Atlas of Human Assisted Reproductive Technologies [1 ed.] 8180619540, 9788180619540

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Atlas of

Human Assisted Reproductive Technologies

Atlas of

Human Assisted Reproductive Technologies Editor

Mangala Telang

MBBS DGO MD FACOG

Senior Fellow of American College of Obstetricians and Gynecologists President of Indian Fertility Society Director Fertility Research and IVF Centre New Delhi

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD. NEW DELHI

Published by Jitendar P Vij Jaypee Brothers Medical Publishers (P) Ltd EMCA House, 23/23B Ansari Road, Daryaganj New Delhi 110 002, India Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021, +91-11-23245672, +91-11-32558559 Fax: +91-11-23276490, +91-11-23245683 e-mail: [email protected] Visit our website: www.jaypeebrothers.com Branches • 2/B Akruti Society, Jodhpur Gam Road, Satellite Ahmedabad 380 015, Phone: +91-079-30988717, +91-079-26926233 • 202 Batavia Chambers, 8 Kumara Krupa Road, Kumara Park East Bangalore 560 001, Phones: +91-80-22285971, +91-80-22382956, +91-80-30614073 Tele Fax: +91-80-22281761 e-mail: [email protected] • 282 IIIrd Floor, Khaleel Shirazi Estate, Fountain Plaza, Pantheon Road Chennai 600 008, Phones: +91-44-28262665, +91-44-28269897 Fax: +91-44-28262331 e-mail: [email protected] • 4-2-1067/1-3, Ist Floor, Balaji Building, Ramkote, Cross Road Hyderabad 500 095, Phones: +91-40-55610020, +91-40-24758498 Fax: +91-40-24758499 e-mail: [email protected] • “KURUVI BUILDING”, 1st Floor, Plot/Door No. 41/3098-B &B1, St. Vincent Road Kochi 682 018, Ph: +91-0484-4036109, +91-0484-2395739, +91-0484-2395740 e-mail: [email protected] • 1A Indian Mirror Street, Wellington Square Kolkata 700 013, Phones: +91-33-22456075, +91-33-22451926 Fax: +91-33-22456075 e-mail: [email protected] • 106 Amit Industrial Estate, 61 Dr SS Rao Road, Near MGM Hospital, Parel Mumbai 400 012, Phones: +91-22-24124863, +91-22-24104532, +91-22-30926896 Fax: +91-22-24160828 e-mail: [email protected] • “KAMALPUSHPA”, 38 Reshimbag, Opp. Mohota Science College, Umred Road Nagpur 440 009, Phones: +91-712-3945220, +91-712-2704275 e-mail: [email protected] Atlas of Human Assisted Reproductive Technologies © 2007, Mangala Telang All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the editor and the publisher. This book has been published in good faith that the material provided by contributors is original. Every effort has been made to ensure accuracy of material, but the publisher, printer or editor will not be held responsible for any inadvertent error(s). In case of any dispute, all legal matters would be settled under Delhi jurisdiction only. First Edition: 2007 ISBN 81-8061-954-0 Typeset at JPBMP typesetting unit Printed at Ajanta Offset

To My parents Aie, Dada who gave us all they had and Ananya, the sparkle in my life and the future

Contributors Mangala Telang MBBS DGO MD FACOG Director, Fertility Research and IVF Centre New Delhi Ajit Saxena MS (Delhi), FRCS (Edin), FICS, MNAMS (Urol), Dipl. Urology (London)

Senior Consultant Urologist and Andrologist, Indraprastha Apollo Hospitals, New Delhi. Ashok Khurana MBBS MD (Radiology) Clinical Director, Senior Fetustician and Consultant in Reproductive Imaging Institute of Fertility Fulfilment and Research New Delhi Rupin Shah MS MCh (Urology) Consultant Andrologist and Microsurgeon Lilavati Hospital, Mumbai Vijay Kulkarni MS Consultant Andrologist and Microsurgeon Bhatia Hospital, Mumbai Narendra Malhotra MD FICOG FICMCH Ian Donald Diploma

Practising Obstetrician and Gynecologist Malhotra Test Tube Baby Centre, Agra Arun Tewari MD Urologist Malhotra Test Tube Baby Centre, Agra Jaideep Malhotra MD Malhotra Test Tube Baby Centre, Agra

Shilpa Shah MD DNB FCPS Lilavati Hospital IVF Centre, Mumbai Rishma Pai MD FCPS DNB FICOG Lilavati Hospital IVF Centre Mumbai and Jaslok Hospital Mumbai Nandita Palshetkar MD FCPS FICOG Lilavati Hospital IVF Centre, Mumbai and Batra Hospital IVF Centre, Delhi Sushma Ved MD Southend Rotunda (Centre for Human Reproduction), Holy Angels Hospital, Vasant Vihar New Delhi Sonia Malik MD Southend Rotunda (Centre for Human Reproduction), Holy Angels Hospital, Vasant Vihar New Delhi Sapna Srinivas MD Embryologist, Infertility Institute and Research Centre, Secunderabad, Andhra Pradesh Alok Teotia MVSc Consultant Reproductive Biologist, Indraprastha Apollo Hospitals, New Delhi Sudesh A Kamat MSc Consultant Reproductive Biologist, Leelavati Hospital Mumbai

Ashok Sharma Malhotra Test Tube Baby Centre, Agra

Vijay Mangoli MSc Reproductive Biologist, Fertility Clinic and IVF Centre, Mumbai

Hrishikesh Pai MD FCPS FICOG Lilavati Hospital IVF Centre, Mumbai and Batra Hospital IVF Centre, Delhi

Ranjana Mangoli MSc Reproductive Biologist, Fertility Clinic and IVF Centre, Mumbai

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Col RK Sharma MD Senior Advisor (Obs., Gynae & ART) Army Hospital (Research & Referal) New Delhi

H Ingolf Nielsen MediCult a/s, Møllehaven 12, DK-4040 Jyllinge Denmark, and Essex Fertility Centre United Kingdom

Prochi Madon Department of Assisted Reproduction and Genetics Jaslok Hospital and Research Centre Mumbai

Anne Lis Mikkelsen The Fertility Clinic, Herlev University Hospital DK-2730 Herlev, Denmark

Arundhati Athalye Department of Assisted Reproduction and Genetics Jaslok Hospital and Research Centre Mumbai Nandkishor Naik Department of Assisted Reproduction and Genetics Jaslok Hospital and Research Centre Mumbai Firuza Parikh MD Department of Assisted Reproduction and Genetics Jaslok Hospital and Research Centre Mumbai

NS Moorthy Medical Director, Asia Cryo-Cell Private Limited Chennai GA Rama Raju Krishna IVF Clinic, Visakhapatnam K Murali Krishna Krishna IVF Clinic, Visakhapatnam G Jaya Prakash Krishna IVF Clinic, Visakhapatnam K Madan Krishna IVF Clinic, Visakhapatnam

