1.66f, no morphological differences between human ES cells originating from ICM and from morula were observed. Nor were differences observed in the pattern of marker expres­

II

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

sion, including alkaline phosphatase (AP), T R IT C im m unofluorescence o f O ct-4 expression, im m uno­ fluorescence o f TR A -2-39 (L-A P), TR A -2-60 and TR A -2-80, detected by m onocloncal antibodies labeled by FITC, antigens SSEA -3 and SSEA -4 (Figure 1.67). The fact that these m arkers are expressed in the sam e colony o f m orula-derived ES cells that are characterized by specific AP expression is shown in Figure 1.68. As can be seen from Figure 1.69, there was no difference in patterns o f marker expression in m orula-derived E S cells cultured in feeder layer-free m edium . The established hum an ES cell lines were m ain­ tained in vitro from 10 to 15 passages before freezing in sufficient am ounts with control thaw out28. Tests for differentiation in vitro through em bryonic bodies, with subsequent disaggregation and cell culture show ed a w ide range o f cell types belonging to ecto­ derm, endoderm and m esoderm . The cells were also shown to spontaneously differentiate in vitro into a variety o f cell types, including neuron-like cells with dendrites and contracting prim itive cardiocyte-like cells (Figure 1.70). The current institutional repository contains m ore than 50 human ES cell lines, including ES cell lines obtained from em bryos with single-gene disor­ ders, such neurofibrom atosis type I, Marfan syndrome, myotonic dystrophy, Becker m uscular dystrophy, fragile-X syndrome and thalassemia. T hese cell lines are currently used for research purposes and are also available on request (RGI, Inc, Chicago, IL).

REFERENCES 1.

Kahraman S, Kumpete Y, Sertyel S, et al. Pronuclear scoring and chromosomal status of embryos in severe male infertility. Hum Reprod 2002;17:3193-200

2.

Gianaroli L, Magli MC, Ferraretti AP, Fortini D, Griego N. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selec­ tion. Fertil Steril 2003;80:341-9

3.

Van Blercom J, Davis P, Alexander S. Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence. Hum Reprod 2000;15:2621-33

4.

12

Ebner T. Yaman C Mose M, Sommergruber M, Feichtinger O, Tews G. Prognostic value of first polar body morphology on fertilization rate and embryo

quality in itracytoplasmic sperm injection. Hum Reprod 2000;15:427-30 5.

Balaban B, Urman B, Isiklar A, Alatas C, Aksoy S, Mercan R. The effect of polar body morphology on embryo quality, implantation and pregnancy rates. Fertil Steril 2001;76(Suppl 1):S8

6.

Miller KF, Sinoway C.E, Fly KL, Falcone T. Fragmentation of the polar body at the time of ICSI does not predict fertilization or early embryo devel­ opment but may be associated with improved preg­ nancy and implantation. Fertil Steril 2001;76(Suppl 1):S201

7.

Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Nuclear abnormlities in series of 6733 human oocytes. Reprod BioMed Online 2003;6:54-9

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Veeck LL. An Atlas of Human Gametes and Conceptuses London, UK: Parthenon Publishing Group, 1999

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Van Blerkom J. Epigenetic influences on oocyte developmental competence: perifollicular vascularity and intrafollicular oxigen. J Assist Reprod Genet 1998;15:226-34

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Gregory L. Ovarian markers of implantation poten­ tial in assisted reproduction. Hum Reprod 1998; 3(Suppl 43:11 7-32

11.

Eppig JJ, O ’Brien M, Wigglesworth K. Mammalian oocyte growth and development. Mol Reprod Dev 1996;44:260-73

12.

Smitz J, Cortvrindt R. Oocyte in-vitro maturation and follicle culture: current clinical achievements and future directions. Hum Reprod 1999;14(Suppl 1): 145—61

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Rosenbusch B, Schneider M. Maturation of binuclear oocyte from the germinal vesicle stage to metaphase II: formation of two polar bodies and two haploid chromosome sets. Hum Reprod 1998;13:1653-5

14.

Verlinsky Y, Lerner S, Illkevitch N, et al. Is there any predictive value of the first polar body morphology for embryo genotype or developmental potential? Reprod BioMed Online 2003;7:336^tl

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Schoolcraft W, Gardner D. Lane M, Schlenker T, Hamilton F, Meldrum D. Blastocyst culture and transfer results in high pregnancy and implantation rates while reducing high order multiple gestations. Fertil Steril 1999;72:604-9

16.

Larsen W. Human Embryology. New York: Churchill Livingstone, 1994:18

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Handyside AH Kontogiani EH, Hardy K, 'Winston RML. Pregnancies from biopsied human preimplan­

N O R M A L A N D A B N O R M A L H U M A N PREIM PLAN TATION DEVELOPM ENT

tation embryos sexed by Y-specific DNA amplifica­ tion. Nature 1990;344:768

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Meldrum DR. Blastocyst transfer - a natural evolu­ tion. Fertil Steril 1999;72:216-17

18.

Munne S, Weier HUG, Grifo J, Cohen J. Chromosome mosaicism in human embryos. Biol Reprod 1994;51:373-9

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Munne S, Alicani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosomal abnormalities. Fertil Stenl 1995;64:382-91

McArthur S, Marshall J, Wright D, de Boer K Successful pregnancies following blastocyst (day 5) biopsy and analysis for reciprocal and Robertsonial translocations. Fifth International Symposium on Preimplantation Genetics, 2003; 5-7 June, Antalya, Turkey: 33

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20.

Delhanty JDA. Chromosome analysis by FISH in human preimplantation genetics. Hum Reprod 1997; 12(Suppl): 153-5

21.

Rechitsky S, Strom C, Verlinsky O, et al. Allele drop out polar bodies and blastomeres. J Assist Reprod Genet 1998;15:253-7

Muggleton-Harris AL, Glazier AM, Pickering S, Wall M. Genetic diagnosis using PCR and FISH analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos. Hum Reprod 1995; 10:183

27.

Pickering S, Braude P, Patel M, Burns J, Bolton V, Minger S. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. Reprod BioMedic Online 2003;7:353-64

28.

Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y. Establishment of RGI Repository of Human Embryonic Stem Cells. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey. 2003:34-5

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23.

Hardy J, Martin KL, Leese HJ, Winston RM, Handyside AH. Human preimplantation develop­ ment in vitro is not adversely affected by biopsy at the 8-cell stage. Hum Reprod 1990'5:708-14 Verlinsky Y, Munne S, Simpson JL, et al. Current status of preimplantation diagnosis. ) Assist Reprod Genet 1997;14:72-5

13

2 Micromanipulation and biopsy of polar bod es and blastomeres

In addition to standard in vitro fertilization (IVF) techniques1, three major micromanipulation proce­ dures are involved in' preimplantation genetic diag­ nosis (PGD), which include intracytoplasmic sperm injection (ICSI)2,3, polar body removal4,5, and embryo biopsy6. The micromanipulation set-up and tool construction needed for performing each of these procedures are presented below, with the equipment, materials and reagents listed in the Tables 1 and 2. To perform procedures involved in PGD at least five basic microtools are constructed: (1)

Holding pipette to fix and position the oocyte or embryo during a procedure;

(2)

Microneedle to create an opening in the zona pellucida;

(3)

Blunt micropipette 15-16 pm in diameter for polar body removal;

(4)

Blunt micropipette 25-30 pm in diameter for blastomere removal;

(5)

Finely pulled micropipette of 7-8 pm in inner diameter beveled to a 30° angle with the tip pulled to form a spike for ICSI.

Three different instruments - a pipette puller, micro­ forge and beveler - are used for microtool construc­ tion. The first step involves creation o f a microneedle by softening the capillary tube glass by heat and pulling it to form an attenuated tip. This can be done by hand (holding pipettes), or by utilizing a Flaming-Brown mechanical pipette puller (Model P-87, Sutter Instrument Co.). The program and heating filament of the mechanical pipette puller is adjusted to give the desired length o f the attenuated portion of the microneedle. The attenuated portion

of the holding pipette is approximately 2 cm, while the microneedle for partial zona dissection (PZD) and micropipettes for biopsy procedures are approx­ imately 1 cm. ICSI microtools are pulled using a Narishige puller (Model PB-7) in two steps, because of the small diameter (7-8 pm) required and the need for a short attenuated tip of 0.7 cm. The holding pipette, after pulling by hand, is prepared by means of the de Fonbrune-type micro­ forge (Model MF-1, TPI Instruments). Under the control of the microforge stereomicroscope, equipped with eyepiece reticule, the tip of the needle is broken to create an opening. This is accom­ plished by placing the part of the needle of 200 pm in diameter on top of the glass ball surrounding the heating filament and increasing the temperature of the filament until the needle is fused to the glass ball. The power is then switched off, to cause an abrupt decrease in temperature and shrinkage of the heating filament, leading to a break in the tip of the microneedle. The pipette tip is then flame-polished by being placed approximately 0.5 mm away from the glass ball while the temperature o f the filament is increased to achieve melting of the glass. The pipette tip is moved away from the heating filament when the opening is almost closed, leaving an inner diam­ eter of 15-30 pm. The attenuated portion of the micropipette is bent by being placed above the glass ball. The filament is heated until the glass begins to soften, so that the pipette tip bends under its own weight (at an angle of approximately 35°). This is done to all microtools so that they are parallel to one another and to the microscope stage during micro­ manipulation.

15

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Table 1 Equipm ent required fo r m icrom anipulation and biopsy Supplier

Model

Laminar flo w hood

Holten

LFM 24725

Inverted m icroscope

N iko n (D ia p ho t) o r O lym pus

IX-70

H ydraulic joystick (2)

N ikon-N arishige

M 0202

Coarse m anipulator (2)

N ikon-N arishige

M N 188

M ounting adapter

N ikon-N arishige o r Olym pus-N arishige

Equipment

M icro in je cto r (2)

Narishige

IM -6 o r IM -16

Stereom icroscope

N ikon o r O lym pus

S M Z-U o r SZH 10

De Fonbrune-type m icroforge

TPI Instrum ents

MF-I

M icroforge

Narishige

MF-9

Pipette puller

Narishige

PB-7

Pipette puller

Sutter

P-87

Pipette beveler (w ith p o w e r supply)

Research Instrum ents

Piezo electronic m icro to rch

Blazer

G B -2 0 0 1

D ouble to o l holder

Leica o r Narishige

520143 o r HD-21

Injector holder (2)

Narishige

H I- 7

A microneedle for PZD is the least complicated tool to create. After a capillary tube is pulled on the Flaming-Brown mechanical pipette puller, the microneedle is simply bent on the microforge, as for all other microtools. The PZD microneedle is not connected to the microsyringe system but is simply installed in a micropipette holder and mounted to the micromanipulator. To prepare blunt micropipettes for polar body removal or embryo biopsy, the same procedure is followed, except for the diameter of the micro­ pipette and the duration of the flame-polishing. A shorter period with less heat will only smooth any rough edges without decreasing the size of the opening. To construct a micropipette with a particu­ lar diameter, after the capillary tube is pulled the newly created microneedle is transferred to the microforge. The attenuated portion o f the micro­ needle is broken with the same steps followed as for the holding pipette, except for breaking at a site that has approximately the desired diameter (1 5-16pm for polar body removal, 25-30 pm for blastomere biopsy). The tool is flame-polished and bent, as previously described. To prepare the ICSI pipette, the first pull accom­ plishes the melting and stretching of the glass to give a tapered tube, while the second accomplishes the attenuation in which the walls are almost parallel, resulting in a break with a final inner diameter of

16

"'-8 pm. The microneedle is then beveled with a Research Instruments MBI microbeveler at a 30° angle to create a beveled micropipette. The tip o f the needle is viewed through a monocular microscope (x400) to ensure proper beveling. Since water is used to wet the grinding wheel, water will enter the micropipette, so the tool is then connected to a syringe to rinse the tip with HPLC H 2O followed by pure grain alcohol, and blown dry by having air forced through it. The Narishigc microforge (MF-9) with compound microscope is used to create a spike at the tip of the micropipette. The spike is necessary for easier breakage of the oolemma when the oocyte is entered. Under the control of a compound micro­ scope (magnification of x300), the micropipette is positioned so that the bevel faces the operator as it is lowered towards the heating filament, which is turned on by a foot pedal. As soon as the tip of the micropipette fuses with the glass ball surrounding the heating filament, the tool is raised, with simulta­ neous releasing of the foot pedal to switch the fila­ ment off, resulting in a spiked tip of the micropipette. Finally, the tool is bent with the use of the de Fonbrune-type microforge, similar to other microtools, to obtain the angle necessary for micro­ manipulations. Microtools are stored in a Petri dish, with clay or foam rubber to hold the pipettes in place, avoiding breakage.

M IC R O M A N IP U L A T IO N A N D B IO PSY O F P O LA R B O D IE S A N D B LASTOM ERES

Equipment is placed in a laminar flow hood with an immersion heater circulator and a stereomicroscope. An inverted microscope with a stage warmer for fertilization assessment and micromanipulation is also placed in the hood. Special measures are taken to maintain the pH of the culture medium even when working under oil. This can be better controlled by use of gas flow meters that control the passage of tri-gas mixture (5% C O 2 , 5% 02, 90% N ;) through humidifiers to incubator chambers located on the working surface o f the hood. For manipulation of gametes and embryos, coarse manipulators are mounted to the inverted micro­ scope. The Narishige M 0 2 0 2 fine manipulator (hanging joysticks) provides good contr ol of both the holding and the ICSI pipettes, as well as of biopsy micropipettes. Hydraulic control for all tools is performed using the Narishige IM-6 or, more recently, the IM-16 microsyringe injector. A Stoelting microsyringe (no. 51222) can also be used for the holding and aspirating pipettes with slight modifica­ tion; the micrometer is connected to the plunger of

the 100 pi microsyringe with epoxy to obtain suction. Th>= whole hydraulic system is filled with light paraffin oil, with special attention paid to elim­ ination of any air bubbles. Microtools, needed for holding, biopsy or injection, are filled with silicone oil (12 500 centistokes) prior to attachment to the micropipette holders. Several solutions are required for culture and micromanipulation procedures: (1)

HTF medium prepared fresh every 2 weeks.

(2)

HTF medium supplemented with plasmanate (5%) for oocyte retrieval, sperm preparation, insemination, ICSI and polar body removal;

(3)

HTF medium supplemented with plasmanate (5%) containing 0.05m ol/l sucrose for polar body removal (optional);

(4)

Polyvinylpyrrolidone (PVP) medium for ICSI;

(5)

HTF medium supplemented with plasmanate (10%) for embryo culture, assisted hatching

(10%) in HTF

Table 2 Materials and reagents required for micromanipulation and biopsy Materials and reagents

Supplier

Catalog number

35 x 10-mm Petri dishes (Falcon)

Fisher Scientific

08-757-IO0A

60 x I 5-mm Petri dishes (Falcon)

Fisher Scientific

08-757-I00B

150 x I 5-mm Petri dishes (Fisher)

Fisher Scientific

08-757-14

4-well culture dishes (Nunc)

Scientific Supply

176740

50-ml culture flasks (Falcon)

Fisher Scientific

10-126-IB

0.2|im filters (I-I flask)

Fisher Scientific

09-740-26A

Receiver flasks (I-I) (Nalgene)

Fisher Scientific

09-740-25F

0 .2|^m filters (150-ml) (Nalgene)

Fisher Scientific

09-740-31F

Sterile Pasteur pipettes (plugged 9-inch)

Invitro Scientific Products

600-900P

Large gloves (powder-free)

Scientific Products

2D7853i

Platinum wire for de Fonbrune microforge

Fisher Scientific

13-766-1OA

Platinum wire for Narishige microforge

Fisher Scientific

13-766-1 IA

Drummond capillary tubes (30|il)

Fisher Scientific

2I-I70J

Chromar HPLC water (Mallinckrodt)

Scientific Products

6795-1

Plasmanate protein supplement (50 ml)

Bayer Biological

61320

10% PVP for ICSI MW 360000

IVFonline

MPVC-5000

Life Global LiteOil™ mineral oil

IVFonline

LGOL-500

Quinn's Advantage'" Ca/Mg-free medium with Hepes

Sage BioPharma

Silicone oil 12 500 centistokes

Sigma Chemical

DMPS-I2M

Sucrose

Sigma Chemical

S-9378

Hyaluronidase

Sigma Chemical

H-3757

17

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

prior to transfer and embryo biopsy. More recently, Global medium (IVFonline,. Guelph, O N N1H 2T3, Canada) has been used for embryo culture. (6)

Life Global LiteO IL™ mineral oil, sterile filtered and equilibrated. This is used for oocyte and embryo eulture and all micromanipulation procedures to reduce the pH and incubation temperature fluctuations of the microdrops of medium. All mineral oil is sterile filtered through a 0.2-pm filter unit prior to equilibra­ tion with unsupplemented HTF medium and then incubated for 3 days before being used for all procedures.

Approximately 35 h from the time of human chori­ onic gonadotropin (hCG) administration, oocytes are aspirated and collected in HTF culture medium supplemented with plasmanate (5%). Two to three hours after retrieval, oocytes are removed from culture and transferred to a four-well dish with meduim containing hyaluronidase (80U /m l) for 30 s and transferred to the adjacent wells of culture medium for denuding. Usually, within these few seconds, the cumulus cells begin to disperse. After 20 min in culture, by means of an attenuated flamepolished pipette with an inner diameter of 150-180 pm, the remaining corona cells are mechan­ ically removed bv pipetting. The oocytes are cleaned thoroughly in order to allow the PB1 to be visualized during micromanipulations. It is also of special importance for PGD of single-gene disorders to eliminate the possibility for corona cell contamina­ tion. Once oocytes have been cleaned, they are collected into one of the four wells, and their matu­ rity is determined by the presence o f PB1, assessed with an inverted microscope. If no PB1 has been extruded, the oocytes are further cultured for possi­ ble maturation in vitro, which may take an additional 24 h in culture for germinal vesicle oocytes. To ensure easy maneuvering on and off the microscope stage without having to realign the microtools for each manipulation, micromanipula­ tion dishes are prepared using Falcon 1008 35 x 10-mm Petri dish lids. The dishes are positioned so that the Falcon label is seen at the top. Microdrops of the required medium are placed in the center of the lid at the 3, 6, 9 and 12 o’clock positions and covered with sterile equilibrated mineral oil. Each dish is kept separately by being placed in a larger

18

60 x 15-mm Petri dish with lid, so that only one dish at a time is removed for a procedure.

FIRST POLAR BODY REMOVAL To perform PB1 removal an opening in the zona pellucida of sufficient size is needed. A device for the mechanical PZD, called the ‘3D -PZD ’, has been developed, which is a modification of two-dimen­ sional PZD that enables the creation o f three types of openings'. A 'cross’ shaped opening is created for ‘assisted hatching’ and ‘V ’ shaped opening for embryo biopsy (routinely used). If necessary, a square hole can also be created by four intersecting cuts performed in the zona pellucida. For PGD o f single-gene disorders, all microtools are constructed in advance and exposed to ultravio­ let radiation for 2 0 min. The medium can be supple­ mented with 0.05 mol/1 sucrose to cause ooplasm shrinkage, increasing the perivitelline space and decreasing the chances of plasma membrane damage during the procedure (optional). Three 15-pi drops of medium without sucrose are placed at the 12, 3 and 6 o’clock positions and one 15-pl drop of medium containing sucrose (optional) is placed at the 9 o’clock position. The drops are overlaid with the sterile equilibrated mineral oil. The number of prepared dishes is equivalent to the number of mature oocytes retrieved, as a separate micromanip­ ulation dish is used for each oocyte to reduce the chance o f D NA contamination. To perform PB1 removal, a holding pipette with an inner diameter of 15-30 pm is filled with silicone oil and connected to the microsyringe hydraulic system with the micropipette holder. A micropipette of approximately 15-16 pm is filled with silicone oil and a PZD microneedle attached to the micropipette holder and the second microsyringe hydraulic system. The aspirating micropipette and the microneedle are mounted onto a modified Leica double tool holder (no. 520143) or Narishige HD-21 and the tools are aligned so that they are parallel not only to the microscope stage but also to one another within the same field. An oocyte is removed from the four-well dish and transferred to a micromanipulation dish into the microdrop at 9 o ’clock. The dish is placed on the microscope stage, the instruments are lowered initially into the 12 o’clock microdrop and the excess oil is removed from the outer surface of the pipettes. A small amount o f medium is aspirated into both the

MICROMANIPULATION A N D BIOPSY OF POLAR BODIES A N D BLASTOMERES

holding and the aspirating micropipettes, to ensure the hydraulic system is easily controlled. The stage is then moved so that the microdrop containing the oocyte is brought into view. Once the oocyte has been secured by the holding pipette with the use of gentle suction, the oocyte is oriented with the microneedle so that PB1 is visualized at the 6 o'clock position (Figure 2.1a). Using the microneedle, the oocyte is rotated horizontally until the polar body is slightly out of focus (Figure 2.1b and c). The slit is made in the zona pellucida at the 4-5 o’clock posi­ tion, passing tangentially through the perivitelline space and out at the 7-8 o'clock position (Figure 2 .Id). The oocyte is released from the holding pipette and held by the microneedle (Figure 2.1e), which is brought to the bottom of the holding pipette and pressed to it, pinching a portion of zona pellucida (Figure 2 .If). By gently rubbing the microneedle against the holding pipette with a sawing motion, the cut is accomplished and the oocyte is released. The oocyte is then rotated so that the opening is at the 5 o ’clock position and PB1 is in focus (Figure 2.2b); the aspirating micropipette is passed through the opening to PB1 (Figure 2.2c) with gentle suction applied to aspirate the polar body into the micropipette (Figure 2.2d and e). Pressure from the hydraulic system is equilibrated prior to withdrawal o f the aspirating micropipette, to avoid damage to the oocyte. The oocyte is released from the holding pipette and all microtools are raised slightly before the microscope stage is moved, so that the drop of medium at the 6 o ’clock position is visualized (Figure 2.2f). The micropipette containing the PB1 is lowered to the bottom of the dish and the PB1 is expelled, with careful attention paid so as not to scratch the bottom o f the dish with the microtool, avoiding PB 1 adherence to the dish and breakage during its trans­ fer to the microcentrifuge tubes containing lysis buffer. Transfer is accomplished by a separate, atten­ uated, sterile Pasteur pipette with its tip heated over a microtorch and hand pulled. The pipette is trans­ ferred to the de Fonbrune microforge, and its atten­ uated portion is broken and flame-polished to a diameter of 40 (im. In a laminar flow hood, under control of the stereomicroscope, each PB1 is aspi­ rated into the attenuated portion of a Pasteur pipette with 5 pi of medium and expelled into the microcentrifuge tube. To ensure that PB 1 was not retained in the pipette, the tip of the Pasteur pipette is repeat­

edly flushed and examined in the same drop in which PB1 was located, under the control of the stereomicroscope. If fluorescence in situ hybridiza­ tion (FISH) analysis is required, the PBI is not trans­ ferred, but removed from the microdrop at the time of fixation.

INTRACYTOPLASMIC SPERM INJECTION ICSI is required for PGD for single-gene disorders to avoid sperm cell contamination when performing polar body removal or embryo biopsy. The procedure m shown in Figures 2.3 and 2.4. On the day o f retrieval, after follicular aspiration, micromanipiulation dishes are prepared, with their number usually determined by the number of mature oocytes retrieved (one oocyte is placed in one micromanipulation dish for ICSI for cases requiring PBI removal on day 0). The lid of a 35 x 10-mm Petri dish is oriented so that the Falcon label is at the top. Three 10-pl drops o f culture medium are placed in the center of a lid at the 6, 9 and 12 o ’clock positions and the fourth drop containing 10% PVP in culture medium at the 3 o'clock position. The drops are covered with sterile filtered and equilibrated mineral oil. Also, with use of 35 x 10-mm Petri dishes, individual culture dishes are prepared. F.ach of these dishes is filled with sterile equilibrated oil, under which a drop (50 pi) of HTF medium or Global medium supplemented with plasmanate (10%) is placed. All micromanipulation and culture dishes are kept at 37°C, under 5% C O 2 and air until use. After 4 h from oocyte retrieval the tools (one holding pipette and one ICSI micropipette) are back filled with silicone oil (12 500 centistokes) and connected to microinjectors via silicone tubing and micropipette holders. They are then installed onto the mounted micromanipulators, the left microma­ nipulator controlling the holding pipette, the right the ICSI micropipettc. Under control of the inverted microscope, tools arc aligned facing one another, parallel to the microscope stage and then raised. Under control o f the stereomicroscope the washed sperm are added to the drop containing 10% PVP, to slow down sperm movement, facilitating selection of a morphologically normal sperm for injection. It also minimizes sperm adherence to the glass surface once it is inside the injection micropipette. The micromanipulation dish, contain­ ing both sperm and oocytes, is transferred to the

19

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

inverted microscope stage, and the microtools are lowered into the drop of culture medium at the 9 o'clock position to ensure complete control of fluid in and out of the microtools. The microtools arc raised slightly and the dish is moved by use of the mechanical stage to the location o f the PVP drop with sperm. The ICSI micropipette is lowered and both sperm and micropipette are brought into focus. A sperm is immobilized by gentle rubbing o f its tail on the bottom of the dish and aspirated into the micropipette, tail first (Figure 2.3a and b), being raised slightly and moved to the drop containing the oocyte. Once the oocyte is brought into focus, the ICSI micropipette containing the immobilized sperm is lowered and brought into focus; once again, the fluid control and sperm movement within the pipette are assessed. Should the sperm become stuck in the micropipette, it is expelled and another sperm is retrieved, or if necessary the microtool is changed. The holding pipette is lowered and using gentle suction the oocyte is held in a semi-fixed position (Figure 2.3c and d), so that it can still be rotated to the appropriate position using the ICSI micropipette. The ICSI micropipette is brought to the 3 o’clock position, the outer edge o f the oocyte is brought into focus and the sperm is brought to the tip o f the micropipette. The micropipette is guided through the zona pellucida into the center of the oocyte (through the slit opening if PBI removal has occurred prior to ICSI) and a small amount of ooplasm is aspirated into the micropipette to ensure breakage of the membrane by slow turning of the micrometer o f the microinjector (Figure 2.3e). Once the membrane has been broken, the contents of the micropipette, i.e. ooplasm and the immobilized sperm, are expelled slowly into the oocyte and the micropipette is slowly withdrawn (Figure 2.3f). Complete control over aspiration and expulsion are needed to diminish the amount of medium deposited along with the sperm. The entire injection procedure for each oocyte takes approximately 2-3 min. After injection, each oocyte is placed in its own dish, and fertilization is assessed 16-18h after ICSI.

FIRST A N D SECOND POLAR BODY REMOVAL PBI removal after ICSI is performed by the same procedure described above, except for some modifi­ cations to avoid the damage of the meiotic spindle

20

during the procedure. Initially, the oocyte is oriented so that PBI is seen at the 12 o’clock position (Figure 2.5a). With the use o f the microneedle, the oocyte is rotated horizontally until the polar body is visualized directly in the center of the oocyte, facing the oper­ ator (2.5b). The first slit is made by entering the zona pellucida at the 1-2 o’clock position and passing tangentially through the perivitelline space and out at the 10-11 o'clock position (Figure 2.Sc)* and is accomplished as previously described (Figure 2.5d). The oocyte is then rotated so that the opening is at the 2 o'clock position and the PBI is in focus (Figure 2.5e). The aspirating pipette is passed through the opening to PBI (Figures 2.5f, 2.6a and 2.6b), which is removed by gentle suction (Figure 2.6c and d). The oocyte is released and the PBI is expelled from the micropipette (Figure 2.6e). The PB2 is removed in the same way as PBI, except that there is no need for PZD, since the opening in the zona pellucida was created prior to PBI removal. The zygote is rotated into position so that the opening is at the 4-5 o’clock position (Figure 2.7a). The opening should be in focus when the micropipette is passed into the perivitelline space (figure 2.7b). Once in the perivitelline space, the PB2 is brought into focus, by advancing the micropipette towards it (Figure 2.7c). Using gentle suction, the PB2 is aspirated into the micropipette and the procedure further continues as for PBI removal (Figure 2.7d-f). Oocytes are returned to their culture dishes and examined for cleavage after 48h. Contrary to PGD for single-gene disorders, in which both PBI and PB2 are removed separately in sequence, PBI and PB2 are removed simultaneously for the purpose of PGD for chromosomal abnormal­ ities. The procedure of simultaneous PBI and PB2 biopsy, demonstrated in Figures 2.8-2.10, is similar to that performed for PBI removal, except that it does not require strict precautions for D NA contam­ ination that are necessary when testing for single­ gene disorders. Although PB 1 and PB2 are not expected to have any biological role in the development of the embryo, the subject has been explored by following up and evaluating the resulting micromanipulated oocytes at different stages of development8,9. No significant decrease in fertilization rate for oocytes after ICSI, or cleavage of the resulting embryos, following PBI removal was observed.The percentage of embryos entering cleavage was similar in biopsied

MICROMANIPULATION A N D BIOPSY OF POLAR BODIES A ND BLASTOMERES

and non-biopsied oocytes. There was also no increase in the percentage of polyspermic embryos. Data on the long-term effect of the procedure, from culturing the embryos to the blastocyst stage, demonstrated that the proportion of embryos reaching the blasto­ cyst stage was similar to that known for non-micromanipulated oocytes. A follow-up study of the viability of the sampled oocytes through implanta­ tion and post-implantation development of the resulting embryos also suggested no detrimental effect. As will be described below, the procedure of PBI removal has already been applied to over 10 000 oocytes in the process o f PGD for age-related aneu­ ploidies and single-gene disorders, and showed no effect of PBI removal on fertilization, preimplanta­ tion or, possibly, post-implantation development. Furthermore, no deleterious effect was observed in the follow-up study of more than 200 children born following PBI and PB2 sampling10.

BLASTOMERE REMOVAL Embryo biopsy is performed as soon as the embryo reaches a minimum o f six cells, so as not to cause a considerable decrease in cell number at later stages of development. As in PBI removal, a mechanical approach to open the zona pellucida has been devel­ oped, called 3D-PZD, allowing the creation of a Vshaped triangular or square flap opening, sufficient in size for a micropipette to pass through in order to remove blastomere(s). Micromanipulation dishes are prepared in the same way as for PB 1 removal, in that all solutions are sterile filtered and equilibrated prior to use. The micromanipulation setup is the same as for PBI removal with two exceptions - the biopsy micropipette has a larger diameter o f 25-30 Jim and biopsy may be performed using a calcium- and magnesium-free medium with HEPES buffer supplemented with plasmanate (5%) (optional) to temporarily disassociate tight cell gap junctions in compacting embryos for ease of cell removal. During aspiration the cell membrane may break, so a blastomere with a clearly visible nucleus is chosen so that its aspiration into the pipette can be controlled and the chance of losing genetic material is reduced. Once a blastomere is chosen, the embryo is rotated so that the blastomere is at the 12 o’clock position (Figure 2.11a). The embryo is rotated slightly, horizontally, and the first slit is made (Figure 2.11b). The microneedle is passed from the 1-2

o'clock position tangentially through the periv­ itelline space and out at the 10-11 o'clock position (Figure 2.11b). The embryo is released and the first cut accomplished by rubbing the microneedle against the holding pipette (Figure 2.11c). The: second cut is completed by entry into the first slit, advancing tangentially through the perivitelline space and ending at the 10-11 o'clock position (Figure 2.1 Id and e). The embryo is released from the holding pipette and held by the microneedlc, and the cut is accomplished as previously described. This results in the creation o f a V-shaped opening (see: below) sufficient for passage of a 25-30-pm micro­ pipette to remove a blastomere. If a larger opening is desired, two additional intersecting slits are made, creating a square opening. After the second cut, the embryo is rotated until the new slit is visible at the 12 o ’clock position and then backward until the slit is slightly out of focus. The microneedle is passed through the slit opening and perivitelline space and out at the 10-11 o ’clock position to make the third intersecting cut (Figure 2.1 le). To complete the square and create a hole in the zona pellucida, the final cut is made by entering the first slit and passing through the perivetilline space (2.1 If). The proce­ dure results in an opening in the shape of a square as seen in Figure 2.12a, after removal o f the cut-out portion by microneedle. To complete the embryo biopsy procedure, the embryo is rotated until the opening is at 3 o ’clock and then held firmly by suction from the holding pipette (Figure 2.12b). The micropipette is inserted (Figure 2.12c) and gentle suction is applied, to aspirate the blastomere slowly into the pipette to avoid breakage and disruption of the surrounding blastomeres (Figure 2.12d). Once the blastomere is aspirated, pressure within the pipette is equilibrated and the micropipette is with­ drawn (Figure 2.12e). The blastomere is slowly expelled into the 6 o ’clock microdrop (Figure 2.12f) and transferred to the microcentrifuge tube as in PB 1 removal for single-gene disorders. The same procedure is applied for embryo biopsy using a triangular opening in the zona pellucida as shown in Figures 2.13-2.18. Slight upward pressure is applied with the micropipette to the triangular flap, followed by insertion of the micropipette to remove a blastomqre (Figure 2.15) As for PBI and PB2 removal, the follow-up studies o f embryos after blastomere biopsy did not show any detrimental effect11. No increase in congenital malformation has been reported among

21

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

m ore than 1000 children born following polar body or blastom ere biopsy1213, although further system ­ atic study will be needed to monitor the clinical outcom es o f P G D using PBI and PB2 sam pling or em bryo biopsy and to collect further data on the safety o f the procedures used in P G D for single-gene and chrom osom al disorders.

of Genetic Disorders: A New Technique for Assisted Reproduction. New York: Wily-Liss, 1993;49— 67

6.

Handyside A. Biopsy of human cleavage stage embryos and sexing by DNA amplification. In Verlinsky Y, Kuliev A, eds. Preimplantation Genetics. New York: Plenum Press, 1991/75-83

7.

Cieslak J, Ivakhnenko V, Wolf G, Sheleg S, Verlinsky Y. Three-dimentional partial zona dissection for preimplantation genetic diagnosis and assisted hatch­ ing. Fertil Steril 1999;71:308-13

8.

Verlinsky Y, Milayeva S, Evsikov S, et al. Preconception and preimplantation diagnosis for cystic fibrosis. Prenat Diagn 1992;12:103-10

9.

Kaplan B, Wolf G, Kovalinskaya L, Verlinsky Y, Viability of embryos following second polar body removal in a mouse model. J Assist Reprod Genet 1995;12:747-9.

10.

Strom C, Levin R, Strom S, et al. Neonatal outcome of preimplantation genetic diagnosis by polar body removal: the first 109 infants. Pediatrics 2000; 106: 650-3

I 1.

Hardy J, Martin KL, Leese HJ, Winston RM, Handyside AH. Human preimplantation develop­ ment in vitro is not adversely affected by biopsy at the 8-cell stage. Hum Reprod 1990;5:708-14

12.

Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340:17

Kuliev A, Verlinsky Y. Current features of preimplan­ tation genetic diagnosis. Reprod BioMed Online 2002;5:296-301

13.

Palermo G. Assisted fertilization by intracytoplasmic sperm injection (ICSI). In Veeck LL, ed. An Atlas of Human Gametes and Conceptuses. Carnforth, UK: Parthenon Publishing Group 1999:76-85

Verlinsky Y, Cohpn J, Munne S, et al. Over a decade of preimplantation genetic diagnosis experience - a multi-center report. Fertil Steril 2004;82:292-4

14.

Muggleton-Harris AL, Glazier AM, Pickering S, Wall M. Genetic diagnosis using PCR and FISH analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos. Hum Reprod 1995; 10:183

15.

McArthur S, Marshall J, Wright D, de Boer K. Successful pregnancies following blastocyst (day 5) biopsy and analysis for reciprocal and Robertsonial translocations. Fifth International Symposium on Preimplantation Genetics, 5-7 lune 2003, Antalya, Turkey:33

BLASTOCYST BIOPSY With the current tendency for blastocyst transfer, there has been a renewed interest in the develop­ m ent o f m ethods for blastocyst biopsy14, which has recently resulted in successful PG D cycles perform ed by blastocyst biopsy for translocations, yielding ongoing clinical pregnancies15. The proce­ dure o f blastocyst biopsy is dem onstrated in Figure 2.14. A lthough blastocyst biopsy is not yet a m ethod o f choice in many centers, its potential is obvious, especially for additional testing required to confirm the polar body or blastom ere diagnosis.

REFERENCES 1.

Cohen

J,

Maker

H,

Talansky

B,

Grifo

L.

Micromanipulation of Gametes and Embryos. New

York: Raven Press, 1992 2.

3.

4.

5.

22

Verlinsky Y, Cieslak J, Evsikov S. Techniques for micromanipulation and biopsy of human gametes and preembryos. In Verlinsky Y, Kuliev A, eds. Preimplantation Genetics. New York: Plenum Press, 1991 ;273—8 Verlinsky Y, Cieslak J. Embryological and technical aspects of preimplantation genetic diagnosis. In Verlinsky Y, Kuliev A, eds. Preimplantation Diagnosis

3

Nuclear transfer techniques for preim plantation diagnos s and prospect for artificial gam ete form ation

VISUALIZATION OF POLAR BODY A N D BLASTOMERE CHROMOSOMES The possibility of reprogramming of the donor cell nucleus by the recipient oocyte makes current devel­ opments in nuclear transfer (NT) techniques useful for improvement o f the methods for preimplantation genetic diagnosis (PGD). For example, it has become possible to convert interphase nuclei of single blas­ tomeres and the second polar body (PB2) into metaphasc, to perform PGD by full karyotyping (Figure 3.1; see description below). This is an essen­ tial improvement, because the current fluorescence in situ hybridization (FISH) technique allows chro­ mosomes to be enumerated on interphase cell nuclei, with the number of chromosomes studied being limited to the number of chromosome-specific probes available. Even with the currently available methods for re-hybridization of interphase nuclei for the second and the third time, complete karyotyping will not be realistic in the near future. In contrast to PBI, which consists of metaphase chromosomes and may be analyzed using whole chromosome-specific fluorescence probes, PB2 forms a nucleus and never transforms into metaphase. We have developed various techniques for converting the PB2 into the metaphase stage. One o f these approaches involved electrofusion of the mouse PB2 with intact and/or enucleated mouse zygotes, and resulted in PB2 nucleus transformation into the metaphase plate in 34% o f cases. The same results were obtained by electrofusion of the PB2 with a foreign one-cell mouse embryo, with the proportion of metaphase plates reaching 65% when the recipient one-cell stage mouse embryo was enucleated1. The other approach involved the treat­

ment o f the one-cell stage mouse embryos with okadaic acid (a specific inhibitor of phosphates 1 and 2A), leading to visualization o f PB2 chromosomes in up to 80% of cases2. The visualized PB2 chromo­ somes were unichromatid G i premature condensed chromosomes of good quality, suitable for differen­ tial staining. However, in contrast to the mouse data, okadaic acid treatment of human PB2 led to further condensation of already pycnotic PB2 nuclei, so we developed a special technique to convert PB2 into metaphase chromosomes. The technique for removal of PB2 was described in a previous chapter, with the only modification here that the biopsy is performed in a medium without sucrose. The PB2 is introduced into oocyte cytoplasts (see Figure 3.2), which are usually obtained by enucleation of metaphase II oocytes, which remained unfertilized after in vitro fertiliza­ tion (IVF) or intracytoplasmic sperm injection (ICSI), or of those metaphase II oocytes that were matured for 24-48 h in vitro from immature oocytes. Prior to enucleation, these ooplast recipients were incubated for 10-15 min in medium with 1 }ig/mi cytochalasin D, 0.3pg/m l nocodazole and 0.5pg/m l Hoechst 33342. As shown in the procedure steps for oocyte enucleation (Figure 3.3), PBI is removed together with the nucleus of th£ oocytes. Because the oocytes with failure of fertilization are used, special attention is paid to remove both the meiotic metaphase II spindle and the sperm chromosomes. The enucleated oocytes are then washed and trans­ ferred into culture medium for at least 1 h to recover, before PB2 injection, which is essentially the same as ICSI. The pipette for intracytoplasmic PB2 injection is prepared in the same way as for biopsy tools, except for modification of the very tip of the needle,

23

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

which is broken using a microforge. The resulting pipette has a tip broken perpendicularly, without any irregularities and with an inner diameter of 7-10 pm. In contrast to the biopsy tools, these pipettes arc not flame-polished. The same microforge is used to bend the tool to the desired angle. Prior to use, the micropipettes are treated with non-ionic detergent (NP10). The procedure of intracytoplasmic PB2 injection is shown in Figures 3.4 and 3.5. PB2 is aspirated into an injection pipette, with care taken that the PB2 plasma membrane is broken, and the pipette is brought into the perivitelline space of the recipient cytoplast through the partial zona dissection (PZD) slit made during oocytc cnucleation, and moved into the center of the cytoplast. Cytoplasm is aspirated into the pipette until the plasma membrane is broken, and the PB2 nucleus is expelled into the cytoplast (Figures 3.4 and 3.5). The reconstructed haploid embryos are cultured in standard medium for at least 1 h. Oocyte activation is performed by electrofusion, with the use of custom-made electrostimulation apparatus (XRO NO S, Chicago, IL, USA; Figure 3.6) in a fusion chamber consisting o f two platinum wire electrodes glued to the bottom o f a glass dish with a gap of 0.33 mm (Figures 3.4e and 3.5d). Electrofusion medium consists o f 0.3 mol/1 mannitol, 0.1 mmol/1 M gS 0 4 , 0.05 mmol/1 CaC b'and 0.5% polyvinylpyrrolidone, dissolved in HPLC-grade water. The pH of the medium is adjusted to 7.4 by titration with 0.1 mol/1 NaOH. The reconstructed embryos are checked every 30 min starting at 24 h after activation, and fixed 45 min after the disappearance of PB2 pronuclei (Figure 3 7). Alternatively, premature chromosome condensation of the resulting PB2 pronuclei in the reconstructed embryos is induced by 1-h exposure to okadaic acid (5-10pg/m l). To prevent embryo cyto­ plasm fragmentation, okadaic acid is diluted to the working concentration with phosphate-buffered saline containing 3 mg/ml bovine serum albumin and 0.5pg/m l cytochalasin D. Hypotonic treatment of the embryos is avoided, to prevent overspreading of chromosomes. Because there is also no need to improve the spreading of chromosomes during fixa­ tion, the embryos transferred from the fixative to the slide are simply left to dry out. The sequence of PB2 nuclear transformation in foreign cytoplasm from the very beginning of its injection to the first mitotic division o f the recon­

24

structed embryo and premature chromosome condensation by okadaic acid is presented in Figures 3.7 and 3.8. As can be seen from immunocytochemical study, as early as 2 h after injection, tubulin microfilaments, initially present only around the PB2 nucleus, start to elongate into the cytoplasm, eventu­ ally forming the metaphase spindle of PB2 (Figure 3.8). The whole procedure is usually completed within 2 days of PB2 removal, and so can be realized well before the embryo transfer. The procedure is started by PB2 removal a few hours after oocyte exposure to sperm or ICSI, followed by its injection into enucle­ ated oocytes, and electrofusion. The following day the embryos without pronuclei are fixed or exposed to okadaic acid prior to fixation, should the PB2 pronucleus still be present. Initially, 18 of 34 recon­ structed embryos were activated1, demonstrating the efficiency o f the method for karyotyping of the PB2, which has also been applied for the PGD of translo­ cations. (Karyotype of PB2 obtained through the above procedure is presented in Figure 3.9). The same principle is used for visualization of individual blastomeres (Figures 3.1 and 3.10). Initially individual blastomere nuclei were trans­ formed into metaphase chromosomes through blas­ tomere fusion with enucleated human oocytes (Figure 3 .11)4. Although metaphases were obtained from 23 of 38 blastomeres treated by this method, its efficiency was not high enough to be applicable for PGD. This was due to inability of a replicating nucleus to form metaphase chromosomes after induction o f premature chromosome condensation (PCC). However, because biopsied blastomeres may be at any stage of the cell cycle at the time of biopsy, the timing of mitosis of the blastomere nucleus should be controlled; this can be achieved by intro­ duction of the blastomere into the cytoplasm of a cell at a known cell cycle. To achieve such repro­ gramming, the individual blastomeres are fused with intact or enucleated mouse zygotes at the pronuclear stage, known to be at the S phase of the cell cycle (Figure 3.12). Frozen mouse zygotes, from Charles River Laboratories (Wilmington, MA), may be used as recipient cytoplast to induce conversion of the blastomere nucleus into metaphase. Although initially mouse sdygotes were enucleated, there is no need for this step for most of the cases, as mouse and human chromosomes may be clearly distinguished.