Foreword The birth of the first human baby conceived in vitro, in July 1978, was not an accident. It was preceded by more than 30 years of intense laboratory and animal experimentation by innumerable scientists. From this long list, I believe that it is imperative to highlight: R Moricard, CR Austin, MC Chang, L Dauzier, C Thibault, JM Bedford, R Yanagimachi, P Soupart, BD Bavister and RG Edwards. Dauzier and Thibault of France were the first to report on “Fertilization in vitro of rabbit oocyte” in 1954. Chang and Bedford confirmed their findings in 1959, following which Yanagimachi and Chang reported on “Fertilization of hamster eggs in vitro” in 1963. Two years later Edwards reported the “Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes.” The first clinician, Patrick Steptoe, entered the scene in 1968 when he started to collaborate with Edwards; this association enabled them to perform important studies using human gametes: “Early stages of fertilization in vitro of human oocytes matured in vitro” (1969), “Fertilization and cleavage in vitro of preovulatory human oocytes” (1970), and “Human blastocysts grown in culture” (1971). The vision and driving force behind the human work was Bob Edwards. The first human pregnancy derived from in vitro fertilization (IVF) was a tubal pregnancy in 1976. The first baby conceived in vitro was born, by cesarean section, on July 25, 1978, just before midnight. Both events were reported by Steptoe and Edwards in the Lancet; the birth of the first IVF baby was reported immediately and with great fanfare by the world media. During the first decade that followed this event, the IVF results remained fairly modest. The first international survey carried out in 1984 by Markku Seppala reported a delivery rate per initiated cycle of only 5.4%. This rate remained under 12% until the end of the decade. My team and I, in Vancouver, were fortunate to have the first baby conceived in vitro in Canada; he was born on December 25, 1983. The nineties brought sunshine to IVF; the success rate improved gradually, and by the end of the decade the rate of delivery per initiated cycle, in the USA, reached 25.4%. In 2003, the last reported year, this rate was 28%. It is interesting to note that improvement in outcomes was realized without any change in the cancellation rate, which remained around 13 to 14%. Intracytoplasmic sperm injection (ICSI) also started with animal experimentation by Gianpiero Palermo and co-workers. Their initial report in 1991 “Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs” was followed by work on human oocytes that led to a report in 1992 “Induction of acrosome reaction in human spermatozoa used for subzonal insemination”. During the same year, Palermo and associates were also able to report on pregnancies in human subjects: “Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte”. The introduction of ICSI has dramatically changed treatment of male infertility. The 2003 USA results clearly confirm the results of previous years, that the delivery per oocyte pick-up (OPU) rates are fairly similar between couples with male factor and those without male factor treated with IVF plus ICSI.

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The significant improvement in outcomes was due to the simplification of clinical and especially the simplification and standardization of laboratory techniques; the improvements in cryopreservation of supernumerary embryos, the advent of intracytoplasmic sperm injection (ICSI), and to the industrialization of IVF services. This improvement came about at the expense of a very high rate of multiple pregnancies, especially triplets and higher order of multiples, as a result of replacement (transfer) of multiple embryos. Multiple pregnancies are associated with a higher incidence of obstetrical complications and neonatal complications and deaths. They incur significant costs to the society, associated with the care of premature and sick infants, and tax the parents financially and emotionally. Fortunately, there is now a growing movement to decrease the number of embryos transferred, and to individualize the number taking into account the woman’s age the quality of available embryos and other pertinent parameters. Assisted Reproductive Technology (ART) which includes IVF has had a tremendous impact in the practice of reproductive medicine. It made IVF possible for a significant proportion of women, who otherwise would not achieve a pregnancy, to bear children. It has permitted the introduction of prenatal genetic diagnosis (PGD), enabling couples who are carriers of genetic disease to have healthy children. We must be reminded that, despite the impressive progress in outcomes, ART fails to yield an offspring for approximately 50% of couples willing to undergo 3 cycles of treatment. And many do not persist that long. The industrialization of IVF proved to be a two edged sword. On the one hand, it has permitted ready access to this form of treatment globally, while on the other it funnels to IVF many couples who would benefit from simpler forms of treatment. It has caused significant reduction in the use and teaching of reproductive microsurgery. The reestablishment of a balanced approach remains the responsibility of the teaching institutions. The selection of the initial and subsequent treatment modalities, for a given infertile couple, must be individualized on the basis of the findings obtained from a proper investigation. Reconstructive surgery and ART must not be viewed as competitive techniques; instead, they should be accepted and used as complementary methods to achieve a greater rate of success in patients presenting with complex fertility problems. There is ample evidence in this regard. IVF has also opened many areas of investigation and progress: stem cell research, gene therapy, therapeutic cloning, etc. It is also opening the Pandora box of human cloning. Such is the destiny of scientific research. Progress is made by visionaries who are willing to push the envelope; visionaries who have the will to stay the course, and the strength to withstand the abuse from those who fear change. I am honored to be asked to contribute a foreword to this “Atlas of Human Assisted Reproductive Technologies” edited by Dr. Mangala Telang. This is a concise, practical and richly illustrated book, designed for those involved in the practice of ART, and the personnel working in ART laboratories. The book is divided into three sections: clinical aspects of ART, laboratory aspects of ART, and new developments. The initial chapter of the first section is authored by Dr. Telang and devoted to the female partner. The chapter commences with a detailed discussion of the evaluation of the female partner, which is so important in selecting the most appropriate treatment. The second part of the chapter covers important clinical aspects of assisted reproduction including controlled ovarian stimulation, ovum pick-up and transfer, etc. The subsequent four chapters that complete the first section ably discuss the evaluation of the male partner, non-surgical and surgical methods of sperm collection, and the role of ultrasound in ART. The second section commences with a chapter that discusses the laboratory, its equipment, quality control and assurance. The subsequent seven chapters in this section provide a detailed description of the various laboratory

Foreword

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techniques: sperm preparation, culture, ICSI, cryopreservation, PGD, etc. The last section, “new developments” include three chapters devoted to in vitro maturation, stem cells, and vitrification. The balance and clarity of the book reflects the expertise and wisdom of its editor Dr. Mangala Telang who selected its authorship, and crafted and edited its contents. I am certain that it will prove to be a very useful guide to those involved in the practice of ART, personnel working in ART laboratories, and undoubtedly residents in gynecology. Professor Victor Gomel Department of Obstetrics and Gynecology Faculty of Medicine, University of British Columbia Vancouver BC. Canada

Preface Assisted Reproductive Technologies (ART) have given a ray of hope to the countless couples who otherwise had no possibility of fulfilling their dream of parenthood. There is hope for women who have lost ovarian function, whose fallopian tubes are blocked, who do not have healthy wombs and even those who develop cancer of the reproductive organs. There is also hope for men who for some reason produce very few sperms or none at all. There is constant addition to the armamentarium needed for ovulation induction and laboratory equipment and techniques. There was no atlas in the Indian market which would explain pictorially all the facets of clinical and laboratory management of ART. The atlases available from outside India were very expensive for Indian laboratories and this is an attempt to make such a book available for all ART laboratories. All the authors are very renowned and experienced in their own fields of expertise which they have shared in this atlas. There is a comprehensive section on clinical aspects, which describes in detail the evaluation of an infertile woman and the management of various problems which could affect her fertility. There are chapters which handle male infertility comprehensively. Apart from the routine ART, latest advances in cryopreservation, in vitro maturation, vitrification have been contributed by very experienced scientists. The latest interest in stem cell research has also been addressed. I hope this atlas finds a place in every IVF laboratory and is found to be useful both by the clinician and the laboratory personnel. Mangala Telang

Acknowledgements I am very thankful to Dr Victor Gomel who is a pioneer in the field of endoscopy and ART for being gracious and for writing the foreword for this atlas. I am grateful to all the contributors for making special efforts to make their text meaningful by adding their personal experiences in the form of pictures. I am thankful to Sudesh A Kamat for helping me to contact contributors from various parts of the country. My special thanks to Dr Ashok Khurana and Dr Narendra Malhotra for lending some of their pictures. I have to acknowledge the valuable help given by Vishal Mittal with the computer work. Last but not the least I have to thank my husband Dinbandhu for his understanding and encouragement always and Nandita and Sucheta for being bare for me. Shri Jitendar P Vij gave me the idea to bring out an atlas and I thank him and Jaypee Brothers Medical Publishers for the excellent work they have done with the editing and printing.

Contents Section 1 Clinical Aspects 1. Clinical Aspects of Human Assisted Reproductive Technologies ........................................ 3 Mangala Telang

2. Investigating an Infertile Male ........................................................................................... 38 Ajit Saxena

3. Ultrasound in Assisted Reproductive Technologies............................................................ 49 Ashok Khurana

4. Techniques of Non-surgical Sperm Retrieval ..................................................................... 71 Rupin Shah, Vijay Kulkarni

5. Surgical Methods of Sperm Retrieval ................................................................................ 76 Narendra Malhotra, Arun Tewari, Jaideep Malhotra, Sudesh A Kamat, Ashok Sharma

Section 2 Laboratory Aspects 6. The ART Laboratory ........................................................................................................... 85 Hrishikesh Pai, Shilpa Shah, Rishma Pai, Nandita Palshetkar

7. Techniques of Sperm Preparation for ART ........................................................................ 93 Sushma Ved, Sonia Malik

8. The Human Oocyte .......................................................................................................... 110 Sapna Srinivas

9. In Vitro Fertilization and Blastocyst Culture ................................................................... 129 Alok Teotia

10. Intracytoplasmic Sperm Injection (ICSI) ......................................................................... 143 Sudesh A Kamat

11. Cryopreservation: Gametes, Oocytes and Ovarian Tissue ............................................... 151 Vijay Mangoli, Ranjana Mangoli

12. Laser in Assisted Reproductive Technologies .................................................................. 159 Col RK Sharma

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13. Preimplantation Genetic Diagnosis .................................................................................. 167 Prochi Madon, Arundhati Athalye, Nandkishor Naik and Firuza Parikh

Section 3 New Developments 14. In Vitro Maturation of Human Oocytes ......................................................................... 177 H Ingolf Nielsen, Anne Lis Mikkelsen

15. Stem Cells: A Brief Overview .......................................................................................... 191 NS Moorthy

16. Vitrification of Embryos ................................................................................................... 195 Rama Raju GA, Murali Krishna K, Jaya Prakash G, Madan K

Index .................................................................................................................................................... 203

Clinical Aspects of Human Assisted Reproductive Technologies

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Clinical Aspects of Human Assisted Reproductive Technologies Mangala Telang

INTRODUCTION The success of Assisted Reproductive Technologies (ART) depends on cooperation between the clinician and embryologist. The clinician’s role is to select the patients, the right protocol for ovulation and decide upon the correct timing for administration of human chorionic gonadotropin. The clinician has also to perform a non-traumatic ovum pick up and perfect embryotransfer. There is also a role for a bonding between the patient and doctor as mind has a big role to play in the reproductive process. There is a need for very understanding and sympathetic paramedical staff and psychological counsellors. A thorough history helps in identifying the suitable patients for ART. Most of the women who are considered for in vitro fertilization (IVF) have had several investigations and various forms of treatments by various doctors. One has to evaluate the quality and adequacy of the investigations and guide the patient so that with minimal interference, she is made suitable to undergo IVF treatment. Patients who have had several drug induced ovulation cycles and several months of clomiphene treatment should not keep on

having a repeat of the same treatment as there have been reports of increased risk of ovarian cancer in these women.1 Usually with no obvious pathology in the female partner, and with an adequate sperm preparation after sperm wash, if intrauterine insemination fails to achieve a pregnancy in four cycles, it is time the couple is advised to go in for IVF. This decision is of course much easier with obvious pathology like tubal blockage or oligospermia.

EVALUATION OF THE FEMALE PARTNER MEDICAL HISTORY History of chronic disease which gives the patient debility must be looked after before undertaking elective procedures like ART. Adequate body weight is necessary for proper ovarian function. Extremely underweight patients as seen in anorexia nervosa should be advised to gain weight. On the other hand obesity has an adverse effect on process of ovulation. Fat can be of central or peripheral distribution. Women with central fat have high levels of LH, androstenedione, estrone, insulin,

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triglycerides, VLDL and apolipoprotein beta and lower levels of HDL. 2 These women also have more menstrual abnormalities, lower conception rates and infertility.3 Success rate of the reproductive process improves significantly by achieving optimal body weight. Thyroid Disorders Hyperfunction of thyroid causes serious effects on the body. Severe weight loss, anxiety, menstrual disorders and disorders of ovulation are all seen in with hyperthyroidism. Hypothyroidism also causes ovulation disorders dysfunctional uterine bleeding. It is also associated with hyperprolactinemia, which disrupts the normal reproductive process.

SURGICAL HISTORY History of surgery in childhood for intestinal disorders like intestinal obstruction or appendectomy have serious implications for future fertility as there can be dense adhesions caused by these surgeries which can affect the Fallopian tubes. Previous surgery for endometriosis resection of ovarian cyst, dermoid, myomectomy, can cause adhesions and distort normal pelvic anatomy . Dilatation and curettage for termination of pregnancy is an important factor in causing endometrial scarring or tubal infection and blockage. This is particularly significant if the pregnancy was advanced and products do not get expelled spontaneously and have to be evacuated. GYNECOLOGICAL AND OBSTETRICAL HISTORY

Diabetes

Menstrual History

History of diabetes in the family or in the woman undergoing treatment for infertility is very significant. Familial diabetes is very commonly associated with polycystic ovarian disease as normal metabolism of insulin is important for the ovulation process.

Hyper and hypopituitary states both affect reproduction. Heavy bleeding during or after delivery can cause Sheehan’s syndrome, which affects reproductive process.

Age of menarche and the menstrual rhythm gives an idea of the hormonal mileau in the body. Primary amenorrhea can be evaluated by FSH and LH levels which helps differentiate between hypogonadotropic hypogonadism and primary ovarian failure. Menstrual pattern is affected in polycystic ovarian disease(PCOD) in which there are long intervals between periods with prolonged irregular bleeding or sometimes no spontaneous periods at all and withdrawal bleeding has to be induced by giving progesterone.

Adrenal Disorders

Obstetrical History

Adrenal hyperplasia and neoplasia have direct effect on reproduction so does adrenal hypofunction.

This is very important as if there has been a pregnancy before, it helps plan the investigative and therapeutic approaches. Recurrent pregnancy loss and proper evaluation of each loss of pregnancy gives many clues to the cause of this condition.

Pituitary Disorders

Pelvic Inflammation Pelvic inflammation has the potential of affecting tubal function and patency.

Age Tuberculosis Tuberculosis has an affinity towards genital organs. Fallopian tubes and endometrium are the first targets. It causes endosalpingitis and leads to irreversible damage of the tubal cilia. Also causes scarring of the endometrium.

This has the biggest impact on outcome of ART. A woman’s physiology is designed to bear children before the age of 35 years. Once that age is reached the success rate declines drastically. Enrolling women above the age of 40 years in the ART programs should be done with proper counselling about poor outcome.

Clinical Aspects of Human Assisted Reproductive Technologies ASSESSMENT OF THE UTERUS AND FALLOPIAN TUBES Congenital abnormalities, septa, fibroids, polyps, all affect implantation. Endometrial scarring, adhesions following vigorous curettage previous intrauterine device, history of endometritis or tuberculosis can all cause irreversible changes in the endometrium which cause implantation failures. For the assessment of the uterus, hysterosalpingography (Figure 1.8) is very useful and is complimentary to the planning of further evaluation and treatment for uterine factor management. Sonohysterosalpingography with saline infusion and 3D ultrasound can give a fair idea of uterine and tubal structure and function. However the information provided by the hysterosalpingography about the fallopian tubes is more useful in assessing the nature of tubal pathology. Ultrasonic assessment of the thickness, character and vascularity of endometrium also helps in assessing the endometrial factor. Hysteroscopy is the ultimate investigation for assessing the uterine cavity. At Fertility research and IVF Center we routinely perform hysteroscopy prior to all ART procedures. Mullerian Defects Uterine septum is one of the commonest Mullerian defect observed in infertility practice. It may be seen as just an arcuate uterus or a partial or complete septum. A septum may cause defective implantation, recurrent early pregnancy loss and premature delivery. It can be detected by hysterosalpingography, 2D or 3D ultrasound(Figure 1.1A). It can be very effectively resected through hysteroscopy (Figures 1.1B and C). Uterus diadelphis or double uterus, bicornuate uterus are also manifestations of Mullerian abnormalities. Sometimes they have no effect on fertility but most often they are causes of infertility, recurrent implantation failures and pregnancy loss. A graphic repesentation of bicornuate uterus is seen in Figure 1.2A. Hysterosalpingograhy and 3D ultrasound can successfully diagnose the condition. Laparoscopy helps to get a correct idea of the defect and the feasibility of a successful pregnancy (Figures 1.2 B to D).