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Blastomere biopsy is performed in the same way as described in Chapter 2, except that only intact blastomeres are chosen, with a clearly visible nucleus. Also, several precautions have to be taken to ensure the integrity o f the blastomere plasma membrane during the biopsy procedure. Although intact blastomeres may be inserted microsurgically into the perivitelline space, this has appeared to be traumatic and is replaced by blastomere-zygote agglutination with phytohemagglutinin (Irvine; Scientific, Santa Ana, CA). Before the procedure, the thawed mouse zygotes are freed of zonae pellucidae with acidic Tyrode’s solution and pipetted through the flame-polished Pasteur pipettes with an internal diameter of 80 pm to separate the PB2. Using the flame-polished Pasteur pipettes with internal diame­ ter of 100 pm, blastomere-zygote pairs are brought together and agglutinated in 300pg/m l of phyto­ hemagglutinin in protein-free human tubal fluid buffered with 20 mmol/1 of HEPES in a four-well plastic dish (Nunc) (Figure 3.12a). Electrofusion is induced in the same way as mentioned in the above section, except for substitu­ tion of 0.5% polyvinylpyrrolidone in the electrofu­ sion medium by 0.1% with molecular weight 36000 0 (kd). Blastomere-zygote pairs are oriented between electrodes by hand, with the final orienta­ tion achieved with alternating current (500 kHz; 0.2kV /cm for 2 s). Cell fusion is induced with a single direct current pulse (1 kV/cm for 500 ps), and the results are assessed in 2 0 min (Figure 3.12b). When human blastomeres are fused with intact mouse zygotes, the heterokaryons entering mitosis are identified under the dissecting microscope (Figure 3.12c-e). Because of the transparency of mouse cytoplasm, the disappearance of pronuclei and the formation of the joint metaphase plate are clearly visible. The heterokaryons with a persisting pronucleus are exposed for 1 h to 5 pmol/1 of okadaic acid in phosphate-buffered saline containing 3 mg/ml of bovine serum albumin and 0.5 pg/ml of cytochalasin D (Figure 3.12f). After 10-15 min of incubation in a hypotonic solution (0.1% sodium citrate and 0.6% bovine serum albuminj the result­ ing mitotic heterokaryons are fixed in a cold 3:1 solution of methanol and acetic acid in a four-well plastic dish. When the cytoplasm clears, heterokaryons are transferred onto slides and airdried. Chromosome plates are assessed by phase contrast (Figure 3.13a) and then used for standard chromosome analysis (Figure 3.14). For FISH analy­

sis the slides are pretreated with formaldehyde and pepsin (Abbott Inc., Downers Grove, IL) (Figure 3.13b). Although the overall success rate of the proce­ dure is as high as 83% (Figure 3.15), its efficiency can be further improved with experience5. Similar results were obtained by using bovine ooplasts for fusion with human blastomeres6. Our data showed that some of the failures were simply due to the absence of the nucleus in biopsied blastomeres, or because the heterokaryons were fixed after they had already cleaved. It is also useful to perform blas­ tomere biopsy no earlier than day 3 or day 4, to avoid the biopsy of two- and four-cell embryos, leading to accelerated heterokaryon cleavage. However, the success rate did not depend on whether mouse zygotes, were enucleated before fusion with blas­ tomeres. This allows simplification of the procedure by using intact mouse zygotes. The procedure is quite simple and includes the following components. Mouse zygotes are thawed and freed of zonae pellucidae and PB2 1-2 h before electrofusion with human blastomeres. Four hours after fusion, heterokaryons are monitored for signs of the disappearance of pronuclei, and fixed at mitosis following hypotonic treatment. To avoid monitoring and a possible missing of mitosis, the heterokaryons may be cultured in the presence of microtubuli inhibitors, vinblastine or podophyllotoxin. All the embryos left in the culture by the 9th hour after fusion are fixed following 1 h of pretreatment with okadaic acid. This method has been applied for PGD of pater­ nally derived reciprocal translocations and for confir­ mation of the PGD of chromosomal abnormalities performed by PBI and PB2 FISH analysis with a success rate of 83% (Figure 3.15). With the current success rate of blastomere nucleus conversion into metaphase, the method was applied to 89 clinical cycles for translocation carriers, resulting in the transfer of balanced or normal embryos in 68 of them, which yielded 22 unaffected pregnancies (see Chapter 5). It has recently been reported that the blastomere metaphase can be obtained without the application of a specific conversion method7. To obtain analyzable chromosomes, the embryos were monitored closely from the second day after ICSI, in order to identify the blastomere with nuclear breakdown, which was biopsied and fixed within 1 h. This method is now modified by employing a 1-h culture

25

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

o f the biopsied blastomere in medium containing vinblastine, which results in a harvest of mPtaphase chromosomes of good quality (see Figure 3.16)

SPERM DUPLICATION As seen above, the genetic composition of oocytes may reliably be tested through removal and exami­ nation of PBI and PB2. However, no method is as yet available for testing the outcome of male meiosis, because genetic analysis destroys the sperm, making it useless for fertilization. To overcome this problem, a new technique has been adopted, allowing dupli­ cation o f a sperm prior to genetic analysis, so that one of the duplicated sperms can be used for testing and the other for fertilization and consequent trans­ fer of the resulting embryo, provided that the genetic analysis o f the corresponding duplicate shows a normal genotype8. To demonstrate the reliability of the technique, over 100 human sperm from chromosomally normal donors, as well as from translocation carriers, were injected into enucleated mouse oocytes, and the duplicated cells resulting from an overnight culture were tested by FISH to compare the chromosomal status of both daughter cells. All but 3% of the haploid cell pairs derived from the normal donors were identical for the chromosomes tested, while, as expected, a high proportion of the paired nuclei derived from the sperm of transloca­ tion carriers were chromosomally unbalanced, suggesting that ooplasm from mature mouse eggs can support the faithful replication of any human sperm genome, irrespective o f the genotype. A similar technique has been developed to dupli­ cate human sperm using human oocytes (Figure 3.17), showing that the duplication of sperm may be performed (Figure 3.18) faithfully in only one-half o f cases, in contrast to when using murine oocytes (Figures 3.19 and 3.20); thus, the technique requires further testing before being applied clinically, with expected important practical implications for PGD of paternally derived conditions, such as transloca­ tions, which are known to produce as much as 7086 abnormal sperm on average. The technique also has potential for research purposes, as shown in preliminary work devoted to the study of mosaicism9. Following duplication of human sperm in cow oocytes, a series of 31 resulting embryos were cultured up to the eight-cell stage, and tested by probes specific to chromosomes 13, 16, 18, 21 and 22. In all, 16% o f the resulting sperm dupli­

26

cates appeared not to be identical, which may further be related to the genetic differences between the donors involved. In fact, one of the three sperm donors for the above experiment produced mostly mosaic embryos in two PGD cycles. However, the rate o f mosaicism in sperm duplicates of the three donors involved in this small series was similar, indi­ cating that the generation of mosaic embryos, at least in the patients previously tested by PGD, may not be related to sperm genotype, but to the sperm centrosom e10.

DEVELOPMENT OF ARTIFICIAL HUMAN GAMETES IN VITRO One recent development in micromanipulation and nuclear transfer has been the progress in the construction of artificial human gametes. Attempts were undertaken to create female and male gametes, both demonstrating strong morphological CSidence for haploidization11-13. However, insufficient cytoge­ netic evidence for haploidization has been presented, which would also be required to ensure the normalcy of the resulting gametes, derived from the somatic cell nuclear transfer into the matured oocytes. The technique is based on inducing nuclei of mitotic somatic cells to skip S-phase of the cell cycle and undergo haploidization when introduced into oocytes, thus allowing artificial gametes to be obtained from somatic cells through the process of haploidization. We have shown that the efficiency of haploidization of donor cumulus cell nuclei differs depending on the stage o f development of the enucleated recipient oocyte. This may be tested using the extruded polar bodies (PB), or generated pronulei, which also allow investigation of thp correctness of chromosomal segregation. As seen from the flow chart (Figures 3.21a,b and 3.22a,b) the first step involves enucleation of in vitro matured metaphase II oocytes under control of UV-luminescence, which is important to ensure the accuracy of chromosomal analysis of the resulting haploid nuclei. Then the cumulus cell nuclei, which are at Go of the cell Cycle, are introduced into ooplasts by injection (Figures 3.21c, d and 3.22c, d) and the oocytes arc activated by electrostimulation delivered by the elec­ trofusion device (X R O N O S (Chicago, IL) (Figure 3.21e, f). Following the oocyte activation the chro­ mosomes of the transferred nuclei segregate with the extrusion of polar bodies (Figure 3 .2 If), or forming

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

two pronudei (Figures 3.21e, 3.22e, f and 3.23) both evidencing the formation of artificial gametes through somatic cell haploidization. Although the resulting pronuclei have similar morphology to pronuclei resulting from natural fertilization (Figures 3.23 and 3.24b), spacial differences were observed, with the pronuclei derived from natural fertilization migrating from the periphery to the center of the oocyte (Figure 3.24a), in contrast to the artificial pronuclei, which were closely positioned to each other from the very beginning, and remain in the same position, despite growing in volume (Figures 3.23 and 3.24b). FISH analysis (Figure 3.25) and DNA fingerprinting (Figure 3.26) of PBI and pronu­ clei resulting from the haploidization procedure showed the haploid chromosomal set, with the resulting D N A originating from the donor nuclei, but up to 90% of these haploid nuclei appeared to have chromosomal aneuploidies (Figure 3.27). This suggests that the: use o f the resulting haploid nuclei in gamete reconstruction procedures may not be acceptable at the present time. To determine whether incubation o f nuclei in ooplast improves chromosomal segregation two groups of a total o f 122 reconstructed metaphase II oocytes were studied, one activated 5-7 h after nuclear transfer, and the other after 12-21 h. A higher activation rate in response to parthenogenetic stimulation was observed in the latter group, suggest­ ing the need for a longer incubation period. An aneu­ ploidy rate as high as approximately 90% was observed irrespective of incubation time, with the majority being of complex nature, suggesting that prolonged incubation would not improve the accu­ racy o f chromosomal segregation. Preliminary results suggest more accurate chromosomal segregation when. PBI and pronuclcus were formed. The data show that although haploidization o f somatic cclls may be achieved using metaphase II oocyte cyto­ plasm, the aneuploidy rate is much higher than in normal meiosis. Our preliminary data show that the aneuploidy rate in haploidization may be improved by substitut­ ing recipient metaphase II oocytes with immature oocytes o f metaphase I stage. This experiment was performed by the electrofusion o f the G 2 human fibroblast nuclei with metaphase I oocytes (Figure 3.28). Twelve hours following maturation two PB ls were extruded (Figure 3.29), one originating from the recipient nucleus, and the other from the donor fibroblast, with the overall efficiency of human

metaphase I oocytes to haploidize the chromosome set of the G 2 somatic cells as high as 80%. According to preliminary FISH lesults, the aneuploidy rate in the oocytes producing two metaphase II metaphases and two P B ls was comparable to control. The available data, therefore, show that despite previous hopes of using nuclear transfer technology to produce female and male gametes through haploidization of somatic cells, the majority of the resulting haploid cells are chromosomally abnormal. Therefore, despite the feasibility of somatic cell haploidization by the use of the metaphase II oocyte cytoplasm, its clinical use cannot be considered at the present time.

REFERENCES 1.

Verlinsky Y, Dozortzev D, Evsikov S, Visualization and cytogenetic analysis of second polar body chro­ mosomes following its fusion with one-cell mouse embryo. J Assist Reprod Genet 1996; 11:123-31

2.

Dyban A, De Sutter P, Verlinsky Y. Okadaic acid induces premature chromosome condensation reflecting the cell cycle progression in one-cell stage mouse embryos. Mol Reprod Dev 1993;34.403-15

3.

Verlinsky Y, Evsikov S. Karyotyping, of human oocytes by chromosomal analysis of the second polar body. Mol Hum Reprod 1999; 5:89-9 5

4.

Evsikov S, Verlinsky Y. Visualization of chromosomes in single human blastomeres. J Assist Reprod Genet 1999;16:133-7

5.

Verlinsky Y, Evsikov S. A simplified and efficient method for obtaining metaphase chromosomes from individual human blastomeres. Fertil Steril 1999;72: 1-6

6.

Willadsen S, Levron J, Munne S, et al. Rapid visual­ ization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Hum Reprod 1999;14:470-4

7.

Tanaka A, Nagayoshi M, Awata S, et al. Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a balstomere biopsied from 4-6 cell stage embryo. Fertil Steril 2004;81:30-4

8.

Willadsen S, Munne S, Schmmel T, Cohen J. Applications of nuclear sperm duplication. Fifth International Symposium on Preimplantation Genetics, 5-7 June 2003 , Antalya, Turkey:35

9.

Munne S, Willadsen S, Schmmel T, Cohen J. Nuclear sperm duplication as a tool to study mosaicism. Fifth

27

ATLAS OF PR EIM PLA N TA TION GENETIC D IAG N O S IS

International Symposium on Preimplantation Genetics, 5-7 June 2003, Antalya, Turkey:55-6 10.

11.

28

Silber S, Sadowy S, Lehahan K, et al. High rate of chromosome mosaicism but not aneuploidy in embryos from karyotypically normal men requiring TESE. Reprod BioMed Online 2002;4(Suppl 2):20 Lacham-Kaplan O, Daniels R, Trounson A. Fertilization of mouse oocytes using somatic cells as

male germ cells. Reprod BioMed Online 2001;3: 205-11 12.

Tesarik T, Mendoza C. Somatic cell haploidization: an update. Reprod BioMed Online 2003;6:60-5

13.

Galat V, Ozen S, Rechitsky L, Verlinsky Y. Is haploidization by human mature oocytes real? Fifth International Symposium on Preimplantation Genetics, 5-7 June 2003, Antalya, Turkey:36-7

4 Preim plantation diagnosis for aneuploidies

INTRODUCTION Although preimplantation genetic diagnosis (PGD) was initially introduced for pre-existing genetic conditions, its application appears to be of particular relevance for sporadic conditions, such as chromoso­ mal abnormalities, which contribute significantly to pregnancy loss and infertility. At least three-quarters o f all PGDs have been performed for age-related aneuploidies, resulting in the birth o f almost 1000 healthy children. PGD is currently performed by two main approaches that involve the testing of either blastomeres biopsied from the cleaving embryos, or polar bodies removed from matured and fertilized oocytes, combined with fluorescent in situ hybridization (FISH) analysis. As described in Chapter 2, embryo biopsy can be performed as early as the 6-8-cell stage and enables testing for both maternally and paternally derived genetic abnormalities. First and second polar body (PBI and PB2) removal (see Chapter 2), on the other hand, allows testing exclusively for maternally derived abnormalities, as can be seen from the schematic presentation of the possible errors in meiosis I and meiosis II shown in Figure 4.1. Each of the two approaches has advantages and disadvan­ tages, and are applied depending on the clinical circumstances. For example, despite a reduction in embryo cell number, which may have an influence on the embryo viability, embryo biopsy is performed for paternally derived chromosomal abnormalities, as well as for gender determination. However, removal of PBI and PB2, naturally extruded from oocytes in the process of maturation and fertilization, respec­ tively, should not have any effect on the embryo viability, although it provides no information on

gender and paternal meiotic errors. The particular value of PB testing for PGD of aneuploidies is obvious from the fact that over 90% of chromosomal errors originate from maternal meiosis. This approach is also of importance owing to a relatively high rate of mosaicism at the cleavage stage, which is recognized as the major limitation of blastomerebased PGD for chromosomal disorders (see below ). PGD for aneuploidies is currently performed by FISH analysis, using commercially available chromo­ some-specific probes (Abbott, Downers Groves, IL, USA). FISH analysis was first applied in 1991 for gender determination using D N A probes specific for either the X or Y chromosome1. Since testing for only one o f the sex chromosomes could lead to misdiagnosis of gender due to a possible failure of hybridization, a dual FISH was introduced, involving the simultaneous detection of X and Y, each with a different color2. Additionally, the dual FISH analysis was combined with a ploidy assessment to improve the accuracy, by adding a centromeric probe specific for chromosome 183,4. Testing was then extended to up to five autosomes, including chromosomes 13, 16, 18, 21 and 22 (Figures 4.2 and 4.3), although it is currently possible to analyze up to a dozen chromo­ somes, using additional rounds of re-hybridization (Figures 4 .4 -4 .7 )5-7. The overall experience of preimplantation FISH analysis currently involves approximately 5000 clin­ ical cycles, resulting in an improved pregnancy rate in poor-prognosis in vitro fertilization (IVF) patients8-11. Approximately half of these cycles were performed by FISH analysis of blastomeres and half by FISH analysis of PBI and PB2, resulting in hundreds of unaffected pregnancies and healthy chil­ dren being born to date. The follow-up confirmation

29

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

studies o f the preselected abnormal embryos, and the babies born following the procedure, demonstrated an acceptable accuracy o f the FISH analysis, which is described below. Examples of normal FISH patterns in PB 1, PB2 and blastomeres are presented in Figures 4.2 and 4.3 for five chromosomes, and Figures 4.4 and 4.5 for seven or nine chromosomes. The reliability o f the FISH technique for aneu­ ploidy detection in blastomeres has been extensively studied12-15. By comparing the FISH results in the cleaving embryos to morphological abnormalities and maternal age, it was established that the observed chromosomal abnormalities were not related to the limitations of the FISH technique, but were due to the: embryo variables. A high rate of mosaicism was observed at the cleavage stage, which was initially suggested to be particularly high in slow embryos exhibiting an arrested development. Furthermore, the accumulated experience showed that up to half of all cleaving embryos are mosaic (Figures 4.6, 4.8 and 4.9), representing a common feature of preimplantation embryo develop­ ment13-17. Mosaicism currently represents a major limitation of the FISH analysis of aneuploidies, performed at the cleavage stage, which may obvi­ ously affect the diagnostic accuracy of PGD, result­ ing in the possible transfer of an affected embryo, one or a few cells of which were found to be normal due to mosaicism. Conversely, when the embryo is erroneously diagnosed as abnormal, while it is actu­ ally normal, the perfectly normal embryo may be untransferred, compromising the chances o f the patient to become pregnant. It has recently been shown that mosaicism also presents diagnostic prob­ lems at the blastocyst stage (Figures 4.6 and 4 .8 )18, involving also inner mass cells, despite the initial prediction that the abnormal cells are deviated mainly to trophectoderm. As shown in Figures 4.6, 4.8 and 4.10-4.13, even embryos with monosomies, including those with double monosomies, are able to reach the blastocyst stage, comparable to trisomies (Figure 4.14). The first attempt to use FISH analysis for testing PBI was undertaken in 199419"21. In this work, 130 unfertilized metaphase II oocytes were tested simul­ taneously with their PB 1, using X chromosome- and chromosome 18-specific probes. It was demonstrated that PBI FISH data allow an exact prediction of the chromosome set in the corresponding oocytes. Each chromosome in PBI was represented by double dots (signals), corresponding to two chromatids in each

30

univalent (Figure 4.2). The data suggested that the number of signals (chromatids) in PBI reliably predicts the corresponding number of signals (chro­ matids) in the metaphase II oocytes, therefore providing an excellent tool for the genetic pre­ selection of oocytes. It was also of interest that, in addition to a normal distribution of signals in PBI and the corresponding metaphase II oocytes, meiotic errors were also detected, confirming the accuracy of PBI diagnosis for predicting the genotype of the corresponding oGcyte. For example, in one PBI four signals for chromosome 18 were detected, perfectly in accordance with the lack of the chromosome 18 signals in the corresponding metaphase II oocyte (chromosome 18 non-disjunction). This suggested that the chiomosomal complements of the oocyte could be inferred from the testing of P B I, which can be removed following its extrusion from the mature oocyte, with no potential influence on the embryo viability. Another interesting phenomenon was the observation of chromatid malsegregation as a possi­ ble cause of chromosomal aneuploidy in the result­ ing mature oocytes (see schematic presentation of possible errors in meiosis in Figure 4.1). In four oocytes, instead of the expected two signals, three were found in the metaphase II oocytes, which perfectly corresponded to a single signal in the corre­ sponding PB 1 (example of the error detected by PB 1 testing, 'leading to trisomy or monosomy 21, are shown in Figures 4.15 and 4.16). Similar results haw been reported by another group, confirming the diagnostic significance of PBI FISH analysis for predicting the genotype of the preimplantation embryo22. However, it bccame clear from the very begin­ ning of the application of PB 1 testing, that the result­ ing genotype of the oocyte cannot be inferred without testing the outcome o f the second meiotic division, by testing the PB2, extruded following fertilization of the oocyte.23 It was shown that in contrast to the paired dots in PB 1,:each chromosome in PB2 was represented by a single dot (Figure 4.2), so the lack or addition o f a signal for a particular chromosome provided evidence of a second meiotic division error. Although only 19 of 55 oocytes in this first study were tested by both PBI and PB2, evidence for a possible error in both PBI and PB2 was presented. These data suggested that some oocytes selected as normal, based on the PBI FISH analysis, could still appear to be abnormal following non-disjunction in the second meiotic division

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

(Figures 4.17 and 4.18). Therefore, FISH analysis for both PB1 and PB2 has become the basic requirement for PGD of aneuploidies, which allows detection of errors in both the first and second meiotic divisions (Figures 4.19-4.25). Currently, more than 10000 oocytes have been tested by FISH analysis, demonstrating the accuracy and reliability of PB1 and PB2 testing for predicting the karyotype of the corresponding oocyte and resulting embryo. The data demonstrated a greater than 50% aneuploidy rate in oocytes from IVF patients of advanced maternal age24-29, resulting from the errors in both the first and second meiotic divisions, in contrast to the previously accepted concept that aneuploidies mainly originate from meiosis I (see below).

P R EP A R A T IO N OF P O L A R B O D IE S A N D B L A S T O M E R E S FO R F L U O R E SC E N C E IN SITU H Y B R ID IZ A T IO N A N A L Y S IS

procedure for preparation of polar bodies is outlined below. (1)

Fresh fixative (methanol and glacial acetic acid, 3:1) is prepared in a 50-ml culture flask and stored in the freezer until needed.

(2)

Micropipettes are pulled over the flame of a microtorch to obtain an attenuated tip with an inner diameter o f approximately 50-70 |im, as a larger diameter may lead to a potential PB loss.

(3)

Precleaned glass slides are cleaned again as is the hanging-drop slide, using fixative to remove any grease or dirt, which may be on the slide. This is achieved by dropping a few drops of fixative and wiping with a lint-free tissue.

(4)

The hanging drop slide is filled with HPLCgrade H 2 O using a Pasteur pipette.

(5)

After oocytes/zygotes have been manipulated and PB removal has been accomplished, the PB(s) can be found in the micromanipulation dish in a 15-20 (J.1 drop of culture medium under oil using an inverted microscope with phase-contrast optics (xlO and x20 objectives).

Preparation of P B I and PB2 Materials and reagents for preparation of PB1, PB2 and blastomeres are presented in Tables 1-3. The

Table I Equipment for fluorescence in situ hybridization (FISH) analysis of polar bodies and blastomeres Microscope (Nikon Microphot F X A ) with epifluorescence attachment, 100-W mercury lamp, phase-contrast optics with xlO objective, oil immersion objectives x63 and x 100 Single-band pass filters (Chrom a Technologies) for all fluorophores: D A P I (blue), FIT C (green), T R IT C (red), A Q U A (aqua), F I5 (yellow) and F6 (blue) Dual bandpass filters - FITC/TRITC (red and green) and Aqua / Blue C C D camera, filter wheel and computer with imaging software (Quips Imaging Workstation, Applied Imaging, Santa Clara, C A ) Inverted microscope with phase-contrast optics, x 10 and x2 0 objectives (Olym pus C K 40, Olym pus America) Stereomicroscope Slide w arm er (capable of consistent temperature, i.e. 68-73°C ) A ir incubator (set at 37°C) W ater bath (capable of maintaining temperatures up to 100°C) Microcentrifuge Vortex Calibrated thermometers (for slide warmer, incubator and water bath) pH meter and standards Micropipettors (2 -20 jxl, 20—200 (j.1) and appropriate sterile tips Microtorch (Blazer Corporation G B 2001) HYBrite denaturation/hybridization system (Vysis 30-144010)

31

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Table 2 Materials fo r fluorescence in situ hybridization (FISH) analysis o f polar bodies and blastomeres Supplier

Materials and reagents

Catalog number

M icroscope ( 2 5 x 7 5 x I-m m ; frosted precleaned slides

Fisher

12-550-14

D rum m o n d I —5 (al pipettes

Fisher

21-176-2A

D rum m o n d 2 5 -5 0

Fisher

2 I- I7 6 - 2 D

(J I

pipettes

5 3/4-inch Pasteur pipettes

Fisher

13-678-20A

35 x I Om m Petri dishes (Falcon 1008)

Fisher

0 8 -7 5 7 -100A

50-m l culture flask (Falcon 353014)

Fisher

10 - 12 6 - 1B

C oplin jars (12)

Fisher

0 8 -8 17

4 glass dishes w ith rem ovable tra y

Fisher

0 8 -8 12

Hanging d rop slides

Fisher

12-560

C arbide m a rker fo r engraving glass

Fisher

13-378

Fisher

12 -5 3 1A

2 2 x 3 0 -m m coverslips (C orning)

M idw est Scientific

TT05B

Forceps (fine tip )

Fisher

0 8-953E

Parafilm

Fisher

13-374-10

Sigma

A -5 177

Fisher

14-085

M icrocentrifuge tubes

Graduated cylinders and serological pipettes M outhpiece w ith tubing to hold m icrop ipe tte Lead pencil and fine point perm anent m arker Rubber bulb fo r b lo w drying

(6)

Once located, using the micropipette, a small amount of water is aspirated to ensure proper control of fluid in and out of the pipette.

(7)

After aspiration o f a small amount of water, the PB is aspirated into the pipette and brought to the hanging drop slide containing water. The PB is released from the pipette by blowing gently until it is visualized in the center circle o f the hanging-drop slide. Do not allow the polar body to settle and stick to the glass surface of the hanging-drop slide.

(8)

(9)

32

Once rinsed in the water, the PB is again aspi­ rated into the pipette. Any oil surrounding the pipette usually is removed by going through the water. The PB is transferred to a clean slide and a drop of water containing the PB is released from the pipette by gently blowing. After partial evaporation of the water, the PB will swell and begin to flatten out onto the slide. Just at the time o f complete drying of the water, a drop of fixative is placed on top o f the PB from the same micropipette to obtain spreading o f the chromatin.

(10] Prior to complete drying of the fixative, another drop of fixative is placed on top of the polar body and this is repeated if necessary until the cytoplasm dissolves. Careful attention is paid to observing this process and to removing thor­ oughly all cytoplasm. (11J The location of the chromatin is defined by encircling using a carbide marker, taking care to engrave the glass lightly to avoid interference from glass fragments. A second heavier circle is engraved on the bottom of the slide for easy location of the smaller circle and thus the chro­ matin at the time of analysis. (12) The slide is marked with the patient’s name and the corresponding oocyte number, along with pertinent information such as the number of pieces of chromatin. (13) Additional drops of fixative are added to the slide and blown dry using a rubber bulb. (14) Slides can be stored at room temperature overnight before hybridization or if hybridized immediately, a pretreatment method is recom-

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

Table 3 Reagents fo r fluorescence in situ hybridization (FISH) analysis o f polar bodies and blastomeres M aterials and reagents

Supplier

Catalog number

MultiVysion PB Panel (chromosomes 13, 16, 18, 21 and 22)

Vysis

32-131085

MultiVysion PGT (chromosomes X , Y, 13, 18 and 21)

Vysis

32-131080

MultiVysion 4 C o lor Custom (chromosomes X ,Y, 17 and 15)

Vysis

32-131086

CEP X ( D X Z I , alpha satellite) spectrum green

Vysis

32-132023

CEP Y ( D Y Z I, satellite) spectrum aqua

Vysis

32-131024

CEP 17 ( D 1721) spectrum aqua

Vysis

32-131017

CEP 15 ( D 15 Z 1) spectrum green

Vysis

32-182015

Sub-telomeric 13q spectrum orange

Vysis

33-260013

Cytocell

LPTI3qg

Sub-telomeric I6q spectrum orange

Vysis

33-260016

Sub-telomeric 16p spectrum green

Vysis

33-252016

Sub-telomeric 18q spectrum orange

Vysis

33-260018

Sub-telomeric 21 q spectrum orange

Vysis

33-260021

Sub-telomeric 22q spectrum orange

Vysis

33-260022

Sub-telomeric Xp/Yp spectrum green

Vysis

33-252023

Vector Labs

H - 1000

DAPI counterstain

Sigma

D-1388

Glacial acetic acid

Sigma

A-6283

Methanol

Sigma

M-3641

Scientific Products

6795-1

Bovine serum albumin

Sigma

A- 331 1

Sodium citrate

Fisher

S279-500

25-mg vials o f pepsin

Vysis

30-806001

Magnesium chloride

Sigma

M-2393

Irvine Scientific

9235

Sub-telomeric 13q (green)

Vectashield antifade

HPLC H 20 (M allinckrodt Baker Inc.)

Phosphate buffered saline Neutral buffered form alin (10% )

VWR

3239-4

20 x SSC

Vysis

32-804850

Ethanol series (70, 85 and 100%'

Fisher

A 407-4

NP-40

Vysis

32-804818

Immersion oil (fluorescence)

Fisher

12-371-IB

Sodium hydroxide solution (1 mol/1)

Sigma

930-65

Hydrochloric acid (2 mol/1)

Sigma

251-2

m ended to include exposure in 2 x S S C at 3 7 °C for 1 0 min for aging (see below ). (15) U sing a perm anent marker, the area in which the PB chrom atin is enclosed is m arked from the back o f the slide. This determ ines the area in which the probe is to be placed.

P re p a r a tio n o f b la s to m e re s

T he procedure for preparation o f blastom eres is outlined below. (1)

Fresh fixative (m ethanol and glacial acetic acid, 3:1) is prepared in a 50-m l culture flask and stored in the freezer until needed.

33

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

(2)

A circle is engraved on the bottom o f a 35 x 10mm Petri dish and 3 ml of hypotonic solution is added. The circle allows easy location of blas­ tomeres.

(3)

Using a microtorch, 25-50-|ll micropipettes are pulled to obtain an attenuated tip approxi­ mately 7 0 -100|im in diameter.

(4)

A small amount of hypotonic solution is aspi­ rated into the pipette.

(5)

The blastomere contained in a microdrop of culture medium is located using a stereomicro­ scope. The size of the pipette opening is checked prior to picking up the blastomere to ensure that it is larger than the blastomere. The blastomere is gently aspirated into the pipette and transferred to the Petri dish containing hypotonic solution.

(6)

After 3-5 min, the blastomere is aspirated into the pipette and transferred to a microscope slide with a small amount of hypotonic solu­ tion.

(7)

Under the control o f the inverted microscope with phase-contrast optics, the blastomere is constantly observed until drying is almost complete. Just at the time of drying (crystal­ lization) o f the hypotonic solution, the fixative is aspirated into the pipette and dropped onto the blastomere. While constantly observing the cell, just before drying occurs, fixative is dropped again. This is done several times at intervals until the cytoplasm has dissolved with only the nucleus remaining. Complete removal of the cytoplasm is essential for hybridization.

(8)

Using a carbide marker, the nucleus is encircled for easy location after hybridization. A second heavier circle is engraved on the bottom of the slide to locate the smaller circle easily and thus the chromatin at the time of analysis.

(9)

After recording pertinent information on the frosted area o f the slide, several drops o f fixa­ tive are added to the slide and blown dry using a rubber bulb.

(10) The slides are pretreated. Just prior to probe application, the exact location of the nuclcus is indicated on the bottom of the slide using a permanent marker. This defines the area in which to apply the probe.

34

PRETREATMENT, PROBE APPLICATION, HYBRIDIZATION A N D WASHING Slides are incubated in 2 x S S C (pH 7-7.5) for 10 min at 37°C, before being fixed for 5 min in 1% formaldehyde at ambient temperature. They are then washed for 5 min in lx P B S (pH 7-7.5) at ambient temperature, before being incubated for 5 min in 0.5 mg/ml pepsin in 0.01 mol/1 HC1 at 37°C. The slides are then washed for 5 min in J jk PBS at ambient temperature, the back of the slide is drained and wiped to remove excess PBS, before being sequentially immersed in 70%, 85% and 100% ethanol at ambient temperature; for 1 min each, air dried and then hybridized. For the purpose o f re­ hybridization, prior to pretreatment, coverslips are removed from the slides and the slides are placed in methanol for 5 min to ensure fixation o f the chro­ matin to the slide. Probe mixtures are prepared according to the manufacturer specifications. A three-color probe mixture is prepared by first centrifuging and then vortexing each of the probes of interest along with a vial o f locus-specific identifier (LSI) hybridization buffer for 10 s. For 10 pi of working probe, 1 |il of each probe is added to 7 (tl o f LSI hybridization buffer in a small microcentrifuge tube, which is then vortexed prior to probe application. For a single chromosome probe mixture, 1 |ll of probe and 2 |il of HPLC grade H 2O is added to 7 (il of hybridization buffer, which is then vortexed and centrifuged for 10s. MultiVysion five-color and four-color custom probe mixtures are ready to use, so that after being brought to room temperature, they arc centrifuged and vortexed prior to probe application. All working probe mixtures should be validated prior to case use on peripheral blood lymphocyte control slides and can be stored at -2 0 °C for several months according to the manufacturer’s expiry date. Hybridization procedure

The hybridization procedure is outlined below. (1)

Prior to probe application, 2 2x30-m m cover­ slips are cut into 8 x 8-mm squares using a carbide marker and stored in a glass Petri dish with lid.

(2)

Parafilm is cut into approximately 2 2x22-m m squares and stored in a Petri dish.

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

(3)

A humidification chamber for hybridization is prepared by placing a paper towel moistened with sterile filtered water into a glass (staining) dish and the slide rack is placed inside and covered. The humidification chamber is warmed in an air incubator at 37°C prior to the application o f the working probe.

(4)

The working probe is removed from the freezer, centrifuged for 10 s and mixed by vortexing.

(5)

Using a micropipette, 2 |tl of working probe is applied to the marked area where the chro­ matin is located. Immediately after application o f the probe, an 8 x 8-mm coverslip is placed on top using forceps. Air bubbles should be avoided, as they interfere with hybridization. If any air bubbles should appear, the coverslip is depressed lightly using forceps.

(6)

Coverslips can be sealed using rubber cement or parafilm. When applying parafilm, adhesion to the slide may be ensured by applying pres­ sure down around the coverslip with fingertips.

(7)

The slides are placed on a slide warmer at 69°C for 8 min for simultaneous denaturation o f both probe and specimen nucleic acids. Afterwards, they are removed from the slide warmer and quickly transferred to the humidification chamber, which is placed in a 37°C air incuba­ tor for hybridization to occur. HYBrite (Vysis) can be used instead o f a slide warmer and humidification chamber for the simultaneous denaturation o f probe and specimen nucleic acids. Water is added to each trough for humid­ ity, which is especially important for overnight hybridizations.

Hybridization times vary with the first hybridization of the MultiVysion PB panel five-color probe mixture at 3 h followed by re-hybridization with mixtures that may include sub-telomeric probes requiring a longer minimum hybridization of 4-5 h. Whole chromosome paints are hybridized for a minimum of 8 h. Washing procedures

MultiVysion PGT for chromosomes 13, 18, 21, X and Y. (1)

A coplin jar is filled with approximately 50 ml of 0 .4 x S S C /0 .3 % NP-40, pH 7.4 and is placed in a 73°C water bath. Using a calibrated ther­ mometer, the temperature of the solution inside the jar is checked before adding slides for the wash procedure. The solution temperature should be 73 ± 1°C.

(2)

A second coplin jar is filled with 50m l of 2 x S S C /0 .1 % NP-40, pH 7.4 and placed at room temperature.

(3)

Slide(s) are placed in the 0 .4 x S S C /0 .3 % NP40 immediately after removing the coverslip, and incubated for 5 min not to exceed more than four slides at a time so as not to decrease dramatically the temperature of the wash solu­ tion.

(4)

After 5 min the slide(s) are removed from the wash solution and placed in the coplin jar, containing 2 x S S C /0 .1 % NP-40 at room temperature and incubated for 1min.

(5)

The slide(s) are removed from the wash solu­ tion, dipped in a coplin jar containing HPLCgrade water and placed vertically to drain on a paper towel in a dark area (such as a drawer).

Rapid wash II This wash is used for MultiVysion PB

panel probe for chromosomes 13, 16, 18, 21 and 22, and the four-color custom probe for chromosomes X, Y, 15 and 17. (1)

A coplin jar is filled with 50 ml of 0 .7 »S S C /0 .3 % NP-40, pH 7.4 and placed in a 73°C water bath. Using a calibrated thermome­ ter, the temperature of the solution inside the jar is checked before adding slides for the wash procedure. The solution temperature should be 73 ± 1°C.

(2)

A second coplin jar is filled with 50 ml of 2 x SSC/0.1% NP-40, pH 7.4 and placed at room temperature.

(3)

Slides are placed in the 0 .7 x S S C /0 .3 % NP-40 immediately after removing the coverslips, not to exceed more than four slides, and incubated for 7 min.

(4)

After 7 min of incubation the slide(s) are removed from the wash solution and placed in

The following two wash protocols may be used, which do not require formamide. R apid wash I This wash is used for 1-3-color probe mixtures including whole chromosome paints and

35

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

the coplin jar, containing 2 x S S C /0 .1 % NP-40 at room temperature and incubated for 1min. (5)

The slide(s) are removed from the wash solu­ tion, dipped in a coplin jar containing HPLCgrade water and placed vertically to drain on a paper towel in a dark area (such as a drawer).

D API counterstain

Approximately 10 p.1 of DAPI counterstain is applied to each slide and covered with a 22 x 30-mm cover­ slip for three-color probe testing. The slides are blotted using a paper towel or paper blotter to spread evenly and remove excess medium from the slide. For hybridizations using MultiVysion five-color and four-color custom probe mixtures, Vectashield antifade mounting medium is applied without DAPI.

FLUORESCENT SIGNAL EVALUATION Signals are determined under x6'30 magnification using fluorescence immersion oil. Fluorophores are readily photobleached by expo­ sure to light. To limit this degradation, handle all solutions containing fluorophores in reduced light. This includes all steps involved in handling the hybridized slide. All steps, which do not require light for manipulation (incubation periods and washes), are carried out in the dark. Fluorescence microscopy is accomplished using a 100-W mercury lamp. The manufacturer's recom­ mendations for the length of time the lamp may be used should be followed since prolonged use may result in dim signals creating the potential for misdi­ agnosis. An adequate supply of replacement bulbs should be on hand. For the MultiVysion PGT probe, chromosome X (alpha satellite D X Z 1) is seen in blue, Y chromo­ some (alpha satellite DYZ3) is seen in gold, chromo­ some 13 (13q 14) is seen in red, chromosome 18 (alpha satellite D 18Z1) is seen in aqua and chromo­ some 21 (LSI 21q22.13-21q22.2) is seen in green. Whereas, for the MultiVysion PB Panel probe, chro­ mosome 13 (1 3 q l4 ) is seen in red, chromosome 16 (satellite II D16Z3) is seen in aqua, chromosome 18 (alpha satellite D18Z1) is seen in violet blue, chro­ mosome 21 (LSI 21q22.13-21q22.2) is seen in green and chromosome 22 (2 2 q ll.2 ) is seen in gold (Figures 4.2 and 4.3).

36

Visualization of signals is performed, using the appropriate single-bandpass filters for the probe fluorophores (red, green, blue, gold, aqua and DAPI). Dual band pass filters are useful in distinguishing signals from non-specific fluorescence or bleedthrough which is sometimes seen with centromeric enumeration probes that hybridize to alphoid repeat sequences resulting in large bright signals. When determining the number of signals present, the size and intensity of each signal is considered especially when in close proximity to another one. Chromosomes 13, 21 and 22 are identified using locus-specific identifier D N A probes that contain specific sequences homologous to the chromosomes o f interest as well as unlabeled blocking D N A to suppress common sequences. This results in smaller round signals when compared to signals from centromeric enumeration probes, which hybridize to a greater number of alpha repeat sequences identify­ ing chromosomes 15, 16, 17, 18, X and Y. To improve the accuracy of diagnosis, especially in cases of split or missing signals, repeat hybridization with a probe that targets a different locus (i.e. sub-telom­ eric probe) for the same chromosomes is required, as shown in Figures 4.26-4.28. Signals for different chromosomes, which are within close proximity to one another to the point of overlapping, especially in cases of highly condensed chromatin are distinguished from non-specific hybridization by close examination using both single- and dual-bandpass filters, or re-hybridization with telomeric probes (Figure 4.26). Upon imaging, the overlapping portion of the signals may appear yellow or even white because of the fluorophore combination. Use of an imaging system is highly recommended when using probe mixtures contain­ ing more than three colors. The PBI contains two chromatids for each chro­ mosome, so the signals for each chromosome are paired, at least one domain apart and of equal size (Figure 4.2). However, if the chromatids are in close proximity to one another, the signals may appear as one fluorescent strip, representative of two fihromatids (Figures 4.16, 4.23 and 4.26). The PB2 contains single chromatids for each chromosome, therefore, resulting in one fluorescent signal for each chromosome tested (Figure 4.2). Two single-dot signals for each chromosome studied are normally observed in each blastomere nucleus (Figure 4.3). However, signals can appear as double dots depending upon the stage of the cell

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

cycle, degree o f chromatin condensation and over­ spreading (Figures 4.27 and 4.28). After replication, a signal may appear as paired dots less than one domain apart or as a strip consisting of two intercon­ nected dots. These signals are counted as one signal according to standard criteria. If signals are of equal size and intensity and are two domains apart they are counted as two separate signals. However, proximity of two separate signals may b e even closer than two domains in highly condensed nuclei and must be considered in order to avoid a misdiagnosis. Size and intensity of the signals of the individual nuclei as well as additional nuclei on the same slide must be taken into consideration when distinguishing non-specific fluorescence from actual signals. Hybridization spots of lower intensity, smaller in size or with a flat, non-fluorescing appear­ ance are not counted. As with all cytogenetic testing, for quality assur­ ance, two independent readers are required for each case.