FIGURES 1.1A TO C: Septum: (A) 3D ultrasound, (B) Hysteroscopy, (C) Resected septum

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C

FIGURES 1.2A TO D: Bicornuate uterus: (A) Graphic, (B) Hysterosalpingography, (C) 3D ultrasound (D) Laparoscopy

Unicornuate uterus (Figures 1.5A to C) can be spacious and there may be a normal uneventful pregnancy but most often there is either difficulty in conceiving , pregnancy loss or premature delivery. A T shaped uterus is either the result of exposure to Diethyl stilbestrol during fetal life or due to Mullerian abnormality. The space inside the uterus gets restricted

and there may be resulting infertility, early or late pregnancy loss. The intrauterine space can be increased by doing a lateral metroplasty (Figure 1.6). Uterine polyps are often missed in an ultrasound though sizable polyps can be easily seen by both 2D and 3D ultrasound (Figures 1.3 A and B). Polyps are best diagnosed with hysteroscopy (Figure 1.3C). It is

Clinical Aspects of Human Assisted Reproductive Technologies

A

7

C

B

FIGURES 1.3A TO C: Polyp

a routine practice in our center to do a hysteroscopy before ART procedures when a polyp can be easily seen and resected. Intrauterine synechiae or Asherman’s syndrome (Figures 1.4A to D) can result from intrauterine infections especially tuberculous infections. Intrauterine devices can also result in endometritis which can then cause intrauterine adhesions. Vigourous curettage of the uterus, intrauterine surgical procedures, retained products of conception can all lead to intrauterine synechiae. If the adhesions are severe, it is difficult to treat as even if they are resected, they reappear and it is difficult to cause regeneration of the endometrium. Calcification of cavity (Figure 1.8) is seen resulting from previous endometrial infections especially tuberculous infection . Pieces of retained fetal bones from previous midtrimester abortions act as foreign bodies inside the uterus and cause infertility. Even if the calcified areas are removed through hysteroscopic resection, the original pathology usually leads to a poor prognosis.

HYSTEROSCOPY, LAPAROSCOPY AND EVALUATION OF PELVIS Hysteroscopy and laparoscopy are together a way to evaluate the cause of infertility in a comprehensive way in patients suffering from infertility. If there is an obvious cause for infertility like male factor and the female partner has no significant history of disease, laparoscopy may be put on a hold but hysteroscopy is

definitely a useful investigation to perform before IVF. Normal hysteroscopy is seen in Figure 1.9 and normal view at laparoscopy in Figure 1.10. At Fertility Research and IVF Center, we prefer to do both laparoscopy and hysteroscopy endometrial biopsy. The procedure is timed so that maximum information and benefit is obtained through it. At our center, the procedure is planned in the third week after menstruation. This is the optimal time for endometrial biopsy for hormonal evaluation. Since there is a good endometrial development, pathology like tuberculosis can be evaluated better. And there is enough tissue for PCR evaluation for tuberculosis. The endometriosis is also more visible in the premenstrual period and can be cauterized more effectively. Mock transfer is also done at this time. Other procedures like adhesiolysis, delinking or removal of hydrosalpinges and ovarian drilling where indicated, can be performed at one sitting. ENDOMETRIOSIS Many studies have shown that endometriosis even in its very mild form comes in the way of fertility due to various biochemical agents and cytokines produced by the endometriotic deposits.4 Other studies have not confirmed such findings.5 The presence of endometriosis may affect duration of ovulation induction, amount of gonadotropin required and number of oocytes retrieved.6 The intrafollicular levels of progesterone were found to have a correlation with severity

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A

B

C

D

FIGURES 1.4A TO D: Asherman’s syndrome: (A) Hysterosalpingography, (B) Ultrasound, (C) Hysteroscopy and (D) Graphic

of the disease being higher in mere severe forms of endometriosis. 7 Even the minimal endometriosis therefore must be cauterized if it is detected during laparoscopy. If there is more severe endometriosis with adhesions, all visible implants should be cauterized. Figure 1.11 shows endometriotic deposits and Figure 1.12 shows cauterization of the deposits. Endometrioma can be also diagnosed by ultrasound with its typical appearance of echogenic material in the chocolate cyst (Figures 1.4A to D). Endometrioma should be evacuated and wall of the cyst wall excised or cauterized with bipolar cautery or laser (Figures 1.13A to C). If there is extensive endometriosis after surgery,

medical treatment with depot GnRH agonist every 4 weeks for 3 months, helps suppress non-visible endometriotic deposits and after that there is a window of about one year during which endometriotic activity is suppressed. IVF can be planned immediately after the three injections are given and has been seen to give better pregnancy rates.8 For moderate to severe endometriosis, intracytoplasmic sperm injection rather than IVF has been found to give better results. POLYCYSTIC OVARIAN DISEASE (PCOD) Polycystic ovarian disease is one of the most common manifestations of a hormonal disorder, which is

Clinical Aspects of Human Assisted Reproductive Technologies

FIGURE 1.5A: Unicornuate uterus laparoscopic

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A

B

B

FIGURES 1.6A AND B: Lateral metroplasty for T-shaped uterus

C

FIGURES 1.5B and C: Unicornuate uterus ultrasound

FIGURE 1.7: Hysterosalpingography

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FIGURE 1.10: Laparoscopy normal

FIGURE 1.8: Calcification of cavity

observed in women of reproductive age. It is characterized by hyperandrogenism seen as increased tendency to gain weight, male type hair growth, acne, frontal balding, irregular ovulation and irregular menstruation. There is generally a history of diabetes in the family and familial clustering is observed. Association of PCO with insulin resistance was reported by Khan et al in 19769 and Burghen et al in 1980.10 The ultrasonic criteria for detection of PCO became streamlined in 1985.11 FIGURE 1.11: Endometriosis deposits

FIGURE 1.9: Hysteroscopy normal

FIGURE 1.12: Cauterization of deposits

Clinical Aspects of Human Assisted Reproductive Technologies

A

B

C

FIGURES 1.13A TO C: (A) Endometrioma. (B) Fulgration of cyst wall (C) Resection of cyst

11

FIGURE 1.14: Ultrasound of endometrioma

PCOS has a strong genetic component to its etiology. Research has suggested involvement of two key genes, the steroid synthesis gene CYP11a and the insulin gene variable number tandem repeats (VNTR). The newest candidate suggested is the follistatin gene.12 PCOS is a syndrome where there is abnormal ovarian steroidogenesis which is influenced by hypersecretion of LH and insulin. There is hyperfunctioning of the thecal compartment of the ovary and there are multiple, small antral follicles which are neither atretic nor apoptotic but are arrested in development. The changes in insulin sensitivity in PCOS causes an increase in ovarian androgen production and interferes with transport of testosterone by decreasing SHBG concentrations. Obesity further exacerbates these changes. The appearance of the ovaries is typical with increased ovarian volume, and a smooth pearly white capsule (Figure 1.15 A). Ultrasound appearance reveals increased ovarian stroma, larger ovarian volume (>10 cubic cm) and 12 or more subcapsular immature follicles less than 10 mm (Figure 1.15B).13 There are increased levels of LH changing the LH: FSH ratio. There is associated insulin insensitivity. Fasting and post-prandial levels of insulin are elevated. Pretreatment with metformin gives much better pregnancy rates. This also brings down the miscarriage rate.14

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A

FIGURE 1.16: Ovarian drilling

HYDROSALPINX

B

FIGURES 1.15A AND B: (A) PCOD at laparoscopy (B) PCOD ultrasound

Ovarian drilling is still a debated treatment. Some studies have shown development of adhesions after this procedure. It should be undertaken with caution and the burr holes should be restricted to 5 to 6 on each side (Figure 1.16). 500 to 1000 ml lactated Ringer’s solution should be left in the pelvis after surgery to prevent adhesions. In a study undertaken at our center, adhesions were found in 92 out of 122 cases in a repeat laparoscopy following ovarian drilling. There were signs of ovarian insufficiency in 4 cases. Ovarian drilling should be done only in selected cases and with care.15 Stimulation protocols have to be selected with caution to avoid ovarian hyperstimulation syndrome.

It is important to diagnose and manage hydrosalpinx before undertaking an ART cycle as it can adversely affect the outcome. 16 Hysterosalpingography can diagnose the condition very easily (Figure 1.17). If the hydrosalpinx can be seen in the ultrasound (Figures 1.18 and 1.19A) or if there is fluid in the endometrial cavity (Figure 1.20), Laparoscopy gives the accurate diagnosis as to whether there is infection and pyosalpinx (Figures 1.18 and 1.19B) and decision can be made whether the fallopian tube can be delinked and retained or resected. Delinking has to be undertaken as there is evidence that the fluid from the tubes can drain intermittently into the uterine cavity around the time of embryotransfer and hinder

FIGURE 1.17: Hydrosalpinx by hysterosalpingography

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FIGURE 1.20: Fluid in the endometrial cavity FIGURE 1.18: Hydrosalpinx by laparoscopy

implantation. The pregnancy rates were seen to go down in these cases.17,18 In these cases the tubes were blocked at the cornual ends with cautery and terminal salpingostomy performed so that drainage of the tubal secretions can be maintained. If there is a large hydrosalpinx, it is better to remove it as the fluid has been found to contain cytokines, inflammatory products and prostaglandins19 which is detrimental to implantation. FIBROIDS

FIGURE 1.19A: Hydrosalpinx by ultrasound

FIGURE 1.19B: Pyosalpinx by ultrasound

Fibroids are the commonest benign tumors of the reproductive tract. They can be intramural, subserous, submucous and combined intramural and either submucous or subserous (Figure 1.21). They may be polypoidal either inside the cavity or subserous. The ones which project into the cavity, polyps in the cavity of the uterus and intramural fibroids which distort the cavity are the ones which affect fertility and cause pregnancy loss. Submucous polyps act as foreign bodies in the uterine cavity and prevent pregnancy. 2D and 3D ultrasound can very accurately delineate the position of the fibroids (Figures 1.23A and B). There may be mechanical compression of the fallopian tubes. The endometrial growth and implantation gets affected due to the abnormal vasculature associated with fibroids. There may be ulceration and infection associated with intracavitory fibroids.

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Atlas of HART

A

FIGURE 1.21: Fibroids—graphic

A

B

FIGURES 1.23A AND B: Fibroids. (A) 2D ultrasound picture of fibroid, (B) 3D ultrasound picture of fibroid B

FIGURES 1.22A AND B: Fibroids. (A) Laparoscopy, (B) Resection of myoma

Whether significant myomas should be routinely resected before in vitro fertilization is a controversial issue. There are studies which show that unless the uterine cavity is distorted, myomectomy does not

help achieving higher pregnancy rates.81 Stovall82 performed a prospective study in 91 women undergoing ART cycle in women who had myomas which did not distort the cavity and were less than 6 cm in diameter. The control group consisted of women without fibroids undergoing ART. He concluded that

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pregnancy rates were much lower 37.4% as compared to 52.7% in the control group. There was no difference in the two groups as far as miscarriage rates were concerned. The other controversy is whether laparoscopic myomectomy is better than the traditional abdominal myomectomy (Figures 1.22A and B show a fundal myoma and its laparoscopic resection). Laparoscopic myomectomy requires high degree of skill in doing the procedure, expensive morsellators to remove the fibroid after resection.The time taken for the surgery is much longer than the abdominal surgery.The strength of the scar may be compromised during a future pregnancy.83,84 Intracavitory polyps can be handled very effectively through a hysteroscope (Figures 1.24A and B). Bipolar cautery (Versapoint, Johnson and Johnson) can be used with saline as the distending medium and reduces the complications due to hypotonic fluid absorption.85 Uterine artery embolization which is otherwise a successful option for patients who need myectomy for indications like menorrhagia, pressure symptoms, large size of fibroids is not a recommended method of treatment before ART as its effect on endometrial and ovarian function is as yet unknown.86

A

B

Medical Treatment Gonadotropin releasing hormone: Filicori first reported the use of GnRH agonist for reduction in the size of myoma.87 He reported that the myoma size went down by 77% in 90 days therapy. There are other studies which also report a 45 to 60% reduction in size occurring two to 3 months after therapy is initiated. This therapy is useful in reducing size of large fibroids before myomectomy so as to reduce the extent of trauma to the uterus.88,89 Otherwise medical therapy does not have a significant role before ART.

HYPERPROLACTINEMIA Prolactin has a very important role to play in the reproductive process.

C

FIGURES 1.24A TO C: Fibroids. (A) Fibroid polyp, (B) resection of polyp and (C) after resection

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Atlas of HART

PITUITARY GLAND INVOLVEMENT • Pituitary tumors • Idiopathic or functional hyperprolactinemia • Secondary hyperprolactinemia due to polycystic ovarian syndrome and hypothyroidism.

In 30% of PCOS patients there is an increased level of prolactin. Recent investigations using serial sampling have ruled out transient elevations of prolactin in PCOS.22 Hence treatment of both these conditions should be distinct from each other.

PITUITARY TUMORS

SYSTEMIC DISORDERS

Microadenoma These are very small tumors less than 1 cm with prolactin levels less than 100 ng/ml and usually respond to medical therapy. Contrast MRI is the best way to diagnose them. Macroadenoma These affect the whole Sella Turcica and even the whole pituitary gland. The size of the tumor is bigger than 1 cm and levels of PRL more than 100 ng/ml. Empty Sella Syndrome This is usually associated with pituitary tumors which expand the sella turcica due to which there is widening of the sellar diphragm and herniation of the arachnoid membrane into the sellar cavity displacing the pituitary gland.This causes displacement of the pituitary gland. Radiologically it appears as a clear area in the place of the sella.20 Acromegaly Functional hyperprolactinemia: The prolactin levels do not go beyond 100 ng/ml. This causes poor folliculogenesis,poor corpus luteal function, amenorrhea or oligomenorrhea, poor endometrial development. Intermittent or trasient hyperprolactinemia: There is a transient preovulatory peak in PRL levels before ovulation coinciding with the E2 peak which lasts 2 to 3 days. In a study by Ben David and Schenker21 they reported 40% success rates in achieving pregnancy by administering bromocryptine in cases of unexplained infertility. These claims are, however, not substantiated by other studies.