CHROMOSOMAL ABNORMALITIES IN POLAR BODIES A t the present time chromosomal abnormalities in polar bodies have been studied in more than 10000 oocytes, obtained from 1551 clinical cycles for IVF patients of advanced reproductive age23-29. Based on the results of PBI and PB2 testing, aneuploidy-free zygotes were pre-selected for transfer back to patients. The embryos resulting from abnormal oocytes, when available, were tested further to confirm the PBI- and PB2-based diagnosis (see examples in Figures 4.21-4.25). O f 10317 oocytes obtained from 1027 patients with an average age of 38.5 years, FISH results were available in 8213 (79.6%). As shown in Figure 4.29, results were not obtained due to either failure of hybridization or because the polar bodies were lost in process of preparation. The results for both PBI and PB2 were obtained in 6037 (73.5%) oocytes. The remaining oocytes had either PBI (1073; 13.1%), or PB2 results (1100; 13.4%) (Figure 4.30). A total of 4365 (53%) oocytes had aneuploidies, of which 1270 (29.1%) had errors in both PBI and PB2, 1704 (39%) in only PBI and 1391 (31.9%) in only PB2. O f 7103 PBI with results, 2973 (41.7%) had errors (Figure 4.31), compared with 2660 (35.1%) errors detected in 7125 PB2 (Figure 4.32). On the one hand, the observed rates should be an

overestimate, as the average maternal age of the IVF patients, from whom the oocytes were obtained, was 38.5 years. On the other hand, this may be an under­ estimate, because 3630 of these oocytes were tested using a tri-color probe specific for chromosomes 13, 18 and 21, with only 4583 analyzed using a fivecolor probe specific for chromosomes 13, 16, 18, 21 and 22, which detected 2830 (61.8%); oocytes with aneuploidies in that series. The types of errors observed in PBI and PB2 are presented in Figure 4.33, showing that at least a three times higher frequency was observed for missing signals (nullisomy) compared to extra signals (disomy) in PBI (53.1 % and 18.1%, respectively; approximately 3:1 ratio), in contrast to a Comparable distribution of missing and extra signals in PB2 (38% and 39.9%, respectively). PBI data also showed a 64.9% chromatid error rate (47.4% missing and 17.5% extra chromatids), compared to a 6.2% chro­ mosome error rate (5.7% missing and 0.5% extra chromosomes) (Figure 4.34). Figure 4.34 shows that, as in chromatid errors, missing chromosomes were more frequent than extra chromosomes, suggesting a possible maintenance of the extra chro­ matid or chromosome material in metaphase II oocytes, which is in agreement with a higher frequency of trisomies over monosomies in pre- and post-implantation embryos. Only 18.1% of PBI abnormalities were disomies, compared to 53.1% nullisomies. with the remaining being of complex origin (Figures 4.33 and 4.34). Although the observed excess of missing signals in PBI may be attributable to technical errors, such as hybridization failure, it is also possible that there is a mechanism preventing extra chromosome material extrusion into the PBI if meiotic errors occur during the oocyte maturation process. A significant proportion of abnormalities in PBI and PB2 (28.8% and 22.1%, respectively) were of complex origin, represented by errors in both meiotic divisions and/or involving more than one chromosome (Figure 4.35). O f 1988 complex abnor­ malities, overall, 1512 (78.5%) involved two or more chromosomes simultaneously, and 476 (21.5%) the same chromosome(s) in both PBI and PB2 (Figure 4.35). O f 1270 oocytes with both PBI and PB2 being abnormal, different chromosomes were involved in 743 (58.5%). In the oocytes with the same chromosome(s) involved, chromosome 21 appeared to be the most frequent (199; 15.5%), followed by

37

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

chromosome 22 (99; 7.8%), chromosome 16 and 13 (63 and 61; 5.0 and 4.8%, respectively), and chro­ mosome 18 (52; 4.1%); two or more same chromo­ somes were observed in 53 (4.2%) cases. Although 413 (32.5%) of the zygotes in the latter group appeared to be balanced (Figure 4.32), the pre-selection and transfer of the embryos resulting from such oocytes is yet to be justified, as incidental abnormal­ ities o: the same or different chromosomes cannot be excluded, as demonstrated below. Chromosome-specific patterns o f meiotic errors are shown in Figure 4.36. As mentioned, the most frequent chromosomes involved in meiotic error were chromosomes 21 and 22 (10.9% and 11.8%, respectively), with the error o f chromosome 21 derived comparably from meiosis I and meiosis II, while the error of chromosome 22 derived predomi­ nantly from meiosis II. Involvement in meiotic error of chromosomes 13, 16 and 18 was much less frequent (6%, 6.4% and 6.8%, respectively), and their error patterns were not similar. Chromosome 16 errors originated predominantly in meiosis II (55.9% meiosis II vs. 28.8% meiosis I), in contrast to chromosome 13 and 1 8 errors, which derived more frequently from meiosis I (44.9% and 61.8% vs. 36.7% and 28.5% in meiosis II, respectively). Based on the above results, 3848 aneuploidy-free zygotes were pre-selected, from which 2945 were transferred in 1295 cycles, resulting in 301 (23.2%) clinical pregnancies in womeji, whose average age was 38.5 years. Sixty-seven (22.7%) pregnancies spontaneously aborted, with the rest resulting in the birth of 234 healthy children at the time o f writing. The data show that testing for only five chromo­ somes has revealed an aneuploidy rate as high as 53%. Although some overestimation attributable to limitations of the FISH technique cannot be excluded, the majority of PBI and PB2 abnormalities were true errors as confirmed by follow-up studies of the embryos resulting from oocytes with meiosis I and meiosis II errors (see examples in Figures 4.21-4.25)- . In contrast to a well-established concept of a female meiosis I origin of chromosomal abnormalities, our results show that the observed errors originate from both meiosis I and II as per the expected patterns of segregation illustrated in Figure 4.1. The results are of clinical significance, suggesting that the genotype of the resulting zygote cannot be predicted without testing the outcomes of both meiotic divisions, inferred from PBI and PB2 (Figures 4.17, 4.18 and 4.21-4.24). For instance,

38

testing of meiosis I errors alone, prior to fertilization, should reduce the aneuploidy rate in the resulting embryos by at least two-thirds. Despite the fact that approximately one-third of these oocytes will be aneuploid following the second meiotic division, PB 1 testing could still sufficiently improve the implanta­ tion and pregnancy rates in poor prognosis IVF patients by selectively applying intracytoplasmic sperm injection (ICSI) to the oocytes with aneu­ ploidy-free PB 1. On the other hand, only half of the abnormalities deriving from the second meiotic divi­ sion may be detected by PBI analysis as complex errors; therefore, to avoid the transfer of all the embryos resulting from aneuploid oocytes, testing of both PBI and PB2 is still required. As PBI and PB2 are extruded during the normal process of oocyte maturation and fertilization, having no biological significance in pre- and post-implantation develop­ ment, their removal and testing may become a useful tool in assisted reproduction practices to identify the oocytes without nuclear abnormalities, which should help in the preselection o f oocytes with the highest potential for establishing a viable pregnancy, improv­ ing significantly the efficiency of IVF. As can be seen from the types of the errors shown in Figure 4.34, the majority o f abnormalities in meiosis I are chromatid errors, in contrast to the expected chromosomal non-disjunction, suggested by most of the previous studies mentioned. However, we still observed chromosomal errors in 6.2% of oocytes, which docs not support the other extreme tlaim that all abnormalities in metaphase II oocytes are o f chromatid origin30. Thus, probably both chromatid and chromosomal errors are involved in producing metaphase II abnormalities, with the frequency of chromatid errors being at least ten times higher than that of chromosomal ones (Figures 4.31 and 4.34). There is no doubt that both of these meiosis I errors lead to aneuploidy in the resulting embryos, as demonstrated by the follow-up study of the embryos resulting from these oocytes, the transfers o f which were avoided25. However, differences in the effect of chromatid and chromoso­ mal errors on the pre- and post-implantation devel­ opment cannot be excluded. Unfortunately, such a differential effect could not be explored in our study, because the embryos resulting from such oOcytes were neither transferred, nor cultured further, as they were used for confirmation o f PBI and PB2 diagnosis instead.

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

As already mentioned, as many as 41.7% of abnormal oocytes deriving from meiosis I errors may be detected by testing of PBI (Figure 4.31). In addi­ tion, this information allows a significant proportion of oocytes with the second meiotic division errors to be predicted: of 2660 oocytes with meiosis II errors, 1269 (47.7%) had also PBI aneuploidies. However, the remaining 1391, which represents approximately one-third (29.9%) of the overall number of abnormal oocytes, became abnormal only following the second meiotic division, which could not be predicted by PBI results and would be missed if testing were limited to PBI (see example in Figures 4.17, 4.18 and 4.21). This may suggest that in order to identify all oocytes with chromosomal abnormalities, the outcome of both the first and second meiotic divi­ sions should be studied, using PBI and PB2 (Figures 4.21-4.25). The data show that the direct testing of meiosis I and meiosis II errors enables the transfer of at least 50% of embryos ^resulting from aneuploid oocytes to be avoided, which should clearly contribute to the pregnancy outcome o f IVF patients participating in this study. In fact, the pre-selection and transfer of the aneuploidy-free oocytes (an average of 2.3 embryos per transfer) resulted in a 23.2% clinical pregnancy rate, which may be considered to be a positive clinical outcome for the IVF patients of maternal age of 38.5 years, involved in the present series, as the clinical pregnancy rate for the compara­ ble age group of patients without testing during the same period of time was 15.5%, despite transferring 2.9 embryos on average. Although randomized controlled studies will still be required further to quantify the clinical impact o f the pre-selection of aneuploidy-free zygotes for embryo transfer, the present results suggest clinical relevance and accu­ racy o f the preselection of aneuploidy-free oocytes by the described approach for the direct testing of the first and second meiotic division errors.

CHROMOSOMAL ABNORMALITIES IN CLEAVING EMBRYOS As shown above, approximately half of meiosis II errors are observed in the oocytes with prior crtois in meiosis I. As a result o f such sequential errors, almost one third of the resulting zygotes may have been considered normal (euploid) (Figure 4.32), provided that the preceding errors in meiosis I and meiosis II have no effect on the further preimplantation devel­

opment of the corresponding embryos. However, the follow-up testing of these embryos at the cleavage stage demonstrated subsequent errors in mitotic divisions in the resulting zygotes, leading to the development of chromosomally abnormal embryos which were unacceptable for embryo transfer. Overall, only 18% of embryos, deriving from the apparently balanced zygotes, were euploid for the five chromosomes analyzed (Figure 4.37), while the remaining majority had chromosomal abnormalities (Figures 4M, 4.38 and 4.39)31-32. All of the chromosomally normal (euploid for five chromosomes tested) embryos appeared to result from zygotes with only one chromosomal error rescue, with none resulting from the zygotes balanced for two chromosomes. The fact that only a few resulting embryos (11%) were abnormal for the same chromosome, for which sequential meiosis 1 and meiosis II led to the balanced set, may suggest that the observed sequential errors in female meiosis may be attributable to a meiotic apparatus abnor­ mality overall, rather than to a single-chromosome segregation defect, and further may lead to a general defect in the mitotic apparatus of the resulting embryos. This seems to be also in agreement with the observed types of aneuploidies detected in the resulting embryos, which in 79.3% of cases were represented by complex errors (Figures 4.40-4.42), including mosaicism, known to be highly prevalent at the cleavage stage. According to the data on PGD for aneuploidies performed at the cleavage stage at least 60%, of embryos tested had chromosomal abnormali­ ties10,33'34. Although the reported types of aneuploi­ dies may differ in different studies, there seems to be no doubt that approximately half of these abnormal­ ities are represented by mosaicism. As there was no information about the initial chromosome comple­ ment of the zygotes from which the mosaic embryos originated in any of these studies, the nature of mosaicism in preimplantation embryos is not known, despite its high prevalence and the potential clinical relevance. There were, however, some indirect obser­ vations, suggesting that the observed mosaicism at the cleavage stage may be of a different nature, with some mosaic types increasing with maternal age35, therefore probably stemming from female meiosis errors, while others are possibly attributable to immaturity of centrosome structures in sperm, expected to be active from the first mitotic divisions of the zygote, as suggested for cases involving

39

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

testicular sperm extraction (T E SE ) patients.36 It may also be suggested, that a significant proportion of m osaic em bryos originate from oocytes that are aneuploid from the outset, through a process of trisom y ‘rescue’. A possible high rate o f further m itotic errors in cleaving embryos, deriving from oocytes with the com plex aneuploidies, m ay also explain the phenom enon o f chaotic embryos, which m ake up alm ost half o f the em bryos with m osaicism . A com parable prevalence o f aneuploidies in oocytes and embryos, with the differences in the types of chrom osom al anomalies, mainly attributable to a high frequency o f m osaicism in cleavage-stage embryos, may also support a prezygotic origin o f the m ajority of em bryo chrom osom e abnormalities, including mosaicism. C om parison o f the chrom osom e-specific aneu­ ploidy rates in oocytes and em bryos m ay also be of relevance in understanding the relationship betw een oocytc and em bryo abnormalities. The data show an alm ost tw o-fold higher rate for each chrom osom e error in oocytes com pared to that in embryos, which may indicate a possible correction o f som e of the aneuploidies through the m echanism o f 'trisomy rescue’, probably resulting in a certain proportion o f m osaic em bryos following the first three cleavage divisions. In fact, the exact data on the m osaicism rate in preim plantation developm ent are not known, because only a lim ited num ber o f the preim planta­ tion em bryos were fully studied, with the majority available from PG D for aneuploidies perform ed on a single biopsied blastomere, which m ay not be repre­ sentative of the whole embryo. A lthough the p ossi­ bility o f postzygotic m itotic errors in cleavage-stage embryos, euploid from the outset, cannot be excluded, the proportion o f the aneuploidy and m osaicism stem m ing from these errors is not known, as well as the im pact o f these postzygotic errors on the pre- and post-im plantation em bryo develop­ ment. Based on the above data, it m ay be suggested that the m ost accurate pre-selection o f em bryos for trans­ fer in P G D for aneuploidies m ay be perform ed by a sequential testing o f meiosis I, meiosis II and mitotic errors, through sequential PBI, PB2 and blastom ere sampling. This m ay enable the transfer o f em bryos with prezygotic chrom osom al errors to be avoided, which seem s to be the m ajor source o f chrom osom al abnorm alities in the embryo, and also detecting possible m itotic errors in embryos resulting from the euploid zygotes, the proportion o f which cannot be

40

evaluated at the present time. The accum ulated data on such sequential sam pling will help to evaluate possible differences in the viability o f em bryos with chrom osom al abnorm alities o f meiotic and mitotic origin.

REFERENCES 1.

Griffin DK, Handyside AH, Penketh, RJA, et a I. Fluorescent in-situ hybridization to interphase mu lot of human preimplantation embryos with X and Y chromosome specific probes. Hum Reprod 1991 ;6: 101-5

2.

Griffin DK, Wilton LJ, Handyside AH, et al. Dual fluorescent in situ hybridization for the simultaneous detection of X and Y chromosome specific probes for the sexing of human preimplantation embryonic nuclei. Hum Genet 1992;89:18-22

3.

Schrurs BM, Winston RML, Handyside AH. Preimplantation diagnosis of aneuploidy using fluo­ rescent in-situ hybridization: evaluation using a chro­ mosome 18-specific probe. Hum Reprod 1993;8: 296-301

4.

Munne S, Weier HUG, Stein J, et al. A fast and effi­ cient method for simultaneous X and Y in-situ hybridization of human blastomeres. J Assist Reprod Genet 1993;10:82-90

5.

Munne S, Lee A, Rozenwaks Z, et al. Diagnosis of major chromosome aneuploidies in human preim­ plantation embryos. Hum Reprod 1993;8:2185-91

6.

Munne S, Magli MC, Bahce M, et al. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16’, 18, 21, 22. Prenat Diagn 1998;18:1459-66

7.

Bahce M, Cohen J, Munne S. Preimplantation genetic diagnosis of aneuploidy: were we looking at the wrong chromosomes? J Assist Reprod Genet 1999;16:154-9

8.

Kuliev A, Verlinsky Y. Thirteen-years experience of preimplantation genetic diagnosis. Reprod BioMed Online 2004;8:229-35

9.

Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in mtro fertilization with poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999;72:837-44

10.

Munne S. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod BioMed Online 2002;4:1 83-96

IEIM PLANTATION D IA G N O S IS FOR ANEU PLOIDIES

11.

Munne S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod BioMed Online 2003;7:91-7

12.

Munn6 S, Alikani M, Tomkin G, et al. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64:382-91

13.

Munne S, Weier HUG, Grifo J, Cohen J. Chromo­ some mosaicism in human embryos. Biol Reprod 1994;51:373-9

14.

Delhanty JDA, Griffin DK, Handyside AH. Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplanta­ tion sex determination by fluorescent in situ hybridi­ sation (FISH). Hum Mol Genet 1993;2:1183-5

15.

Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal monospermic preimplanta­ tion human embryos. Prenat Diagn 1994;15:41-9

16.

International Working Group on Preimplantation Genetics. Current status of preimplantation diagno­ sis. J Assist Reprod Genet 1997;14:72-5

17.

International Working Group on Preimplantation Genetics. 10th Anniversary of preimplantation genetic diagnosis. J Assist Reprod Genet 2001; 18: 66-72

18.

Evsikov S, Verlinsky Y. Mosaicism in inner cell mass of human blastocysts. Hum Reprod 1998;13:3151-5

19.

Dyban A, Fredine M, Severova E, et al. Detection of aneuploidy in human oocytes and corresponding first polar bodies using FISH. Presented at the 7th

common aneuploidies by polar body FISH analysis. Fertil Steril 1966;66:126-9 24.

Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Pre-pregnancy genetic testing for age-related aneuploidies by polar body analysis. Genet Test 1998; 1:231 —5

25.

Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Preimplantation diagnosis of common aneuploidies by the first and second polar body FISH analysis. J Assist Reprod Genet 1998;15:285-9

26.

Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Prevention of age-related aneuploidies by polar body analysis. J Assist Reprod Genet 1999;16:165-235

27.

Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Polar body preimplantation diagnosis in aging IVF patients. Ref Gynecol Obstet 2000; 7:191—4

28.

Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Chromosomal abnormalities in the first and second polar body. Mol Cell Endocrinol 2001; 183:S47—9

29.

Kuliev A, Cieslak J, Ilkevitch Y, et al. Chromosomal abnormalities in a series of 6733 human oocytes in preimplantatron diagnosis for age-related aneuploi­ dies. Reprod BioMed Online 2003;6:54-9

30.

Angel R. First meiotic division nondisjunction in human oocytes. Am J Hum Genet 1997;65:23-32

31.

Kuliev A, Cieslak J, Zlatopolsky Z, et al. Origin of aneuploidies in preimplantation embryos. 2003 Fifth International Symposium on Preimplantation Genetics, 5-7 June 2003, Antalya, Turkey. 16-17

32.

Kuliev A, Cieslak J, Zlatopolsky Z, et al. Aneuploidy rescue after female meiosis I and follow up analysis of its outcome in resulting preimplantation embryos. Am J Hum Genet 2003;73(Suppl):189

33.

Gianaroli L, Magli MC, Ferraretti AP. The in vivo and m vitro efficiency and efficacy of PGD for aneu­ ploidy. Mol Cell Endocrinol 2001; 183 :S 13—18

34.

Munne S, Bahce M, Sandalinas M. Differences in chromosome susceptibility to aneuploidy and survival to first trimester. Reprod BioMed Online 2003:webpaper 1058

35.

Munne S, Sandalinas M, Escudero T, et al. Some mosaic types increase with maternal age. Reprod BioMed Online 2002:webpaper 382

36.

Silber S, Sadowy S, Lehahan K, et al. High rate of chromosome mosaicism but not aneuploidy in embryos from karyotypically normal men requiring TESE. Reprod BioMed Online 2002,4 (Suppl 2):20

International Conference on Early Prenatal Diagnosis

Jerusalem, Israel, 22-27 May, 1994: abstract 97 20.

21.

22.

23.

Verlinsky Y, Cieslak J, Freidine M, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod 1995;10:1923-7 Dyban A, Fredine M, Severova E, et al. Detection of aneuploidy in human oocytes and corresponding first polar bodies by FISH. J Assist Reprod Genet 1996;13:72-7 Munne S, Daily T, Sultan KM, et al. The use of first polar bodies for preimplantation diagnosis of aneu­ ploidy. Hum Reprod 1995;10:1014-120 Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Birth of healthy: children after preimplantation diagnosis of

41

5 Preim plantat on d agnosis for translocations

As described in the previous chapter, cytogenetic testing of single cells obtained from preimplantation embryos is currently performed by interphase fluorcsccnt in situ hybridization (FISH) analysis, which allows chromosome enumeration on interphase cell nuclei. However, the number of ghromosomes studied by FISH is limited to the number o f chro­ mosome-specific probes available. Even with the currently available methods for re-hybridization of interphase nuclei for a second and third time, complete karyotyping will not be realistic in the near future. The technique also has clear limitations for the detection o f some rearrangements such as chro­ mosome insertions and inversions making it very important to develop methods for visualization of chromosomes in single cells, including polar bodies (PB) and individual blastomeres, which would clearly improve the accuracy of preimplantation genetic diagnosis (PGD) for chromosomal disor­ ders '~4. One of the approaches for PGD of maternally derived translocations is first polar body (PBI) testing, based on the fact that PBI never forms an interphase nucleus and consists of metaphase chro­ mosomes. It has been shown that PBI chromosomes are recognizable when isolated 2-3 h after in vitro culture, with degeneration beginning 6-7 h after extrusion5, thus whole-chromosome painting or locus-specific probes may be applied for testing6-8. As shown in Figures 5.1-5.6, demonstrating segrega­ tion during meiosis I, by testing PBI using specific probes for chromosomes involved in translocation, unbalanced chromosome complements may be detected and avoided. Although the method resulted in a significant reduction in the rate of spontaneous abortions in the patients carrying translocations,

yielding unaffected pregnancies and the birth of healthy children, it has been shown to be sensitive to malsegregation and/or recombination between chro­ matids (Figures 5.7-5.9), requiring a further followup analysis of the second polar body (PB2) in order to accurately predict the meiotic outcome following the second meiotic division®;9. However, despite the progress in transforming PB2 into metaphase chro­ mosomes via electrofusion of the PB2 nucleus with a foreign one-cell human embryo (see Chapter 3), the proportion of mctaphase plates obtained did not exceed 65%, which would be necessary to be useful in clinical practice2. Therefore, alternative methods were developed to convert single blastomere nuclei into metaphase chromosomes, following the fusion of single blastomeres with murine: (see Chapter 3 )3, or bovine zygotes4. These methods appeared to be useful for PGD for both maternally and paternally derived translocation. Initially individual blastomere nuclei were trans­ formed into metaphase chromosomes through blas­ tomere fusion with enucleated human oocytes3, but currently this is accomplished by the fusion of indi­ vidual blastomeres with intact mouse zygotes at the pronuclear stage9, as described in Chapter 3. To date, the method has been applied for PGD of paternally derived reciprocal translocations, chromosome inver­ sions and for confirmation of PGD of chromosomal abnormalities identified by PBI and PB2 FISH analy­ sis (Figures 5.10-5.14). A total of 100 patients carry­ ing balanced translocations were offered PGD, using a standard IVF protocol combined with PBI and PB2 removal and/or embryo biopsy and FISH analysis. Thirty-five PGD cycles were performed for Robertsonian translocations, maternally derived in 18 cycles and paternally derived in 17 cases. PGD for

43

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

reciprocal translocations were performed in 10 cycles, 64 for maternal derivation and 43 for paternal derivation. O f 146 PGD cycles, 49 were performed using PBI and PB2 testing, eight by interphase blas­ tomere analysis and the remaining 89 cycles using a blastorrterg nucleus conversion technique to meta­

phase chromosomes (see below). The list of translo­ cations and the probes applied is presented in Table

£

Examples of PGD cycles for translocations performed utilizing the nuclear conversion method are presented in Figures 5.10-5.14. Overall, the

Table I List o f translocations and fluorescent in situ hybridization (FISH) probes used in preim plantation genetic diagnosis (PGD) Patients

Cycles

Karyotype

FISH probes

9

II

4 5 ,X X ,ro b (l 3; l4 )(q 10;q 10)

W C P I3 .W C P 14, LSI 13, Tel I4q

1

1

4 5 ,X X ,ro b (l 3;22)(q 10;q 10)

W C P 13, W C P 22, PB panel (chrom osom es 13,16,18,21,22)

1

1

4 5 ,X X ,ro b ( 14 ;2 1)(q 10;q 10)

W C P 14, W C P 21, LSI 21

1

5

4 5 ,X X ,ro b ( 14;22)(q 10;q 10)

W C P 14, W C P 22, Tel I4q, LSI 22

11

4 5 ,X Y ,ro b (l 3; I4)(q 10;q 10)

W C P 13, W C P 14, LSI 13, Tel I4q

2

4 5 ,X Y ,ro b (l 3; I5)(q 10;q 10)

W C P 13, W C P 15, LSI 13, Tel I5q

4

4 5 ,X Y ,ro b ^ l4 ;2 l)(q I0;q 10)

W C P 14, W C P 21. LSI 21

1

1

4 6 ,X X ,t(l ;4 )(q 2 3 ;q 3 1.1)

W C P 1, W C P 4, (CEP 18 as a co ntro l)

1

1

4 6 ,X X ,t( l;7 )(q 2 3 ;p l 1)

W C P l,W C P 7, CEP 1

1

3

4 6 ,X X ,C (i;8 )(q 4 2 ;p l 1.2)

W C P 1, W C P 8, CEP 1, CEP 8, Tel Iq, Tel 8p

1

2

4 6 ,X X ,t( 1; 15)(q32;q26)

W C P I.W C P 15, Tel Iq , Tel 15 q, CEP 1, CEP 15

1

2

4 6 ,X X ,t(2 ; 10 )(q 3 1;q 1 1.2)

W C P 2, W C P 10, CEP 2, Tel 2q W C P 2, W C P 13, CEP 2, Tel 13q, LSI 13

1

1

1

4 6 ,X X ,t(2 ; 13)(q22;q33)

1

1

4 6 ,X X ,t(2 ; 16 )(q3 7 ;p l 3.1)

W C P 2, W C P 16, CEP 16, Tel 2q, Tel 16p

1

1

4 6 ,X X ,t(2 ;2 0 )(q 3 7 ;q l 1.2)

W C P 2, W C P 20, CEP 2,Tel 20q

4 6 ,X X ,t(3 ;6 )(p 2 6 .2 ;q 2 l)

W C P 3, W C P 6, CEP 6, Tel 3p, Tel 6q

1

1

4 6 ,X X ,t(3 ;7 )(p 2 l;q 2 2 )

W C P 3, W C P 7, Tel 3p, Tel 7q

1

1

4 6 ,X X ,t(3;8)(q 2 7;q 22 )

W C P 3, W C P 8, Tel 8q, CEP 3, CEP 8

1

1

4 6 ,X X ,t(3 ;IO H p 2 5 ;q l5 .l)

W C P 3, W C P 10, Tel 3p, Tel lOq, CEP 3, CEP 10

1

2

4 6 ,X X ,t(3 ;l2 )(q 2 l;q 2 4 .3 3 )

W C P 3, W C P 12, CEP 3, CEP 12, Tel 3q

4 6 ,X X ,t(3 ; 19)(p25;p 13.3)

W C P 3, W C P 19

1

1 1

1

4 6 ,X X ,t(4 ; 13)(p 14 ;q 3 1)

W C P 4, W C P 13, Tel I3q

1

2

4 6 ,X X ,t(4 ; 13 )(q 2 3 ;q 2 1)

W C P 4. W C P 13, LSI 13, CEP 4, Tel 4q

1

2

4 6 ,X X ,t(4 ;l4 )(q 2 l.3 ;q 2 4 .3 )

W C P 4, W C P 14, CEP 4, Tel I4q

1

1

4 6 ,X X ,t(5 ;7 )(p 14 ;p 2 1)

W C P 5, W C P 7, Tel 7p

1

2

4 6 ,X X ,t(5 ; 10)(q 1 1;q 2 2 .1)

W C P 5, W C P 10, CEP 10

1

1

4 6 ,X X ,t(5 ; I4)(q35;q32.2)

W C P 5, W C P 14, Tel 5q

1

2

4 6 ,X X ,t(5 ;2 l)(q 1 I.2;q22)

W C P 5, W C P 21, LSI 21, Tel 21 q, Tel 5p fo r enum eration, "lei 5q

3

4 6 ,X X ,t(5 ,2 1)(q 3 1;q 2 2 .1)

W C P 5, W C P 21, LSI 5, LSI 21

1

4 6 ,X X ,t(6 ;l3 )(q 2 l;q 2 2 )

W C P 6, W C P 13, LSI 13, Tel 6q, Tel 13q W C P 6, W C P 14, CEP 6, Tel 6q, Tel I4q

1

1*

4 6 ,X X ,t(6 ; 14)(q 15;q 13.1)

1

1

4 6 ,X X ,t(6 ;l5 )(p 2 l.3 ;q 2 6 .l)

W C P 6, W C P 15, CEP 15, Tel I5q

1

1

4 6 ,X X ,t(7 ;l l) ( p l I.2;q24,2)

W C P 7, W C P 1 1, CEP 1 1, Tel 1 1q

1

1

4 6 ,X X ,t(7 ; 13)(q36;q 12)

W C P 7, W C P 13, LSI 13, CEP 7, Tel 7q

1

1

4 6 ,X X ,t(7 ;l8 )(q 3 2 ;q 2 3 )

W C P 7, W C P 18. CEP 18

1

1

4 6 ,X X ,t(7 ; I9)(q32;p 13.2)

W C P 7, W C P 19, CEP 7, Tel 7q, Tel I9 p

1

1

4 6 ,X X ,t(7;22 )(p 22 .3;p 1 1.2)

CEP 7, Tel 22q, Tel 7p

1

3

4 6 .X X .t(8 ;IO )(q 2 4 .3 ;q 2 4 .l)

W C P 8, W C P 10, CEP 10, Tel lOq, Tel 8p fo r enum eration, Tel 8q continued

44

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

Table I Continued Patients

Cycles

Karyotype

FISH Probes

1

1

46,X X ,t(8; 10)(p 10;p 10)

W CP 8, W CP 10, CEP 10, Tel 1Op, Tel 8p

1

1

46,X X ,t(8; 12 )(q 2 1.2;p 13.3)

W CP 8, W CP 12, Tel 12p, CEP 12

1

2

46 ,X X ,t(8;22 )(q2 4.1;q 1 1.2)

W CP 8, W CP 22, CEP 8

1

i

46,X X ,t(9; 1 l)(q 13;q 13.5)

W CP 9, W CP 1 1, CEP 1 1, Tel 1 Iq

1

i

46,X X ,t(9; 1 1)(P24;q23.1)

W CP 9, W CP 1 1, CEP 1 1, Tel 9p, Tel 1 Iq

4 6 ,X X ,t(9 ;l3 )(q 2 2 ;q l4 )

W CP 9, W CP 13, CEP 9, Tel 9q, LSI 13

1

i

4 6 ,X X ,t( 10; 1 1)(p 15;q23)

W CP 10, W CP 1 1, CEP 10, Tel 1Op, Tel 1 1q

1

i

4 6 ,X X ,t( 10,15)(q23;q21)

W CP 10, W CP 15, CEP 15

1

i

4 6 ,X X ,t( I I ; 14)(p 15;q24)

W CP 1 1, W CP 14, CEP 1 1, Tel l i p

1

1 1

i

1 1

4 6 ,X X ,t( 1 1;22)(q23;q 1 1.2)

W CP I I , W CP 22, CEP I I , Tel 1 Iq, LSI 22

46 ,X X ,t( 12; 15)(p 13.3;q 13.3)

W CP 12, W CP 15, Tel I2p, Tel I5q, CEP 15

4 6 ,X X ,t( 12; 18)(p 13.31 ;q 2 1.32)

W CP 12, W CP 18, CEP 18, Tel I2p, Tel I8q

4 6 ,X X ,t(l4 ;2 l)(q 2 4 .l;q 2 2 .l)

W CP 14, W CP 21, Tel I4q

1

i

46,XX,inv(X)(p22.3;q 13)

CEP X, Tel Xp/Yp, Tel Xq/Yq

1

i

46 ,X Y ,t(l;3)(p 36 .l;q 27)

W CP 1, W CP 3, CEP 1, Tel 1p, Tel 3q

46,XY,t( 1;8)(p 13;q23)

W CP 1, W CP 8, CEP 8, CEP 1,Tel 1q

1 1

4 6 ,X Y ,t(l;2 l)(p l3 ;q l 1.2)

W CP 1, W CP 21, CEP 1

1

i

46,XY,t(2;3)(q21;p 13)

W CP 2, W CP 3, CEP 2,Tel 2q, Tel 3p

1

i

46,XY,t(2;l IX p l3 .3 ;p lS .I)

W CP 2, W CP 1 1, CEP 2, CEP 1 1, Tel 2p

46,XY,t(2; 18)(p 13;q 1 1)

W CP 2, W CP 18, CEP 18

1 1

i

4 6 ,X Y ,t(2 ;l9 X p 2 5 .l;p l3 .l 1)

W CP 2, W CP 19, CAP 2, Tel 2p, Tel I9p

1

i

46,XY,t(3;7)(p24;p21)

W CP 3, W CP 7, Tel 3 p

46 ,X Y ,t(3,IO )((q 2l;pl4)

W CP 3, W CP 10, CEP 10

1

i

46,XY,t(4;l IX q3 3.3;pl3)

W CP 4, W CP 1 1, CEP 4, Tel 4q, Tel l i p

1

1

i

46,XY,t(4; 17)(q 31.1 ;p 13)

W CP 4, W CP 17, CEP 4, Tel 4q, Tel 17p

1

i

46,XY,t(5;7)(q2l.2;q36)

W CP 5, W CP 7, Tel 7q, CEP 7, Tel 5p fo r enumeration

1

i

46,XY,t(6;7)(q23;q36)

W CP 6, W CP 7, CEP 7

46,XY,t(6;8)(q21;q32.1)

W CP 6, W CP 8

1 1

i

46,XY,t(7; 10)(q36;p 1 1.2)

W CP 7, W CP 10, CEP 10, Tel 7q, Tel lOp

1

i

46 ,X Y ,t(8;IO )(q22.l;q22.l)

W CP 8, W CP 10, CEP 10, Tel 8q

46,XY,t(8; 19)(p23.1;q 13.3)

W CP 8, W CP 19, CEP 8, Tel 8p, Tel I9q

46,XY,t(9; 1 I)(p22;q23)

W CP 9, W CP I I , CEP I I , Tel 9p, Tel 1 Iq

i

46,XY,t( 10; 13)(p 12;p 11.2)

W CP 10, W CP 13, CEP 10

i

46,XY.t( 11 ;20)(p 15;q 13.12)

W CP 1 1, W CP 20, Tel l i p

1 1

i

1 1 1

i

1

46,XY,t(l 1;22)(q23.3;q 1 1.2)

CEP 1 1, le i 1Iq, LSI 22

46,XY,t( 1 1;22)(q25;q 12)

W CP 1 1, W CP 22, CEP 1 1, Tel 1 Iq W CP 10, W CP 22, LSI 22, CEP 10, Tel lOq

1

i

46,XY,t( 10;22)(q24.1;p 12) 46,XY,t( 13;20)(q22;q 1 1.2)

W CP 13, W CP 20

1

i

46,XY,t( 15; 16)(q 13;q 13)

W CP 15, W CP 16, CEP 15

1

i

46,XY,t( 16;20)(q 12.3;q 13.1)

W CP 16, W CP 20, CEP 16, CEP 20, Tel I6q, Tel 20q

i

i

46 ,X Y ,t(l8 ;2 0)(p l I.2 ;q l3 .3 )

W CP 18, W CP 20, CEP 18, Tel I8p, Tel 20q

46,XY,inv(5)(p 13 ;q 13)

X cyte 5 (Metasystems), Tel 5p, Tel 5q

1

i

47 ,X X ,+ fis ( 19)(p 10),+ fis( 19)(q 10)

Tel I9p, Tel I9q

101*

147*

1

WCR whole-chrom osom e paint; LSI, locus-specific identifier; Tel, sub-telomeric; CER centrom eric-enum eration probe. * ( I } Patient/cycle not included in te xt data

45

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

technique was applied to 767 blastomeres, which included 582 for reciprocal and 185 for Robertsonian translocations, resulting in successful nuclear metaphase conversion in as many as 635 (83%). This made possible the preselection of normal embryos or those with a balanced chromo­ somal complement for transfer in 73% o f the cycles, resulting in a 32% pregnancy rate per transfer, with only under 20% spontaneous abortions, compared to a close to 90% spontaneous abortion rate before PGD in the same patients. In 49 PGD cycles from couples with maternally derived translocations, testing was performed using PBI and PB2 FISH analysis (see Figures 5.7-5.9; 5.13-5.14], From these cycles; more than 500 oocytes were tested and FISH results were obtained from 79% o f the oocytes, allowing preselection of normal or balanced embryos for transfer in 63% of cycles, resulting in clinical pregnancies and the deliv­ ery of nine unaffected children. Confirmatory testing was possible in two of three spontaneously aborted embryos, showing the presence o f de novo transloca­ tions which were different from the expected meiotic outcom es10. The proportion of abnormal oocytes and embryos detected varied depending on the type of transloca­ tions and their origin. The meiotic outcomes of female reciprocal translocations based on PB and blastomere analysis are presented in Figure 5.15, showing the most frequent segregation patterns which are mainly in agreement irrespective of the method of analysis, except for chromatid exchange, which can be detected only by sequential PBI and PB2 analysis (Figures 5.7-5.9, 5.13). Overall, complex errors are identified more frequently with PB analysis using a combination of centromeric and sub-telomeric probes, because it detects non-disj unc­ tion events, which occur in meiosis I and II involving single chromatids (Figure 5.14). This may also explain the higher rate of 3:1 segregation observed with blastomere analysis, since these may have been the product of a different segregation pattern in conjunction with anaphase II non-disjunction. The segregation patterns for paternally derived transloca­ tions, when compared to patterns observed from blastomere analysis of maternally derived transloca­ tions, showed similar tendencies, predominantly represented by alternate and adjacent I segregation, with a lower adjacent II pattern observed (Figure 5.16-5.20). In embryos tested for maternally derived reciprocal translocations 76.1% unbalanced embryos

46

were predicted, leaving only 23.9% suitable for transfer, which included 12.6% balanced and 11.3% normal. On the other hand, the testing o f embryos for paternally derived reciprocal translocations resulted in the prediction of 69% unbalanced embryos, leaving 31.0% of embryos suitable for transfer, including 16.1% balanced and 14.9% normal. Testing of embryos for maternally derived Robertsonian translocations (Figures 5.21—5.23), resulted in the prediction of 69.7% unbalanced embryos, with the remaining 30.3% embryos suit­ able for transfer, which included 16.8% balanced and 13.5% normal. Similarly, the testing of embryos for paternally derived Robertsonian translocations allowed identification of 52.1% unbalanced embryos, with the remaining 47.9% being suitable for transfer, including 28.6% balanced and 19.3% normal. The overall results for the total of 100 PGD patients carrying chromosome rearrangements show that normal/balanced embryos were available for transfer in 71.2% of the cases. Clinical pregnancies were obtained in 33.3% o f transfer cycles in which the mean number transferred was 1.7 embryos, resulting in a 26% live-birth rate. The pregnancy history of each of the 100 couples prior to undertak­ ing PGD was available, which included the birth of eight affected children and a spontaneous abortion rate of up to 90%. This is in direct contrast to the spontaneous abortion rate of under 20% and 31 healthy children born after PGD (Figure 5.24), demonstrating the tremendous positive impact that PGD has on the clinical outcome of pregnancies in couples carrying balanced chromosome transloca­ tions. As previously mentioned, the detection rates of embryos suitable for transfer depended on the types of the translocations tested. For example, the clinical outcomc is poorer in reciprocal than Robertsonian translocations,: with the pregnancy rates o f 23% and 28%, respectively. As may be predicted, there was also a relationship between the poorer clinical outcome and the proportion of unbalanced embryos in these PGD cycles. In PGD cycles with the propor­ tion of unbalanced complements under 75%, the pregnancy rate was 30% per cycle. Compared to 11% in PGD cycles with an unbalanced rate of over 75%. Although the application of the conversion tech­ nique to visualize chromosomes in single blas­ tomeres improves the accuracy of the diagnosis by

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

analysis of metaphase chromosomes using a combi­ nation of commercially available probes, a high frequency o f mosaicism in the cleavage-stage embryos, arising from anaphase lag or nuclear frag­ mentation, still presents problems for diagnosis (Figure 5.26}. Follow-up analysis of unbalanced embryos, including those in which chromatid malsegregation or recombination was identified by the analysis of PBI, and subsequent testing of PB2 interred a balanced or normal embryo, revealed a mosaicism rate o f 40%. Different cell lines were present, including normal or balanced, that if inves­ tigated only by embryo biopsy, may have led to misdiagnosis. Therefore, our PGD strategy for mater­ nally derived translocations is still based on PBI and PB2 testing, applying the blastomere nucleus conver­ sion technique only if further testing is required. To avoid transferring the embryos with aneuploidy, which may be prevalent in prcimplantation embryos resulting from carriers o f balanced translocations, re­ hybridization with a five-color probe for chromo­ somes 13, 16, 18, 21 and 22 may be useful, as shown in Figure 5.25. It was also demonstrated, that embryos with unbalanced chromosome complements have the potential to reach the blastocyst stage o f embryo development in extended culture. O f 420 unbal­ anced embryos cultured for a further period, 151 (36%) reached the blastocyst stage, confirming that some of the detected chromosomal rearrangements may not be lethal in preimplantation development, and may be eliminated either during implantation or post-implantation development11, explaining an extremely high spontaneous abortion rate in couples carrying translocations. As mentioned, our data suggest a more than four-fold reduction o f sponta­ neous abortions following PGD cycles for transloca­ tions, in agreement with previous data6-8. In addition to avoiding the use of expensive and time-consuming customized breakpoint-spanning probes12, the conversion technique used in the present study was highly accurate based on the follow-up testing of embryos predicted to be abnor­ mal, and confirmatory prenatal diagnosis of the ongoing pregnancies or testing of the newborn babies. The method also allows balanced and normal embryos to be distinguished, which cannot be achieved by currently available interphase FISH analysis (Figures 5.10-5.12J. Because PGD is almost the only hope for couples with translocations to be able to have an unaffected

child without fear of repeated spontaneous abor­ tions, increasing numbers of PGD cycles for this indi­ cation have been performed. To date, more than 500 clinical cycles have been performed, resulting in more than 100 clinical pregnancies and births of unaffected children1,8'13. The presented data suggest that patients with translocations, who are at an extremely high risk for spontaneous abortions, should be informed about the availability of PGD for this indication. Awareness of the availability of PGD will permit such couples to establish pregnancies, that are unaffected from the onset, and offer them the opportunity to have chil­ dren of their own, instead of multiple unsuccessful attempts of prenatal diagnosis and subsequent termi­ nation of pregnancy. The application of the method for conversion of interphase nuclei from blastomeres will enable PGD to be performed for a wide variety o f structural .rearrangements independent of the availability of specific fluorescent probes, which amplifies the limitations of interphase FISH analysis.

REFERENCES 1.

International Working Group on Preimplantation Genetics. Preimplantation gcnctic diagnosis - experi­ ence of three thousand clinical cycles: report of the 11th Annual Meeting International Working Group on Preimplantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 15, 2001. Reprod BioMed Online 2001;3:49-53

2.

Verlinsky Y, Evsikov S. Karyotyping of human oocytes by chromosomal analysis of the second polar body. Mol Hum Reprod 1999;5:89-95

3.

Verlinsky Y, Evsikov S. A simplified and efficient method for obtaining metaphase chromosomes from individual human blastomeres. Fertil Steril 1999; 72: 1-6

4.

Willadsen S, Levron J, Munne S, et al. Rapid visual­ ization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Hum Reprod 1999; 14:470-4

5.

Verlinsky Y, Kuliev A. Preimplantation Diagnosis of Genetic Diseases: a New Technique for Assisted Reproduction. New York: Wiley-Liss, 1993

6.

Munne S, Morrison L, Fung J, et al. Spontaneous abortions are significantly reduced after preconcep­ tion genetic diagnosis of translocations. J Assist Reprod Genet 1998;15:290-6

47

ATLAS O F PREIM PLAN TATION GENETIC D IAG N O S IS

7.

Munn£ S, Sandalinas M, Escudero T, et ill. Outcome of preimplantation genetic diagnosis of transloca­ tions. t'ertil Steril 2000;73:1209-18

8.

Munne S. Preimplantation, genetic diagnosis of numerical and structural chromosome abnormalities. Reprod BioMed Online 2002;4:183-96

9.

Verlinsky Y, Cieslak J, Evsikov S, et al. Nuclear trans­ fer for full karyotyping and preimplantation diagno­ sis of translocations. Reprod BioMed Online 2002;5: 302-7

10.

Scriven PN, Handyside AH, Mackie Ogilvie C. Chromosome translocations: segregation modes and

48

strategies for preimplantation genetic diagnosis. Prenat Diagn 1998; 18:143'7—49 11.

Evsikov S, Cieslak J, Verlinsky Y. Survival of unbal­ anced translocations to blastocyst stage. Fertil Steril 2000;74:672-6

12.

Cassel MJ, Munne S, Fung J, Weier HUG. Carrierspecific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells. Hum Reprod 1997;12:2019-27

13.

Kuliev A, Verlinsky Y. Thirteen-years experience of preimplantation genetic diagnosis. Reprod BioMed Online 2004;8:229-35

6 Preim plantation diagnosis for single-gene disorders

Preimplantation genetic diagnosis (PGD) for single­ gene disorders is based on the testing of oocytes or embryos to preselect and transfer only normal embryos back to the patient, and to achieve an unaf­ fected pregnancy and the birth o f a healthy child. As in PGD for chromosomal disorders, it is performed by testing either single blastomeres removed from eight-cell preimplantation embryos or female gametes, based on the removal and genetic analysis of the first and second polar bodies (PBI and PB2). However, the latter approach cannot be applied for the testing o f paternally derived abnormalities and gender determination, which can be detected by genetic analysis of single blastomeres, removed from the cleavage-stage embryos. Therefore, both methods are complementary and their application is depen­ dent upon the PGD objectives in the individual patient. Overall, the experience of using PGD for single­ gene disorders is now clo.sc to 1500 caseS,: showing that it is an established alternative to traditional prenatal diagnosis, and can be reliably applied as an integral part of genetic practice *~3.

U P D A T E D P R O C E D U R E O F SIN G LE-C ELL D N A A N A L Y S IS FO R PG D O F S IN G L E -G E N E D IS O R D E R S Principles of P G D using first and second polar bodies

The principle of PGD of single-gene disorders using PBI and PB2 analysis is presented in Figure 6.1. As seen from this figure, there are three genetic possi­ bilities for the PBI genotype from a heterozygous

woman. If no crossover occurs, the PBI will be homozygous (either normal or affected), but in the event of a crossover, the PB 1 will be heterozygous. If a crossover does not occur and the PBI is homozy­ gous for the mutant gene, the oocyte must contain two copies of the normal gene and any embryo from this oocyte can be transferred. If the PBI is homozy­ gous for the normal gene, the maternal contribution to the embryo must be the mutant gene. In the event that a crossover occurs, the PB 1 will be heterozygous and analysis of the PB2 is required to predict which maternal allele will be present in the fertilized oocyte (Figure 6.1). If the PB2 contains the normal gene, the oocyte will be affected, and if the PB2 contains the mutant gene the oocyte will have the unaffected gene. The crossover rate varies with the distance of the locus being studied from the centromere and perhaps with other, as yet, undelin­ eated mechanisms. Our experience has shown a high rate of crossover, limiting the number o f embryos available for transfer based on PBI analysis, thus sequential two-step analysis of the PBI and PB2 in genetic analysis of oocytes is required4. As will be demonstrated below, the highest accuracy of diagno­ sis can be achieved if a crossover occurs, as in this case both mutant and normal alleles are detected, so that the finding of the mutant allele in the PB2 indi­ cates that the resulting oocyte is free of the muta­ tion. D N A analysis using conventional PCR analysis in single cells

Genetic analysis in single cells, allowing performance o f PGD for single-gene disorders, became possible after the introduction of the polymerase chain reac­

49

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

tion (PCR) technique5 which is an in vitro technique for the amplification o f specific D N A sequences. PCR is a cycling process, in which the number of D N A targets doubles in each cycle (a flow chart for single-cell genotyping is shown in Figure 6.2). The basic PCR components include a D N A template (10p g-500n g), D NA polymerase (0.5-2.5 unit), primers (0.1-1 .Opmol/1), dNTPs (50-200 pmol/1 of each), buffer (pH 8.3-9.5), and M g C lj (1.5-3.0 pmol/1). PCR allows a single gene to be copied more than a billion times in a matter of hours. It consists o f three basic steps: denaturation, anneal­ ing and extension. (1)

(2)

(3)

Denaturation is carried out at 94—96 °C for 0.5-2.0 min during regular cycles and leads to melting of the template into single strands, eliminating the secondary structure. The annealing temperature allows primers to hybridize to the template, varying with the strand-melting temperature of the primers, with standard reactions using 4 5 -6 0 °C for 1-2 min. Extension is carried out at 72CC, which is optimal for Taq polymerase, completing the synthesis of the new D N A strand. PCR can be efficiently carried out using only two tempera­ tures - one for denaturation and the other for annealing and extension (also called two-step PCR or turbo PCR).

One important factor to avoid amplification failure in the PCR of single cells is the application of an adequate cell lysis procedure. Several methods, such as proteinase K treatment, freeze-thaw technique or KOH lysis procedure, have been used to lyse PBI, PB2, blastomeres or somatic cells6. According to our observations, proteinase K treatment is the optimal method of single-cell lysis for PGD. Single Cells are placed directly into a lysis solution, consisting of 0.5 pi 1 0 x PCR buffer, 0.5 pi l%Tween 20, 0.5 pi 1% Triton X-100, 3.5 pi H^O and 0.05 pi proteinase K (20m g/m l in a 0 .5 m l PCR tube). After centrifuga­ tion at a low speed in a microfuge for a few seconds, the samples are covered with one drop of mineral oil and incubated at 4 5 CC for 15 min in a thermal cycler (a heating block can also be used). Proteinase K is then inactivated at 96°C for 20 min, which is the beginning the hot-start PCR (see below). In case the samples have to be sent by mail, they are Covered with one drop of mineral oil and frozen in vertical position.

50

The other critical feature of single-cell PCR is preventing DNA contamination. Because standard sterile techniques are not sufficient to avoid DNA contamination, a separate laboratory for preimplan­ tation D NA analysis with its own laminar flow hood, refrigerator, freezer, microcentrifuge and thermal cyclers is used. To identify possible contamination by extraneous DNA, a multiplex PCR system, involving polymorphic markers, is applied. The following rules are strictly adhered to: (1)

Tubes containing PCR products are never opened in the D N A laboratory;

(2)

Gloves are worn at all times;

(3)

No traffic is allowed in the D NA laboratory when analysis is being performed;

(4)

Positive' displacement pipettes containing pipette tips are used;

(5)

PCR tubes and micromanipulation tools are decontaminated prior to use under ultraviolet light inside a Template Tamer for at least 30 min;

(6)

Reagents are stored as small aliquots to mini­ mize the number of times the tubes are opened;

(7)

All solutions with the exception of Taq poly­ merase and dNTPs are millipore filtered;

(8)

All reagents, including those used in the embry­ ology laboratory, are tested for D NA contami­ nation prior to every PGD case, as it has been observed that culture media, human serum and immersion oil can become contaminated with jExtraneous DNA.

and

filter-

Any PGD assay contains several control assays with no added cells or D N A and false biopsies in fully reconstituted PCR mixtures, in order to ensure that there is no contamination with extraneous D NA sources. Positive controls containing 100 pg of homozygous abnormal, homozygous normal and heterozygous D NA are also included in each case. Single cells of known origin are used as a positive control, if available. Pseudo-contamination is eliminated: by increas­ ing the stringency of the PCR reaction, by lowering concentrations of dNTPs, Mg2+, primers and Taq polymerase, and increasing annealing temperature. A Template Tamer (ultraviolet decontaminating hood) is used to reduce contamination during the sample preparation for the nested PCR (the list o f equip-

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

Table I Equipment, materials and reagents fo r single-cell D N A analysis Equipment

Supplier

M odel

Therm al cycler

MJR

DYAD D N A Engine (96 V and 30/30 block)

Laminar air flo w workstation

Forma Scientific

Model 1849

D N A contamination-free workstation

MJR

CleanBox D N A W orkstation

Capillary D N A sequencer/genotyper

ABI

3100 Genetic Analyzer

Capillary D N A sequencer/genotyper

ABI

3 10 Genetic Analyzer

Real-time PCR cycler

C o rbe tt Research

RotorGene 2000

Horizontal gel electrophoresis system

Owl

A 3 -I Gator

Vertical gel electrophoresis system

Bio-Rad

Mini PROTEAN 3 Electrophoresis System

DC pow er supply

LabNet

PowerStation 300

Gel Documentation System

UVP

G elD oc-lt System

Microcentrifuge

Beckman

Microfuge 12

Vortex

LabNet

V X I0 0

W ater bath

Fisher Scientific

Model 2LS

Single-channel adjustable pipettor

Finnpipette

Model 4500

Multichannel adjustable pipettor

Brandlech

Transferpette-8

(0 .5 -1 0 //I, 5.0-50 fj\, 20-200 jL/l, 200-1000/71)

Reagents and disposables

Rox size standard

Applied Biosystem

Taq-polymerase

Bioline

Biolase D N A Polymerase

dN TP mix

Bioline

dN TP M IX

D N A molecular weight marker (100-1000 bp)

Bioline

HyperLadder IV

Oligonucleotide primers

MWG Biotech Inc.

D N A contaminant removal solution

CPG

D N A-O FF D0500

W ater

Irvine Scientific

9307

"Iween-20

Amresco

M l47 (1 L)

Triton X - 100

Amresco

M l47 (1 L)

Formamide

Amresco

M l43 (1 L)

Dimethylsulfoxide (DMSO)

Sigma

D-5879

Proteinase K

Invitrogen

Proteinase K Solution RNA grade N e w Fngland Biolabs, MBI Fermentas, ROCHE, Promega

Restriction enzyme Agarose 3:1 high-resolution blend

Amresco

E776 (lOOg)

Am m onium persulfate

Amresco

0486 (25 g)

TEMED

Amresco

0761 (100 ml)

Acryl/Bis 19:1 solution 40%

Amresco

0496 (500 ml)

TBE 10 x buffer liquid concentrate

Amresco

0658 (4

TE buffer pH 8.0

Amresco

El 12 (500m l)

Ethidium brom ide 0.625 mg/ml

Amresco

E406 (5 ml)

Ethidium brom ide destaining bag

Amresco

E732-Q-I

Powder-free latex gloves

Microflex

Evolution One

Aerosol resistant filtered pipette tips

Midwest Scientific

l)

Software

Baby sentry fe rtility database management

BS Ltd 2002

Primer prem ier 5

Biosoft International

51

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

m ent and reagents used for single-cell D N A analysis is presented in Table 1). The sensitivity and specificity of D N A amplifica­ tion are dramatically im proved by using th e nested PCR m ethod (Figure 6.2). The larger fragm ent produced by the first round o f PCR is used as a tem plate for th e second round of PCR, involving nested PCR primers, i.e. those internal to the first prim er pair. This also significantly increases th e effi­ ciency of D N A am plification from a single cell. The best results are obtained by using lower stringency, 5% dim ethyl sulfoxide (D M SO ) and a longer anneal­ ing time, during th e first round of PCR, and higher stringency in the second round. Also, use of a single inside nested prim er (heminesting), w ith the appro­ priate original outside primer, is as efficient as using tw o inside prim ers (full nesting) for the second reac­ tion. In addition, nested PCR allows m ore reactions to be carried out, especially w hen m ultiplex PCR is applied during th e first round of am plification (Figure 6.2). As shown in Figure 6.2, following th e first round of PCR containing outside prim ers for all the genes tested, separate aliquots are am plified using the specific inside prim ers for each site. Only w hen both th e polym orphic allele and the m utation analysis agree, are embryos transferred (Figure 6.3). This dual am plification allows detection of m ost cases of allele dropout (A D O ) and prevents misdiagnosis in PGD (Figures 6.4 and 6.5). To prevent non-specific annealing of prim ers and subsequent elongation of spurious D N A fragments, a hot-start PCR is used for th e first round of amplifi­ cation. A hot-start PCR involves w ithholding one or m ore of th e required reagents of the PCR m aster m ix until th e sample has reached a tem perature of at least 72°C (Figure 6.2). Lysed samples are placed into a therm al cycler and am plified (first round) at th e following therm al cycler program: 45°C for 15m in, 96°C for 20m in, 72°C for 7 m in (hot start); then: 96°C for 20 s, 50°C for 1 m in and 30 s, 72°C for 20 s; 95°C for 20 s, 45°C for 1 min, 72°C for 20 s; for 10 cycles and for 18 cycles 72°C for 7 min. W hen the therm al cycler reaches the 72°C step, each sample in a tube is brought up to a final volum e of 50 pi, using th e first-round PCR m aster mix, consisting of PCR buffer, dNTPs, M gC ^, D M SO and th e set of outside upstream and downstream prim ers for the gene tested, under the mineral oil. A fter th e com pletion of the first round of am pli­ fication th e samples are rem oved from the therm al

52

cycler and spun down in a microcentrifuge to prevent an aerosol splash of PCR product. An aliquot of 1-2 pi of th e resulting first-round PCR product is transferred into 0.5-ml tubes w ith 48 pi of th e fresh second-round PCR m aster mix, containing PCR buffer, dNTPs, MgCl?, Taq polymerase, H 2 O and nested upstream and downstream prim ers for the genes tested. In contrast to th e first round, each tube contains singular pairs of specific nested primers. A drop of mineral oil is added in each tube, and the samples are am plified using the therm al cycler program as follows: 95°C for 5 min, then 95°C for 20 s, 60°C for 20 s for 25 cycles; and 72°C for 7 min. Following nested amplification, PCR products are analyzed either by restriction digestion, real-time PCR, direct fragm ent size analysis or minisequencing (Figure 6.2) (see below). D epending on the m u ta­ tion studied, different prim er systems are designed, w ith special emphasis on the elim ination of false prim ing to possible pseudogenes, such as in PGD dem onstrated for long-chain 3-hydroxyacyl-CoA deficiency (LCHAD ) and congenital adrenal hyper­ plasia, for which purpose the first-round prim ers are designed to anneal to th e regions of non-identity w ith a pseudogene (Figures 6.6 and 6.7). W hen breakpoints are known, a similar approach may be used to test for large deletions, such as alphathalassemia and large deletion forms of betathalassemia (Figure 6.8). Fluorescence PCR

A direct fragm ent size analysis of PCR product is perform ed by fluorescent PCR (F-PCR)7. Instead of using gel electrophoresis and ethidium brom ide staining, F-PCR uses fluorescently labeled prim ers in the PCR mix, and the sample is analyzed on a fluo­ rescent capillary detector, as shown in Figure 6.9. Because F-PCR is m uch m ore sensitive than th e stan­ dard gel detection, fewer PCR cycles are run, w ith the m ultiplex reaction being perform ed and analyzed in one tube using different color labels on each primer. F-PCR is particularly useful for direct sequencing of the PCR product for detection of point m utations and also for distinguishing preferen­ tial amplification from A D O (see below). Examples of the application of F-PCR in parallel w ith conven­ tional PCR are shown in Figures 6.10 and 6.11. Because of the possibility of preferential amplifi­ cation or A D O in single-cell PCR analysis, highly polym orphic linked markers, such as short tandem repeats (STRs), are sim ultaneously am plified w ith

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

the gene to which these STRs are strongly linked (Figure 6.12). The conditions o f PCR analysis are different for each diagnostic system and require a preliminary work-up. R eal-tim e PCR

Real-time PCR detects specific nucleic acid amplifi­ cation products as they accumulate by using a fluorescently labeled oligonucleotide probe, which elim­ inates the need for post-PCR processing. A reporter fluorescent dye and a quencher dye are attached to the hybridization probe. In an intact probe, the reporter dye's emission is minimal due to energy transfer to the quencher dye, resulting in negligible fluorescence being observed. During the PCR amplification, the probe anneals to its target sequence on the sample DNA. As the newly synthesized D NA strand reaches the site of probe hybridization, the D NA polymerase cleaves the probe, releasing the reporter dye from the quencher dye. At this point an increase in fluores­ cence is detected. The replication of the D N A strand continues undisturbed until the amplification cycle is complete. This process is known as TaqMan® chemistry (Figure 6.13). Since the reporter dye molecules are cleaved from the probes during each amplification cycle, the fluorescence intensity of the overall system increases proportionally to the amount of D N A amplified (Figure 6.14). Real-time PCR allows screening for genes with a single basepair variation between the normal and mutant geno­ types, and detection of sequence changes, deletion or insertion. Two hybridization TaqMan probes are designed for this purpose: one being a perfect match to the normal sequence and the other containing a mutant sequence. The perfect match probe binds to its target and is cleaved to produce a typical real­ time PCR signal. The amplification product from heterozygous cells gives rise to two signals (Figure 6.15). The fluorescence emission is detected every few seconds, and data are plotted in real time as a func­ tion of the cycle number. Rather than monitoring the final amplification product, as is performed in tradi­ tional PCR, the real-time PCR reaction is character­ ized by the point in time when amplification o f a PCR product is first detected. This time point is referred to as cycle threshold (Ct). The larger the amount of initial sample, the sooner this threshold value is achieved. Single-cell amplification with TaqMan probes requires at least ten cycles more than

extracted genomic D NA amplification (Figures 6.14 and 6.15) The advantage of real-time PCR is that the PCR tubes do not need to be opened after the ampli­ fication reaction is complete since all the data have already been collected. This prevents contamination by PCR products and reduces the amount o f 'false positive’ results. Real-time PCR can also be performed with mole­ cular beacons (MBs). In contrast to the linear probes used in TaqMan systems, MB is a stem-and-loop hairpin structure design with a reporter fluorescent dye on one end and a quencher moleculc on the other. The loop portion of the molecule is a probe sequence, which is complementary to a target sequence, and the stem is. formed by short comple­ mentary sequences located at opposite ends of the molecule (Figure 6.16). The hairpin structure causes the MB probe to fold when not hybridized. This brings the reporter and quencher molecules into close proximity with little or no fluorescence being emitted. However, when the MB hybridizes to the template, the hairpin structure is broken and the reporter dye is no longer quenched. At this point, the real-time instrument detects fluorescence. The hairpin shape o f the probe causes mismatched probe/target hybrids to dissoci­ ate easily at a lower temperature than perfectly matched hybrids. The thermal instability of mismatched hybrids increases the specificity of MBs, enabling them to distinguish targets by only a single nucleotide. When conjugated to different fluo­ rophores, two allele-specific MBs can simultaneously discriminate the three possible genotypes (Figure 6.17). As already mentioned, the PCR product after the second round may also be analyzed by minisequenc­ ing (Figure 6.18). As will be described in Chapter 7, the feasibility o f applying microarray technology at the single-cell level is currently also being assessed with the clear prospect of PGD for single-gene disor­ ders using microarrays, especially with simultaneous detection of mutations and surrounding single­ nucleotide polymorphisms.

D IA G N O S T IC A C C U R A C Y

Because the PGD for single-gene disorders is based on single-cell genetic analysis, its accuracy depends largely on the limitations of single-cell D N A analysis, which may potentially cause misdiagnosis. One ol the key contributors to misdiagnosis is the phenom­

53

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

enon o f preferential amplification (Figure 6.9), also known as A D O (Figures 6.9 and 6.19), requiring the application o f special protocols to ensure the highest A DO detection rate8. A few previously reported misdiagnoses, involving PGD for beta-thalassemia, myotonic dystrophy (DM), fragile-X syndrome (FMR1) (Figure 6.20) and cystic fibrosis (CF), could have been due to this phenomenon1,3, which initially was not fully recognized. It has been demonstrated that ADO rates in single cells might be different for different types of heterozygous cells (Figure 6.21)9. The A D O rate may be as high as between 10 and 20% in blas­ tomeres, compared to the A D O rate in single fibro­ blasts and PBI, which is under 10%, A high rate of A D O in blastomeres may lead to an obvious misdi­ agnosis, especially in compound heterozygous embryos. As previously mentioned, most misdiag­ noses, especially those at the initial stage of applica­ tion of PGD for single-gene disorders, have occurred in cases of blastomere biopsy from apparently Compound heterozygous embryos. To avoid misdiag­ nosis due to preferential amplification, a simultane­ ous detection o f the mutant gene together with up to three highly polymorphic markers, closely linked to the gene tested, was introduced (Figure 6.22)8. Each additional linked marker may reduce' the misdiagno­ sis rate by approximately half, so that with one linked marker amplified together with mutation a misdiagnosis risk in blastomere analysis may be reduced from 20% to 10%, with two from 10% to 5%, and with three from 5% to practically zero. The number o f markers required might be reduced with the use of real-time PCR, because of the consider­ ably lower A DO rate observed in real-time PCR (Figure 6.23). Therefore, the most reliable method to date is multiplex nested PCR analysis, with the initial PCR reaction containing all the pairs of outside primers, so that following the first-round PCR, sepa­ rate aliquots of the resulting PCR product can be amplified using the inside primers specific for each site (Figure 6.24). Only when the polymorphic sites and the mutation agree, are embryos transferred. Thus, multiplex amplification allows detection of A D O and prevents the transfer of misdiagnosed affected embryos, as shown in the example of PGD for autosomal dominant early-onset torsion dystonia (DYT) (Figure 6.25). Another efficient approach to avoid misdiagnosis may be a sequential genetic analysis o f the PBI and PB2 in PGD for maternally derived mutations.

54

Detection o f both mutant and normal alleles in the heterozygous P B I, together with the mutant allele in the corresponding PB2, leaves no doubt that the resulting maternal contribution to the embryo is normal, even without testing for the linked markers as a control, as shown in the case of PGD for famil­ ial posterior fossa brain tumor (hSNF5) (Figure 6.26). However, as demonstrated in these cases, it is ideal to test simultaneously for at least one linked marker to confirm the diagnosis. Alternatively, muta­ tion-free oocytes can also be predicted when the corresponding PBI is homozygous mutant, in which scenario the corresponding PB2 should be hemizygous normal, similar to the resulting maternal pronu­ cleus. However, the genotype of the resulting mater­ nal contribution may be quite the reverse, i.e. mutant, if the corresponding PB 1 is in fact heterozy­ gous, but erroneously diagnosed as homozygous normal because of A D O of the normal allele (see Figures 6.3, 6.4 and 6.27). In the above scenario, the extrusion of the normal allele with the PB2, would lead to the mutant allele being left in the resulting oocyte. Therefore, the embryos resulting from the oocytes with homozygous mutant PBI cannot be acceptable for transfer, unless the heterozygous status of the PBI is excluded by the use of linked markers as described. Thp example of misdiagnosis, due to A D O of the normal allele in PBI has been described earlier in a PGD cycle performed for FMR1 (Figure 6 .2 0 )l0. To avoid misdiagnosis completely, sequential PBI and PB2 analysis may be required to combine with multiplex PCR to exclude the possibility of an undetected A D O in a heterozy­ gous PBI (see Figures 6.3, 6.4 and 6.27). The analy­ sis of more than 1000 oocytes tested by sequential PBI and PB2 analysis showed that more than half of A D O s were detected by sequential analysis o f PBI and PB2, with the remaining cases detected by multiplex PCR8. The particular value of linked marker analysis combined with sequential PBI and PB2 testing is obvious from the presented case of PGD for spinal muscular atrophy (SMA) (Figure 6.28), caused by mutations in survival motor neuron gene (SMA/), present in two homologous copies [SM N 1 and SM N 2 ), the loss of only one of them [SM N 1] leading to SMA. The accuracy of this approach may also be demonstrated by the reports of PGD for thalassemia and familial dysautonomia (FD), resulting in the transfer of three unaffected embryos in each case, which were confirmed by the

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

birth of the two sets of triplets free from thalassemia and FD (Figures 6.29—6.31311• The other method with the proven potential for the detection and avoidance o f misdiagnosis due to preferential amplification is fluorescence PCR (FPCR), which can allow detection of some of the heterozygous PBI or blastomeres misdiagnosed as homozygous in conventional PCR, and therefore, has the potential to reduce the A D O rates to some extent (Figures 6.9 and 6.23)8. In addition, the method also allows simultaneous gender determina­ tion, D N A fingerprinting and detection of common aneuploidies (Figures 6.32-6.34). With further improvement and simplification, F-PCR combined with a multiplex system and sequential PBI and PB2 analysis in cases of maternally derived mutations can almost completely exclude the risk for misdiagnosis due to preferential amplification. The accuracy of PGD has been further improved with the application of F-PCR with the expand long template (ELT) kit, which reduces the A D O rate from as high as 30-35% in both conventional and FPCR to as low as 5% in testing for myotonic dystro­ phy (D M )12. Another development to improve the accuracy o f single-cell PCR analysis involves the application of real-time PCR, which reduces the A D O rate almost by half, as shown in Figure 6.23, compared to conventional or F-PCR. The application of these approaches, together with simultaneous testing for the causative mutation along with at least one or two linked markers can reliably avoid the risk for misdiagnosis. Finally, because of the high rate of aneuploidies in oocytes and embryos, including mosaicism at the cleavage stage, testing for the chromosome in which the gene in question is mapped, is of obvious value, to exclude the lack or extra mutant allele caused by monosomy or trisomy of this chromosome in the biopsied blastomere (Figures 6.35—6.38). As shown in Figures 6.32, 6.34 and 6.40, aneuploidy testing is technically feasible and can be performed by adding primers for chromosome-specific microsatellite markers to the multiplex PCR protocols worked out for specific genetic disorders. The development of multiplex nested PCR systems will also allow PGD to be performed simultaneously for different condi­ tions, as attempted for PGD for CFTR or SMN mutations together with aneuploidy testing and gender determination (Figures 6.28, 6.32 and 6.34), and mutation testing together with H LA typing (Figures 6.41 and 6.42), for which purpose different

strategies m aybe chosen (Figure 6.43). For example, H LA typing, demonstrated an approximately 6% aneuploidy rate for chromosome 6, which has a clear impact on the correct H LA typing as shown in Figure 6.44. There is strong indication for aneuploidy testing in PGD for patients'of advanced reproductive age, such as in many cases of preimplantation HLA typing. The relevance o f aneuploidy testing in HLA typing for patients of advanced reproductive age is demonstrated in Figure 6.33. Owing to the requirement to develop a custommade PGD design for each mutation and each couple, a preparatory work has become an integral part of PGD for single-gene, disorders to ensure; potential misdiagnoses are avoided. For example, in some cases a particular set of outside primers has to be designed to eliminate false priming to a pseudo­ gene, as described in PGD for long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and also presented above for congenital adrenal hyperplasia (Figures 6.6 and 6 .7 )13. Furthermore, the prepara­ tory work can frequently involve single-sperm typing which is required to establish paternal haplotypes, so that linked marker analysis can be performed in addition to mutation analysis, especially in cases of paternally derived dominant conditions (Figure 6.45). The availability of the parental haplotypes allows not only the absence of the mutant gene to be confirmed, but also the presence of both maternal and paternal wild alleles in PGD by blastomere analysis, especially when only one informative marker is available (Figure 6 .4 6 ).14 Finally, haplotype analysis allows PGD for those conditions for which no exact causative gene status is available, such as autosomal dominant polycystic kidney disease type 1 and 2 (PKD1 and PKD2), the first experience of which is presented in Figures 6.47 and 6.48. This may have implications for PGD of other similar conditions, which, to date, cannot be performed by traditional approaches.

P G D FO R SPEC IFIC G E N E T IC D IS O R D E R S

The list of disorders, currently comprising approxi­ mately 100 different conditions, for which PGD has been applied is being extended beyond the indica­ tions for prenatal diagnosis, although the most frequent ones are still CF and hemoglobin disorders’~3,15. According to our experience o f approxi­ mately 400 PGD cycles for single-gene disorders, almost half of these cycles were performed for CF

55

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

and hemoglobin disorders, followed by DM and FMR1, similar to the experiences in other active centers1-3. The list of conditions for which PGD has been performed to date is presented in 'Fable 2. Specific diagnosis fo r X -lin ke d diseases

More than half of PGD cases for single-gene disor­ ders have been performed by gender determination for X-linked conditions, which have been the most straightforward application from the very beginning, either using PCR or FISH techniques*"®* This was not only because the sequence information was not always available, but also because it was technically easier to identify female embryos by D N A analysis or FISH, despite the obvious cost of discarding 50% o f healthy embryos because they were male. On the other hand, testing for X-linked genetic disorders may be limited entirely to oocytes, because the mutations involved are fully maternally derived. Therefore, testing of oocytes for maternally derived specific mutations makes it possible to avoid further testing of the resulting embryos, which may be trans­ ferred irrespective o f gender or any contribution from the father. Initially this approach was applied for ornithine transcarbamylase deficiency (O T C )20, then it was extended to the specific diagnosis of other X-linked disorders10, and presently comprises the experience o f specific diagnosis in dozens of cycles performed for O TC (Figure 6.49), FMR1 (Figure 6.20), myotubular myotonic dystrophy, Alport syndrome, Charcot-Marie—'footh disease type X , hemophilia A, hemophilia B, Hunter syndrome, incontincntia pigmenti, Duchenne muscular dystro­ phy (DM D), Becker type muscular dystrophy (HMD), Norrie disease, Pelizaeus-Merzbacher disease, retinitis pigmentosa, Wiskott-Aldrich syndrome, X-linked adrenoleukodystrophy, X-linked hyper-IgM syndrome, X-linked incontinentia pigmenti and X-linked hydrocephalus. This resulted in the transfer of mutation-free embryos in most of the cycles and yielded many unaffected clinical preg­ nancies, demonstrating the clinical usefulness of the specific polar body testing for X-linked disorders as an alternative to PGD by gender determination. Couples w ith a homozygous affected p a rtn e r

PGD has also been provided for couples with one homozygous or double-heterozygous affected partner, affected with thalassemia, cystic fibrosis or phenylketonuria (PKU), resulting in an unaffected

56

pregnancy and the birth o f healthy children (Figures 6.34, 6.50 and 6.51)21. Although the risk of produc­ ing an affected child in such couples is as high as 50%, irrespective of the maternal or paternal affected status, the strategy of PGD in such cases will depend on whether the father or mother is affected. In couples with affected fathers, PGD may conccntrate on the preselection of mutation-free oocytes, while with affectcd mothers, cleavage-stage PGD is required to identify those few embryos containing the normal gene. The testing is particularly complicated if the parents carry different mutations. In one such case performed for thalassemia, the affected mother was double heterozygous (IVSI-110; IVSI-1-6), while the male partner was a heterozygous carrier of the third mutation (IVSII-745) (Figure 6.52). This required a complex PGD design to exclude preferential ampli­ fication of each o f the three alleles tested in biopsied blastomeres, as has been described in PGD for PKU21. In this case the affected father was compound heterozygous for R408 and Y414C muta­ tions in exon 12 o f the phenylalanine hydroxylase (PAH) gene, and the carrier mother was heterozy­ gous for the R408W mutation in the same exon (see Figure 6.50). The PGD strategy was based on the preselection of the mutation-free oocytes using sequential PBI and PB2 D N A analysis. Based on the multiplex heminested PCR analysis, four embryos resulting from the zygotes predicted to contain no mutant allele of the PHA gene were transferred, yielding an unaffected twin pregnancy and the birth of healthy twins. With improvement in the treatment and life expectancy for an increasing number of genetic disorders, an increasing number of couples with affected maternal or paternal partners may require PGD as the only means to be able to have unaffected children of their own. C ancer predisposition

Traditionally, cancer predisposition has not been considered as an indication for prenatal diagnosis, as this would lead to pregnancy termination, which can hardly be justified on the basis of genetic predisposition alone. However, the possibility of choosing embryos free from the genetic predisposition for transfer would obviate the need to consider pregnancy termi­ nation, as only potentially normal pregnancies would be established. PGD for such conditions appears to be acceptable on ethical grounds because only a

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

Table 2 List o f diseases fo r which PGD has been perform ed as o f January 2004 Achondroplasia

Li-Fraumeni syndrome ^mutations in p53 gene)

Adenosine aminohydrolase (ADA) deficiency

Long-chain hydroxyacyl-CoA dehydrogenase

a i-antitrypsin deficiency

Malignant autosomal recessive osteopetrosis

ai-iduronidase (IDUA)

Marfan syndrome

A lp o rt syndrome

Metachromatic leukodystrophy

Alzheim er’s disease

Multiple exostoses type I

Aneuploidies by STR genotyping

muscular dystrophy, Duchenne type (D M D )

Autosomal dominant polycystic kidney disease type I

Muscular dystrophy, Becker type (BMD)

Autosomal dominant polycystic kidney disease type 2

Myotonic dystrophy

Autosomal recessive polycystic kidney disease

Myotubular myopathy

C harcot-M arie-T ooth disease type IA

Neurofibrom atosis type I

C harcot-M arie-T ooth disease type IB

Neurofibrom atosis type 2

C harcot-M arie-T ooth disease type 2

Neuronal ceroid lipofuscinoses

C harcot-M arie-T ooth disease type X

N o rrie disease

Choroideremia

Oculocutaneous albinism type I

Citrullinemia

Oculocutaneous albinism type 2

Congenital adrenal hyperplasia

O ptic atrophy

Crouzon syndrome

O rnithine transcarbamylase (O TC) deficiency

Currarino triad

Osteogenesis imperfecta

Cystic fibrosis

Pelizaeus-Merzbacher disease

Diamond-Blackfan anemia

Phenylketonuria

Dystonia torsion

Polycystic kidney disease autosomal dominant type I

Epidermolysis bullosa

Polycystic kidney disease autosomal dominant type 2

Epiphyseal dysplasia, multiple

Polycystic kidney disease, autosomal recessive (ARPKD'*

Familial adenomatosis polyposis

Retinitis pigmentosa

Familial dysautonomia

Retinoblastoma

Familial posterior fossa brain tum or

Sickle cell anemia

Fanconi anemia A

Spinal muscular atrophy (SMA)

Fanconi anemia C

Spinocerebellar ataxia type 2

Fragile-X A & E syndromes

Spinocerebellar ataxia type 3

Galactosemia

Spinocerebellar ataxia type 6

Gaucher disease

Spinocerebellar ataxia type 7

Glycogen storage disease type VI

Symphalangism

G orlin syndrome

Tay-Sachs disease

Hemophilia A

Thalassemia alpha

Hemophilia B

Thalassemia beta

Hereditary non-polyposis colorectal cancer

Treacher Collins syndrome

H LA genotype matching

Tuberous sclerosis type I

Holoprosencephaly

Tuberous sclerosis type 2

H unter syndrome

Von Hippel-Lindau disease

Huntington disease

W iskott-A ldrich syndrome

H urler syndrome

X-linked adrenoleukodystrophy

Hypophosphatasia

X-linked hydrocephalus

Incontinentia pigmenti

X-linked hyper-IgM syndrome

Kell disease

X-linked incontinentia pigmenti

Krabbe disease STR, short tandem repeat

57

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

limited number of the embryos available from hyperstimulation are selected for transfer. The first PGD for inherited cancer predisposition was performed in a couple carrying p53 tumor suppressor gene mutations, which are known to determine a strong predisposition to the majority of cancers22. The couple had the paternally derived missense mutation due to a transversion of a G to an A in exon 5 of the p53 tumor-suppressor gene (Figure 6.53). The carrier was a 38-year-old proband with Li-Fraumeni syndrome (I.FS), diagnosed with rhabdomyosarcoma of the right shoulder at the age of two years followed by right upper extremity amputation. At the age o f 31, he was also diagnosed with a high-grade leiomyosarcoma of the bladder and underwent a radical cystoprostatectomy. His mother was diagnosed with leiomyosarcoma at the age of 37. PGD was performed by blastomere biopsy and multiplex nested PCR analysis with simultaneous testing for the p53 tumor suppressor gene mutation and linked polymorphic markers, allowing the prese­ lection and transfer back to the patient of only muta­ tion-free embryos. This resulted in a singleton preg­ nancy and the birth o f a mutation-free child who at the time of writing was healthy. PGD has also been applied for other cancers, including familial adenomatous polyposis coli (FAP) (Figure 6.54), von Hippel-Lindau syndrome (Figure 6.55), retinoblastoma, neurofibromatosis type I and II (Figures 6.56-6.59) and hSNF5 (Figure 6.26)23. Overall, 20 PGD cycles have been performed for ten couples, resulting in the preselection and transfer of 40 mutation-free embryos, which to date have yielded five unaffected clinical pregnancies and four the birth of healthy children. Despite the contro­ versy of PGD use for late-onset disorders, the data demonstrate the usefulness of this approach as the only acceptable option for couples at risk to avoid the birth o f children with an inherited predisposition to cancer and have a healthy child. O th e r late-o n set disorders w ith a genetic predisposition

One of the first experiences of PGD for late-onset disorders was PGD for genetic predisposition to one of the forms of Alzheimer disease (A D )24, caused by an autosomal dominant familial predisposition to the presenile form of dementia, determined by a nearly completely penetrant autosomal dominant mutation in the amyloid precursor protein (APP) gene, for

58

which no treatment is available despite a possible predictive diagnosis. A 30-year-old woman had no signs o f AD, but was a carrier o f V 717L mutation, resulting from G to C substitution in exon 17 of the APP gene (Figure 6.60). Predictive testing in the patient was performed because of the early onset of AD in her sister who also carried this mutation and developed symptoms o f AD at the age of 38. At the time of publication, this sister was still alive, but her cognitive problems had progressed to the point that she was placed in an assisted living facility (Figure 6.61). Her father had died at the age of 42 and also had a history of psychological difficulties and marked memory problems. The V 717L mutation was also detected in one of her brothers, who had experi­ enced mild short-term memory problems as early as age 35, with a moderate decline in memory, new learning and sequential tracking in the next 2-3 years. The other family members, including one brother and two sisters were asymptomatic, although predictive testing was performed only in the sisters, who appeared to be free from the APP gene muta­ tion. PGD was performed by D NA analysis of the PBI and PB2, to preselect and transfer back to the patient only the embryos resulting from mutation-free oocytes (Figure 6.60). Based on both mutation and the linked marker analysis, unaffected embryos resulting from mutation-free oocytes were prese­ lected for transfer back to the patients, resulting in a singleton clinical pregnancy and the birth of an unaf­ fected mutation-free child in the first PGD cycle and healthy twins in the second PGD cycle (Figure 6.61). Therefore, PGD provides a non-traditional option for patients who may wish to avoid the transmission of the mutant gene predisposing to late-onset disor­ ders in their potential children. Because such diseases that present beyond early childhood and even later may not be expressed in 100% of the cases, the application of PGD for this group of disorders is still controversial. However, for diseases with no current prospect for treatment, arising despite presymptomatic diagnosis and follow-up, PGD may be offered as the only relief for the at-risk couples. Blood group incom patibility

The first PGD for maternal fetal incompatibility resulting in a healthy pregnancy outcome was performed for the Kell (K l) genotype, which is one

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

of the major antigenic systems in human red blood cells, comparable in importance to RhD, causing maternofetal incompatibility leading to severe hemolytic disease o f the newborn (H D N) in sensi­ tized mothers25. K1 allele is present in 9% of the population, in contrast to its highly prevalent allelic variant K.2. The gene is located on chromosome 7 (7q33), consisting of 19 exons, with only C to T base substitution in exon 6 in K1 compared to K 2 antigen. In the case of a pregnancy with a K1 fetus in a K2 mother, antibodies to K1 can develop leading to maternofetal incompatibility causing severe HDN. Although prenatal diagnosis is available for identifi­ cation of pregnancies at risk for HDN, this cannot always prevent the potential complications for the fetus, stillbirth or neonatal death, making PGD a possible option for preventing both Kell and Rhesus hemolytic diseases. PGD for Kell disease was performed for two atrisk couples with a history of neonatal death in previ­ ous pregnancies due to H D N (Figures 6.62 and 6.63). The preselection and transfer of the embryos free from the K1 allele of the KEL gene was possible in each case, yielding a clinical pregnancy and the birth o f healthy twins, confirmed to be free of the K 1 allele (Figures 6.62 and 6.63). A number of attempts have also been undertaken to perform PGD for Rhesus disease; however, they have not yet resulted in clinical pregnancy26. Both of these conditions are quite prevalent, taking into consideration the approximately 15% frequency for RhD and 9% for KEL antigen, presenting a risk for alloimmunization that may lead to H D N in some of the at-risk couples. Therefore, PGD may be a useful option for these couples to avoid the establishment of an RhD or K1 pregnancy in sensitized mothers. Although the at-risk pregnancies detected by prenatal diagnosis may be treated by intrauterine transfusion, potential complications for the fetus cannot be completely excluded even after this proce­ dure. Pregnancy termination in such cases is unac­ ceptable, as the antibodies to K l, for example, are developed only in 5% of those obtaining incompati­ ble blood. On the other hand, some o f the at-risk couples have had the unfortunate experience of HDN, resulting in neonatal death, as had both of our couples, so that they regard PGD as their only option to plan another pregnancy. This makes PGD attrac­ tive for patients at risk for alloimmunization,

although such conditions have rarely been an indica­ tion for prenatal diagnosis. Congenital m alform ations

Congenital malformations are highly prevalent (29.3/1000 live births) and are usually sporadic. However, with the progress o f the human genome project, an increasing number of inherited forms are being described, which, therefore, may he avoided through PGD. For example, sonic hedgehog ( SH H ) gene mutation, for which the first PGD has recently been performed7 , causes failure of the cerebral hemispheres to separate into distinct left and right halves and leads to holoprosencephaly (HPE), which is one of the most common developmental anom­ alies of forebrain and midface. Although the majority of HPE are sporadic, familial cases are not rare, with clear autosomal dominant inheritance. Wide intrafamilial clinical variability of HPE from alobar HPE and cyclopia, to cleft lip and palate, microcephaly, ocular hypertelorism, and even normal phenotype, suggests the interaction o f the SH H gene with other genes expressed during cranio­ facial development and the possible involvement of environmental factors. This may explain the fact that almost one-third o f carriers of SHH mutations may be clinically unaffected. Therefore, even in familial cases, the detection of SHH mutations in prenatal diagnosis might not justify pregnancy termination, making PGD a more attractive, option for couples at risk for producing progeny with HPE, as demon­ strated by the first PGD for this mutation mentioned27. A couple presented for PGD because of their previous two children who had the clinical signs of HPE (Figure 6.46). One of them, a female, with severe HPE and cleft lip and palate, died shortly after birth. Both the child and the parents were chromo­ somally normal, but D N A analysis of th€ child's autopsy material demonstrated the presence of SHH nonsense mutation due to GAG>TAG sequence change leading to premature termination of the protein at position 256 (Glu256stop). The same mutation was found in their 5-year-old son, who was born after a full-term normal pregnancy. This child had less severe facial dysmorphisms, which included microcephaly, Rathke’s pouch cyst, single central incisor and choanal stenosis. There was also clinodactyly of the fifth fingers and incurved fourth toes bilaterally. The child’s growth was slow during the

59

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

first 2 years, but after that time he maintained reasonably good growth. PGD was performed by blastomere biopsy and multiplex nested PCR analysis, involving specific; mutation testing simultaneously with linked marker analysis (Figure 6.64). O f nine tested embryos four embryos were free of the mutant gene, two of which were transferred back to the patient, resulting in a singleton pregnancy and the! birth of a healthy child following confirmation of the mutation-free status by amniocentesis. A similar approach was used for PGD of Crouzon syndrome (Figure 6.27) and Currarino triad, representing inherited congenital malformations for which PGD may be an attractive approach to ensure that at-risk couples can have an unaffected offspring. The data suggest that PGD for familial cases of congenital malformations is clini­ cally useful. Because of the high prevalence of congenital anomalies, the approach may have practi­ cal implications for at-risk couples as a preventive measure to be employed in genetic practice. P re im p lantatio n H L A m atching com bined w ith causative gene te s t

Preimplantation HLA matching was first introduced in combination with mutation analysis for Fanconi anemia, with the objective of establishing an unaf­ fected pregnancy yielding potential donor progeny for transplantation in an affected sibling28. This resulted in a clinical pregnancy and the birth of an unaffected child, whose cord blood was transplanted to the affected sibling saving her life (Figures 6.41 and 6.42). This strategy would probably not be clinically acceptable using traditional prenatal genetic diagno­ sis because o f a possible clinical pregnancy termina­ tion after H LA matching. However, PGD for such a purpose should be acceptable because only a limited number of the embryos are usually preselected for transfer, which in this case will represent unaffected embryos with a perfect match for affected siblings in need of a transplant. Since the multiplex single-cell PCR used in PGD presents the opportunity for combined PGD and HLA testing, it has become a useful way to preselect an embryo, which may be an HLA match to the affected sibling requiring stem cell transplantation (Figure 6.65). As can be seen from Figure 6.66, because of the possibility of recombination within the HLA region, which was detected in 4.3% of cases and can lead to misdiagno­ sis in the preselection of HLA-matched embryos, a

60

set of markers covering the whole H LA cluster (Figure 6.65) is required to detect and avoid the transfer of misdiagnosed non-matched embryos caused by recombination within the H LA region. To date, this method has been applied for the HLA genotyping in 24 cycles in combination with PGD for thalassemia (Figures 6.67-6.69), Fanconi anemia (Figures 6.41 and 6.42), hyper-IgM syndrome (Figure 6.70) and Wiskott-Aldrich syndrome, confirming the usefulness of preimplanta­ tion H LA matching as part of PGD, with the prospect o f applying this approach to other inherited conditions also requiring an HLA-compatible donor for bone marrow transplantation. This provides a realistic option for couples desiring to avoid the birth of an affected child, together with the establishment of a healthy pregnancy, potentially providing an HLA-matched progeny for the treatment of an affected sibling. Most recently, preimplantation H LA typing has been performed without testing for a causative gene, for the sole purpose of identification of HLAmatched embryos in order to have a potential donor for stem cell transplantation for siblings with bone marrow disorders. Overall, 13 clinical cycles have already been performed for H LA typing without PGD, which resulted in the embryo transfer of the HLA-matched embryos, yielding the birth of five HLA-matched healthy children (Figures 6.38 and 6.39). Although this is still controversial, it appears to be very attractive for the couples who need an HLA-matched bone marrow transplantation for siblings with leukemia or sporadic DiamondBlackfan anemia29.