Hypothyroidism: Low levels of T4 lead to a negative feedback on the hypothalamic-pituitary axis. This causes increased secretion of TRH which stimulates both the thyrotrophs and lactotrophs and causes increase in PRL. Stress: Severe stress, illness, exercise can cause elevated levels of PRL. Renal disease: Acute or chronic renal disease causes reduced clearance of PRL. Chest surgery: Patients with previous breast surgery or even breast implants can have galactorrhea due to peripheral nerve stimulation. Clinical Features Menstrual irregularities, amenorrhea, oligomenorrhea delayed puberty, decreased libido, galactorrhea, infertility , visual defects. HYPERPROLACTINEMIA AND INFERTILITY High prolactin levels affect the hypothalamic neurotransmitter content through a direct feedback mechanism resulting in a decrease in GnRH and LH pulsatility. Prolactin may have direct effect at the gonadal level.23 Sometimes there may be increased sensitivity to prolactin with normal hormone levels. Patients who have galactorrhea but normal prolactin levels or those who show luteal phase deficiency may benefit from imperical bromocriptine therapy. Investigations PRL is secreted in a circadian rhythm in both men and women. 24 Circadian rhythm is sleep related. It increases shortly after the onset of sleep and is highest between 3 and 5 AM. The PRL should be tested in the

Clinical Aspects of Human Assisted Reproductive Technologies morning after 7-8 hours of fasting in the follicular phase as there may be an increase of almost 30% in the luteal phase. Serum prolactin levels should be a part of normal infertility work up.Thyroid function must also be tested simultaneously. The normal range for prolactin is 0-20 ng/ml. If it is more than three times the normal level investigations should be carried out for pituitary disorders. MRI with contrast is the best investigation to evaluate microadenomas. Cone down view of the sella tursica and X-ray used to be done but MRI is very much more sensitive. Peripheral visual fields should be mapped. Medical Management Bromocriptine is a dopamine receptor agonist which activates the postsynaptic dopamine receptors in the pituitary gland and inhibits prolactin secretion.25,26 It can be given orally, vaginally and parenterally. Initially it is better to start with a small dose as there are side effects. A starting dose of 1.25 is given after dinner. The dose is gradually increased as per the level of prolactin by 1.25 mg every week till desired dose is achieved. Side effects: Nausea, vomiting, dizziness, constipation, postural hypotension can cause discontinuation of therapy. Gradual increase in dosage helps to reduce the side effects. Gastrointestinal side effects are relieved if the drug is administered vaginally. Long-term side effects: Raynaud’s disease, Psychiatric problems and chronic constipation. Cabergolin: This is a long acting ergot derivative 0.5 mg is given once or twice a week. The side effects are less as compared to bromocriptine. It is the drug of choice in treating tumors. Quinagolide: This is an agonist not derived from ergot with better D2 receptor affinity. It is used in cases where there is intolerance for other medicines. The dosage is 75 mcg daily.

17

Hydergine: This is a mixed ergot alkaloid which inhibits prolactin secretion and induces ovulation. It is not very effective if prolactin levels are higher than 100 ng. It can be used. Luteal phase deficiency should be anticipated and treated by using HCG for induction of ovulation and with micronized progesterone intravaginal supplements during the luteal phase.

INFECTIONS Infection of the reproductive system play a very significant role in causing infertility both in males and females. In 15% couples infection is the major cause of infertility. Pelvic inflammatory disease causes tubal blockage in 20% of cases.27 INFECTIONS IN MALE PARTNER The effect of infection on semen parameters and male fertility is controversial.28 The presence of leukocytes, in a concentration of >1 × 106/mL in the ejaculate, is often used for the determination of an infection of the male sex glands 28. Several studies showed that semen samples of subfertile men contain more leukocytes than fertile controls and that semen quality is decreased with elevated concentrations of leukocytes. Other studies, however, found no correlation between leukocyte counts, semen quality, and the presence of microorganisms.29,30 Ureaplasma urealyticum and Chlamydia trachomatis are the two most frequently studied microorganisms in relation to male fertility. The prevalence of U. urealyticum is approximately 40% in subfertile men and 28% in fertile controls.28 This microorganism has often been associated with decreased sperm motility, poor sperm morphology, and an increase in the percentage of coiled sperm tails, probably because these organisms attach to spermatozoa . C. trachomatis is a common cause of urethritis and acute epididymitis in men 50% vascularity. Pulsatility index values of perifollicular flow do not correlate with pregnancy outcome. However, the peak systolic velocity (PSV) is an excellent parameter to assess the chances of obtaining mature oocytes and high grade preimplantation embryos. The chances of producing a Grade I or II embryo is 75% if the PSV is > 10 cm/second. Interestingly, during the ovulatory process there are prominent changes in the regional blood flow of the follicle with a marked increase in flow to the base of the follicle and a concomitant decrease of blood flow to the apex. These changes may be essential for the release of a mature oocyte. Patients with a PSV of > 10 cm/second in ovarian stromal arteries after pituitary suppression yield

significantly higher mature oocytes and achieve a higher clinical pregnancy rate. Ovarian stromal pulsatility index does not correlate with these parameters. Interestingly pulsatility index, resistive index and systolic/diastolic ratio of ovarian stromal arteries during ovulation induction correlates with the chances of developing ovarian hyperstimulation syndrome. If the resistive index < 0.48, more than two thirds of patients will develop a pleural effusion. Over one half of patients with a pulsatility index < 0.75 and a systolic/ diastolic ratio < 1.92 develop pleural effusions. Thin walled clear cysts discovered in baseline scans on the day of commencement of induction of ovulation have traditionally been aspirated. Evidence is building up, however, that apart from a small increase in the

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amount of gonadotropin required and a minimal increase in the number of days of stimulation no other detrimental effects have been established.55 These include no significant differences in E2 levels on the day of the ovulation trigger, number of oocytes retrieved, number of embryos transferred and clinical pregnancy rates.

OVULATION MONITORING Ultrasound assessment of follicular growth was introduced in the 1970s when a linear relationship between follicle size and estradiol levels was first observed. Pelvic ultrasound permits an assessment of the presence, size, number and echo pattern of developing follicles. Transabdominal ultrasound is completely reliable in assessing the size and number of follicles. For the prediction of peri-ovulatory events, however, transvaginal scanning with its superior resolution is useful. In the evaluation of the ovary which is located at the pelvic brim, transabdominal scanning especially with a 5 MHz frequency is not infrequently necessary. color Doppler is useful in assessing periovulatory and postovulatory events and should be used whenever possible. The sequence of events that occurs in the natural menstrual cycle has been well characterized. The initiation of follicular growth is a continuous process that is independent of gonadotropin stimulation. Gonadotropin-independent growth proceeds until the follicle reaches 5 mm. Sonography of the ovaries will always demonstrate follicles of this size in all women in the reproductive age group (Figure 3.28). Further growth of the follicle occurs only in an appropriate gonadotropin environment. A decline in follicle stimulation hormone (FSH) occurs by day 6 to 9 in spontaneous cycles and is responsible for the selection of the single most mature follicle. Follicles with fewer FSH receptors on their surface will become atretic. Once the leading follicle reaches a diameter of 14 mm (Figure 3.29), it shows a daily growth of 1.5 to 2.3 mm per day until just before ovulation. Estradiol (E2) is produced by the granulosa cells and ovulation usually

FIGURE 3.27: Follicular maturation is accompanied by perifollicular neovascularization. This can be recognized by the appearance of occasional perifollicular vascular signals when the follicle reaches 12-14 mm in size, which then progress to 50-100% of perifollicular vascularization as the cycle progresses. This phenomenon correlates well with parameters of oocyte quality such as the levels of follicular fluid estradiol, pH, follicular fluid pO2 and absence of oocyte aneuploidy. This neoangiogenesis, however, does not correlate well with endometrial receptivity

FIGURE 3.28: Multiple immature follicles are evident in a healthy ovary between Day 2 and Day 9 with a decline in FSH, follicles with fewer FSH receptors become atretic

occurs after the serum E2 reaches 150 to 400 pg/ml. Extrusion (Figure 3.30) takes place consequent to a surge of luteinizing hormone (LH) at 18-24 mm size. In this size range, follicular growth is exponential and stands at 2.6 to 6.0 mm in 24 hours. The cumulus oophorus (Figure 3.31) can be identified within a mature follicle frequently. This visualization is related to equipment resolution, operator skill and operator technique. Non-visualization often means an empty follicle but it is important to keep in perspective the time interval between scans for optimal visualization of the cumulus. It is seen as a mural echogenic focus 1

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B

A

C

D

FIGURE 3.29: By Day 10 of a 28 day unstimulated cycle one follicle becomes the leading follicle and grows at the rate of 1.5 to 2.3 mm per day. The figure shows a 14 mm leading follicle. Note the clear contents and the thin wall

FIGURES 3.30A TO D: Follicle extrusion is evidenced by a combination of several features. These include (A) Reduction in the size of the follicle, (B) Filling up of the follicle with internal echoes, (C) Capsular hemorrhage seen as increased

to 3 mm in maximum diameter. Following follicular extrusion, the corpus luteum can be visualised in the ovary (Figures 3.32 to 3.36). A baseline evaluation, as deduced from the above events, is necessary on Day 1, 2 or 3 of the cycle with a follow-up on Day 5 if a cystic area is seen. Follow-up visits can then be scheduled depending on spontaneous cycle length and medication. Typically, for a 28 day cycle, this would mean daily monitoring from Day 10 upto extrusion. Luteal phase studies should be done on Day 07, 11 and 14 postextrusion. In stimulated cycles the essential difference is the number of follicles (Figures 3.37 and 3.38) that undergo maturation and the rate of follicular growth. Follicular

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echogenecity of the ovarian surface, and, (D) Free fluid in the pelvis FIGURE 3.32: Following extrusion the corpus luteum can be visualized in the ovary. It is frequently thin-walled and has occasional fine internal echoes

FIGURE 3.31: The cumulus oophorus can be seen as an echogenic mural focus 1 to 3 mm in maximum diameter. Its visualization depends on operator expertise, time spent on the examination and equipment resolution

FIGURE 3.33: Internal echoes within a corpus luteum may be fine or coarse as seen here

FIGURE 3.34: Dense hemorrhage is not unusual in a corpus luteum and presents as variable increased echogenecity of the contents

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Atlas of HART INTERVENTIONAL PROCEDURES

FIGURE 3.35: Occasionally a hemorrhagic corpus luteum may be isoechoic with ovarian stroma and may only be recognized as rim vascularity on color Doppler studies

Oocyte recovery is now entirely an ultrasound guided transvaginal procedure. The technique has come a long way since Lenz and his colleagues first obtained oocytes by a transabdominal, transcutaneous, transvesical approach. This approach was soon replaced by a transvaginal recovery but with transabdominal monitoring and has now been supplanted universally by a transvaginal guidance and transvaginal pick-up. This has the advantages of high resolution, an easy to learn and easy to use technique, easy ovarian access and few complications. After the patient is put into a lithotomy position the vagina is prepped with only a sterile saline wash. The guide is then mounted onto the transducer and checked to see if the recovery needle passes through. The needle is thrust into each follicle with a minimum number of puncture sites on the ovary. If the follicular fluid does not yield an oocyte the aspiration is repeated after injecting warmed flushing medium or warmed saline. All visible follicles should be aspirated. At our center all procedures are carried out under general anesthesia and antibiotic cover. If the cervical route is inaccessible, embryotransfer may be done by an ultrasound guided transabdominal or transmyometrial approach. Ultrasound is also indispensable in multifetal pregnancy reduction.

CONCLUSION FIGURE 3.36: The corpus luteum shows progressive rim vascularization in the luteal phase of the cycle. This can be seen as dramatic color signals on color Doppler studies. The new vessels show low impedance to flow with an ideal resistive index of 0.55 or less by Day 11 post-extrusion

growth is generally accelerated to 1.8-2.9 mm per day. Since endometrial disease is more evident in a thicker endometrium, the appearance of polyps or submucous fibroids, should be looked for at each sitting for follicular monitoring. Ovarian hyperstimulation correlates best with the observed number of smaller (10-14 mm) follicles in the late proliferative phase and not to the total number of follicles and with stromal flow.

Ultrasound has emerged as an indispensable component of treatment cycles and its role in ART is continuously expanding. The modality permits an unsurpassed view of follicles, the endometrium, needle paths and real time guidance.

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33. Hammadieh N, Afnan M, Evans J, Sharif K, Amso N, Olufowobi O. A postal survey of hydrosalpinx management prior to IVF in the United Kingdom. Hum Reprod 2004; 19(4): 1009-12. 34. Savaris RF, Pedrini JL, Flores R, Fabris G, Zettler CG.Expression of alpha 1 and beta 3 integrins subunits in the endometrium of patients with tubal phimosis or hydrosalpinx. Fertil Steril 2006; 85(1): 188-92. 35. Kupesic S, Kurjak A. Predictors of IVF outcome by threedimensional ultrasound. Hum Reprod 2002; 17(4): 9505. 36. Hendriks DJ, Mol BW, Bancsi LF, Te Velde ER, Broekmans FJ. Antral follicle count in the prediction of poor ovarian response and pregnancy after in vitro fertilization: A metaanalysis and comparison with basal follicle-stimulating hormone level. Fertil Steril 2005; 83(2): 291-301. 37. Scheffer GJ, Broekmans FJ, Dorland M, Habbema JD, Looman CW, te Velde ER. Antral follicle counts by transvaginal ultrasonography are related to age in women with proven natural fertility. Fertil Steril 1999; 72(5): 845-51. 38. Frattarelli JL, Lauria-Costab DF, Miller BT, Bergh PA, Scott RT. Basal antral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles. Fertil Steril 2000; 74(3): 512-7. 39. Bancsi LF, Broekmans FJ, Looman CW, Habbema JD, te Velde ER. Impact of repeated antral follicle counts on the prediction of poor ovarian response in women undergoing in vitro fertilization. Fertil Steril. 2004; 81(1): 35-41. 40. Scheffer GJ, Broekmans FJ, Bancsi LF, Habbema JD, Looman CW, Te Velde ER. Quantitative transvaginal twoand three-dimensional sonography of the ovaries: Reproducibility of antral follicle counts. Ultrasound Obstet Gynecol 2002; 20(3): 270-5. 41. Ng EH, Tang OS, Chan CC, Ho PC. Ovarian stromal blood flow in the prediction of ovarian response during in vitro fertilization treatment. Hum Reprod. 2005; 20(11): 3147-51. 42. Pierson RA. Imaging the endometrium: are there predictors of uterine receptivity? J Obstet Gynaecol Can 2003; 25(5): 360-8. 43. Baruffi RL, Contart P, Mauri AL, Peterson C, Felipe V, Garbellini E, Franco JG. A uterine ultrasonographic scoring system as a method for the prognosis of embryo implantation. J Assist Reprod Genet 2002; 19(3): 99-102. 44. Schild RL, Knobloch C, Dorn C, Fimmers R, van der Ven H, Hansmann M. Endometrial receptivity in an in vitro