C O N C L U S IO N

PGD has become an established procedure for avoiding the birth of affected children with singlegeng disorders and having healthy unaffected offspring. PGD is performed through PB or blas­ tomere biopsy, which has no deleterious effects on the pre- and postimplantation development. The present PGD experience includes approximately 1500 clinical cycles performed for single-gene defects, with the majority of cycles resulting in embryo transfer, and more than one-quarter in clini­ cal pregnancy and the birth of unaffected and appar­ ently healthy children. Therefore, PGD for single­ gene disorders may be considered to be a safe,

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

accurate and reliable technique, with growing value for prevention o f genetic disorders. Available experience provides the basis for the wider application o f P G D for any genetic disease currently diagnosed by prenatal diagnosis, and also indications for which prenatal diagnosis has not previously been practiced, such as late-onset and com plex disorders, congenital m alform ations, blood group incom patibility and preselection o f unaffected and H LA -m atched embryos. This extends the practi­ cal value o f PGD , with its utility being no longer lim ited to the prevention o f single-gene disorders, by expanded it to the treatm ent o f siblings requiring stem cell transplantation.

9.

Rechitsky S, Strom C, Verlinsky O, et al. Allele drop out m polar bodies and blastomeres. J Assist Reprod Genet 1998;15:253-7

10.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Polar body based preimplantation diagnosis for X-linked genetic disorders. Reprod BioMed Online 2002;4:38-42

1 1.

Rechitsky S, Verlinsky O, Kuliev A, et al. Preimplantation genetic diagnsis for familial dysautonomia. Reprod BioMed Online 2003;6:488-93

12.

Sermon K, Seneca S, De Rycke M, et al PGD in the lab for triplet diseases - myotonic dystrophy, Huntington's disease and fragile-X syndrome Mol Cell Endocrinol 2001; 183: S77-S85

13.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Reprod BioMed Online 2001;2:17-19

14.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for sonic hedgehog muta­ tion pausing familial holoprosencephaly. N Engl J Med 2003;348:1449-54

15.

Handyside AH, Lesko JG, Tarin JJ, et al. Birth of a normal girl after in I’itro fertilization and preimplan­ tation diagnosis testing for cystic fibrosis. N Engl J Med 1992;327:905-9

16.

Verlinsky Y, Kuliev AM, eds. Preimplantation Diagnosis of Genetic Diseases: a New Technique in Assisted Reproduction, New York: Wiley-Liss, 1993

17.

Xu K, Shi ZM, Veeck LL, et al First unaffected preg­ nancy using preimplantation genetic diagnosis for sickle cell disease. J Am Med Assoc 1999; 281:1 701-6

18.

Kuliev A, Rechitsky S, Verlinsky O, et al. Birth of healthy children after reimplantation diagnosis of thalassemias. J Assist Reprod Genet 1999;16:207-11

19.

Goossens V, Sermon K, Lissens W, et al. Clinical application of preimplantation genetic diagnosis for cystic fibrosis. Prenat Diagn 2000;20:571-81

20.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for ornithine transcarbamylase deficiency. Reprod BioMed Online 2000; 1: 45-7

21.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for PKU. Fertil Steril 2001;76:346-9

22.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for p53 tumor suppressor gene mutations Reprod BioMed Online 2001 ;2; 102-5

REFERENCES

1.

2.

3.

4.

5.

International Working Group on Preimplantation Genetics (IWGPG). Preimplantation genetic diagno­ sis - experience of three thousand clinical cycles. Report of the 11th Annual Meeting International Working Group on Preimlantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 15, 2001. Reprod BioMed Online 2001;3:49-53 Kuliev A, Verlinsky Y. Current features of preimplan­ tation genetic diagnosis. Reprod BioMed Online 2002;5:296-301 European Society of Human Reproduction and Embryology. Preimplantation Genetic Diagnosis Consortium. Data Collection III, May 2002. Hum Reprod 2002; 17,233-46 Verlinsky Y, Rechitsky S, Cieslak J, et al. Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body. Biochem Molec Med 1997;62:182-7 Saiki R, Scharf S, Faloona F, et al. Enzymatic amplifi­ cation of beta-globin genomic sequences and restric­ tion site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-4

6'.

Li H, Cui X, Arnheim N. Analysis of DNA sequence variation in single cells. Meth Enzymol 1991;2:49-59

7.

Findlay I, Quirke P. Fluorescent polymerase chain reaction: Part I. A new method allowing genetic diag­ nosis and DNA fingerprinting of single cells. Hum Reprod Update 1996;2:137-52

8.

Rechitsky S, Verlinsky O, Amet T, et al. Reliability of preimplantation diagnosis for single gene disorders. Mol Cell Endocrinol 2001;1 83:S65-S68

61

ATLAS OF PRE IMPLANTATION GENETIC DIAGNOSIS

23.

Rechitsky S, Verlinsky O, Chistokina A, et a l Preimplantation genetic diagnosis for cancer predis­ position. Reprod BioMed Online 2002;5:148-55

24.

Verlinsky Y, Rechitsky S, Verlinsky O, Masciangelo Q Lederer K, Kuliev A. Preimplantation diagnosis for early onset Alzheimer disease caused by V717L mutation. J Am Med Assoc 2002;287:1018-21

25.

Verlinsky Y, Rechitsky S, Verlinskv O, et al. Preimplantation diagnosis for Kell genotype. Fertil Steril 2003;80:1047-51

26.

Van Den Veyver IB. Prenatal and Preimplantation Genetic Diagnosis for RhD Alloimmunization. 10th International Conference on Prenatal Diagnosis and

62

Therapy Barcelona (Spain), June 19-21, 2000; Barcelona: University of Barcelona, 2000; 181-5 Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for sonic hedgehog muta­ tion causing familial holoprosencenphaly. N Engl J Med 2003;348:1449-54 _c

29

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. J Am Med j4s.soc 2001 ;285;3130-3 Verlinsky Y, Rechitsky S, Sharapova T, et al. Preimplantation HLA typing. J Am Med Assoc 2004; 291:2079-85

7 Future perspectives for preim plantation diagnosis

With the expanding indications for preimplantation genetic diagnosis (PGD] and its wider application to assisted reproduction practices, the annual numbers of PGD cycles performed in the past 2 years has almost doubled. To date, more than 1000 unaffected children have been born following PGD, indicating a further improvement in the accuracy and reliability o f this relatively novel procedure1. Introduced initially as an alternative to prenatal diagnosis, PGD has now become an important complement to the currently available approaches for prevention o f genetic disorders, which can allow some couples at risk to forgo pregnancy only because an unaffected pregnancy can be established. As previously described, PGD has also stimulated improvements in the accuracy of single cell genetic analysis. As seen from specifically designed sections, significant progress has been achieved in PGD for aneuploidies and translocations, owing to the intro­ duction o f novel and improved techniques.

IM P R O V IN G A C C U R A C Y O F P G D FO R S IN G L E -G E N E D IS O R D E R S

At least 1500 PGD cycles have been performed for single-gene disorders, resulting in the birth o f more than 300 unaffected children1-3. Among the new groups of disorders for which PGD has been performed, since the first edition o f the Atlas, are maternal-fetal incompatibility, congenital malforma­ tions, late-onset disorders with a genetic predisposi­ tion and human leukocyte antigen (H LA) testing without diagnosis for a causative gene4-9. The accu­ mulated experience: shows that some of the prob­ lems previously known to lead to misdiagnosis of single-cell genetic analysis, such as contamination by

extraneous D N A or failed amplification, can now be well controlled, with reliable methods currently available. However, some centers are still working on the application of these methods to achieve a PGD clinical outcom e10-12, or are concentrating on the development of PGD protocols13' 15!; therefore the methods described in this edition can be of practical value in the integration of PGD within assisted reproduction and genetic practice. As was demon­ strated in Chapter 6, considerable progress has also been made in the detection of preferential amplifi­ cation and allele drop-out (A D O ), which can currently be avoided through the application of multiplex nested polymerase chain reaction (PCR) analysis, involving the simultaneous mutation testing with a number of closely linked polymorphic markers16-19. The available experience strongly suggests that PGD protocols for single-gene disor­ ders may no longer be appropriate for clinical prac­ tice without a set of closely linked polymorphic markers being tested simultaneously with the causative gene. Because the availability of a sufficient number of closely linked informative markers cannot be predicted, especially in certain populations or ethnic groups, the introduction of single nucleotide poly­ morphisms (SNPs) as linked markers has consider­ ably improved the chance of finding such markers. An adequate number of informative linked markers, together with the availability of a sufficient number of fluorochromes and the introduction of real-time PCR, have substantially improved the accuracy of PGD, also making possible the detection and avoid­ ance of misdiagnosis due to recombination between the gene and markers, and aneuploidy, which presents potential sources for misdiagnoses. A

63

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

crossover rate of 4.3% within the H LA area has been observed in the recent experience of preimplanta­ tion H LA typing involving a series of 330 preim­ plantation embryos. The fact that aneuploidies are highly prevalent in oocytes and embryos is also well known20-22, but their impact on the accuracy of PGD has not previously been fully appreciated. There is, however, no doubt that the accuracy of PGD may be compromised by the copy number of the chromosome(s), in which the causative gene tested is localized, which is particularly important in PGD for single-gene disorders offered to patients of advanced reproductive age, when the same biopsied single cell has to be simultaneously tested for the causative genii and specific chromosome number. Preliminary data, involving preimplantation HLA typing o f 371 embryos, demonstrated that as many as 6.4% of tested cells were aneuploid for chromosome 6, including 2.2% trisomic and 4.2% monosomic embryos, which may have led to the misdiagnosis of H LA alleles, without evaluating the copy number of chromosome 6 (unpublished data). The data suggest that reliable H LA typing, as well as PGD for single­ gene disorders, should involve testing for the copy number and the origin of the missing or extra chro­ mosomes, in which the genes tested are localized.

R E C O N C E P T IO N D IA G N O S IS FO R PA TER N A LLY D E R IV E D G E N E T IC D IS O R D E R S

As previously described in the first edition, the genetic composition of the oocyte can be reliably tested before conception through removal and testing of the first and second polar bodies. However, no method is yet available for testing the out. nine of male meiosis, as genetic analysis destroys the sperm, making it useless for fertilization. As described in Chapter 3, a new technique has been introduced, allowing the duplication o f a sperm before genetic analysis, so that one of the duplicated sperm can be used for testing and the other for fertilization with the consequent transfer of the resulting embryos23,24. The technique was tested using mouse recipient oocytes, and appeared to produce identical haploid pairs in 97% of cases. However, preliminary data using human oocytes as recipient’ cells for duplica­ tion revealed a higher aneuploidy rate, therefore more data will be needed to investigate whether human oocytes are able to duplicate sperm as faith­ fully as murine oocytes, before the technique can be

64

implemented in the routine practice o f PGD for paternally derived conditions, such as translocations.

D E V E L O P M E N T S IN S A M P L IN G PR O C ED U R ES

There have also been developments in the biopsy procedures applied for removing samples from oocytes and embryos, which may be required to improve the accuracy of PGD. For example, the current PGD practice frequently requires the removal o f a sample from both the oocytes and the resulting embryos, such as in PGD combined with preimplantation H LA typing, and a combined PGD for single-gene and chromosomal disorders. It is of interest that a preliminary evaluation of the outcome of such a double biopsy appeared to have no effect on the viability o f the embryos25. The approach has currently been applied on a regular basis, as a tool for avoiding misdiagnosis due to mosaicism, and has resulted in a 26% pregnancy rate in in vitro fertiliza­ tion (IVF) patients of advanced reproductive age (38.5 years on average)26. With the current tendency towards blastocyst transfer, there has been renewed interest in the development of methods for blastocyst biopsy, as demonstrated by successful PGD cycles performed by blastocyst biopsy for translocations, which has resulted in ongoing clinical pregnancies27. Although blastocyst biopsy is not yet a method of choice in many centers, its potential is obvious, especially for the additional testing required to confirm the polar body or blastomere diagnosis.

T O W A R D S PC R -BASED K A R Y O T Y P IN G

There has been progress in the development of microarray technology to examine the gene-expression profiles of individual human oocytes and embryos, although, to date, no reliable correlation has been identified between molecular markers and the developmental competence of oocytes and preimplantation embryos. By testing the expression profile of over 8000 genes examined in single oocytes and embryos, using Affymetrix GeneChip Microarrays, differences in expression were evident even between oocytes of the same developmental stage, which could be due to underlying genetic differences that Contribute to the patient’s pheno­ type28,29. Comparison o f embryos at different stages

FUTURE PERSPECTIVES FOR PREIMPLANTATION DIAGNOSIS

of development revealed large divergences o f gene expression, therefore microarray technology may help to reveal genes that play a critical role in specific infertile conditions and normal preimplanta­ tion development, thus providing potential targets for diagnosis. Customized microarray technology is currently being developed for the testing of chromosomal aneuploidies and translocations. However, the method is not yet sufficiently reliable for aneuploidy testing, requiring the improvement of the detection of ratio changes o f 0.530. The method was applied for testing genomic D NA for gains and losses of chro­ mosomal material in blocked morulas, showing different levels of mosaicism and aneuploidy for different chromosomes . Also, a prototype microar­ ray was developed for the detection o f Robertsonian translocations, which may be applied in clinical prac­ tice, after further improvements to whole genome amplification to reduce the loss of heterozygosity on low template D N A 3“. On the other hand, no significant progress has been reported in the application of comparative genome hybridization (C G H ), which is still a highly labor-intensive procedure, incompatible with the current laboratory framework o f PGD, even with efforts to perform it on the polar body, or to acceler­ ate the procedure to be completed within 38 h33,34. Also, despite the expected higher aneuploidy detec­ tion rates by CGH, no significant differences were reported35. In the majority o f embryos tested by both fluorescent in situ hybridization (FISH) and C G H , the results were reported to be mainly in agreement, although a few inconsistencies were also reported34,36. As for the available clinical experience of CG H , it is still limited to a series of 20 poor-prognosis IVF patients, involving CG H analysis of 141 embryos, which resulted in preselection and transfer of only 20 aneuploidy-free embryos in 14 cycles, yielding three clinical pregnancies35. This suggests that, to date, C G H may be impractical for clinical application, and requires further improvement.

P R O D U C T IO N O F M ALE A N D FEMALE G A M E T E S F R O M H U M A N S O M A T IC CELLS

Despite previous hopes of using nuclear transfer technology to produce female and male gametes through haploidization of somatic cells37,3>, testing o f the resulting presumably haploid cells by FISH analysis revealed chromosomal abnormalities in all

but 10% of haploid cells in preliminary results39. This may be due to the enucleated recipient metaphase II oocyte and the somatic donor nuclei being at conflicting stages of the cell cycle, therefore synchronization of the cell cycles prior to activation may help to reduce the frequency of chromosomal errors, as was demonstrated in preliminary results in which the number of normal cells almost doubled if the transferred somatic cells were activated 8-14 h after nuclear transfer. This may suggest that, despite the feasibility of somatic cell haploidization by the use of the metaphase II oocyte cytoplasm, its clinical use will require further research to avoid an extremely high aneuploidy rate.

STEM CELL T R A N S P L A N T A T IO N A N D A V A IL A B IL IT Y O F H U M A N E M B R Y O N IC S TEM CELLS

Preimplantation H LA matching has been used during the past 3 years for the preselection of mutation-free embryos, which may also be potential donor progeny for bone marrow transplantation40,41. Preimplantation HLA genotyping in combination with PGD was applied in more than 24 cycles, resulting in the preselection and transfer of the HLA-matched unaffected embryos in 17.5% of the embryos tested, as expected. Overall, the number of requests to perform PGD in combination with H LA typing has been increasing, with the recent emer­ gence o f a considerable proportion of cases involving preimplantation H LA typing without PGD. The currently available experience in the Chicago center includes more than 400 PGD cycles performed for approximately 100 different conditions, including single-gene defects, dynamic mutations and some medically relevant genetic variations, of which 12% cycles have been performed for HLA typing. In 1999, when preimplantation HLA typing was first introduced, only four cycles (0.05%) were performed for this indication however; in 2002, 23 cycles (19%) were undertaken. While the majority of these cases were undertaken for preimplantation H LA genotyping in combination with PGD for causative genes, including thalassemia, Fanconi anemia, hyperimmunoglobulin M syndrome, Xlinked adrenoleukodystrophy and WiskottAldrich syndrome, 13 clinical cycles have already been performed for HLA typing without PGD, i.e. with the only objective of preselecting HLAmatched progeny for transplantation treatment of

65

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

siblings with bone marrow disorders42. As described in Chapter 6, the present experience of preimplanta­ tion H LA typing as the sole indication has already resulted in the birth of five HLA-matched healthy children to becomc potential HLA-compatible donors for siblings requiring bone marrow transplan­ tation. The data provide a realistic option for couples desiring to establish a pregnancy with the potential to provide an HLA-matched progeny for the treat­ ment of an affected family member, with the prospect o f applying the approach to other inherited or acquired conditions, also requiring HLA-compatible donors for bone marrow transplantation. Although preimplantation H LA typing is still controversial, and is not allowed in certain countries, it appears to be so attractive for couples in need, that they are prepared to achieve their goal to provide the affected sibling with HLA-matched donor progeny even by traveling out of the Country. For example, PGD for genetic disease combined with H LA typing for sibling bone marrow rescue has been allowed in some countries, but H LA typing in the absence of high-risk genetic transmissible disease is still allowed only rarely. Thus these couples currently request preimplantation H LA typing outside their own countries, with examples already available in achiev­ ing the goal of producing the birth of HLA-matched progeny for siblings with leukemia or sporadic Diamond-Blackfan anemia42. Similarly, the law in some countries condones the use of embryos for stem cell research and therapeutic cloning, which is still the subject of intense discussion in many other countries. In addition to the provision of HLA-matched stem cells for bone marrow disorders, PGD provides a novel source for the establishment of embryonic stem (ES] cells43. Although ES cells are usually derived from culture of the inner cell mass of the preimplantation blastocyst, a highly efficient and original technique was developed for the establish­ ment of ES cell lines from human embryos at differ­ ent stages of preimplantation development, without isolation and culture of the inner cell mass, which has been applied in the establishment of dozens of ES cell lines44. These ES lines, as well as numerous other ES lines established from spare human blasto­ cysts obtained after PGD for chromosomal disorders, were characterized as typical pluripotent ES cells and were shown spontaneously to differentiate in vitro into a variety of cell types, including neurons and contracted cardiocytes. The established reposi­

66

tory of ES cells is currently used for research purposes and is also available on request. As PGD allows genetic characterization of preim­ plantation embryos, prior to the formation of ES cells and their directed differentiation into different ES cell lineages, this may also improve the efficiency o f the establishment of ES cells, which still varies greatly between laboratories. O f particlar relevance is the identification o f the factors influencing the differentiation of human ES cells, which is an essen­ tial prerequisite for their therapeutic application in regenerative medicine. For example, in the presence of an inhibitor of bone morphogenic proteins (N O G G IN ), human ES cells were reported to form neurospheres that expressed classical neuronal markers45. Under defined culture conditions, neurospheres were capable of differentiating into a variety o f neural cell types, including astrocytes and oligodendrocytes. The fact that human ES cells can be induced to differentiate in vitro into somatic progeny and that highly enriched cultures of prolif­ erating neural progenitor cells can be isolated from differentiating human ES cells was also reported46. The neural progenitors were differentiated in vitro into astrocytes, oligodendrocytes and mature neurons. Following transplantation to the developing brain, the human neural progenitors responded to host signals and were capable of constructing neuronal and glial lineages. Controlling differentia­ tion into pure populations of specific neural cells may eventually form the basis of therapy for some neurodegenerative:; disorders and spinal injuries.

IN T R O D U C T IO N O F P G D A S T H E F U T U R E IVF S T A N D A R D

An increasing number of centers have been involved in PGD for chromosomal disorders, including translocations and aneuploidies. As previously mentioned, significant reproductive impact has been demonstrated following the use of PGD for chromo­ somal translocations. The experience accumulated to date further confirms the previous observations of more than a four-fold reduction in the spontaneous abortion rate in translocation carriers, making PGD a preferred option for chromosomal translocations over traditional prenatal diagnosis20,47. Further evidence has been accumulated on the positive clinical impact of PGD for aneuploidy, which is currently performed for advanced repro­ ductive age, repeated IVF failures and repeated spon­

FUTURE PERSPECTIVES FOR PREIMPLANTATION DIAGNOSIS

taneous abortions. Overall, experience of approxi­ mately 5000 PGD cycles for aneuploidy has been accumulated, with the majority o f cases (approxi­ mately 4000 cycles) undertaken by centers in Chicago, St Barnabas, Bologna and Istanbul20-22,48. The positive clinical impact of aneuploidy testing has recently been documented in terms of doubling the implantation rate in IVF patients aged 40 years and over, provided that there was a sufficient number of zygotes for testing49. The improvement in reproductive outcome was particularly obvious from the analysis o f the repro­ ductive history o f PGD patients50. Analyzing the outcome of transfer of 318 FISH-normal embryos, an implantation rate of 65.1% in pregnant patients was observed, with a total o f the 161 clinical pregnancies generated, which resulted in the birth of 153 chil­ dren from 125 couples, and 26 spontaneous abor­ tions, giving a take-home baby rate of 82.3%. O f the 161 couples involved in the study, 41 were in their first cycle, 14 had experienced 31 spontaneous preg­ nancies with 29 abortions and two deliveries, with only one birth occurring in the remaining 27 couples. A total of 367 cycles were performed in 120 o f these couples before undertaking PGD, with 30 pregnan­ cies, five term and 25 aborted. These couples had also experienced 50 spontaneous pregnancies, three term and 47 aborted. The overall implantation rate derived from their reproductive experience was 13.0% and the take-home baby rate was 6.8%. This makes obvious the clinical usefulness of PGD for aneuploidy for IVF patients with poor reproductive performance. The potential o f preselecting euploid embryos for transfer is in agreement with the fact that more than half of the oocytes and embryos tested by PGD for poor-prognosis IVF patients were shown to have chromosomal abnormalities20-22. At least half of chromosomally abnormal embryos have mosaicism, which is the major challenge in improving the accu­ racy of PGD for aneuploidies. As the overall preva­ lence o f chromosomal abnormalities in oocytes and embryos seems to be comparable, suggesting that mosaic embryos may originate from the aneuploid oocytes through the process known as ‘trisomy rescue', the further improvement o f PGD accuracy mav, in the future, require testing of both oocytes and the resulting embryos. As previously mentioned, this can be achieved by a sequential biopsy of both the polar bodies and the single blastomere from the resulting embryo, so that both meiotic and mitotic

errors can be excluded. In addition, the information of both the oocyte and the embryo chromosome sets will make it possible to detect potential uniparental disomy cases, which may be expected from the detection of normal disomic embryos, originating from trisomic oocytes. Collecting this unique infor­ mation may also be useful in finding a possible explanation for at least some of the cases of Beckwith-Wiedemann syndrome (BWS) reported recently in association with assisted reproductive technology51-53. Because more than half of IVF patients are 35 years and older, and more than half of their oocytes have aneuploidies, avoiding the transfer of the embryos resulting from these oocytes through PGD for aneuploidies should be useful, in addition to potentially improving implantation and pregnancy rates, for avoiding the transfer of embryos with potential uniparental disomies, as possible contribu­ tors to imprinting disorders. The presented data provide strong evidence that PGD is currently an important alternative to prena­ tal diagnosis, as it widens the options available for couples wishing to avoid the birth o f an affected child, also providing the possibility of having chil­ dren for those who would remain childless because of their objection to termination o f pregnancy following prenatal diagnosis. At the same time, PGD has also become an integral part of assisted repro­ duction, allowing avoidance of the transfer of chromosomally abnormal and potentially non-viable embryos. In the future, this may contribute to a significant increase in the implantation and preg­ nancy rates in IVF and to a general improvement in the standards o f assisted reproduction practices.

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Verlinsky Y, Cohen J, Munne S, et al. Over a decade of preimplantation genetic diagnosis experience - a multi-center report. Fertil Steril 2004:82:292-4

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International Working Group on Preimplantation Genetics Preimplantation Genetic Diagnosis. Experience of three thousand clinical cycles. Report o f the 11 th Annual Meeting International Working Group on Preimlantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 1 5 , 2001. Reprod BioMed Online 2001;3:49-53

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European Society o f Human Reproduction and Embryology. Preimplantation Genetic Diagnosis

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ATLAS O F PR EIM PLA N TA TION GENETIC D IAG N O S IS

Consortium. Data Collection III, May 2002. Hum

16.

Piyamongkol W, Bermudez MG, Harper JC, Wells D. Detailed investigation of factors influencing amplifi­ cation efficiency and allele drop out in single-cell PCR: implications for preimplantation genetic diag­ nosis. Mol Hum Reprod 2003;9:411-20

Reprod 2002;17:233-46

4.

Rechitsky S, Verlinsky O, Chistokina A, et al. Preimplantation genetic diagnosis for cancer predis­ position. Reprod BioMed Unline 2002;5:148-55

5.

Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for early onset Alzheimer disease caused by V717L mutation. J Am M ed Assoc 2002;287:1018-21

17.

Rechitsky S, Verlinsky O, Amet T, et al. Reliability of preimplantation diagnosis for single gene disorders. Mol Cell Endocrinol 2001; 183:S65-8

18.

Verlinsky Y, Rechitskv S, Seckin O, et al. Preimplantation diagnosis for Kell genotype. Fertil Steril 2003;80:1047-51

Goossens V. Sermon K, Lissens W, et al. Clinical application of preimplantation genetic diagnosis for cystic fibrosis. Prenat Diagn 2000;20:571-81

19.

De Vos A, Sermon K, De Rijcke M, et al. Preimplantation genetic diagnosis for CharcotMarie-Tooth disease type 1 A. Mol Hum Reprod 2003;9:429-35

20.

Munn£ S. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod BioMed Online 2000;4:183-96

21.

Gianaroli L. Magli MC, Ferraretti AP. The in vivo and in vitro efficiency and efficacy of PGD for aneu­ ploidy. Mol Cell Endocrinol 2001;183:S13-18

22.

Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Nuclear abnormalities in series o f 6733 human oocytes. Reprod BioMed Online 2003;6:54-9

23.

Willadsen S, Munn£ S, Schmmel T, Cohen J. Applications of nuclear sperm duplication. Fifth

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Rechitsky S, Verlinsky O, Kuliev A, et al. Preimplantation genetic diagnosis for familial dysautonomia. Reprod BioMed Online 2003;6:488-93

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Marshall J, Leigh D, McArthur S, de Boer K. Preimplantation genetic diagnosis for facioscapulo­ humeral muscular dystrophy (FSHD). Fifth International Symposium on Preimplantation Genetics,

5-7 June, Antalya, Turkey, 2003:19-20 9.

10.

Malcov M, Yosef DB, Schwartz T, et al. Preimplantation genetic diagnosis for Canavan disease. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:43-4 Hellani A, Al-Aqueel A, Jaroudi K, et al. Pregnancy after preimplantation genetic diagnosis for Sanjad-Sakati syndrome. Fifth International Symp­ osium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:52

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Moutou C, Gardes N, Viville S. Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy. Prenat Diagn 2003;23:685-9

12.

Girardet A, Hamamah S, Dechaud H, et al Specific detection o f non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy. Mol Hum Reprod 2003;9:421-8

13.

Jiao Z, Zhou C, Li J, et al. Birth of healthy children after preimplantation diagnosis of beta-thalassemia by whole-genome amplification. Prenat Diagn 2003;23:646-51

14.

Fiorentino F, Magli MC, Podini D, et al. The minise­ quencing method: an alternative strategy for preim­ plantation genetic diagnosis of single gene disorders. Mol Hum Reprod 2003:9:399-410

15.

68

Bermudez MG, Piyamongkol W, Tomaz S, et al. Single-cell sequencing and mini-sequencing for preimplantation genetic diagnosis. Prenat Diagn 2003;23:669-77

International Symposium on Preimplantation Genetics,

5-7 June, Antalya, Turkey, 2003:35 24.

Munne S, Willadsen S, Schmmel T, Cohen J. Nuclear sperm duplication as a tool to study mosaicism. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:55-6

25.

Magli MC, Ferraretti A, Gianaroli I,, etal. The combi­ nation of polar body and embryos biopsy does not affect embryo viability. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:24

26.

Verlinsky Y, Kuliev A. PGD - dozen-years experi­ ence. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:13

27.

McArthur S, Marshall J, Wright D, de Boer K. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:33

28.

Steuerwald N, Bermudes MG, Wells D, et al. The use of microarray technology to examine the gene expression profiles of individual human oocytes and Fifth International Symposium on embryos. Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:14

FUTURE PERSPECTIVES FOR PREIM PLAN TATION D IAG N O S IS

29.

Bermudes MG, Wells D, Malter H, et al. Expression profiles of individual human oocytes using microar­ ray technology. Reprod BioMed Online 2004;8: 325-37

30.

Leigh D, Wright D, Stojanov T. Development of customized microarrays for testing of chromosomal aberrations in human preimplantation embryos. Fifth International Symposium on Preimplantation Genetics,

41.

Kuliev A, Verlinsky Y. Preimplantation HLA typing and stem cell transplantation. Reprod BioMed Online 2004;9:205-9

42.

Verlinsky Y, Rechitsky S, Sharapova T, et al. Preimplantation HLA typing. J Am Med Assoc 2004;291:2079-85

43.

Pickering S, Braude P, Patel M, et al. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. Reprod BioMed Online 2003;7: 353-64

44.

Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y. Establishment of RGI repository of human embry­ onic stem cells. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:34-5

45.

Cram D, Hawes S, Pera M, Trounson A. Human embryonic stem cells. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:34

46.

R^ubinoff B. Human ES cells - a potential platform for cell and gene therapy. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:34

47.

Verlinsky Y, Cieslak J, Evsikov S, et al. Nuclear trans­ fer for full karyotyping and preimplantation diagno­ sis o f translocations. Reprod BioMed Online 2002;5:302-7

48.

Kahraman S. PGD impact on IVF efficiency. Fifth International Sy?nposium on Preimplantation Genetics, 5-7 June, Antalya. Turkey, 2003:25-6

49.

Munne S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod BioMed Online 2003;7:91-7

50.

Gianaroli L, Magli MC, Ferraretti A. PGD for aneu­ ploidies: clinical outcome. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:25

51.

DeBaun MR, Niemitz EL, Feinberg AP. Association of in intro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and HI 9. Am J Hum Genet 2003;72:156-60

52.

Gicquel C, Gaston V, Maldenbaum J, et al. In vitro fertilization may increase the risk of BeckwithWiedemann syndrome related to the abnormal imprinting of the K C N Q IO T gene. Am J Hum Genet 2003;72:1338-41

53.

Maher ER, Brueton LA, Bowdin SC, et al. BeckwithWiedemann syndrome and assisted reproduction technology (ART). J M ed Genet 2003;40:62-4

5-7 June, Antalya, Turkey, 2003:21 31.

Benkhalifa M, Mucci M, Kahraman S, et al. Genome profiling of blocked morulas: technical validation using bacterial artificial chromosomes microarray. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:21-2

32.

Osborne EC, Trounson AO, Cram DS. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:31

33.

34.

Wells D, Escudero T, Levy B, et al. First clinical appli­ cation of comparative genomic hybridization and polar body testing for preimplantation genetic diag­ nosis of aneuploidy. Fertil Steril 2002;78:543-9 Ozen S, Rechitsky S, Chmura M, Verlinsky Y. Highresolution genomic hybridization (CGH ) using single-cell standards in single blastomeres deriving from oocytes with known PBI and II FISH results. Fifth International Symposium on Preimplantation Genetics, 5-7 June, Antalya, Turkey, 2003:45-6

35.

Wilton L, Voullaire L, Sargent RM, et al. Pre­ implantation diagnosis of aneuploidy using compara­ tive genomic hybridization. Fertil Steril 2003;80: 860-8

36.

Trussler JL, Pickering SJ, Ogilvie CM. Investigation of chromosome imbalance in human embryos using comparative genomic hybridization (CGH ). Fifth International Symposium on Preimplantation Genetics,

5-7 June, Antalya, Turkey, 2003:46 37.

Lacham-Kaplan O, Daniels R, Trounson A. Fertilization of mouse oocytes using somatic cells as male germ cells. Reprod BioMed Online 2001;3: 205-11

38.

Tesarik J, Mendoza C. Somatic cell haploidization: an update. Reprod BioMed Online 2003;6:60-5

39.

Galat V, Ozen S, Rechitsky L, Verlinsky Y. Is haploidization by human mature oocytes real? Fifth International Symposium on Preimplantation Genetics,

5-7 June, Antalya, Turkey, 2003:36-7 40.

Verlinsky Y, Kuliev A, Tur-Kaspa I, et al. Preimplantation genetic diagnosis with H LA match­ ing. Reprod BioMed Online 2004;9:210-21

69

Section 11 Preimplantation Genetic Diagnosis Illustrated

Index

abortion risk with chromosome rearrangements 47, 209 acute lymphoid leukemia 249 adenomatous polyposis coli, familial 58, 265 adrenal hyperplasia, congenital 52, 55, 219, 256 alkaline phosphatase staining, embryonic stem cells 12, 99-101 allele-specific amplification failure (allele dropout; A D O ; preferential amplification) 54-55, 216-217, 231-234 cystic fibrosis transmembrane conductance regulator gene, AF508 mutation 224, 227, 233-234 allele-specific primers, HLA detection 253 Alzheimer's disease, early-onset 58, 271-272 amyloid precursor protein (APP) gene 58, 271 androgenetic embryo 129, 130 aneuploidy 29-41, 141 177, 243-248 haploid somatic cells 2 7 ,1 3 8 pie chart showing proportion and types 171 sperm duplication into aneuploid cells 132 testing ix, 29-41, 141-177, 210-211, 243-248 clinical impact 66-67 combined mutation and H LA testing and 254 microarray technology 64 PBI morphology related to 9 PCR-based 5 5 ,2 4 3 - 2 4 8 ,2 5 1 ,2 5 4 annealing temperature in PCR 50 APC gene (familial adenomatous polyposis coli) 58, 265 APP gene 58,271 assisted reproduction 1 oocytes without nuclear abnormalities in, identification 38 see also specific methods

atretic metaphase II oocytes

77

Beckwith-Wiedemann syndrome 67 binovular complex, metaphase II oocytes 77 biopsy (embryo) 15, 21-22, 64, 112-116 blastocyst 11, 22, 64, 116 blastomere 1 1 ,2 1 -2 2 ,2 5 holoprosencephaly 60, 274 for chromosome analysis 29 inversion 190 ploidies 146-147,210-211 translocations 189, 1 9 1 ,2 0 2 -2 0 8 ,2 1 0 -2 1 2 recent/prospective developments 64 blastocyst 1 1 ,9 3 -9 7 biopsy 1 1 ,2 2 ,6 4 ,1 1 6 formation, normal and abnormal 11, 93-97 hatching 75 PB 1 morphology related to potential to reach blastocyst stage 9 ploidy analysis (with FISH) 30, 146, 148-153 stem cell cultures from 11, 11-12, 98 blastomere 2 1 -2 2 ,2 4 -2 6 ,9 1 -9 2 abnormalities 91-92 biopsy see biopsy inversion diagnosis by nuclear conversion 190 nuclear conversion to metaphase chromosomes 24-26, 123-128, 210-212 by blastomere-zygote fusion 25, 43, 126 translocation diagnosis 44-46, 46-47, 189, 191, 202-208, 210-212 ploidy analysis (using FISH) 30, 143-145, 210-211

preparation of blastomeres signal evaluation 36-37 removal 21-22 translocation diagnosis

33-34

281

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

by interphase analysis 46, 196', 208 by nuclcar conversion 44-46, 4 6 -4 1 , 189, 191, 202-208, 210-212 blood group incompatibility 59, 272-273 blunt micropipettes 1 5 ,1 6 bone marrow transplantation 65, 66 brain tumor, familial posterior fossa tumor (and the hSNF5 gene] 54, 58, 237 breakpoint-spanning probes 4 7 cancer predisposition 56-58, 264-270 cardiocyte-like from embryonic stem cells 12, 102 cerebellar ataxia, spinal, type-2 246 CFTR see cystic fibrosis transmembrane conductance regulator chromatids (in FISH analysis] 36 errors (missing or extra) 37, 38, 141, 149, 170 exchange in carriers of translocations 181-182 chromatin staining, blastocyst 94-96 chromosomc(s) abnormalities (testing for/diagnosis of) 29-41 in embryos 2 9 ,3 9 -4 0 ,1 4 3 -1 5 3 in haploid somatic cells 27, 65, 141-142 non-disjunction 30, 38, 170 in oocytes related to PB morphology 8-10 PB removal and testing for see polar bodies sex chromosomes see X ; Y see also specific abnormalities e.g. aneuploidy; translocations and specific chromosomes below polar body visualization see polar bodies

segregation in carriers of translocations during meiosis 42, 179-184 see also karyotyping

chromosome-1, translocations involving

186-188,

200-201

chromosome-2, translocations involving 192, 204, 212 chromosome-3, translocation involving 210-211 chromosome-5 inversion 190 chromosome-6 aneuploidy testing 55, 244, 255 translocation involving 210-211 chromosome-7 monosomy 246 chromosome-8, translocation involving 200-202 chromosome-9 monosomy and trisomy 247 translocations involving 191, 194-195, 197 chromosome-10, translocations involving 192, 202 chromosome-11, translocation involving 191 chromosome-12

282

translocation involving 185 trisomy 21 chromosome-13 FISH signal evaluation 36 ploidy analysis 29, 37, 38, 142-145, 150, 152, 156, 159, 166, 171, 177, 210-211, 244, 251 translocations involving 189, 194-195, 197, 205-208, 212 chromosome-14, translocation involving 205-208 chromosome-15 ploidy analysis 143-146 translocation involving 188 chromosome-16 FISH signal evaluation 36 ploidy analysis 29, 37, 38, 142-145. 147, 149-150, 160, 162, 164, 171, 174, 176, 251 chromosome-17, ploidy analysis 143-146 chromosome-18 FISH signal evaluation 36 ploidy analysis 29, 30, 37, 38, 142-145, 150, 152, 161, 163, 171, 176-177, 210-211, 244 translocations involving 185, 204 chromosome-19, aneuploidy testing 248 chromosome-20, translocation involving 189 chromosome-21 FISH signal evaluation 36 ploidy analysis 29, 30, 37, 37-38, 38, 142-145, 148-149, 149, 150, 154-155, 158, 171, 174-176, 210-211, 244, 251 chromosome-22 FISH signal evaluation 36 ploidy analysis 29, 37, 38, 142-145, 150-153, 157, 171, 174-176, 244 cleavage see embryo comparative genomic hybridization 2, 65 congenital adrenal hyperplasia 52, 55, 219, 256 congenital malformations 59-60 Crouzon syndrome 60, 238 cultures embryonic stem cell 11-12, 66 oocyte and embryo, solutions 17, 17-18 cumulus cells haploidization 13 7 inclusions (in perivitelline space) 79-80 avoidance 8 oocyte-cumulus cell complex 7-8 removal prior to insemination 8 Currarino triad 60 CYP21A2 gene 2 1 9 ,2 5 6

INDEX

cystic fibrosis transmembrane conductance regulator (CFTR) gene 55-56, 221-224, 233-234, 246 AF508 mutation 221-222, 224, 226, 233-234, 262 AI507 mutation 262 G 542X mutation 245 N 1303K mutation 2 1 7 ,2 2 3 R117H mutation 2 4 5 ,2 6 2 cytogenctic testing see chromosomes; karyotyping DAPI counterstain 36 deletions (gene) 52 familial adenomatous polyposis coli 265 spinal muscular atrophy 239 [i-thalassemia 220 denaturation in PCR 50 development (preimplantation) x, 7-11 abnormal 11 PBI and PB2 role in 20-21 PBI morphology related to potential to reach various stages o f 9-10 developmental malformations 59-60 Diamond-Blackfan anemia 250 diseases late-onset ix-x, 58-59 single-gene see single-gene disorders disomies in PBs 37 DMPK gene and myotonic dystrophy 248 D N A analysis for single-gene disorders 49-55 diagnostic accuracy 63-65 updated procedure 49-53 D N A contamination in PCR 50-52 D NA minisequencing 52, 53, 230 dominant mutation and aneuploidy 254 double heterozygous-affected partner, couples with 56, 245 dysautonomia, familial 54-55, 240-242 dystrophia myotoniea (myotonic dystrophy; DM) 55, 56, 248 DYT1 gene (autosomal dominant early-onset torsion dystonia) 54, 236, 247 electrofusion apparatus (X R O N O S) 2 4 ,2 7 ,1 2 0 blastomere-zygote pairs 25, 43 oocyte activation by 24, 27, 120, 132 embryo androgenetic 129, 130 biopsy see biopsy

chromosomal abnormalities in 29, 39-40, 143-153 cleavage normal and abnormal 11, 89-92 PBI removal and 20-21 development see development mosaicism see mosaicism transfer ix, 46, 67 translocation testing 46 embryonic stem (ES) cells 11-12, 66, 95-102 equipment/instruments for FISH analysis of PBs and blastomeres 31 for micromanipulation 15-18 for PCR 51 expand long template kit 55 expression profiling 64-65 extension reaction in PCR 50 FAC gene and Fanconi anemia, H LA typing 60, 252 familial adenomatous polyposis coli 58, 265 familial dysautonomia 54-55, 240-242 familial posterior fossa tumor (and hSNF5 gene) 54, 58, 237 Fanconi anemia (and FAC gene), HLA typing 60, 252 FBN1 gene 246 female-derived translocations see maternally-derived translocations fertilization (oocyte) 10-11 normal and abnormal 10-11, 86-89 rate PBI morphology related to 9, 9-10 PBI removal and 20-21 in vitro see in vitro fertilization FGFR2 gene and Crouzon syndrome 238 fibrillin-1 (FBN1) gene 246 fibroblast growth factor receptor-2 (FGFR2) gene and Crouzon syndrome 238 FISH see fluorescence in situ hybridization fluorescence PCR 52-53 in avoidance of misdiagnosis due to preferential amplification 55 cystic fibrosis transmembrane conductance regulator gene 221-224 fluorescence in situ hybridization (FISH) 29—41 interphase chromosomes 23, 43, 143, 146-147, 166-167 limitations 43 PB 1 morphology related to chromosomal abnormalities detected by 9, 82-85

283

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

ploidy assessment 29-41 preparation of PBs and blastomeres 31-34 pretreatment/probe application/hybridization/washing 34-36 signal evaluation 36-37 translocations 43-48, 185-195, 197-208,

homozygous-affected partner, couples with 261 hot-start PCR 52 hSNF5 54 HTF medium 1 7 ,1 7 -1 8 ,1 8 hyper-IgM syndrome, X-linked 60, 280

56,

210-212

FMR1 gene 54, 56, 232, 23~ fragile X syndrome (and FMR1 gene) 237

54, 56, 232,

gametes, artificial human, in vitro 26-27, 132-135 see also oocyte; sperm gene expression profiling 64-65 genomic hybridization, comparative 2, 65 germinal vesicle (G V ) 8 breakdown (G V BD ) 7 P-globin gene 220, 231, 233, 263 H LA typing and 277, 279 see also hemoglobin disorders an d specific disorders

haploidization, somatic cell (for artificial gametes) 26-27, 65, 132-136 haplotype analysis 55 Alzheimer’s disease of early onset 272 fragile X syndrome 232 neurofibromatosis type-1 267 neurofibromatosis type-2 269 hatching, blastocyst 75 heminested PCR 52 spinal muscular atrophy 243 hemoglobin disorders 55-56 see also globin gene an d specific disorders heterokaryons with blastomere-zygote pairs 25, 125 heterozygous-affected partner, double, couples with 56, 245 H LA typing x, 55, 64, 65-66, 235, 244, 252-256, 275-278 with causative gene testing 60, 252-253, 275-278 combined mutation and aneuploidy testing and 254 developments in 64, 65-66 stem cell H LA matching 65-66’ without causative gene testing 60, 249-250 holding pipettes 15 PB 1 removal 18 holoprosencephaly 59-60, 257, 274

284

ICSI see intracytoplasmic sperm injection IKBKAP gene 240-242 immunosurgery blastocyst 97 embryonic stem cell establishment 11-12 implantation rates, PBI morphology related to 9 in vitro fertilization (IVF) ix, 1, 66-67 aneuploidy testing for 38, 39 INI 1 (hSNF5) gene, human 54 inner cell mass (ICM), blastocyst, stem cell cultures from 11 ,1 1 -1 2 , 99, insemination blastocyst following 93 oocyte following binovular complex 78 fertilization abnormalities or failure 86, 88 mature 78 instruments see equipment interphase FISH 23, 43, 143, 146-147, 166-167 intracytoplasmic PB2 nucleus injection 24, 119-120, 122, 123 intracytoplasmic sperm injection (ICSI) 19 fertilization following 10, 86, 87 PBI morphology and rate of 8, 10 micropipettes for 15, 16, 19, 20, 24 PB removal after 20, 107-109 PB removal before 19 ,1 0 5 -1 0 6 polyvinylpyrrolidone (PVP) for 17, 18 inversion, chr-5 190 K1/K2 and Kell genotype 59, 272-273 karyotyping blastomere converted to metaphase 126 by nuclear transfer technique 117 PB2 converted into metaphase 23, 117 following intracytoplasmic injection 24, 123 PCR-based 64-65 see also chromosomes Kell genotype 59, 272-273 late-onset diseases ix-x, 58-59 leukemia, acute lymphoid 249 Li-Fraumeni syndrome 58

INDEX

Life Global LiteOIL™ 18 linked marker (incl. linked polymorphism] analysis 54-55, 215 Cystic fibrosis 21 7, 246 polycystic kidney disease-1 258 polycystic kidney disease-2 259 sickle cell disease 215 long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency 5 2 ,5 5 ,2 1 8 lymphoid leukemia, acute 249 male-derived disorders see paternally-derived disorders malformations, congenital 59-60 malignancy predisposition 56—58, 264-270 Marfan syndrome 246 materials and reagents (incl. solutions) FISH analysis o f PBs and blastomeres 32 micromanipulation 17 ,1 7 -1 8 PCR 51 maternally-derived translocations reciprocal translocation 4 4 ,4 6 , 183-188, 189-201, 210-212 Robertsonian translocations 46, 205-208 meiosis (females = oocyte) chromosome segregation in carriers of translocation during 42, 179-184 errors 30, 38 meiosis I 31, 38, 39, 141, 149, 154-155, 158-159, 161, 163-164, 169, 171-172, 175-176, 246 meiosis II 30-31, 38, 39, 141, 149, 153, 156-157, 160, 162-163, 169, 171-172, 176-177, 246 PCR-based detection 246 oocyte in meiosis I 7-8 meiosis (male = sperm) testing outcome 26 metaphase blastomere in with nuclear conversion method see blastomere, nuclear conversion without specific conversion method 26 oocytes in metaphase I 8, 74, 78 aneuploidy testing/FISH analysis 169 haploidization of somatic cells using 27, 139-140 oocytes in metaphase II 8, 76-80 aneuploidy testing/FISH analysis 30, 169 haploidization of somatic cells using 27, 65, 134-136 sperm duplication using 26, 129, 131

PB2 nuclear conversion into

23, 24, 117-118,

121-122

microarray technology 53, 64-65 microforge 15, 16, 24 micromanipulation x, 15-22 tools and materials 15-18 see also specific techniques

microneedles 1 5 ,1 6 micropipettes 16 blunt 16 for PBI removal 15, 18 finely pulled (for ICSI) 15, 16, 19, 20, 24 for FISH analysis of PBs and blastomeres 31, 32, 34 microsyringe injectors 17 mineral oil 18 minisequencing 52, 53, 230 miscarriage (abortion) risk with chromosome rearrangements 47, 209 misdiagnosis 2 D NA analysis of single cells with single-gene disorders 5 3 - 5 5 ,6 3 ,2 1 8 ,2 3 1 due to allele dropout see allele-specific amplification failure mitochondria blastocyst 96 metaphase II oocytes 76 mitotic errors in cleaving embryos 40 molecular beacons 53, 228-229 monosomies 30, 141 chr-6 2 4 4 ,2 5 5 chr-7 246 chr-9 247 chr-13 1 5 2 ,1 5 6 ,2 4 4 ,2 5 1 chr-16 1 4 9 ,1 6 0 ,1 6 2 ,1 6 4 ,2 5 1 chr-18 152, 161 chr-19 248 chr-21 30, 148-149, 155, 158, 175, 244, 251 chr-22 1 5 1 -1 5 2 ,1 5 7 ,2 4 4 PCR-based detection methods 55, 243-244 morula-derived stem cells cultures 11, 98-99, 101-102 mosaicism 26, 244 ploidy analysis (by FISH) and 30, 39—40, 55, 174, 176, 244 translocation analysis (by FISH) and 47, 212 multiplex PCR 52, 214 cystic fibrosis transmembrane conductance regulator gene 223-224, 233 diagnostic accuracy 54 HLA typing 235

285

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

spinal muscular atrophy 243 mutation analysis see single-gene disorders myotonic dystrophy [DM] 55, 56, 248 Narishigc instruments fine manipulator (M 0 2 0 2 ) 17 microforge (MF-9) 15, 16 microsyringe injectors (IM-6 and IM-16) 17 pipette puller (PB-7) 15 neoplasms see tumor nested PCR 5 2 ,2 1 4 diagnostic accuracy 54 H LA typing 235 see also heminested PCR neurofibromatosis (NF) type-1 5 8 ,2 6 7 type-II 5 8 ,2 6 8 -2 6 9 neuron-like from embryonic stem cells 12, 66, 102 NF see neurofibromatosis nuclear transfer techniques 23-27, 117-127 blastomeres see blastomere, nuclear conversion somatic cell 26-27, 65, 132-133 nullisomies 37, 170 okadaic acid, nuclear transformation using 24, 25 oocyte (s) 7-11 abnormalities 8, 7 6 -7 1 activation by electrofusion 24, 27, 120, 132 fertilization see fertilization mature 8, 76-77 meiosis 1 7-8, 74-75 metaphase see metaphase PBI morphology related to chromosomal abnormalities in 8-10 in PBI removal, micromanipulation 18-19 prophase I 8, 74 sperm duplication using 26, 64, 129 see also gametes oocyte-cumulus cell complex 7-8, 73 ornithine transcarbamylase gene 56, 260 O TC gene 56, 260 p53 58, 264 PAH (phenylalanine hydroxylase) gene 56, 261 paternally/male-derived disorders inversion 190 preconception diagnosis 64 translocations reciprocal 43, 44, 189, 196, 202-204 Robertsonian 46 PCR see polymerase chain reaction

286

perivitelline space cumulus cells in see cumulus cells in partial zona pellucida dissection 21 phenylketonuria (phenylalanine hydroxylase deficiency) 56, 261 phytohemagglutinin, zygote—blastomere agglutination 25 pipette(s) see holding pipettes; micropipettes pipette pullers and bevellers 15, 16 ploidy assessment see aneuploidy polar bodies (PB) 8-11, 15-22 extrusion o f PB2, fertilization following 10-11, 87 morphology (and morphological abnormalities) chromosomal abnormalities in oocytes related to H o grading of PBI 7 in PBI with metaphase II oocytes 77, 81-85 ploidy analysis (using FISH) 30, 37-39, 142, 145, 149, 154-155, 157-158, 160, 170, 171-177 preparation of PB I and PB2 31-33 signal evaluation 36 removal of PB ICSI after see intracytoplasmic sperm injection pie chart of FISH (for chromosomal abnormalities) 1 6 8 ,1 7 0 removal of PBI 18-19, 20, 103-104 avoidance of cumulus cell inclusions 8 and diagnosis of chromosome abnormalities 30, 155, 157-158, 160, 168, 171-172 removal of PBI and PB2 simultaneously 20-21, 110-112

and diagnosis of chromosome abnormalities 20, 29-30, 142, 145, 154, 159, 161-165, 168, 173-174, 176 removal of PB2 20, 23 and diagnosis of chromosome abnormalities 30, 157-158, 160, 171-172 sampling of PBI and PB2 2 with single-gene disorders see single-gene disorders translocation diagnosis (PBI and PB2) 43, 46, 185-188, 192-201 variations in PBI formation 8 visualization of chromosomes 23-24 polycystic kidney disease (autosomal dominant) PKD1 55, 258 PKD2 55, 259 polymerase chain reaction (PCR) analysis of single cells 49-53

INDEX

diagnostic accuracy 53-55 fluorescence PCR see fluorescence PCR heminested PCR 52 hot-start PCR 52 karyotyping 64-65 multiplex PCR see multiplex PCR nested PCR see nested PCR real-time PCR see real-time PCR polymorphisms see linked marker; single-nucleotide polymorphisms polyvinylpyrrolidone [PVP] for electrofusion 24, 25 for ICSI 1 7 ,1 8 posterior fossa tumor (and hSNF5 gene) 54, 58, 237 preconception diagnosis o f paternally-derived disorders 64 preferential amplification see allele-specific amplification failure prenatal diagnosis, PGD as alternative to 67 pronuclear morphology 10-11 prophase I, oocytes 8, 74 pseudogenes 52, 55, 218-219 reagents see materials and reagents real-time PCR 53, 225-230, 234 diagnostic accuracy 54 recessive mutation and aneuploidy 254 reciprocal translocations 46 carriers, meiosis in 179—184 female/maternally-derived 4 4 ,4 6 , 183-188, 189-201, 210-212 male/paternally-derived 43, 44, 189, 196, 202-204 reproduction, assisted see assisted reproduction an d specific methods

rhesus disease 59 Robertsonian translocations 44, 46 maternally-derived 46, 205-208 microarray technology 65 paternally-derived 46 sex chromosomes see X chromosome; Y chromosome SHH gene 5 9 -6 0 ,2 5 7 ,2 7 4 short tandem repeats (STRs) 52-53 aneuploidy testing 244 HI.A typing 275 phenylalanine hydroxylase gene 261 sickle cell disease 2 1 5 -2 1 6 ,2 3 3 single-cell PCR see polymerase chain reaction

single-gene disorders 49-62, 213-251 embryonic stem cell lines with 12 ICSI for 19 late-onset ix-x, 58-59 PB analysis 49, 58 PBI 18 ,4 9 PB analysis, sequential 49 in avoidance of misdiagnosis for maternallyderived mutations 54, 55 cystic fibrosis transmembrane conductance regulator gene 222 single-nucleotide polymorphisms (SNPs) (5-globin gene 230 von Hippel-Lindau gene 266 single-sperm typing 55, 256 SM N genes 5 4 ,2 3 9 ,2 4 3 SNF5 gene, human 54 solutions see materials and reagents somatic cell, haploidization (for artificial gametes) 26-27, 65, 132-136 sonic hedgehog gene 59-60, 257, 274 sperm duplication 26, 64, 129-132 in ICSI, addition 19 single-sperm typing 55, 256 see also gametes spinal cerebellar ataxia type-2 246 spinal muscular atrophy (SMA) 54, 239, 243 split FISH 166-167 SSEA-3 and SSEA-4 expression, embryonic stem cells 12, 100, 101 staining, blastocyst 11, 94-96 stem cells 65-66 embryonic (ES) 1 1 -1 2 ,6 6 ,9 5 -1 0 2 transplantation 65-66 structural defects, congenital 59-60 survival motor neuron (SM N) gene 54, 239, 247 Taq polymerase 50 TaqMan® 5 3 ,2 2 5 -2 2 7 tetraploid blastomere-zygote pairs 25, 125, 126 thalassemia 22, 52, 54-55, 213 HLA typing 6 0 ,2 7 7 -2 7 9 3D-PZD blastomere removal 21 PBI removal 18 torsion dystonia, autosomal dominant early-onset (DYT) 54, 236, 247 TRA-2-39 expression, embryonic stem cells 12, 99 TRA-2-60 expression, embryonic stem cells 12, 100 , 101

287

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

TRA-2-80 expression, embryonic stem cells

12,

100 , 101

translocations ix, 2, 43-48, 179-212 developments in detection 66-67 microarray technology 65 transplantation, stem cell 65-66 trisomies 30, 37, 141 chr-6 255 chr-9 247 chr-13 1 5 9 ,1 6 6 ,1 7 6 ,2 0 8 ,2 5 1 chr-14 208 chr-16 14 chr-18 163, 177 chr-19 248 chr-21 3 0 ,1 5 4 ,2 1 0 - 2 1 1 ,2 4 6 ,2 5 1 chr-22 153, 175 PCR-based detection methods 55, 243-244 trisomy rescue 67 trophectoderm cells 96-97 biopsy 11 trophoblastic vesicle 93 tubulin staining, blastocyst 94-95 tumor malignant, predisposition 56-58, 264-270 posterior fossa, familial (and hSNF5 gene) 54, 58, 237 tumor suppressor gene, p53 58, 264 tyrosine hydroxylase (TH O ) polymorphic site 263

288

von Hippel-Lindau syndrome 58, 266 von Recklinghausen's disease (neurofibromatosis type I) 58, 267 X chromosome, FISH analysis 29, 30, 143-144, 146, 167, 210-211 signal evaluation 36 see also fragile X syndrome X chromosome-linked disorders 56 hyper-IgM syndrome 60, 280 XR O N O S 2 4 ,2 7 ,1 2 0 X X Y 251 Y chromosome, FISH analysis 167, 210-211 signal evaluation 36 see also X X Y

29, 143-144, 146,

zona pellucida dissection/removal, partial (PZD) for embryo/blastomere removal and biopsy 21, 112-114 for embryonic stem cell culture 11-12 for PBI removal 18, 20 zygote blastomeres fused with 25, 43, 126 first cleavage 11 abnormal 90

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

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w e ll dispersed. However, corona cells remain com pact in some

retrieval w ith an apparently norm al oocyte. Cumulus cells are w ell dispersed. C orona cells form a ‘sun-ray’ appearance

areas around the oocyte. The oocyte is round in shape, but the

around the oocyte, w hich is round in shape (x 100)

retrieval w ith an apparently normal oocyte. Cumulus cells are

first polar body is not visible. Removal o f corona cells is required to verify the exact stage o f m aturation (x20 0)

73

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.5 Binuclear prophase I oocyte, an extrem ely rare abnormality observed in human oocytes. The oocyte, 40 h after injection o f human chorionic gonadotropin, is slightly irregular in shape and significantly larger than the rest o f the oocytes from the same cohort. The cytoplasm is coarse, w ith a dark area in the center often seen in prophase I oocytes. Two germinal vesicles are located on opposite sides at the periphery o f the cytoplasm containing one and tw o nuclear organizers. The perivitelline space is clear and the zona pellucida is intact and evenly thick along the circumference (x200)

Figure i.7 Early metaphase I oocyte, 4 0 h after injection of human chorionic gonadotropin, is slightly irregular in shape and regular in size. The cytoplasm is less coarse than is usually observed at the germinal vesicle stage. The perivitelline space is clear. N either a first polar body nor a germinal vesicle is present (x400)

74

Figure 1.6 Early prophase I oocyte, 40 h aiter injection of human chorionic gonadotropin, is slightly irregular in shape and regular in size. The cytoplasm is coarse. A germinal vesicle is located at the periphery o f the cytoplasm and contains one nuclear organizer. The perivitelline space is w ith o u t debris and the zona pellucida is intact and evenly th ick along the circumference (x400)

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

Figure 1.8 G erminal vesicle breakdow n (GVBD): im m unocytochem ical staining (bright field view o f the oocyte is shown in red, tubulin in green, chrom atin in blue), (a) Germinal vesicle, (b) G VBD: disappearance o f nucleoli, (c) GVBD: chrom atin com paction, (d) GVBD: early metaphase I stage: chrom atin is com pacted but no spindle is form ed

75

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

i a

b

e

d

Figure 1.9 Metaphase II oocytes w ith an apparently normal first polar body. Oocytes are round in shape and regular in size. The cytoplasm is homogeneous (a) o r contains a few cytoplasmic inclusions (b) and (c) o r a coarse-appearing region (d). The perivitelline space is even, except in the area where the first polar body is located. One oval, round o r flattened, non-fragmented polar body is visible at the 12 o ’clock position in each. For preconception genetic diagnosis, the flattened polar body (c) is removed in culture medium containing 0 .1 mol/1 sucrose solution in ord er to increase the perivitelline space by slight shrinkage o f the ooplasm (x200)

Figure 1.10 Distribution o f m itochondria in metaphase II oocytes: rhodamine staining (bright-field view o f the oocyte is shown in red, m itochondria in green, chromatin in blue;, (a) The first polar body does not have a significant number o f m itochondria detectable at the light microscopy level, (b) A cumulus cell attached to the oocyte serves as a control that the observed phenomenon is not due to an artifact o f the staining procedure o r due to the polar body size

76

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

c

(' ) Figure 1.1 I Metaphase II oocyte w ith a fragmented first polar body. The oocyte is round in shape and regular in size. The cytoplasm is granular and contains m ultiple inclusions tightly positioned in the center o f the ooplasm. The perivitelline space is clear. The first polar body, consisting of a cluster of fragments, is visible at the 12 o ’clock position (x 200)

( Figure 1.12 Metaphase II oocyte w ith a fragmented first polar body and additional cytoplasmic fragments. The oocyte is round in shape and regular in size. The cytoplasm has a slightly coarse area in the center o f the oocyte. The perivitelline space has a grainy appearance and is enlarged in the area o f the fragmented first polar body and the additional cytoplasmic fragments. The zona pellucida is intact and evenly thick along the circumference (x 200)

V

Figure 1.13 Metaphase II oocyte w ith an enlarged first polar body. The oocyte is round in shape and slightly smaller in size. The cytoplasm is coarse and the perivitelline space is enlarged. One large nonfragmented polar body is visible at the 12 o ’clock position (x200)

(\

(7 ( \ Figure 1.14 Metaphase II oocytes w ith abnormal first polar bodies (a) and (b). Oocytes from a harvest of I I , retrieved 36 h after injection o f human chorionic gonadotropin. Three of the I I oocytes w ere at prophase I, while the remaining eight were w ith large cytoplasmic structures almost four times the size o f a normal first polar body. In tw o oocytes these structures were divided into tw o parts. The structures w ere removed from all oocytes w ith the aid o f a large pipette and analyzed by FISH, revealing chromatin contents similar to those o f the first polar body. Sixteen hours after ICSI the oocytes w ere unevenly divided into tw o , three o r four fragments w ith varying numbers o f nuclei, suggesting abnormalities in cytokinesis ((a) x200; (b) x 100)

Figure 1.15 A tretic metaphase II oocyte, 40 h after injection o f human chorionic gonadotropin, is irregular in shape and reduced in size. The plasma membrane is rough; the cytoplasm is grainy and dark brown in color. The perivitelline space is w ith o u t debris and an intact first polar body is visible at the I I o ’clock position. The zona pellucida is intact (x200'i

77

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.16 Binovular com plex w ith one mature metaphase II and one immature oocyte. The smaller oocyte is at the germinal vesicle stage, and has coarse cytoplasm. The other oocyte is o f normal size, w ith homogeneous cytoplasm w ith a few inclusions and the first polar body at the I o ’clock position. Both oocytes are surrounded by a conjoined zona pellucida of an ‘8-shape1appearance and do not share the same perivitelline space (x200)

Figure 1.18 Binovular com plex consisting o f im m ature metaphase I and mature metaphase II oocytes. Both oocytes are irregular in shape, but have a normal size. The oocyte on the left contains several vacuoles and appears to be at metaphase I, since no polar body can be seen. The oocyte on the right has a few inclusions and has completed the first meiotic division, evident by the presence o f a polar body at the I o ’clock position. Both oocytes are surrounded by a conjoined zona pellucida (x200)

78

Figure 1.17

Binovular complex following insemination. The

smaller oocyte is at the germinal vesicle stage and has coarse, dark cytoplasm. The second, metaphase I, oocyte has a normal size and homogeneous cytoplasm w ith a few inclusions. No polar body is visible in the perivitelline space. Both oocytes are surrounded by a conjoined zona pellucida, each w ith a separate perivitelline space. Multiple spermatozoa are seen attached to the zona pellucida (x200)

Figure 1.19 Apparently mature oocyte following insemm ation.The metaphase II oocyte is small and round, containing vacuole-like structures. A round first polar body (PB I) is visible at the 10 o ’clock position. There is a dark cytoplasmic structure occupying one-quarter o f the subzonal space next to the PB I and is intimately attached to the plasma membrane o f the oocyte. The zona pellucida is intact, unevenly thick along the circumference w ith multiple spermatozoa attached (x 200)

NORMAL AND ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

Figure 1.20 Metaphase II oocytes (a; and (b) with multiple cumulus cells within the perivitelline space. The oocytes are round in shape and small in size. The cytoplasm is homogeneous with several inclusions. The perivitelline space is significantly enlarged, containing multiple cumulus cells. The presence of the first polar body cannot be determined, making the oocytes useless for preconception genetic testing. The zona pellucida is intact, slightly irregular in shape and unevenly thick along the circumference (x 200)

Figure 1.21 Metaphase II oocyte with a few cumulus cell inclusions in the perivitelline space (PVS). The oocyte is round in shape and regular in size. The cytoplasm appears coarse, w ith several cytoplasmic inclusions. The PVS is slightly enlarged, containing at least four cumulus cells attached to the internal surface of the zona pellucida. The first polar body can be identified at the 12 o’clock position (x 200)

Figure 1.22 Metaphase II oocyte with single cumulus cell inclusion in the perivitelline space (PVS). The oocyte is round in shape and regular in size. The cytoplasm appears coarse and granular along the periphery, w ith several cytoplasmic inclusions. The first polar body can be identified at the 9 o’clock position. The PVS is slightly enlarged containing one cumulus cell attached to the internal surface of the zona pellucida (x200)

79

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.23 Apparently mature metaphase II oocytes w ith granularity o f the perivitelline space (PVS). The oocytes are round in shape. The cytoplasm appears coarse, w ith many inclusions. One polar body can be identified w ith difficulty. The PVS is slightly enlarged, containing m ultiple dark granules and debris, possibly the remnants o f corona cells o r an extracellular m atrix comprising granules and filaments. Although this has no effect on fertilization, cleavage rate and em bryo quality, the first polar body removal is complicated in such oocytes, making them o f little value fo r genetic testing (x 100)

Figure 1.24 Enlarged view o f one o f the metaphase oocytes from Figure 1.23

r Figure 1.26 Apparently mature metaphase II oocyte w ith m ultiple cumulus cells (fluorescent D N A staining). The fluorescence analysis confirms the presence o f D N A in multiple cell inclusions localized in the perivitelline space, which can lead to misdiagnosis in genetic testing ix4 00 )

Figure 1.25 Apparently mature metaphase II oocyte w ith m ultiple cumulus cells in the perivitelline space (PVS). The oocyte is round in shape and small in size. The cytoplasm is homogeneous w ith a few inclusions. A questionable first polar body can be seen at the 4 -5 o ’clock position. The PVS is significantly enlarged, containing multiple cumulus cells, which can be a source o f D N A contamination in genetic testing. The zona pellucida is intact and slightly irregular in shape (x400)

80

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

VVw' V

Grade 3 Figure 1.27 Three categories o f first polar body (PBI) m orphology at day 0 oocyte recovery. Grade I , an intact, round o r ovalshaped PB I ; grade 2, an intact (non-fragmented) irregularly shaped PB I ; grade 3, a partially o r totally fragmented PB I . Observations w ere made using Hoffman phase-contrast optics at a magnification o f x200. Images o f both the side and top view positions were captured

81

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

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cl

Figure 1.28 Fluorescence in situ hybridization (FISH) images o f grade 2, slightly irregularly shaped first polar body (PBi). fa) FISH images o f PBI and second polar body (PB2) after a 3-h hybridization w ith MultiVysion PB panel probe for autosomes 13 (red), 16 (aqua), 18 (violet blue), 21 (green) and 22 (.gold), removed simultaneously on day I at the pronuclear stage o f development (c) following fertilization assessment o f an oocyte retrieved from a 40-year-old woman. A normal number of signals are present fo r four o f the five autosomes tested in PBI w ith the exception o f chromosome 16, in which one o f tw o signals in close proxim ity is split (white arrow). PB2 contains a normal number of signals w ith the exception o f chromosome 16 in which no conclusion can be made owing to tw o areas o f diffuse aqua background, necessitating a second round of testing ( a l) Rehybridization o f PBI and PB2 for chromosome 16 targeting a different locus using subtelomeric 16p (green) in which tw o signals are present in PB I (white arrow ) and one signal in PB2 indicating a normal chromosome com plement in the oocyte, (b) Mature metaphase II oocyte w ith a grade 2 PE (black arrow ) corresponding to the FISH images in (a) and fa I). A bilayer defect in the zona pellucida can be seen at the top right (yellow arrow ). The oocyte is round w ith a homogeneous cytoplasm, (b I ) Top view o f the slightly irregular-shaped PB I (black arrow), (c) The same oocyte as shown in (b), 18h post-intracytoplasmic sperm injection (ICSI). Top view o f PBI and PB2 (black arrows; located in close proxim ity to one another and both o f which are irregular in shape, (c l) Two pronuclei, both of which consist of partially aligned nucleoli o f differing sizes, are visible along w ith tw o polar bodies (black arrows;

82

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

*

» %

•

«

•

Mi • PB1

PB2

a

Figure 1.29

Fluorescence in situ hybridization (FISH) images o f a grade 3 (fragm ented) first polar body (P B I). (a) FISH images o f

PBI and second polar body (PB2) after a 3-h hybridization w ith MultiVysion PB panel probe fo r autosomes 13 (red), 16 (aqua), 18 (violet blue), 2 I (green) and 22 (gold), rem oved simultaneously on day I at the pronuclear stage o f developm ent (d) and (e) follow ing fertilization assessment o f an oocyte retrieved fro m the same 40-year-old wom an as in Figure 1.28, showing a norm al num ber o f signals in both PB I and PB2. (b) M ature metaphase II oocyte w ith a grade 3 PB I (black a rro w ) corresponding to the FISH image in (a). The oocyte is round w ith a homogeneous cytoplasm surrounded by a thick zona pellucida. (c) Top vie w o f the fragm ented PB I appearing as a grape-like cluster (black arro w ), (d) The same oocyte shown in (b), 18 h post-intracytoplasm ic sperm injection (ICSI). Two pronuclei w ith non-aligned nucleoli are visible along w ith PB I and PB2 at 5 o ’clock (black arrow s), (e) Top view o f PB I and PB2. PB2 has an irregular ‘S’ type shape; a faint outline o f the pronuclei can be seen in the center

83

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

d

e

F igu re 1.30 Fluorescence in situ hybridization (FISH) images o f a grade I (round) first polar body (PB I ). (a) FISH images o f PB I and second polar body (PB2) after a 3-h hybridization w ith MultiVysion PB panel probe fo r autosomes 13 (red), 16 (aqua), 18 (violet blue), 21 (green) and 22 (gold), removed simultaneously on day I at the pronuclear stage o f development (d) and (e) following fertilization assessment o f an oocyte retrieved from the same 40-year-old woman as in Figure 1.28, showing a normal number o f signals in PB I , but additional signals fo r chromosomes 16 (white arrows) and 18 (yellow arrows) in PB2 indicating a nullisomic oocyte for chromosomes 16 and 18 which w ill result in a double monosomic embryo, (b) Mature metaphase II oocyte w ith a grade I PBI corresponding to the FISH image in (a). The oocyte is irregular in shape w ith a homogeneous cytoplasm surrounded by a thick zona pellucida. (c) Top view o f the grade I PBI. (d) Side view of PBI and PB2 both appearing to be round while the fertilized oocyte containing tw o pronuclei w ith scattered nucleoli is grossly irregular in shape, (e) Top view o f both polar bodies in close proxim ity of one another

84

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

•

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f Figure 1.31

First polar body (P B I) m orphology w ith corresponding fluorescence in situ hybridization (FISH) results, (a) Phase-

contrast image o f a m ature metaphase II oocyte w ith a grade 2 PBI (slightly irregular in shape)(white a rro w ) retrieved fro m a 42year-old patient, (b) FISH image o f the same PBI chrom atin and corresponding PB2 nucleus obtained on day I , follow ing fertilization. Hybridization was perform ed using MultiVysion PB panel probe fo r chrom osom es 13, 16, 18, 21 and 22, showing com plex errors evident fro m an additional signal fo r chrom osom e 22 (yellow arrow s) and only one signal fo r chrom osom e 18 (w hite a rro w ) in PBI. A norm al num ber o f signals (one fo r each chrom osom e) are present in PB2. Based on the PB findings the resulting oocyte is expected to be nullisomic fo r chrom osom e 22 and disom ic fo r chrom osom e 18, w hich w ill result in an em bryo w ith a trisom y 18 and m onosom y 22. (c) Phase-contrast image o f a m ature metaphase II oocyte w ith a grade 3 PBI (fragmented) (w hite a rro w ) obtained fro m a 37-year-old wom an, (d) FISH image o f this PBI chrom atin and the corresponding PB2 nucleus obtained on day I , follow ing fertilization. Hybridization was perform ed using MultiVysion PB panel probe fo r chrom osom es 13, 16, 18, 21 and 22, showing a norm al num ber o f signals fo r each of the autosomes tested in both PB I and PB2. (e) Phase-contrast image o f a m ature metaphase II oocyte w ith a grade 3 PBI (fragm ented) (w h ite a rro w ) retrieved fro m a 38-year-old wom an, (f) FISH image o f this PB I chrom atin and corresponding PB2 nucleus obtained on day I follow ing fertilization. H ybridization was perform ed using MultiVysion PB panel probe fo r chrom osom es 13, 16, 18,21 and 22, showing a normal num ber o f signals fo r fo u r o f the five chrom osom es tested w ith the exception o f chrom osom e 2 1 in w hich only one signal is seen (w h ite arro w ). Based on this and also a normal num ber o f signals present in the PB2 nucleus, this oocyte is predicted to contain an extra chrom atid 2 I leading to trisom y 21 in the em bryo

85

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure

1.32

N orm al fertilization after ICSI. A prezygote o f smaller size and

irregular shape I 8 h after ICSI. The cytoplasm contains a fe w inclusions. Two equal sized pronuclei are visible in the cytoplasm. Six nucleoli can be counted inside the left pronucleus and approxim ately fo u r in the right pronucleus. The perivitelline space (PVS) is slightly enlarged, containing tw o polar bodies at the 1-2 o ’clock position. The first polar body is larger and round, and lies freely in the PVS, com pletely separated fro m the plasma membrane. The second polar body is almost half as small, round in shape, and attached to the plasma m em brane (x200)

Figure 1.33

Fertilization failure after insemination o r ICSI. Metaphase II oocytes I 8 h after ICSi o r insemination; no pronuclei are

visible. A round vacuole containing an intact sperm atozoon can be seen just inside the periphery (a) and (b), which suggests that the plasma m em brane o f these oocytes was not broken during ICSI. O nly one polar body is visible in each oocyte, corresponding to inactivation o f the oocyte. A cumulus cell inclusion is visible at the 5 o ’clock position (a) in the perivitelline space. Fluorescence D N A analysis (Hoechst staining) (d) o f the same oocyte shown in (c) confirm s the presence o f the first polar body chrom atin juxtaposed to the chrom osom es of the oocyte. T here is no additional fluorescence corresponding to the presence o f spermatozoa (x40 0)

86

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

%

*

c Figure 1.34

d

Fertilization failure follow ing initial activation and extrusion o f the second polar body (PB2) (actin is seen as red, tubulin

as green and chrom atin as blue), (a) First polar body (P B I), oocyte metaphase and spindle around prem aturely condensed sperm chrom atin (PCC) can be seen. A fte r activation and decondensation o f the sperm head, the oocyte returns to its initial metaphase IIlike state (metaphase lll-phase) w ith o u t extruding the PB2. (b -d ) T hree focal planes o f an oocyte, w hich extruded the PB2 body after activation, before entering the metaphase III state, (b) Focal plane at the PB I . (c) Focal plane at the metaphase III spindle and sperm PCC spindle, (d) Focal plane at the PB2

87

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.35 Abnorm al fertilization after ICSI. An oocyte I 8 h after ICSI is o f regular size and shape. A single, large pronucleus

Figure 1.36 Abnorm al fertilization after ICSI. An abnormal prezygote o f smaller size 18 h after ICSI. The cytoplasm is

is present w ith three visible nucleoli. The cytoplasm appears

homogeneous, containing a few inclusions. T hree pronuclei are

grainy around the pronucleus and in the cen ter o f this abnormal

present: tw o pronuclei o f regular and equal size and one small

zygote. The perivitelline space is slightly enlarged, containing

pronucleus. D ifferent numbers o f nucleoli are visible in each

m ultiple small granules. Five o r six different-sized cytoplasmic

pronucleus. The perivitelline space is slightly enlarged, w ith

extrusions including polar bodies are visible at the

11-12

tw o polar bodies visible at the I 2 o ’clock position. Both intact

o ’clock position. Prior to ICSI, an intact first polar body was

polar bodies are o f equal size and round, and seem attached to the plasma membrane. The zona pellucida is intact, unevenly

seen (x20 0)

thick along the circum ference w ith significant deform ation on the left side (x200)

Figure

1.37

Abnorm al fertilization after insemination. An

abnormal prezygote, 18 h after insemination, has fo u r regular pronuclei o f equal size. D iffere nt numbers of nucleoli are visible w ith in each pronucleus. The perivitelline space is almost absent. O ne fragm ented polar body is present at the I 2 o ’clock position (out o f focus). The zona pellucida is intact and evenly th ick along the circum ference, w ith m ultiple spermatozoa attached (x40 0)

88

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

-

X J

Jfef-T

V

'

a-

w

Figure 1.38

Fertilization and developm ent o f oocytes m atured in vitro fo r 24 -48 h from the germinal vesicle o r metaphase I stages,

(a) Fluorescence in situ hybridization o f in Wtro-matured metaphase II oocyte (chrom osom e 18, CEP aqua; chrom osom e 21, LSI red). Euploid metaphase II oocyte w ith paired red and aqua signals is seen on the left, and the corresponding euploid first polar body chrom osom es w ith an identical num ber o f signals is seen on the right, (b) N orm al fertilization o f in vitro-m atured metaphase II oocytes after ICSI, resulting in a perfectly norm al diploid zygote (tubulin is immunostained in green and chrom atin DAPI in blue), (c) Poor in vitro developm ent o f zygotes resulting from in W tro-matured metaphase II oocytes. These embryos are extrem e ly sensitive to

suboptim al in vitro conditions, (d) The same em bryos after chrom atin visualization w ith Hoechst stain. Anucleate fragments are seen

89

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

a

(%

3

Figure 1.39 Day 2 embryo w ith failed first cleavage. Uneven distribution of zygote cytoplasm is seen in this em bryo originating from a two-pronucleate zygote. A single, larger multinucleated blastomere, containing five small nuclei, is present along w ith m ulti-

Figure 1.40 Day 2 embryo w ith asymmetrical first cleavage. The day 2 em bryo is still in process o f the first cleavage, w ith uneven distribution o f zygote cytoplasm. Further developm ent o f this em bryo is unlikely (x200)

ple cytoplasmic fragments. Further development o f such an embryo is unlikely (x 200)

F ig u r e 1.41 T w o -c e ll em bryo w ith slightly asymm etric division and fragmentation. Slightly uneven distribution o f zygote cytoplasm is seen in this day 2 embryo, consisting of tw o blastomeres, one slightly larger than the other, w ith an additional fragmented portion. N o nuclei are visible in the blastomeres (x 2001

Figure 1.42 Typical tw o cell em bryo. Two equal sized blastom eres are present in this day 2 em bryo resulting from an even distribution of zygote cytoplasm. A first polar body is visible at the 5 o ’clock position. The other polar body is obscured by the blastomere. Each o f the blastomeres contains one nucleus (x200)

W j F ig u re 1.43 T h re e -c e ll embryo exhibiting asynchronous division and m ultinucleation. Three blastomeres, one large and tw o of equal size, are seen in this day 2 em bryo w ith slight fragmentation at the 5 o ’clock position. Three nuclei are visible in the larger blastomere, while no visible nuclei are present in the others. This may be the result o f asynchrony in the cell cycle between tw o original blastomeres in which the second division was completed by only one (x 200)

90

Figure 1.44 M orphologically abnormal four-cell em bryo w ith fragmentation. Four round blastomeres, slightly different in size, are observed in this day 2 em bryo w ith an extensive cytoplasmic fragmentation occupying nearly half o f the subzonal space. The viability of such embryos is extrem ely poor; they usually undergo developmental arrest (x200)

Figure 1.45 Morphologically normal four-cell embryos (a) and (bl. Four equal sized blastomeres are seen in these day 2 embryos. All blastomeres have nuclei, w ith a polar body visible at the 8 o ’clock position (b). Although both embryos seem to be normal, they differ considerably in cleavage symmetry (x400)

NORMAL AN D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

Figure 1.46 Slow five-cell em bryo w ith cytoplasmic blebs. Four round equal sized blastomeres and one smaller blastom ere are visible in this day 3 embryo. A few small, round cytoplasmic blebs are also present. The odd number o f blastom eres present suggests asynchronous cleavage and/or cytoplasm ic fragm entation. In many instances such slow embryos undergo developmental arrest (x 400)

a

Figure 1.47 Compacting o f a four-cell embryo. Four stretched blastomeres, each w ith a visible nucleus, are seen in this day 3 em bryo. T igh t contacts between blastomeres indicate the beginning of com paction. C om paction on day 3 o f em bryo developm ent is not uncommon, particularly in the com m ercially available G 1.1 medium. Compacting embryos pose a problem when perform ing blastom ere biopsy, because o f tig h t gap junctions. An attem pt at removal o f one blastomere may result in loss o f the whole embryo, therefore decompaction is necessary and perform ed by exposure o f the embryo to m edium devoid of calcium and magnesium prio r to biopsy (x400)

b

F ig u r e 1 . 4 8 Irre g u la r shaped eight-cell embryo. Eight round, equal sized blastomeres are seen in this day 3 embryo. The embryo and zona pellucida are oblong. Developm ent o f embryos w ith this abnormal m orphology is com promised and such embryos usually undergo developmental arrest. Blastomere biopsy is also complicated by the irregular shape

F i g u r e 1. 4 9 S ix - c e ll em bryo w ith cytoplasmic fragments. Six round, slightly unequal sized blastomeres are seen in this day 3 embryo. A t least tw o large cytoplasmic fragments are visible at the I and 9 o ’clock positions, which usually have no chromatin and may not interfere w ith furthe r development o f the em bryo (x 200)

(x200)

F ig u re 1.50 S ix - c e ll em bryo w ith cytoplasmic blebs. Six round, equal sized blastomeres are present in this day 3 em bryo w ith cytoplasmic blebs between blastomeres located in the center o f the embryo. Cytoplasmic blebs can distort contacts among blastomeres and mechanically prevent compaction o f the embryo (x400)

F ig u re 1.51 E xte n sive cytoplasmic fragmentation comprising 50% o f the subzonal space is seen in this day 3 embryo. Five to six round blastomeres, which vary in size, are present. Embryos w ith a similar abnormality usually undergo developmental arrest (x200)

F ig u re 1. 52 E ig h t-c e ll embryos (a), (b) and (c) w ith a few cytoplasmic blebs. Eight round, equal sized blastomeres are seen in each o f these day 3 embryos. A few small cytoplasmic blebs present do not affect development and compaction of the embryo ((a; and (b) x400; (c) x200)

91

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

1m

V

Figure 1.53

i

M orphologically normal day 3 embryos. Eight round, equal sized blastomeres are seen in the embryos. Cytoplasm is

homogeneous. T h e ir m orphology is considered o f grade I quality, since no fragm entation o r cytoplasmic blebs are present in any o f these em bryos. Loose contacts between the blastomeres and th e ir symm etrical positioning provide acceptable material fo r perform ing blastom ere biopsy. O ne o f tw o slit openings after rem oval o f first and second polar bodies can be seen at the 4 o ’clock position in (a) (x400)

92

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

Day 5 early

Figure 1.54

Day 5 early

Figure 1.55

Day 5 early

Figure 1.56

blastocyst,

120 h

blastocyst,

120 h

after

blastocyst. Cells are polygo-

insemination, w ith a w ell-

insemination, in w hich the

by the large oval cells o f the

blastocele comprises onehalf o f the conceptus.

nal and tightly connected to the neighboring cells in this

cyst, 120 h after insemina-

defined blastocele form ed

conceptus,

tocele form ed by a reduced

developing

T ro p h e cto d e rm

after

tro p h o b la st.

cells

are

120 h

insemination.

after

Nuclei

F ig u re 1. 57 A p p a re n tly abnormal day 5 early blastotion, consists of a small blas-

are

num ber o f large, flattened cells. Round cells, different

Round cells accumulating in

flattened and stretched to

visible in the m ajority o f

the lo w e r pole are involved

accom m odate the expan-

cells ( x 400)

in the form ation o f the inner cell mass. T h e re is no

sion. The inner cell mass is distinguishable inside the

the blastocele and are not

perivitelline space present. The zona pellucida is

blastocele cavity (x400)

the

in size, are scattered inside participating in form ation of blastocyst.

A

periv-

itelline space is still visible. Norm al

thinning (x400)

this

developm ent

blastocyst

of

is unlikely

(x 400)

Jr > F ig u re 1. 58

A b n o rm a l

■■/ J

Figure 1.59

7

Day 6 blasto-

Fig u re 1.60

cyst. The very beginning o f

an

6. T he tro p h o b la s tic vesicle, 144 h after insem-

the hatching process is seen

130 h

in

th ro u g h

ination, consists o f a large

blastocyst. A fe w tr o p h ectoderm cells are present

opening created in the zona

outside the zona pellucida at the 12 o ’clock position.

m ere biopsy. Hatching o f embryos w ith the opening

The inner cell mass is also visible on the left (x40 0)

earlier than in em bryos w ith

blastocelic cavity form ed by a single layer o f tro p h ectoderm cells. T here is no identifiable inner cell mass. The zona pellucida

this

fully

expanded

expanded

H atching o f

em bryo developm ent, day

the

blastocyst insem ination V-shaped

pellucida p rio r to

blasto-

in the zona pellucida occurs in ta ct

is ve ry thin (x40 0)

a fte r

zonae

pellucidae

(x200)

F ig u re 1.61 C o m p le te ly hatched, m o rp h o lo g ica lly norm al

blastocysts,

I SO-

HO h after insemination (a) and (b). T he V-shaped opening was created in the zonae pellucidae p rio r to blastom ere biopsy. An inner cell mass is clearly seen in each blastocyst (x20 0) a

b

93

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.62 Human blastocysts stained fo r tubulin and chromatin, (a; Human blastocyst w ith abnormal nuclei and cytoskeleton initially graded as normal on the basis of morphological appearance under the bright-field optics. Tubulin stained green, nuclei blue, bright-field view o f the blastocyst red. (b) Analysis o f the same blastocyst after fixation and nucleus staining (blue) demonstrates the abnormal development o f the embryos resulting from ‘chaotic cleavage’, (c) and (d) Norm al human blastocysts. Nuclei are blue, tubulin microfilaments green

94

NORMAL AND ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

I J

c

d

Figure 1.63 Human blastocysts stained fo r tubulin and chromatin, (a) Apparently abnormal blastocyst w ith a few cells extruded into the perivitelline space and not included in the process o f blastocyst form ation (nuclei are stained blue, tubulin microfilaments green), (b-d ) Morphologically normal blastocysts

95

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

b

Figure 1.64

D istributio n o f m itochondria in the human blastocyst (rhodam ine stain in green), (a) Inner cell mass in focus.

M itochondria are stained green, chrom atin stained w ith DAPI in blue, (b) Trophectoderm in focus. Trophectoderm cells have a similar num ber of m itochondria to that o f the inner cell mass cells

96

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

r

i

. • y I H

nhit&'i**JeP v^S ► v /“‘jc w

y


*

Figure 1.65 Separation o f the inner cell mass and tro p h e cto d e rm by blastocyst immunosurgery. (a) and (b) Blastocysts before immunosurgery, after zona pellucida removal, (c) and (d) Bright-field vie w o f the dissociated blastocyst cells after immunosurgery. (e) and (f) The same em bryos, under epifluorescence. The inner cell mass cell nuclei are stained blue com pared to lysed troph ectod erm cell nuclei stained pink

97

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.66

Establishment o f em bryonic stem (ES) cells from m orula and blastocyst, (a) Human em bryo at m orula stage is placed

under mouse em bryonic fibroblasts to establish ES cells (xlO ). (b) Initial g ro w th o f m orula cells under feeder layer (xlO ). (c) M orphology o f human m orula-derived ES cells (xlO ). (d) Initial o u tg ro w th o f ES cells from isolated inner cell mass (ICM ) (x20). (e) Primary colony of ES cells derived from ICM (x20). (f) M orphology o f human IC M -derived ES cells (x l 0)

98

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

Figure 1.67

Expression o f markers in em bryonic stem cell lines derived from m orula (left colum n) and inner cell mass (ICM ) (right

colum n): generic enzymatic activity o f alkaline phosphatase (a) and (b); human em bryonic carcinoma l-alkaline phosphatase, prim ary antibodies TRA-2-39 against epitope 2 102 (b) and (e); O ct-4 (e) and (f); and beta-tubulin (g) and (h). (a) Enzymatic activity o f alkaline phosphatase in m orula-derived ES cells grow ing on the m urine feeder layer (x20). (b) Matched fluorescein isothiocyanate (FITC): im m unofluorescence cell surface staining o f TRA-2-39 detected by m onoclonal antibodies in the same colonies o f m orula-derived ES cells grow ing on the m urine feeder layer (x20). (c) Tetram ethyl rhodam ine isothiocyanate (TRITC): im munofluorescence cell-surface staining o f O ct-4 expression in the same colonies o f m orula-derived ES cells grow ing on the m urine feeder layer (x20). (d) Enzymatic activity o f alkaline phosphatase in IC M -derived

ES cells grow ing on the m urine feeder layer (x20). (e) Matched FITC:

imm unofluorescence cell-surface staining o f TRA-2-39 detected by monoclonal antibodies labeled w ith FITC in the same colonies o f IC M -derived ES cells grow ing on the m urine feeder layer (x20). (f) TRITC: immunofluorescence cell surface staining o f O ct-4 expression in the same colonies o f IC M -derived ES cells grow ing on the m urine feeder layer (x20). (g) Expression o f beta-tubulin in m orula-derived ES cell line (x40). (h) Expression o f beta-tubulin in ICM -derived ES cell line (x40)

99

e Figure

1.68

*

Expression o f markers SSEA-3, SSEA-4, T R A -l-6 0 , T R A -I-8 I

and corresponding enzymatic activity o f alkaline

phosphatase (AP) in a m orula-derived em bryonic stem (ES) cell line, (a) Expression o f enzym atic activity o f alkaline phosphatase in colony o f ES cells grow ing on the m urine feeder layer (x20). (b) C orresponding im m unofluorescence cell-surface staining o f SSEA-3 detected by monoclonal antibodies labeled w ith fluorescein isothiocyanate (FITC) in the same colony o f ES cells as (a) grow ing on the m urine feeder layer, (c) Expression o f enzymatic activity o f alkaline phosphatase in colony o f ES cells grow ing on the m urine feeder layer, (d) Corresponding im m unofluorescence cell-surface staining of SSEA-4 detected by monoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (c) grow ing on the m urine feeder layer, (e) Expression of enzymatic activity o f alkaline phosphatase in colony o f ES cells grow ing on the m urine feeder layer, (f) C orresponding im m unofluorescence cell-surface staining o f T R A -l-6 0 detected by m onoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (e) grow ing on the m urine feeder layer, (g) Expression o f enzymatic activity o f alkaline phosphatase in colony o f ES cells grow ing on the m urine feeder layer, (h) Corresponding im m unofluorescence cell-surface staining o f T R A -l-8 0 detected by m onoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (g) grow ing on the m urine feeder layer (x20)

100

NORMAL A N D ABNORMAL HUMAN PREIMPLANTATION DEVELOPMENT

d

* g Figure

1.69

h

Expression of m arkers SSEA-3, SSEA-4, T R A -l-6 0 , T R A -l-8 0 and corresponding enzymatic activity o f alkaline

phosphatase (AP) in m orula-derived em bryonic stell (ES) cell line grow ing on feeder-free system, (a) Expression o f enzymatic activity o f alkaline phosphatase in the colony o f ES cells grow ing on the feeder-free system (M atrigel) (x20). (b) Corresponding im m unofluorescence cell-surface staining o f SSEA-3 detected by m onoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (a) grow ing on the feeder-free system (M atrigel). (c) Expression o f enzymatic activity o f alkaline phosphatase in the colony o f ES cells grow ing on the feeder-free system (M atrigel). (d) C orresponding im m unofluorescence cell-surface staining o f SSEA-4 detected by monoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (c) grow ing on the feeder-free system (M atrigel). (e) Expression o f enzymatic activity o f alkaline phosphatase in the colony o f ES cells grow ing on the feeder-free system (M atrigel). (f) Corresponding immunofluorescence cell-surface staining of TRA-1 -60 detected by monoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (e) grow ing on the feeder-free system (M atrigel). (g) Expression o f enzymatic activity o f alkaline phosphatase in the colony o f ES cells grow ing on the feeder-free system (M atrigel). (h) Corresponding im m unofluorescence cellsurface staining o f T R A -l-8 0 detected by m onoclonal antibodies labeled w ith FITC in the same colony o f ES cells as (g) grow ing on the feeder-free system (M atrigel) (x20)

101

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 1.70

D ifferentiation o f m orula-derived em bryonic stem (ES) cells in to cardiocyte-like cells (a) and neuron-like cells (b). (a)

D ifferentiation in vitro o f human ES cells into contracting prim itive cardiocyte-like cells (x20; captured fram e fro m video file), (b) D ifferentiation in vitro o f human ES cells in to neuron-like cells (x20; captured fram e from video file)

102

MICROMANIPULATION A N D BIOPSY OF POLAR BODIES AN D BLASTOMERES

Figure 2.1 First polar body (PBI) removal, (a) Once the oocyte has been secured by the holding pipette by gentle suction, the oocyte is oriented using the microneedle so that the PBI is visualized at the 6 o ’clock position, (b) and (c) Using the microneedle, the oocyte is rotated horizontally, until the polar body is visualized directly in the center o f the oocyte, facing the operator. This orientation o f the oocyte, p rior to creating the opening, is im portant when perform ing ICSI afterwards, (d) A slit is made in the zona pellucida at the 4 -5 o ’clock position, passing tangentially through the perivitelline space and out at the 7 -8 o ’clock position, (e) The oocyte is released from the holding pipette and held by the microneedle, (f) The partial zona dissection microneedle w ith the oocyte is brought to the top o f the holding pipette and pressed to it, pinching a portion o f the zona pellucida. By gently rubbing the microneedle against the holding pipette, w ith a sawing motion, partial zona dissection is accomplished

103

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 2.2 First polar body (P B I) removal (continued), (a) The oocyte is positioned so that the slit opening in the zona pellucida (3 o ’clock) and the PB I (6 o r 12 o ’clock) are clearly seen, (b) The oocyte is rotated so that the opening in the zona pellucida is at the 5 o ’clock position and the polar body is in focus. In some cases, sucrose (0.05 mol/1) is added to the medium to increase the size o f the perivitelline space by shrinkage o f the ooplasm, (c) The aspirating m icropipette is passed through the opening to the polar body, (d) and (e) Gentle suction is applied to aspirate the polar body into the m icropipette. Pressure from the hydraulic system is equilibrated p rio r to the w ithdraw l of the aspirating m icropipette, to avoid oocyte damage. Removal o f the PB I is postponed when it is still attached to the oocyte, in ord er to prevent possible enucleation o f the oocyte, (f) The oocyte is released from the holding pipette and all the m icrotools are raised slightly from the bottom o f the dish. The microscope stage is moved so that another drop o f medium at the 6 o ’clock position is visualized. The m icropipette containing the polar body is lowered to the bottom o f the dish and the PBI is expelled into the drop o f medium

104

MICROMANIPULATION A N D BIOPSY OF POLAR BODIES AN D BLASTOMERES

Figure 2.3 ICSI after polar body removal, (a) A morphologically normal spermatozoon is chosen and immobilized by being pressed against the bottom o f the dish w ith the injection needle, and the membrane of the tail is broken after a few sawing movements w ith the needle, (b) The spermatozoon is aspirated into the injection needle and then raised above the bottom o f the dish, (c) The microscope stage is moved so that one o f the drops o f medium containing an oocyte appears in view. The holding pipette is lowered to the bottom , next to the oocyte. Using the injection needle, the oocyte is rotated until the slit opening in the zona pellucida is visible, (d) The oocyte is positioned and held by the holding pipette w ith the slit opening at the 3 o ’clock position. In this position, the first poiar body, p rio r to its removal, would have been located at the 6 o r 12 o ’clock position, (e) The injection needle is passed through the opening in the zona pellucida and slowly continued to almost three-quarters o f the oocyte’s diameter deep into the ooplasm. A t this point, movement has stopped and a small amount o f the ooplasm is aspirated until a sign is visible o f plasma membrane breakage. Contents o f the injection needle w ith the spermatozoon are gently expelled into the ooplasm, (f) A fte r the spermatozoon has been deposited into the ooplasm, the injection needle is slowly w ithdraw n

105

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

b Figure 2.4 ICSI (continued), (a) Bright-field (N om arski) optics. Entrance o f the m icro pip ette containing a sperm atozoon. The first polar body (P B I) is located at the I I o ’clock position, (b) Epifluorescence after chrom atin staining w ith Hoechst. Three areas o f fluorescence can be seen: the PB I chrom atin; the oocyte chrom osom es near the area o f the PB I ; and the sperm chrom atin

106

MICROMANIPULATION AN D BIOPSY OF POLAR BODIES AN D BLASTOMERES

C

►( e

t

f

Figure 2.5 First polar body (PBI) removal after ICSI. (a) Once secured by the holding pipette, the oocyte is oriented by means of the microneedle so that the PB I is visualized at the 12 o ’clock position, (b) and (c) By means of the microneedle, the oocyte is rotated horizontally until the PB I is slightly out o f focus. In this case, the slit in the zona pellucida is located closer to the PB I , providing easier access to it. If the zona pellucida is cut right above the PB I it w ill lead to deformation o r damage o f the polar body by the biopsy pipette when it passes through the opening. The microneedle is passed through the zona pellucida at the 1-2 o ’clock position, tangentially through the perivitelline space and out at the 10-11 o ’clock position, (d) The oocyte is released from the holding pipette and held by the microneedle. The microneedle w ith the oocyte is brought to the bottom o f the holding pipette and pressed to it, pinching a portion o f the zona pellucida. By gently rubbing the microneedle against the holding pipette w ith a sawing motion, partial zona dissection is perform ed and the oocyte is released, (e; The oocyte is rotated so that the opening in the zona pellucida is at the 2 o ’clock position and the polar body is in focus, (f) The aspirating m icropipette is passed through the opening to the PB I

107

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

d

Figure 2.6 First polar body (PBI) removal after ICSI (continued), (a) The aspirating m icropipette is furthe r passed through the opening to the PBI. (b), (c) and (d) Gentle suction is applied to aspirate the polar body into the m icropipette. Pressure from the hydraulic system is equilibrated p rior to withdrawal o f the aspirating m icropipette, to avoid damage to the oocyte, (e) The oocyte is released from the holding pipette and all the m icrotools are raised slightly from the bottom o f the dish. The microscope stage is moved so that another drop o f medium at the 6 o ’clock position is visualized. The m icropipette containing the polar body is lowered to the bottom o f the dish, and the PB I is expelled to the middle o f the drop o f medium. In this picture, however, the polar body was expelled immediately fo r demonstration and can be seen in the center

108

MICROMANIPULATION A N D BIOPSY OF POLAR BODIES A N D BLASTOMERES

Figure 2.7 Second polar body (PB2) removal, (a) Since an opening has already been created in the zona pellucida p rior to the first polar body removal, the prezygote is rotated so that the opening in the zona pellucida is positioned conveniently enough to access the PB2. (b) The opening should be in focus when passing the m icropipette into the perivitelline space (PVS). (c) Once in the PVS the PB2 is brought into focus and the m icropipette is advanced to the polar body. W ith gentle suction the polar body is aspirated into the m icropipette, (d) and (e) A t this point, the m icropipette w ith the PB2 is w ithdraw n from the PVS. Because the PB2 is almost always attached by a cytoplasmic bridge to the prezygote, positive pressure in the m icropipette is maintained during withdrawal until the cytoplasmic bridge is pinched off. (f) The prezygote is released from the holding pipette and all the m icrotools are raised slightly from the bottom o f the dish. The microscope stage is moved so that another drop o f medium at the 6 o ’clock position is visualized. The m icropipette containing the second polar body is lowered to the bottom o f the dish and the PB2 is expelled to the middle o f the drop o f medium. For demonstration, the PB2 can be seen in the center; the prezygote is out o f focus

109

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

a

c

Figure 2.8 Simultaneous first (PBI) and second (PB2) polar body removal, (a) Once secured by the holding pipette, the prezygote is oriented by the microneedle to visualize the PB I and PB2 side by side at the 12 o ’clock position. A t this position, the morphology and number o f polar bodies are evaluated, (b) and (cl The prezygote is released and gently rotated so that both polar bodies are still at the 12 o ’clock position, one behind the other, (d) The prezygote is rotated until the polar bodies are slightly out o f focus. A slit is made by entering the zona pellucida w ith the partial zona dissection needle at the 1-2 o ’clock position, passing tangentially through the perivitelline space and out at the 10-11 o ’clock position. The opening in the zona pellucida is made similarly to the PBI removal procedure. The prezygote is released from the holding pipette and held by the microneedle. The microneedle w ith the prezygote is brought to the bottom o f the holding pipette and pressed to it, pinching a portion o f the zona pellucida. By gently rubbing the microneedle against the holding pipette w ith a sawing m otion, a partial zona dissection is perform ed and the prezygote is released, (e) and (f) The prezygote is oriented by the microneedle to visualize both polar bodies at the 12 o ’clock position, side by side, and the opening in the zona pellucida. In this position, both polar bodies and slit opening in the zona pellucida are positioned on the same straight line so that the polar bodies are easily accessed by the aspirating pipette

110

MICROMANIPULATION AN D BIOPSY OF POLAR BODIES A N D BLASTOMERES

Figure 2.9 Simultaneous first and second polar body removal (continued), (a) and (b) The aspirating m icropipette is passed through the opening to one o f the polar bodies, (c) and (d) Gentle suction is applied, to aspirate the first available polar body into the m icropipette. Once pressure from the hydraulic system has been equilibrated, the m icropipette is moved forw ard to the remaining polar body, gentle suction is applied and the second polar body is aspirated. If one o r both polar bodies are fragmented, all o f the fragments are removed in similar fashion, focusing on the target object and redirecting the position of the m icropipette towards it. Pressure from the hydraulic system is equilibrated p rio r to the withdrawal o f the aspirating m icropipette, to avoid damage to the prezygote. The prezygote is released from the holding pipette and all the m icrotools are raised slightly from the bottom of the dish. The microscope stage is moved so that another drop o f medium at the 6 o ’clock position is visualized. The m icropipette containing all the polar bodies is lowered to the bottom o f the dish and the polar bodies are expelled in the middle o f the drop of medium

III

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

b

Figure 2.10 Simultaneous first (PBI) and second (PB2) polar body removal (continued), (a) Bright-field (Nomarski) optics. Simultaneous PB I and PB2 removal from an abnormally fertilized prezygote, (b) Epifluorescence after chromatin staining with Hoechst D N A stain

Figure 2 .1 I (opposite) Embryo biopsy following the making of a square opening in the zona pellucida. (a) The embryo is held loosely by gentle suction from the holding pipette and rotated using the microneedle until the area w ith the largest perivitelline space (PVS) is visible at the I 2 o ’clock position, (b) Additional suction is applied, to hold the embryo firm ly in this position. The microneedle is passed through the zona pellucida starting at the 1-2 o'clock position, advanced tangentially through the PVS and out the other side at the 10-1 I o’clock position, (c) The embryo is released from the holding pipette and held by the microneedle. The microneedle is brought to the bottom o f the holding pipette and pressed to it, pinching a portion of the zona pellucida. By gently rubbing the microneedle against the holding pipette with a sawing motion, the cut is accomplished and the embryo is released, (d) and (e) To perform the second cut, the embryo is rotated vertically until the slit is clearly visible at the 12 o'clock position and then rotated horizontally backwards until the slit is slightly out of focus, reaching the end of the slit, which is now at the 12 o’clock position. The second intersecting cut is positioned at the end of the first cut to create the V-shaped opening. This cut is completed by entry into the first slit, advancing tangentially through the PVS and ending at the 10-1 I o’clock position. The embryo is released from the holding pipette and held by the microneedle. A fter the second cut is accomplished, the embryo is again rotated vertically until the new slit is visible at the 12 o’clock position and then rotated horizontally backwards until the slit is slightly out of focus, reaching the end of the slit. The microneedle is passed through the slit opening, through the PVS and out at the 10-1 I o’clock position. The third intersecting cut makes up the third side of the square, (f) To complete the square and create a hole in the zona pellucida, the fourth intersecting cut is made by rotating the embryo vertically until tw o slits are visible at the 12 o ’clock position. The embryo is rotated horizontally backwards until both slits are slightly out of focus and the final cut is made by entering the first slit, passing through the PVS and out at the I I o ’clock position. The embryo is released from the holding pipette and is held by the microneedle. The microneedle is brought to the bottom of the holding pipette, pressed slightly and, w ith a sawing motion, the final cut is accomplished

112

MICROMANIPULATION A N D BIOPSY OF POLAR BODIES AN D BLASTOMERES

Figure 2 .1 I

113

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

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Figure 2 . 12 Embryo biopsy following the making o f a square opening in the zona pellucida (continued! (a) An opening in the shape of a square can be seen after removal o f the cut-out portion using the microneedle. The size of the hole created is dependent on the length o f the first tw o cuts, (b) The em bryo is positioned and held by the holding pipette w ith the opening in the zona pellucida at the 3 o ’clock position. A square piece can be seen adhering to the zona pellucida above the opening, (c) The blastomere biopsy needle is freely passed through the opening towards the nearest blastomere. (d) The blastomere is slowly aspirated into the pipette, (e) Aspiration is continued slowly until the whole blastomere is inside the aspiration needle. The m icropipette containing the blastomere is w ithdraw n from the perivitelline space, (f) The embryo is released from the holding pipette and all the m icrotools are raised slightly from the bottom o f the dish. The microscope stage is moved so that the drop of medium at the 6 o ’clock position is visualized. The m icropipette containing the blastomere is lowered to the bottom o f the dish and the blastomere is expelled to the middle o f the drop o f medium

114

MICROMANIPULATION AN D BIOPSY OF POLAR BODIES AN D BLASTOMERES

Figure 2.13 Embryo biopsy following polar body removal and creation of the ‘V ’-shaped opening in the zona pellucida. (a) To perform the second cut, the em bryo is rotated vertically until the first slit created during polar body removal is clearly visible (5 o ’clock position), (b) The second intersecting slit is accomplished by entry into the first slit, advancing tangentially through the perivitelline space and ending at the 7 o ’clock position, (c) The em bryo is released from the holding pipette and held by the microneedle. The microneedle is brought to the bottom o f the holding pipette and pressed to it, pinching a portion o f the zona pellucida. By gently rubbing the microneedle against the holding pipette w ith a sawing m otion, partial zona dissection is perform ed and the em bryo is released, (d) The em bryo is rotated until the ‘V ’-shaped opening is at 3 o ’clock and then held firm ly by suction from the holding pipette, (e) The em bryo biopsy m icropipette is then brought into focus and by application o f a slight downward pressure, freely passes through the opening tow ard the nearest blastomere. The blastomere is slowly aspirated into the pipette. Aspiration continues slowly until the whole blastomere is inside the m icropipette, which is then w ithdraw n slowly from the perivitelline space. The blastomere is removed w ith little distortion to the embryo, (f) The em bryo is released from the holding pipette and all the m icrotools are raised slightly from the bottom o f the dish. The microscope stage is moved so that another drop o f medium at the 6 o ’clock position is visualized. The m icropipette containing the blastomere is lowered to the bottom o f the dish and the blastomere is expelled to the middle o f the drop of medium

115

ATLAS OF PRE IMPLANTATION GENETIC DIAGNOSIS

C\ Figure 2.14 Blastocyst biopsy on day 5 of embryo development, (a) Image of a poor-quality blastocyst prior to beginning the biopsy procedure. The embryo is held loosely by gentle suction from the holding pipette and rotated using the microneedle, in order to position the embryo so that the opening is positioned opposite the inner cell mass (ICM), if visible, (b) The embryo is then held firmly in position using more suction from the holding pipette and the microneedle is passed through the zona pellucida, entering at approximately I o'clock and exiting at I I o’clock, (c) Once the microneedle has passed through the zona pellucida, a slit-type opening is created by releasing the embryo from the holding pipette, bringing the portion of the zona pellucida under which the microneedle has passed to the holding pipette, gently pressing and rubbing against the holding pipette in order to cut the zona pellucida. (d) After an hour or more in culture, trophectoderm cells will begin herniating through the opening, (e) Depending on the cohesiveness of the cells, they may be aspirated into the micropipette as seen in this example or may be cut using a microsurgical blade, (f) Image of the tw o trophectoderm cells that were removed, (g) Phase-contrast image of the nuclei isolated from the corresponding cells (f). (h) Fluorescence in situ hybridization (FISH) image after hybridization with MultiVysion polar body (PB) panel probe for chromosomes 13, 16, 18, 21 and 22. One normal diploid and one tetraploid cell are evident by the number of signals present for the five chromosomes tested, (i) Image of the same embryo in which blastocyst biopsy was performed (a) after several hours in culture, hatching through the artificial opening created during biopsy

116

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

KARYOTYPING BY CONVERSION

Enucleated oocyte

/ Zygote

Blastomere 2nd polar body Tetraploid heterokaryon Haploid ntti eo-cytoplasfftic hybrid

METAPHASE CHROM OSOM ES

F ig ure 3 .1 Scheme o f karyotyping by nuclear transfer technique

F ig ure 3.2

Flow chart o f second polar body nucleus conversion into metaphase chromosomes

117

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 3.3 Enucleation of human oocytes as a source of cytoplasts fo r second polar body nucleus conversion into metaphase chromosomes, (a) and (b) A fte r partial zona dissection, a flame-polished biopsy tool is used to remove the first polar body (P B I) from the perivitelline space, as described in Chapter 2 (page 15). (c) and (d) Chrom atin epifluorescence after staining w ith vital Hoechst stain, (ej and (f) Enucleation is verified by a second brief exposure to epifluorescence, showing that both the PBI and metaphase chromosomes are inside the pipette

I 18

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

e Figure 3.4 Micromanipulation steps for intracytoplasmic second polar body fPB21 nucleus injection into human ooplasts. (a) Injection tool next to previously biopsied PB2. (b) PB2 pycnotic nucleus is visualized under epifluorescence after chromatin staining with vital Hoechst stain, (c) Intracytoplasmic PB2 injection into the enucleated human oocyte, (d) Epifluorescence of haploid nucleocytoplasmic hybrid immediately after polar body injection, confirming the success of the procedure, (e) Activation of the resulting haploid nucleocytoplasmic hybrids by electrostimulation

19

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

C

d

Figure 3.5 Intracytoplasmic second polar body (PB2) nucleus injection into human ooplasts w ith o u t Hoechst staining and epifluorescence verification, (a) PB2 p rio r to aspiration into the injection pipette, (b) Human ooplast before intracytoplasmic PB2 injection, (c) Reconstructed haploid nucleocytoplasmic hybrid immediately after PB2 injection into the ooplast. (d) Haploid nucleocytoplasmic hybrid activation, one at a tim e

Figure 3.6 O ocyte activation and cell electrofusion device (XRONOS, Chicago, IL, USA), (a) Electrofusion unit, (b) rem ote control and (c) chamber fo r electrofusion

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NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Figure 3.7 Conversion o f the second polar body (PB2) nucleus into metaphase chromosomes, (a) Haploid nucleocytoplasmic hybrid between human PB2 and enucleated oocyte at the pronuclear stage (differential interference contrast using Nomarski optics), (b) The same embryo, at the first metaphase. Chromosomes (blue) are visualized by Hoechst staining; Hoffman modulation contrast optics gives the general view o f the em bryo

121

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 3.8 Second polar body (PB2) nucleus transform ation follow ing its injection into foreign cytoplasm (im m unocytochem ical investigation), (a) Haploid nucleocytoplasmic hybrid im m ediately after intracytoplasm ic PB2 injection. A ctin stained red, tubulin green, chrom atin blue. The PB2 nucleus is inside the cytoplast. A nti-actin antibodies (labeled red) stain the subcortical layer o f actin and are used fo r visualizing membranes. The cavity is still not relaxed after injection. T here is no plasma membrane around the PB2 nucleus since it was broken by the injection too l. However, it is surrounded by tubulin microfilam ents, abundant in the PB2 (unpublished observations), (b) "Iwo hours after PB2 injection (bright-field vie w o f an em bryo is shown in red, tubulin green, chrom atin blue). Tubulin m icrofilam ents, initially present only around the nucleus, started to elongate into the cytoplasm, (c) First m ito tic division o f the reconstructed em bryo, w ith metaphase spindle o f the PB2 clearly seen. Bright-field view o f an em bryo is shown in red, tubulin green, chrom atin blue, (d) Prem ature chrom osom e condensation induced by okadaic acid. PB2 chrom osom es (blue) are scattered all over the cytoplasm, w ith no spindle form ed; staining fo r tubulin (green) also reveals cytoplasm stratification, caused by cytochalasin D

122

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Blastomere biopsy

Zona pellucida removal

Figure 3 .1 0

Flow chart o f blastom ere nucleus conversion into metaphase chrom osom es. PHA, phytohemagglutinin

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ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

-

—

......

F ig ure 3 .1 1 Enucleation o f haploid human zygote as a source fo r blastomere nucleus conversion into metaphase chromosomes, (a-e) Stages o f human one-pronuclear zygote enucleation, (f) Blastom ere-cytoplast electrofusion

124

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Figure 3 .12 Tetraploid heterokaryons between human blastomeres and mouse zygotes, (a) Human blastomere-mouse zygote pairs are kept together by phytohemagglutinin, (b) Blastomere-zygote pairs in the electrofusion chamber, immediately after electric pulse, (c) "letraploid interspecies hybrids immediately after fusion. Human nuclei and tw o mouse pronuclei are visible in all heterokaryons. (d) Five hours after fusion. Some embryos start entering metaphase of the first cleavage division, (e) Hybrids 9h after fusion. Most o f the heterokaryons have entered mitosis by this time. An irregular shape o f an embryo is an indicator o f anaphase o r telophase o f the first mitosis, (f) Okadaic acid is used to induce premature chromosome condensation. Heterokaryons after I h o f treatm ent by okadaic acid. The pronuclei have disappeared. Since okadaic acid disrupts the spindle, no metaphase formation is evident prior to fixation

125

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

r

f

Figure 3.13

Metaphase o f an individual human blastom ere follow ing conversion, (a) H um an-m ouse tetraploid hybrid im m ediately

after fixation (phase contrast), (b) The same metaphase hybridized using w hole chrom osom e painting fo r chrom osom es 16, 18, 2 I and 22 (C orel Photo-Paint was used during final image processing)

Figure 3.1 4

126

Karyotype o f individual human blastom ere obtained follow ing conversion

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

n-anslocation

Blastomeres Anudeate loss Reciprocal 582 26 Robertsonian 185 6

Metaphases 415 119

S-FCC b9 32

conversion 72 28

byW Q V 484 151

Total #(% )

F ig ure 3.15 Efficiency o f the blastomere nucleus conversion. Based on a study o f 767 embryos, fro m 89 preim plantation genetic diagnosis (PGD) cycles fo r chrom osomal translocations. S-PCC, S-phase prem ature chrom osom e condensation; WCR w holechrom osom e paint

127

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

? Figure 3 . 16

Metaphase chrom osom es obtained by periodic observation o f the natural cell cycle o f day 3 em bryos w ith o u t nuclear

conversion, (a) Phase-contrast image o f chrom osom es fro m a haploid em bryo obtained after em bryo biopsy and fixation o f a blastom ere after disappearance o f the nucleus. "Iwo distinct chrom osom e spreads are present, (b) Fluorescence in situ hybridization (FISH) image o f the corresponding chrom osom e spreads seen in (a), using a probe cocktail consisting o f W C P 10 (green), CEP 10 (aqua), Tel I Oq (red) and W C P 22 (orange), showing one chrom osom e 10 (W CP 10, green) and one chrom osom e 22 (W CP 22, orange) in each chrom osom e spread, (c) Phase-contrast image o f metaphase chrom osom es from a haploid em bryo obtained after em bryo biopsy and fixation o f a blastom ere after disappearance o f the nucleus, (d) FISH image o f the metaphase chrom osom es (c) using the probes in (b), showing an extra chrom osom e 22, and one chrom osom e 10 in this haploid set. (e) DAPI-stained metaphase chrom osom es from a trip lo id em bryo obtained after em bryo biopsy and blastom ere exposure to an antim itotic agent blocking spindle form ation, follow ed by fixation, (f) FISH image o f these metaphase chrom osom es using the same probe cocktail previously m entioned (b), in w hich the expected three chrom osom es 10 (green) and three chrom osom es 22 (orange) are present

128

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Metaphase II human oocyte

Enucleation of oocyte

Cytoplast

Single sperm injection

Androgenetic em bryo w ith one pronucleus

n rogenetic 2 cell em bryo

| 1

Analysis o f genetic material o f one blastomere

I

I

O l ] | WMI j J

I

Cryopreservation of another blastomere for future zygote

0

Figure 3.17 Flow chart o f sperm duplication in human metaphase II cytoplast. Step I - enucleation o f human metaphase II oocyte; Step 2 - injection o f single human sperm into the cytoplast; Step 3 -- reconstructed haploid androgenic em bryo w ith one pronucleus; Step 4 - development o f this pronucleus into 2-cell embryos; Step 5 - testing o f one o f the cells, w ith the oth er one available for furthe r zygote construction o f known male contribution

129

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 3.18

Sperm duplication resulting in an androgenetic five-cell cleaving human em bryo, (a) Reconstructed one-cell em bryo

follow ing enucleation and single sperm injection; (b)—(d) D evelopm ent of the reconstructed one-cell em bryo in cleavage stage fivecell em bryo, (a) One-cell em bryo w ith a male pronucleus (20 h after intracytplasmic sperm injection (ICSI)); (b) tw o -cell em bryo (29 h after ICSI); (c) four-cell em bryo (45 h after ICSI); and (d) five-cell em bryo (45 h a fte r ICSI)

130

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Figure 3.1 9

Human sperm duplication into tw o identical chrom osom ally normal haploid cells using human metaphase II oocytes,

(a) and (c) Two identical cells derived follow ing sperm duplication, shown by fluorescence in situ hybridization (FISH) analysis o f chrom osom es 13, 16, 18, 21 and 22. (b) and (d) Re-hybridization of the same cells w ith chrom osom e-specific probes fo r chrom osom es 16 and Y

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ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 3.20 Sperm duplication into tw o aneuploid haploid cells. Single signal in one o f the duplicated cell (a), w ith three signals in the corresponding cell (b), demonstrating non-disjunction o f the X chromosome (tw o green signals)

Figure 3.21 Development o f artificial human gametes in vitro. Flow chart o f haploidization procedure fo r obtaining gametes from somatic cells, (a) In vitro matured metaphase II oocyte w ith extruded first polar body (P B i). (b) Both PBI and metaphase II chromosomes are removed (shown in pipette), to prepare a recipient enucleated ooplast. (c) Single diploid cumulus cells w ere prepared as nuclei donors, from which a nucleus was mechanically isolated, fo r the introduction into ooplast. (d) Reconstructed oocyte converting somatic cell nucleus into metaphase chromosomes, (e) Iw o pronuclei o r (f) pronucleus and PBI obtained after electrostimulation using electrofusion device (Chicago, IL). (g) Fluorescent in situ hybridization analysis, o r (h) polymerase chain reaction (PCR)-based chromosomal analysis, confirm ing the form ation o f tw o haploid sets o f chromosomes in both (e) and (f) scenario

132

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Figure 3.22 Developm ent o f artificial human gametes in vitro (continued). Microsurgery procedure fo r obtaining gametes from somatic cells, (a) Enucleation o f recipient matured human oocytes using flame-polished blunt m icropipette, following the opening perform ed by partial dissection o f zona pellucida (PZD). (b; The removed first polar body (PBI) and small ooplast w ith metaphase plate are shown in the m icropipette, stained w ith a vital Hoechst stain, (c) A single nucleus is isolated from individual donor human cumulus cell, (d) Microinjection o f the cumulus nucleus into the recipient enucleated oocyte (ooplast! through the same zona opening, (e) Reconstructed by nuclear transfer oocyte showing tw o foci, representing metaphase chromosomes, following activation by electrostimulation, (f) Microsurgically removed pronuclei fo r subsequent analysis

133

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 3.23 Developm ent o f artificial human gametes in vitro (continued). In vitro development o f reconstructed oocytes following haploidization. (a), (b) and (c) Reconstructed by somatic cell nucleus injection metaphase II oocytes I Oh after electrostimulation, (d) Isolated pronuclei (x 400)

134

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

0 © ID a

C3 C) 5W b

Figure 3.24 Developm ent o f artificial human gametes in vitro (continued). Flow chart o f pronuclei form ation after natural fertilization and somatic cell haploidization. (a) The sequence of events in the matured metaphase II oocyte, following sperm penetration (upper left), resulting in polar body (PB) extrusion and pronuclei form ation (upper ro w middle and second ro w middle), migrating from the periphery to the middle (upper right), (b) Reconstructed metaphase II oocytes following somatic cell nuclei injection 6 h after electrostimulation, resulting in tw o haploidization pronuclei positioned close to each other despite the volume gro w th (shown from upper left to upper right) (x 200)

135

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

e Figure 3.25

f N orm al segregation o f chrom osom es 13, 16, I 8, 2 I and 22 o f reconstructed somatic cell nuclei, (a) and (b) Two

identical pronuclei obtained fro m the reconstructed somatic cell injected into an enucleated metaphase II oocyte, showing a single signal fo r all the chrom osom es analyzed by fluorescence in situ hybridization (FISH): chrom osom es I 3 (red), 16 (aqua), 18 (blue), 2 1 (green) and 22 (yellow ) (x 600). (c) and (d) and (e) and (f) Two m ore examples o f norm al chrom osom e segregation in the resulting PB and pronucleus (c) and (d) o r haploid pronuclei (e) and (f) obtained through injection into enucleated metaphase II oocytes (chrom osom es 16 and 21 in (c) and chrom osom e 16 in (e and f) are pulverized

136

NUCLEAR TRANSFER TECHNIQUES FOR PREIMPLANTATION DIAGNOSIS

Jl

1i ' ' 1 1i ‘ 1 ' ' i ’ 1 1' i 1' 11 i 1 ' ' ' i ' ' ' ' i ' ' J 1i ' ' ' ' i ' ’ ’ ' i 200

205

210

PN1

215

220

D21S2S0

225

230

235

240

245

1

-6000 -4000 -2000

[22527]

-6000 -2000 |233.41| DONOR

NUCLEUS

D21S290 -6000 4000 -2000

JLJl

122i.32|

OOCYTE

NUCLEUS

|233.3gj

D21S280

-6000 4000 2000

|209B1

•

0 PB2

PB1

PB2

F igure 4.26 First polar body (PB I ) and second polar body (PB2) fluorescence in situ hybridization (FISH) analysis o f highly condensed chromatin, requiring rehybridization, (a) FISH image of PB I and PB2 after hybridization w ith a five-color chromosome cocktail for chromosomes 13 (red), 16 (aqua), 18 (violet blue), 2 1 (green) and 22 (gold), removed from an oocyte obtained from a 36-year-old woman. A normal number of signals are present in PBI fo r chromosomes 13, 18 and 2 1, but close proxim ity and overlapping of signals for chromosome 22 (white arrow) and chromosome 16 (strip-like signal indicated by aqua arrow) render an inconclusive result. PB2 contains a normal number of signals. Interpretation of PB I results in this PBI cannot be undertaken w ithout a second round o f hybridization targeting the terminal region o f each chromosome, (b) FISH image following re-hybridization of PB I and PB2 chromatin w ith a sub-telomeric 16q probe in orange (aqua arrows) and a sub-telomeric 22q in green (white arrows), showing tw o signals for each of these chromosomes tested in PB I along w ith one signal for each chromosome in PB2 (representing a control in this system), suggesting the resulting zygote is normal and can be transferred, (c) FISH image o f highly condensed PB I and PB2 chromatin from the same patient as (a) and (b). "Iwo separate signals are seen for each of the chromatids present in PBI w ith the exception of chromosome 2 1 which appears as a single signal (green arrow); however, it is located near o r overlapping w ith the aqua signal for chromosome 16. Additionally, signal overlap and artifact interference (red arrow) are seen in the PB2 nucleus in which no conclusion can be made regarding chromosomes 13 and 2 1. (d) Re-hybridization performed fo r chromosomes 13 and 2 1 using sub-telomeric I 3q (green) and 2 1q (orange), showing tw o signals for chromosome 13 (representing an internal control) and chromosome 2 1 (located in close proxim ity to one another) in PB I ; one signal for chromosome 13 and chromosome 2 1 are now evident in PB2 following the second round of hybridization, along w ith the same artifactual interference seen w ith the first hybridization, (e) FISH image of an interphase nucleus from the resulting embryo, confirming a normal number of signals for each of the chromosomes tested including chromosome 2 1 (white arrows). Also present is a large, bright artifact (red arrow) causing a dimming effect fo r the chromosome signals due to its intensity when attempting to capture the image

165

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

\ ‘

/

F igu re 4.27 Split fluorescence in situ hybridization (FISH) signals requiring an additional round o f hybridization, (a) FISH image o f an interphase nucleus after hybridization w ith MultiVysion PB panel probe, showing tw o signals fo r each o f the chromosomes tested w ith the exception o f chromosome 13 (white arrows) in which both signals are split consisting o f tw o and three dots, (b) FISH image after a second round o f hybridization, using a probe cocktail targeting the sub-telomeric region of chromosomes 13 (green) and 16 (orange; appears red through a red single bandpass filter), showing tw o signals fo r both o f the chromosomes re-investigated, indicating that the embryo is normal fo r the five autosomes tested (c) FISH image o f an interphase nucleus after hybridization w ith MultiVysion PB panel probe, showing split signals fo r chromosomes 13,18 and 2 1, caused by overspreading during fixation. Because o f the decondensed state o f the chromatin and the fact that signal dots are less than one domain apart, split signals are counted as one. However, in this particular nucleus a single signal fo r chromosome 13 (red) is seen in the low er left area, while the second, located on the right, appears pulverized (white arrow ), making it difficult to draw any conclusion regarding chromosome 13 w ith ou t furthe r testing, (d) FISH image after a second round o f hybridization, using a probe cocktail targeting the sub-telomeric region o f chromosomes 13 (green) and 18 (orange), showing tw o signals present fo r chromosome 18 (internal control), but three signals fo r chrom osom e 13 (w hite arrows), all o f which are m ore than tw o domains apart, indicating a trisom y 13 in the embryo

166

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

Figure 4 .2 8

Value o f re-hybridization in split signals, (a) Fluorescence in situ hybridization (FISH) image o f an interphase nucleus

a fter a second round o f hybridization fo r chrom osom es X (CEP X ; green), Y (CEP Y; aqua) and 16 (sub-telom eric I 6q; orange), revealing a single signal fo r chrom osom e X (yellow arro w ), tw o signals fo r chrom osom e 16, and a double d o t signal fo r chrom osom e Y (w h ite arrow ), requiring fu rth e r testing o f chrom osom e Y to avoid a misdiagnosis, (b) FISH image after a third round o f hybridization w ith a sub-telom eric X p/Y p in green which hybridizes to com m on sequences o f the sub-telom eric region o f the p arm fo r both chrom osom es X and Y, showing three signals (w hite arrow s) w hich indicates an em bryo abnormal fo r the sex chrom osom es by the presence o f an extra Y chrom osom e, (c) FISH image o f an interphase nucleus after hybridization w ith MultiVysion PB panel probe, showing tw o signals fo r each o f the chrom osom es tested w ith the exception o f chrom osom e 21 (w h ite arrow s), in which one signal is large and the o th e r is split, consisting o f tw o separate smaller dots, preventing a conclusion fro m being made regarding chrom osom e 2 I . (d) FISH image follow ing re-hybridization o f the same nucleus (c) fo r chrom osom e 2 I w ith a sub-telom eric 2 1q in orange, showing at least three small signals indicating a possible triso m y 21, despite close p ro xim ity o f signals

167

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Results

(17.2%)

Lost No signal/inconclusive

(3.2%)

(79.6%)

Figure 4 .2 9

Pie chart o f polar body fluorescence in situ hybridization (FISH) analysis efficiency in prediction o f oocyte chrom osom al

constitution. Results w e re obtained in approxim ately 8 0 % o f oocytes tested. N o o r inconclusive results w e re due to eith er loss o f polar bodies during m icrom anipulation procedures (3.2% ), o r failure o f adequate hybridization, resulting in inconclusive results (17.2% )

Polar body 1 Polar body 2

(13.1%)

Polar body 1 and polar body 2

(13.4%)

(73.5%)

Figure 4 .3 0

Pie chart dem onstrating the p ro p o rtio n o f oocytes in which results w e re obtained by one o r both polar bodies. Results

w e re obtained by first polar body (PB I ) only in 13 .1% , second polar body (PB2) only in I 3.4% and by both PBI and PB2 in 73.5% . W hile the detection o f abnorm alities by only one polar body (PBI o r PB2), may avoid transfer o f the corresponding em bryo, no decision can be made if inform ation is available fo r only one polar body w ith a norm al pattern, since erro rs may occur during meiosis I and/or meiosis II. Embryos may be transferred only if both PB I and PB2 w e re found to have normal fluorescence in situ hybridization (FISH) patterns

168

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

Figure 4 .3 1 Schematic representation o f segregation patterns and relative proportions o f each type o f errors occurring in meiosis I. U pper panel shows an overall proportion o f the resulting normal and abnormal metaphase II oocytes. Low er panel shows the types and proportion of each error, providing evidence of non-random distribution, w ith the m ajority representing missing chromatids in first polar body (PB I )

Figure 4.32 Schematic representation o f segregation patterns and relative proportions o f each type of errors occurring in meiosis I!. Left panel shows the types and proportions o f errors, resulting from normal metaphase II oocytes. Right panel shows the expected outcom e o f second m eiotic division from a metaphase II oocyte w ith extra chromatid resulting from meiosis I error. O f the resulting normal oocytes 32.5% have a single chromatid, including all the types o f sequential errors resulting in a balanced oocyte

169

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Nullisomies Complex abnormality (18.1%)

Disomies

(38.0%)

(39.9%)

(2 8 8 % )

(53 1% )

( 22 . 1% )

Figure 4.33 Pie chart dem onstrating the distribution o f the types o f chrom osom al abnorm alities observed in first polar body (P B I) (a) and second polar body (PB2) (b). Nullisomies (missing chrom osom es o r chrom atids) in PBI (a) w e re m ore prevalent com pared to o th e r types o f abnormalities, w h ile the re was no such difference in PB2 (b). C om plex erro rs are comparable in both PB I and PB2

(17.5%)

Figure 4 .3 4

Pie chart dem onstrating the distribution o f types erro rs in first polar body (PB I ). Missing chrom atids (47.4% ) w e re far

m ore prevalent, follow ed by com plex abnorm alities (28.8% ) involving m ore than one chrom osom e, com pared to o th e r types o f errors. Chrom osom al non-disjunction (yellow and green) was almost tenfold less frequ en t than chrom atid malsegregation (blue and orange)

170

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

(19.5%)

(21.5%)

(59.0%)

Two chromosomes Same chromosomes in each polar body

Complex errors

> Two chromosomes

Figure 4 .3 5

Pie chart showing pro po rtions and types o f com plex aneuploidies. (a) O verall prevalence o f com plex errors, involving

eith er e rro rs o f the same chrom osom e in both m eiotic divisions o r involvem ent o f tw o o r m ore chromosomes; (b) relative distribution o f different types o f com plex erro rs

Chromosome 21

Chromosome 13

(1 8 .1 *

(3 6 .5 *

Chromosome 16

Meiosis I errors Meiosis II errors Both meiosis I and meiosis II errors Chromosome 18

(1 8 4 *

< 36.7 *

Chromosome 22

(1 6 6 *

< 50.5 *

Figure 4 .3 6

Pie charts o f m eiotic origin o f chrom osom e 13, 16, 18, 2 1 and 22 aneuploidies detected by first polar body (PB I ) and

second polar body (PB2) and fluorescence in situ hybridization (FISH) analysis. Meiosis I erro rs shown in aqua, meiosis II (pink) o r both m eiotic divisions (orange). C hrom osom e 16 and 22 erro rs predom inantly originate fro m meiosis II (bo tto m left and bo tto m right); chrom osom e 18 e rro r predom inantly originates from meiosis I, and chrom osom e I 3 and 2 1 erro rs com parably in meiosis I and meiosis II

I7 I

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 4.37

Sequential chromosome 21 errors in meiosis I and II resulting in a chromosomally normal embryo, (a) Fluorescence in

situ hybridization (FISH) image o f first polar body (PB I) and second polar body (PB2) after hybridization w ith the MultiVysion PB panel

probe, showing a normal number o f signals (tw o per chromosome) in PBI w ith the exception o f three signals fo r chromosome 21 ^green arrows) indicating chromatid e rro r in meiosis I. The same four o f five chromosomes show a normal number o f signals present in PB2, again w ith the exception o f the chromosome 21 signal, which was missing, indicating a possible normal chromosome complement, including chromosome 2 1. (b), (c) and (d) Follow-up FISH analysis o f the resulting embryo, confirming a normal pattern o f chromosomes in all three nuclei including chromosome 21 (green arrows), obtained after w hole em bryo fixation

172

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

•

*

■

/ tt PB1

PB2

Figure 4 .3 8 Sequential chrom osom e 21 erro rs in meiosis I and II resulting in an abnormal mosaic em bryo. First polar body (P B I) and second polar body (PB2) w e re rem oved simultaneously follow ing fertilization o f an oocyte retrieved from a 39-year-old wom an, (a) Fluorescence in situ hybridization (FISH) image o f PBI and PB2 after hybridization w ith the MultiVysion PB panel probe, showing a normal num ber o f signals fo r each chrom osom e (double dots) in PB I w ith the exception o f three signals fo r chrom osom e 2 1 (w hite arrows). Four instead o f five signals are detected in PB2 (shown in the right lo w e r corner), w ith a missing green signal fo r chrom osom e 21, suggesting the normal num ber fo r all five chrom osom es in the resulting oocyte, (b) FISH image o f an interphase nucleus from the resulting em bryo w ith norm al num ber o f signals, including chrom osom e 21 (w hite arrows), (c) FISH image o f a second interphase nucleus from the same em bryo as (a) and (b), in w hich three signals fo r chrom osom e 22 (yellow arrow s) and only one signal fo r chrom osom e 13 (red a rro w ) are present, tog ether w ith tw o signals fo r chrom osom es 16,18 and 21. (d) FISH image of a th ird nucleus from the same em bryo as (a), (b) and (c) in w hich three signals fo r chrom osom e I 3 (red arrows) and chrom osom e 2 1 (w h ite arrow s) are present w ith a normal num ber o f signals fo r chrom osom es 16, 18 and 22. The observed mosaicism may be associated w ith the sequential erro rs o f chrom osom e 2 I in meiosis I and meiosis II

173

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

/

\

PB1

c Figure 4 .3 9

PB2

d C om plex erro rs involving m ore than one chrom osom e resulting in mosaicism. First polar body (PB I) and second polar

body (PB2) w e re rem oved simultaneously follow ing fertilization o f an oocyte retrieved fro m a 36-year-old woman, (a) Fluorescence in situ hybridization (FISH) image of PBI and PB2 after hybridization w ith the MultiVysion PB panel probe, revealing an abnormal

num ber o f signals (three) in PB I fo r chrom osom e 2 1 (green arrow s) and chrom osom e 22 (yellow arrow s), w ith a normal pattern o f signals (tw o ) fo r each o f the remaining chrom osom es tested, 13, 16 and 18. A norm al num ber o f signals are present fo r fo u r o f the five chrom osom es tested in PB2 (lo w e r right corner), w ith evident absence o f a gold signal fo r chrom osom e 22. (b) FISH image o f an interphase nucleus from the resulting em bryo, showing only one signal present fo r chrom osom e 2 1 (green a rro w ) as predicted by the num ber o f chrom atids extruded during meiosis. T here are a norm al num ber o f signals fo r chrom osom e 22 also expected, tog ether w ith an unexpected three signals fo r chrom osom e 16 (aqua arrows), (c) FISH image o f a second interphase nucleus from the same em bryo as shown in (a) and (b). "two signals fo r each chrom osom e tested including chrom osom e 2 1 (green arrows) are present w ith the exception o f chrom osom e 16 (aqua arrow s), represented by only one signal, (d) FISH image o f a th ird interphase nucleus from this em bryo, in which tw o signals fo r each o f the chrom osom es tested are present including chrom osom e 2 1, but three instead o f expected tw o signals are present fo r chrom osom e 16, indicating mosaicism in the em bryo

174

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

•

I

%

♦

m

\

«•

#

/ —

•

I1 • PB1 a

\

*

«

t

/

,

PB2 b

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Figure 4.40 Com plex errors in meiosis I resulting in monosomy 21 and trisom y 22. (a) Fluorescence in situ hybridization (FISH) image o f first polar body (PBI) and second polar body (PB2) after hybridization w ith the MultiVysion PB panel probe, revealing a double e rro r in PBI, represented by three (instead o f tw o ) chromosome 21 signals (w hite arrows) and only one split chromosome 22 signal (yellow arrow). A normal number o f signals (one each) are present in PB2 (right lo w er corner), (b) and (c) FISH images of nuclei obtained from the resulting embryo, showing three chromosome 22 signals and one chromosome 21 signal, confirming the predicted double aneuploidy. (N o te that chromosome 16 is not seen due to strong aqua background from the nuclear staining as a result of fluorophore labeled aqua human placental D N A incorporated into this probe cocktail)

175

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

•

%

PB1

PB2

A

F ig u re 4.41

C om plex erro rs in meiosis I and II resulting in a mosaic em bryo. First polar body (P B I) and second polar body (PB2)

w e re rem oved simultaneously follow ing fertilization o f an oocyte retrieved fro m a 35-year-old w om an suffering from secondary in fe rtility w ith a reproductive history that included fo u r previous spontaneous abortions, (a) Fluorescence in situ hybridization (FISH) image o f PBI and PB2 follow ing hybridization w ith the MultiVysion PB panel probe, showing only one signal (instead o f tw o ) fo r chrom osom es 18,21 and 22 in PBI. Additional e rro rs are seen in PB2, evident from tw o signals present fo r chrom osom es 18 (red arrow s) and 22 (yellow arrow s) and the absence o f a signal fo r chrom osom e 16. (b) FISH image o f an interphase nucleus from the resulting em bryo, showing only one chrom osom e 2 1 signal (green a rro w ) and three signals fo r chrom osom e I 3 (three red arrows) and 16 (not indicated by arrow s), (c) FISH image o f a second interphase nucleus from the same em bryo, corresponding to (a) and (b), showing tw o signals fo r each o f the chrom osom es tested including chrom osom e 18 (purple arrow s), w ith the exception o f chrom osom e 16 (w h ite arrow s) in w hich three signals are seen, (d) FISH image o f a th ird interphase nucleus fro m the same em bryo, in w hich tw o signals fo r each o f the chrom osom es tested are present w ith the exception o f three signals fo r chrom osom e 16 and 22 (gold arrow s), revealing extensive mosaicism in the em bryo, which may be caused by com plex erro rs in meiosis I

176

PREIMPLANTATION DIAGNOSIS FOR ANEUPLOIDIES

F ig u re 4.4 2 C om plex e rro rs in meiosis II resulting in double trisom ies I 3 and 18. (a) Fluorescence in situ hybridization (FISH) image o f first polar body (PB I ) and second polar body (PB2) follow ing hybridization w ith the MultiVysion PB panel probe, showing a normal num ber o f signals (tw o ) fo r each o f the chrom osom es tested in P B I. H o w e ve r double e rro rs in meiosis II are evident from missing signals fo r chrom osom es 13 and 18 in PB2. (b) F ollow -up FISH analysis o f the em bryo by w h ole em bryo fixation confirm s the predicted double triso m y I 3 and I 8, evident from the presence o f three signals fo r chrom osom e I 3 (w h ite arrow s) and chrom osom e 18 (purple arrow s) in tw o o f the three cells shown (upper left and lo w e r m iddle nuclei). However, three chrom osom e I 3 signals w ith a norm al num ber o f signals (tw o ) fo r the o th e r chrom osom es, including chrom osom e 18, are present suggesting mosaicism fo r chrom osom e 18 (upper right)

177

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

F igu re 5.1 Schematic represention o f alternate chromosome segregation during meiosis I and II in an oocyte from a carrier o f a reciprocal translocation. Following the vertical path, an alternate segregation pattern in which both derivative chromosomes are extruded during meiosis I is depicted. A fte r fertilization and, w ith the exception o f anaphase II non-disjunction, the resulting maternal contribution w ould be that o f a normal chromosome complement. Depicted along the horizontal path, is an alternate segregation pattern in which both normal chromosomes are extruded during meiosis I. Following fertilization and barring anaphase II nondisjunction, the resulting maternal contribution o f both derivative chromosomes would result in an em bryo w ith a balanced chromosome complement. Both outcomes are suitable fo r em bryo transfer, one being both genotypically and phenotypically normal, w hile the oth er balanced outcom e results in a phenotypically normal conceptus, however, w ith the same reproductive risk of producing unbalanced gametes in the future as a carrier. PB I (first polar body); PB2 (second polar body)

179

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 5.2 Schematic represention of adjacent chromosome segregation during meiosis I and II in an oocyte from a carrier o f a reciprocal translocation. Following the horizontal path, an adjacent-1 segregation pattern is depicted in which a normal chromosome and the other derivative chromosome are extruded during meiosis I resulting in an unbalanced oocyte consisting o f excess genetic material fo r one chromosome and a deficiency fo r the other. A fte r fertilization, regardless o f w hat is extruded during meiosis II, the resulting em bryo carries an unbalanced chromosome complement and is unsuitable fo r em bryo transfer. Following the vertical path, an adjacent-2 segregation pattern is depicted in which both a normal chromosome and its derivative are extruded during meiosis I. This results in an unbalanced oocyte consisting o f an excess o f genetic material from one chromosome w ith a deficiency o f the other. Once again, following fertilization, regardless of w hat is extruded during meiosis II; the resulting em bryo carries an unbalanced chromosome complement and is unsuitable fo r transfer. Depending on the chromosomes involved both adjacent-1 and adjacent-2 segregation can result in a viable unbalanced conceptus. PBI, (first polar body); PB2, second polar body

180

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

Figure 5.3 Schematic represention o f chromosome segregation after chromatid exchange (CE) (recombination) has occurred involving one chromosome and its derivative (CE o f I), during m eiotic prophase I in an oocyte from a carrier of a reciprocal translocation. This exchange results in homologous chromosomes in which the sister chromatids consist o f both normal and derivative chromatids. This is identified in the first polar body (PB I ) through fluorescence in situ hybridization (FISH) analysis using a combination o f probes to include w hole chromosome paints, centrom eric enumeration and sub-telomeric probes. Second polar body (PB2) analysis is absolutely required in ord er accurately to predict the chromosome complement o f the oocyte and subsequent em bryo follow ing fertilization. Vertically depicted is CE o f I, after which, following meiosis I and II, a balanced chromosome com plement is present. Following the horizontal path is a depiction o f CE o f I , in which after chromosome segregation during meiosis I and II, an unbalanced chromosome com plement is realized and following the diagonal path, after CE o f I and segregation during meiosis I and II, a normal chromosome complement is present. Obviously this occurrence can only be identified through sequential PB analysis since blastomere analysis shows only the final outcome and may infer a totally different segregation pattern (i.e. alternate segregation when the resulting chromosome complement consists o f either normal o r both derivative chromosomes)

181

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

9 j m f b —ffira© U nbalanced C hrom osom e C om plem ent

Normal Chromosome Complement Ralanced Chromosome Complement Figure 5.4 Schematic represention of chromosome segregation after chrom atid exchange (CE) (recombination) has occurred involving both chromosomes and the ir derivatives (CE o f both), during m eiotic prophase I in an oocyte from a carrier o f a reciprocal translocation. This exchange results in all fou r chromosomes in which sister chromatids consist o f a normal and derivative chromatid. As w ith CE o f I , this is identified in first polar body (PB I ) through fluorescence in situ hybridization (FISH) analysis using a combination o f probes to include whole chromosome paints, centrom eric enumeration and sub-telomeric probes. Second polar body (PB2) analysis is absolutely required in ord er to predict accurately the chromosome complement o f the oocyte and subsequent embryo following fertilization. Vertically depicted is CE of both, after which, following meiosis I and II, a balanced chromosome complement is present. Following the horizontal path is a depiction o f CE o f both, in which after chromosome segregation during meiosis I and II, an unbalanced chrom osom e complement is realized and following the diagonal path, after CE o f both and segregation during meiosis I and II, a normal chromosome com plement is present. Once again, this occurrence can only be identified through sequential PB analysis

182

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

U nbalanced C hrom osom e C om plem ent

me Complement Figure 5.5 Schematic representing o f 3 :1 and 4:0 chromosome segregation during meiosis I and II fo r a maternal carrier o f a reciprocal translocation. Following the vertical path, depicted is a 4:0 segregation pattern in which both normal and derivative chromosomes are extruded from o r retained in the oocyte during meiosis I resulting in a m ore lethal chromosome imbalance. Following fertilization and w ith the exception o f anaphase II non-disjunction, the resulting unbalanced em bryo w ould consist o f a double trisom y o r double monosomy. Following the horizontal path, 3:1 segregation is depicted in which only one normal o r derivative chromosome is retained in the oocyte o r extruded in first polar body (PB I ) resulting in an unbalanced oocyte. Following fertilization the chromosome com plement o f the em bryo could consist o f a tertiary trisom y/m onosom y o r an interchange trisom y/m onosom y as depicted in this example o f interchange trisom y in which extra genetic material from both chromosomes is present. N either outcom e o f these types o f segregation patterns is suitable fo r transfer. Depending on the chromosomes involved, a 3 :1 segregation pattern may result in a viable unbalanced conceptus

183

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Figure 5.6 Schematic represention o f complex chromosome segregation during meiosis I and II and an alternate chromosome segregation during meiosis I w ith anaphase II non-disjunction fo r a maternal carrier of a reciprocal translocation. Following the vertical path, complex segregation is depicted in which chrom atid exchange and chromatid non-disjunction during meiosis I is identified resulting in an unbalanced oocyte. As a result, a textbo ok segregation pattern cannot be identified. Second polar body (PB2) analysis is required accurately to predict the chromosome complement o f the oocyte and resulting embryo, which if studied by blastomere analysis alone, in this example, may appear as if adjacent-1 segregation had occurred. M ore often this type o f abnormal segregation leads to an unbalanced oocyte and would not be considered fo r em bryo transfer. Following the horizontal path, alternate segregation is depicted in which both normal chromosomes have been extruded during meiosis I; however, by analysis o f PB2, anaphase II nondisjunction has been identified in which extra chromosome material has been retained in the oocyte resulting in an unbalanced oocyte. This illustrates the necessity fo r analysis o f both first polar body (PB I ) and PB2 when perform ing PGD fo r maternal translocation carriers

184

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

\ PB1

PB2

♦* ™

PB1

PB1 d

e

PB2 f

Figure 5.7 PGD fo r a maternally derived reciprocal translocation, (4 6 ,X X ,t(l 2; I8 )(p l 3 .3 1;q2l .32)) by polar body (PB) analysis, (a) Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes o f a peripheral blood lymphocyte from the carrier. Chromosome 12 is identified w ith whole chromosome paint (WCP) in green in conjunction w ith the sub-telomeric (Tel) probe 12p in green fo r easy visualization o f the small-translocated portion o f chromosome 12 located on derivative 18. Chrom osom e 18 is identified w ith W CP in orange, which appears as red due to visualization through a red single-bandpass filter, in conjunction w ith the centrom eric-enum eration probe (CEP) 18 in aqua and a Tel I8q in orange, added to the probe cocktail fo r easy visualization of the terminal segment o f chromosome 18 located on derivative 12. Derivative chromosomes are identified by a combination o f both red and green fluorescence (w hite arrows). Derivative 18 is not only distinguished from derivative 12 by size, but also by the presence o f the CEP 18 in aqua, (b) Clumped metaphase chromosomes o f a first polar body (PBI) removed on the day o f oocyte recovery. Two chromatids fo r chromosome 18 are present evident by tw o CEP aqua signals (white arrows). Two chromatids fo r chromosome 12 are present identified by the tw o areas of W CP in green. Further testing through re-hybridization w ith sub-telomeric probes would be required to distinguish derivative chromatids from normal chromatids because the translocated segments are small and difficult to see when chromosomes are poorly spread, (c) Additionally, investigation o f the second polar body (PB2)by FISH w ith CEP 18 (aqua), Tel I2p (green) and Tel I8q (orange) is essential fo r accurate prediction o f the chromosome status o f the oocyte. An unbalanced oocyte is easily recognized by the presence o f an additional CEP 18 signal (w hite arrows) indicating that anaphase II nondisjunction had occurred, which resulted in the extrusion o f both normal and derivative chromatids fo r chromosome 18 so that after fertilization the resulting em bryo is monosomic fo r chromosome 18. (d) A nother PB I removed from an oocyte from the same carrier (a). Individual normal and derivative (der) chromatids are seen by visualization o f the sub-telomeric signals (yellow and w hite arrows), (e) Re-hybridization using only CEP 18 and sub-telomeric probes confirms the findings from the first round o f hybridization which was shown in (d); there is one chromatid present fo r each o f the chromosomes o f interest ( 12,der( 12), 18,der( 18)). Norm al chromatid 12 is identified by a single Tel 12p signal in green, der( 12) is identified by a single Tel 18q signal in orange ^yellow arrow), normal chromatid 18 is identified by a single aqua CEP 18 signal and a single orange Tel 18q signal and der( 18) is identified by a single signal fo r both CEP 18 and Tel I2p (white arrow). Based on these findings, the PB2 must be studied in ord er to rule out anaphase II non-disjunction and to predict accurately the chromosome complement of the oocyte, (e) FISH of the PB2 nucleus. Three signals (expected normal number o f signals representing 12, 18 o r der( 12), der( 18)) are present in PB2 suggesting a normal o r balanced chromosome complement in the oocyte

185

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

F igu re 5.8 (opposite) PGD fo r a maternally derived reciprocal translocation (4 6 ,X X ,t(l ;8)(q42;pl 1.2)) by polar body analysis, (a) Fluorescence in situ hybridization (FISH) analysis o f metaphase chromosomes o f a peripheral blood lymphocyte from the carrier. Chrom osom e I is identified w ith whole-chrom osom e paint (WCP) in green. Chrom osom e 8 is identified w ith W CP in orange, which appears as red due to visualization through a red single-bandpass filter, in conjunction w ith a centrom eric enumeration probe (CEP) 8 in aqua. Derivative chromosomes are identified by a combination o f both red and green fluorescence (white arrows). Since the translocated segments are small, the addition o f Tel Iq (orange) and le i 8p (green) w ere added to the initial probe cocktail to ensure identification o f chromatid exchange (CE). (b) Phase-contrast image o f metaphase chromosomes from first polar body (PB I), (c) FISH image o f PBI shown in (b) in which CE o f both is observed showing tw o CEP signals fo r chromosome 8 indicating tw o chromatids are present; however, only one chromatid carries the signal fo r Tel 8p (green arrow ) indicating that the oth er chromatid contains the translocated segment from chromosome I . Chromosome I is identified by W CP in green; however, only a single signal fo r the Tel I q probe (red arrow ) is seen indicating that the sister chrom atid carries the translocated segment fo r chromosome 8. (d) FISH image o f second polar body (PB2) after hybridization w ith Tel Iq (orange;, Tel 8p (green) and CEP 8 (aqua). A single signal fo r CEP 8 and tw o signals fo r Tel I q (red arrows) are present indicating that the normal chromosome I and the der(8) are present. Based on PB2 findings, it was predicted that this oocyte carried an unbalanced chromosome com plement by the presence o f d e r(l) and the developing em bryo was om itted from em bryo transfer, (e) Follow-up FISH analysis o f metaphase chromosomes obtained after em bryo biopsy and blastomere nucleus conversion o f the corresponding unbalanced em bryo identified by PB analysis (c) and (d). PGD by polar body analysis is confirmed by the presence o f der( I ) (w hite arrow )

186

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

b

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

% #

Jfc PB1

* / t

%

PBI

PB2

9

4 ft

F ig u re 5.9

PGD fo r a m aternally derived reciprocal translocation (46,X X . t ( I ; 15)(q32;q26)) by polar body analysis, (a) Fluorescence

in situ hybridization (FISH) analysis o f metaphase chrom osom es o f a peripheral blood lym phocyte from the carrier. C hrom osom e I

is identified w ith w hole-chrom osom e paint (W CP) in green in conjunction w ith a centrom eric-enum eration probe (CEP) I in aqua. C hrom osom e I 5 is identified w ith W C P in orange (visualization through a red single-band pass filter), in conjunction w ith Tel I 5q in orange since the translocated segment o f chrom osom e 15 on to chrom osom e I is small. D erivative chrom osom es are identified by a com bination o f both red and green fluorescence (w h ite arrows), (b) FISH image of metaphase chrom osom es fro m first polar body (PB I ) in w hich chrom atid exchange (CE) o f both is observed. C hrom osom e I is identified by W C P in green showing tw o CEP signals in aqua indicating both chrom atids are present; however, only one chrom atid carries the signal fo r Tel I 5q (derivative chrom atid, w h ite a rro w ) w hile the o th e r chrom atid does not (norm al chrom atid, green arro w ). C hrom osom e 15 (orange) is visualized in red w ith one chrom atid seen as only red (norm al chrom atid, red a rro w ) and one chrom atid seen as red and green (der( 15), w h ite arro w ), (c) Re-hybridization o f PBI metaphase chrom osom es shown in (b) w ith CEP I (aqua), Tel Iq (orange) and CEP 15 (green). C e n tro m e ric probes confirm the num ber o f chrom atids present w h ile Tel I q confirm s the presence o f one norm al and one derivative chrom atid fo r each o f the chrom osom es o f interest. H ybridization o f second polar body (PB2) w ith the same probe cocktail is seen (lo w e r right) showing a balanced / normal num ber o f signals, indicating that the oocyte carries eith er a normal o r balanced chrom osom e com plem ent, (d), (e) and (f) Em bryo follow -u p analysis by em bryo biopsy and blastom ere nucleus conversion to metaphase chrom osom es confirm s the prediction by PB analysis showing a normal chrom osom e com plem ent. N orm al chrom osom es I (green arrow s) and chrom osom es 15 (red arrow s) are present

188

PREIMPLANTATION DIAGNOSIS FOR TRANSLOCATIONS

fpr * s. 0

i:

%

r

F ig u re 5.10

\

>

PGD fo r paternally derived reciprocal translocation (46,XY, t( I 3;20)(q22;p I 1.2)) by em bryo biopsy and blastomere

nucleus conversion to obtain metaphase chromosomes, (a) Phase-contrast image o f metaphase chrom osom es obtained a fter fixation and pre tre atm en t o f the hum an-m ouse heterokaryon (see C hapter 3). (b) Fluorescence in situ hybridization (FISH) image o f the same metaphase chrom osom es as shown in (a) showing a normal chrom osom e com plem ent consisting o f tw o chrom osom es I 3 in green (green arrow s) and tw o chrom osom es 20 in orange (red arrow s) indicating the corresponding em bryo is suitable fo r transfer, (c) Phase-contrast image o f metaphase chrom osom es from a second em bryo fro m the same couple obtained after fixation and p re tre atm en t o f the hum an-m ouse heterokaryon. (d) FISH image o f the metaphase chrom osom es seen in (c), showing an unbalanced chrom osom e com plem ent consisting of tw o chrom osom es I 3 in green (green arrow s) and one chrom osom e 20 in orange (red arro w ) and one der(20) seen in red and green (w h ite a rro w ) indicating the corresponding em bryo is not suitable fo r transfer

189

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

6

A \

>
T

*

b

Normal allele Mutant allele 3 ' end of 4R prim er Seal restriction site

#

-St. . GTGAGTATTTAATA.... 5'. . . GTGTGTATTTAATA.... 3' CATGAATTATA.... AGT'ACT

3' 3' 5'

Seal

131

Mutant

Seal

23

108

Normal

R estriction digestion

c

M u ta n t 131 bp N o rm a l 108 bp E m b ry o

L

12

3 4 5 0 7

8 9 10 11 12 13 14

ET

Figure 6 .4 1 PGD fo r IV S 4+4 A - T m utation in Fanconi’s anemia co m ple m en t C (FAC) gene, (a) Map o f human FAC gene, showing sites and location o f IV S 4+4 A - T m utation and restrictio n fragm ent length polym orphism , (b) P rim er design and restriction map fo r norm al and abnorm al alleles, (c) Polyacrylamide gel analysis o f Scat restrictio n digestion, show ing one hom ozygous affected (em bryo 6), th re e homozygous norm al (em bryos 7, 9 and 10) and th e rem aining eight heterozygous unaffected em bryos (em bryos 8 and I ! did n o t amplify). O ne (em bryo 3) o f the la tte r unaffected heterozygous em bryos was also an H LA match fo r a sibling w ith Fanconi anemia (see Figure 6.42) and transferred back to th e patient, resulting in an unaffected pregnancy. ET, em bryo transfer

252

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

»g.VVA).

(GAAAI. I

QAAL, Asp 3

C e n tl

H L A -B j

P M

■■

B

mm

Allele-specific prim ers

A sp 5

Allele-specific primers

L P M B 2

3 4 5 6 7 9 10 12 13 14

F igu re 6.42 HLA detection system by allele-specific primers. (Top) Schematic representation o f HLA genes studied. (Bottom ) (a) Results o f HLA typing o f single blastomeres from I I embryos demonstrating that only one o f the unaffected embryos (embryo 3) corresponds to the HLA type o f the affected sibling (S). (b) Polyacrylamide gel analysis of chorionic villus sampling (CVS) confirming the results o f preimplantation HLA typing. ET, em bryo transfer; L, size standard; colored arrows indicate specific prim er positions; R paternal D N A; M, maternal D N A

253

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

a

b

Maternal dominant mutation and aneuploidy

e

Paternal dominant mutation Recessive disorder and aneuploidy and aneuploidy

d

Mutation and HLA and aneuploidy

1

HLA and aneuploidy

or

1 F igu re 6.43 Strategies fo r combined mutation, aneuploidy and HLA testing, (a) First and second polar body (PBI and PB2) testing fo r a maternally derived dominant mutation was perform ed fo r advanced maternal age. Embryos predicted to be free from maternal mutation are furthe r tested fo r aneuploidies using fluorescent in situ hybridization (FISH) analysis, while those w ith inconclusive results fo r the mutation are subjected to m ultiplex nested o r heminested PCR, to test simultaneously fo r the mutation site, linked to its polym orphic markers and STRs on chromosomes 13, 16, 18, 2 1, 22, X and Y fo r aneuploidies. (b) In case o f a paternally derived dominant mutation and advanced maternal age, PB I and PB2 testing fo r aneuploidy is perform ed first, followed by blastomere analysis o f aneuploidy-free embryos fo r paternal mutations and polymorphic markers, (c) In case o f an autosomal recessive disorder and advanced reproductive age, PB I and PB2 aneuploidy testing is done first, follow ed by blastomere analysis o f aneuploidy-free embryos fo r mutations and polymorphic markers. Maternal mutation is also assessed by PB I and PB2 analysis once the parents are known to have different mutations, so that the maternal m utation-free embryos can be furthe r tested fo r the paternal mutation together w ith the linked markers. As above, all embryos deriving from oocytes w ith inconclusive results by PB I and PB2 analysis are subjected to m ultiplex nested o r heminested PCR fo r simultaneous study o f the mutation site, linked to its polym orphic markers and STRs on chromosomes 13, 16, 18, 2 1, 22, X and Y fo r aneuploidies. (d) The strategy is similar to the one applied fo r recessive disorders and aneuploidy, except HLA typing is perform ed on all blastomeres in combination w ith mutation analysis o r aneuploidy STR typing for some embryos, (e) The same approach as in paternal dominant mutations and aneuploidy testing. HLA typing is perform ed for embryos normal by FISH results. HLA typing combined w ith PCR aneuploidy testing is done fo r inconclusive embryos

254

P R E IM P L A N T A T IO N D IA G N O S IS F O R S IN G L E -G E N E D IS O R D E R S

AA

«h

M e io s is I

Meiosis I

PB1

Meiosis II

Meiosis

( MaUlird ooo Ir

Oocy te with disomy 6

^

I

«

PB2

B io p s y

B io p s y

Mo n os om y 6

M on os om y 6

I M a tern al non-m atch

PB2

Dissociated blastomeres

)fT )

f \

f

.Maternal match T R IS O M Y 6 Paternal match

l x

i

i

F igu re 6.44 The value o f chrom osom e 6 aneuploidy testing fo r accuracy o f preimplantation HLA typing, (a) Matched maternal chromosome 6 is shown in pink and non-matched in blue. Maternal disomy 6 was predicted based on the heterozygous first polar body (P B I) and ‘em p ty’ (absence o f D N A material) second polar body (PB2). Blastomere biopsy confirms the presence o f tw o maternal and one paternal set o f markers, evidencing trisom y 6 in the resulting embryo, (b) Sequential PBI and PB2 analysis, suggesting maternal chromosome 6 matching, while the lack o f the paternal markers in the blastomere indicates a potential monosomy 6. The follow -up analysis o f the dissociated single blastomeres from this em bryo revealed monosomy 6 in one o f the blastomeres w ith the presence o f the paternal matching chromosome 6. The rest o f the blastomeres had both maternal and paternal matching chromosomes 6. (c) Monosomy 6 o f maternal origin is inferred from the heterozygous PB I and PB2, in agreement w ith the finding o f only a paternal matching chromosome in the blastomere biopsy. This is furthe r evidenced by the follow -up study o f the dissociated blastomeres o f this embryo, showing monosomy 6 in all the cells

255

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Class III

Class II DR DQ DP DQ C A k

CYP21A1F

1.1

1. 2

Class I HLA-B C A F

CYP21A2

9N 2

TNF A

1. 4

1. 3

1 1

1

171 Q 149 1 189 S 266 I Q318X 126 99 \ 134 \ 117

A ff e c t e d

169 166 183 274 N 130 93 143 117

N o rm a l

2.1

2. 2

PG[ 151 166 195 268 N 136 91 128 119

M IC A

£

M IB

1. 5

151 166 195 268 N 136 91 128 119

N o rm a l

2. 3

PGD

157 151 185 278 Nt 656 132 93 132 123

DQ CAR LH DN D6S273 CYP 9N2 TNF A MIC A MIB

A ffe c te d

2. 1

PGD

171 149 189 266 Q318X 126 99 134 117 C a r r ie r

F igu re 6.45 PGD fo r congenital adrenal hyperplasia (CAH). (Top) Location of the CYP2IA2 gene, C Y P 2 IA IP pseudogene and tightly linked polym orphic markers on chromosome 6 p 2 1.3. (Bottom ) Pedigree o f a family, undergoing PGD fo r CAH: husband (1.4) and w ife (1.5) are carriers o f tw o different mutations in the CYP2IA2 gene (the maternal nephew (2 .1) is affected w ith CAH). As per the strategy o f the mutation detection described on Figure 6.7, the paternal haplotype was established by multiplex single-sperm analysis, showing seven informative polymorphic markers wrapping the mutation site, w ith the maternal haplotype detected being based on sequential analysis o f the first and second polar bodies (PB I and PB2). The m other was informative fo r eight markers. High similarity between the gene and pseudogene makes the application o f polym orphic markers extrem ely im portant fo r testing accuracy. Following tw o PGD cycles, a healthy boy (2.2) was born after the first, and an ongoing tw in pregnancy (2.3 and 2.4) has resulted after the second

256

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

F ig u re 6.4 6

Family pedigree o f a couple undergoing PGD fo r sonic hedgehog (SHH) m utation. (Top) The father has a gonadal

mosaicism fo r the SHH m utation, w hich is linked to a 156-bp dinucleotide C A repeat allele o f D7S550 polym orphic marker, w hile the m o th e r is normal, w ith one normal allele linked to a 158-bp repeat and the o th e r to a I 38-bp repeat allele. (B ottom ) Reproductive outcom es o f this couple, including three previous pregnancies, one resulting in the birth o f an affected child w ith holoprosencephaly, carrying the m utant gene (lo w e r left), one in perinatal death, also carrying the m utant gene (lo w e r cen ter (circle)), and one in a spontaneously aborted fetus w ith Turner syndrom e, free fro m SHH m utation (lo w e r center (triangle)). The lo w e r right (PGD) shows th e outcom e o f preim plantation diagnosis, resulting in an unaffected clinical pregnancy and the birth o f a healthy child, follow ing confirm ation o f the m utation-free status by amniocentesis

257

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

M a rk e r ord e r Kg 8

O 1.1

A C 2.5 N ik Exon 45 D I6S664

127

SM 7

131

I I Oft

96 I3S

I)

106 193

o h 1.3

96

|| I

133 I

96

I

135

' I I 10«

112'

142 I

142

I

2.4

2.3

«. I I

96

135 1 112

133 | I 142

90 137 |

96 135 I

108 129

112

142

F igu re 6.47 Preimplantation linked marker analysis fo r polycystic kidney disease (PKD)-I resulting in the birth of unaffected twins. (Top) A DPKD mutation (mutant chromosome 16 is shown in red) originating from the affected maternal father (1.2), from whom the patient (2.2) inherited a possibly mutant chromosome 16 (red); m utation-free chromosomes o f maternal m other (1.3) are shown in green. (Middle) The affected patient (2.2) had one affected bro th er (2.3), w ho also inherited the mutant chromosome from his father, and the same normal chromosome 16 from his mother. H er unaffected husband’s (2 .1) normal chromosomes 16 differ by one of the markers ( I 3 I bp vs. 133 bp), which makes it possible to identify twins, resulting from PGD (3.1 and 3.2) (Bottom )

258

PREIMPLANTATION DIAGNOSIS FOR SINGLE-GENE DISORDERS

M a r k e r o rd e r 1.

D4S2922

2.

D4S1538

3.

D4S2361

4.

D4S1534

5.

D4S2929

6.

D4S2458

P K D -2

7.

D4S423

8.

D4SI557

1.2

1. 1

123 135 109 122 129 91 133 100

F ig u re 6.4 8

O 129 133 106 113 119 97 129 105

1.3

123 135 109

131 135 10*

122 129

III 117 tl 131 (•7

91 133 100

o

129

135 109 122 129 93 129 107

121 135 109

122 129 93 129 105

Preim plantation linked m arker analysis fo r polycystic kidney disease (PKD)-2 resulting in the birth o f unaffected tw ins

(same pedigree). (Top) An A D P K D m utation (m utant chrom osom e 4 is shown in green) originating from the affected maternal father (1.2), from w h om the patient (2.2) inherited a possibly m utant chrom osom e 4 (green); m utation-free chrom osom es o f maternal m othe r (1 .3) are shown in red and black. (M iddle) The affected patient (2.2) had one affected b ro th e r (2.3), w h o also inherited the m utant chrom osom e from his father, but follow ing recom bination between D4S2929 and D4S24S8, w hich resulted in a recom binant chrom osom e 4 (blue and green), and the same norm al chrom osom e 4 fro m the mother. H e r unaffected husband’s (2.1) normal chrom osom es 4 cannot be distinguished due to sharing the same m arkers, so the fact that the resulting PGD tw ins are dizygotic cannot be dem onstrated (3.1 and 3.2) (B ottom )

259

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

R260 AlwNI c

a

Otc3

P

Exon 1 .

_

Otc6

[ G >A

A >G

Otc2

K46R-4

Restriction maps A lw N I 18

117

Mutant Normal

+ sits - site

106

135

Polar body genotyping —

— — rs — — rs — r^ — CGCCCQCGCCCQCCCGCCQSCQCC

I L

I L

I I I C

l

— |i •

3 reps j±=

In t r o n 12

A

J. 2411 bp

-Site

r-

co

Cvj

■»—

R408W

CM

T-

C\J

T-

(M

COCOCQCQCQCCCO

Q - Q - C L C L Q - Q - C L C L

i 1

240 bp

3 reps

r-

CN CQ CL

cd Q_

^

100 Q.

CM CQ CL

Site

tO M t- C \ J * - C \ I t- C M C O C O C O C O C Q O Q C O C O Q . Q . Q . Q . Q . Q . Q . H

L

3 reps

R 408W

* HI 1

- C M CD CD Q _ Q _

t

N orm al

ADO

R408W

Mutant

(Styl) Intron 12

Mutant

k.

k. W

k

- site

RFLP

+ site

(Xmnl) Intron 8

+ site

VNTR

________ —

-

J-

~ ---- — — ---- ---- “

_ m m m . '

STR Intron 3 Oocyte

~

1

2

3

4

5

7

9

11

'— “ 12

13

__

\|)( |

6 re p e a ts 3 re p e a ts

240 hp 232 h p

15

Figure 6.50 PGD fo r R408W mutation in phenylalanine hydroxylase (PAH) gene in a couple w ith the male partner being homozygous affected. (Top) Schematic representation o f maternal (left) and paternal (right) haplotypes. The heterozygous m other has an R408W mutation linked to short tandem repeats (STR) in intron 3, a variable number o f tandem repeats (VNTR) close to the 3' end o f the gene (3 reps) and restriction fragment length polymorphism (RFLP) in intron 8 ( - site;. The homozygous affected father has tw o different mutations, one the same as the maternal (R408W) mutation, and the other (Y414C mutation), w ith its ow n linkage pattern. (Bottom ) Genotyping of oocytes by sequential analysis o f first (PB I ) and second (PB2) polar bodies fo r R408W mutation and informative linked markers (STR, VNTR and RFLP). All series include the PBI followed by the PB2 in the lane to its immediately to its right, corresponding to I I oocytes studied (oocytes are numbered at the bottom ). As the R408W mutation creates a restriction site fo r Styl enzyme, oocytes 2, 3, 4, 7, 9 and 15 w ere predicted to be normal based on heterozygous PB I and a homozygous mutant PB2. O ocyte 5 was also predicted to be normal, but this was based on a homozygous mutant PB I and a normal PB2 (which was in agreement w ith marker analysis), excluding the possibility fo r allele dropout (AD O ) in the corresponding PB I . A D O o f the mutant allele is evident from the identical genotype o f the PBI and PB2 in oocyte I I (confirmed by all three markers), suggesting affected status o f this oocyte. Three other oocytes w ere predicted to be affected based on a heterozygous PB I and a normal PB2 (oocytes I and 12), and a homozygous normal PBI and mutant PB2 (oocyte 13). A D O was also detected in intron 3 STR in oocytes 7 and 15 (identical genotype o f PB I and PB2). These data are not in conflict w ith the unaffected genotype o f the resulting embryos, which w ere transferred together w ith tw o other unaffected embryos (2 and 4). This resulted in tw in pregnancy and the birth of tw o healthy children, following confirm ation o f PGD by prenatal diagnosis

261

ATLAS OF PREIMPLANTATION GENETIC DIAGNOSIS

Marker order AI507 106 + : m

NI 98l - 1 1

Exon 10(A 15 07) Intron 17 Intron 18 Intron 20

2 .1

§ N 98

| 98B n

-

Oocyt©

1

AI5071 106 I +

A I507| 106

I

+

A15071 106 I

N Il 98

+ I

♦ I1

*11

1

2

4

7

8