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fertilization program as assessed by spiral artery blood flow, endometrial thickness, endometrial volume, and uterine artery blood flow. Fertil Steril 2001; 75(2): 3616. Ardaens Y, Gougeon A, Lefebvre C, Thomas P, Lerov M, Lerov JL, Dewailly D. Contribution of ovarian and uterine color Doppler in medically assisted reproduction techniques (ART). Gynecol Obstet Fertil 2002; 30(9): 66372. Chien LW, Lee WS, Au HK, Tzeng CR. Assessment of changes in utero-ovarian arterial impedance during the peri-implantation period by Doppler sonography in women undergoing assisted reproduction. Ultrasound Obstet Gynecol 2004; 23(5): 496-500. Bourgain C. Devroey P. The endometrium in stimulated cycles for IVF. Hum Reprod Update 2003; 9(6): 515-22. Kupesic S, Bekavac I, Bjelos D, Kurjak A. Assessment of endometrial receptivity by transvaginal color Doppler and three-dimensional power Doppler ultrasonography in patients undergoing in vitro fertilization procedures. J Ultrasound Med 2001; 20(2): 125-34. Ng EHY, Chan CCW, Tang OS, Yeung WSB, Ho PC. Comparison of endometrial and subendometrial blood flow measured by three-dimensional power Doppler ultrasound between stimulated and natural cycles in the same patients. Hum Reprod 2004; 19(10): 2385-90. Raine-Fenning NJ, Campbell BK, Kendall NR, Clewes JS, Johnxon IR. Quantifying the changes in endometrial vascularity throughout the normal menstrual cycle with three-dimensional power Doppler angiography. Hum Reprod 2004; 19(2): 330-8. Yokota A, Nakai A, Oya A, Koshino T, Araki T. Changes in uterine and ovarian arterial impedance during the periovulatory period in conception and nonconception cycles. J Obstet Gynaecol Res 2000; 26(6): 435-40. Kupesic S. Three-dimensional ultrasonographic uterine vascularization and embryo implantation. J Gynecol Obstet Biol Reprod (Paris) 2004; 33(1 Pt 2): S18-20. Buckett WM, Chian RC, Tan SL. Human chorionic gonadotropin for in vitro oocytes maturation: Does it improve the endometrium or implantation? J Reprod Med 2004; 49(2): 93-8. Carbillon L, Perrot N, Uzan M, Uzan S. Doppler ultrasonography and implantation: A critical review. Fetal Diagn Ther 2001; 16(6): 327-32. Levi R, Ozcakir HT, Adakan S, Goker EN, Tavmergen E. Effect of ovarian cysts detected on the beginning day of ovulation induction to the success rates in ART cycles. J Obstet Gynaecol Res 2003; 29(4): 257-61.

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Techniques of Non-surgical Sperm Retrieval Rupin Shah, Vijay Kulkarni

INTRODUCTION Non-surgical sperm retrieval procedures are indicated when a man suffers from anejaculation (i.e. inability to produce an antegrade ejaculate) even though there is no organic obstruction to the passage of sperms. Anejaculation may be situational or total; situational anejaculation is always psychological in origin, while total anejaculation may be psychological (anorgasmic) or physical (orgasmic) in origin.1 a. Situational anejaculation: In this condition, a man is able to ejaculate in some situations (usually at home during intercourse), but has difficulty in doing so on other occasions. Typically, this failure may occur in the clinic (but not at home), during masturbation (but not during intercourse), or during ovulation / egg pick-up (but not when he is not under pressure). b. Non-organic total anejaculation: Here the man never reaches orgasm in the waking state (either by masturbation or during intercourse), and therefore does not ejaculate (hence Anorgasmic Anejaculation). However, nocturnal emissions are usually present.

c. Organic total anejaculation: In this case the man reaches and experiences orgasm but there is no antegrade ejaculate (hence Orgasmic Anejaculation), either due to failure of emission (because of anatomical block or damage to the sympathetic nerves) or due to retrograde ejaculation. In situational anejaculation sperm can be obtained by vibratory stimulation of the penis, which will produce orgasm and ejaculation in 90% of men. Anorgasmic anejaculation can also be treated by vibratory stimulation but the sperm retrieval rate is lower at around 60%. If vibrator therapy fails, electroejaculation can be performed; this will usually succeed in men with situational or anorgasmic anejaculation since there is no physical defect. However, often the sperm quality obtained by electro-ejaculation is inferior to that obtained by natural ejaculation or by vibratory stimulation .2 Men with organic anejaculation due to neurogenic failure of emission (e.g. after spinal cord injury, diabetes or lumbar sympathectomy) can be treated by vibratory stimulation or electro-ejaculation to retrieve sperm.3

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However, when the failure of emission is due to an anatomical block (e.g. after genitourinary tuberculosis) then non-surgical attempts at sperm retrieval will not succeed and operative sperm retrieval will be required. Organic anejaculation due to retrograde flow of semen into the bladder, when due to diabetic neuropathy, may respond to medical treatment with sympathomimetic drugs (ephedrine-25 mg four times a day) in combination with anticholinergics (imipramine-50 mg at night). But when the retrograde ejaculation is due to damage to the bladder neck because of surgery or trauma, then medical therapy will not help, and sperm will have to be retrieved from the bladder after alkalinizing the urine.

A

VIBRATOR THERAPY The vibrator acts by providing a high-intensity, vibratory stimulus to the penis for a long duration, sufficient to overcome any situational/neurogenic inhibition and trigger off the ejaculatory reflex.

B

FIGURES 4.2A and B: High intensity body massagers can also be used successfully for stimulation of the penis. These are inexpensive and can be purchased by the patient for home use

FIGURE 4.1: Dedicated vibrators made for penile stimulation allow modulation of frequency and amplitude of vibration, but are expensive

The procedure is carried out in a room with complete privacy. Preparatory counseling is important: The procedure is explained and it is emphasized that ejaculation will occur automatically as a result of the vibratory stimulation - the patient should not try and force ejaculation. The patient passes urine, takes off his clothes and sits on a bed with his legs apart. The vibrator (Figures 4.1 and 4.2) is positioned between the legs, facing upwards. The penis is placed upon the vibrating head such that the undersurface of the glans and distal shaft are stimulated. Once the patient is comfortable with the vibratory sensation, the glans is pressed down

Techniques of Non-surgical Sperm Retrieval upon the vibrator such that the penis receives the maximum amount of stimulation. Keeping the vibrator in place the patient then closes his eyes and fantasizes sexually (this may be aided with erotic magazines/ videos). Stimulation is continued till ejaculation occurs. This usually occurs in 10 to 30 minutes but some patients with anorgasmic anejaculation, who have never experienced orgasm, may take up to 2 hours of stimulation before they reach orgasm for the first time! This period shortens during subsequent sessions. Some patients require a second or third session before they succeed. If failure persists beyond this, electroejaculation is indicated.

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FIGURE 4.4: The Seager electro-ejaculator has one central electrode, flanked by two other electrodes that function as one unit

ELECTRO-EJACULATION Electro-ejaculation involves the direct, electrical stimulation of the efferent nerves innervating the seminal vesicles and terminal vasa. The most commonly used device is the Seager electro-ejaculator (Figure 4.3) which delivers a sine wave alternating current. The procedure is carried out under general anesthesia (paraplegic men with no sensations do not need anesthesia) with the patient in the left lateral position. The electrodes are mounted on a cylindrical rod (Figure 4.4); this is lubricated and introduced per rectum with the electrodes facing the prostate gland (Figures 4.5A and B). The voltage is turned up to 5

A

B

FIGURES 4.5A and B: Electro-ejaculation—The probe is introduced per rectum, with the electrodes facing anteriorly

FIGURE 4.3: The Seager electro-ejaculator

volts, held for a second and then turned back to 0 volts. For the next stimulus the voltage is increased to 6 volts. The stimuli are progressively increased till ejaculation occurs (Figure 4.6). Stimulation is then continued at

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FIGURE 4.6: Electro-ejaculation-the ejaculate is collected in a beaker and processed for IUI or ICSI. Several beakers may be used to collect the semen as a split ejaculate since the quality may vary between the first and the subsequent ejaculates

that level until there is no more ejaculation. Men with psychogenic anejaculation usually ejaculate between 5 and 15 volts, with a current of 100 to 300 mamps, but some men require a much higher current. If the antegrade ejaculate is scanty, the bladder is catheterized to check for retrograde ejaculation. The sperm retrieved are used for IUI or ICSI, depending on the semen quality.5 Most men with aspermia can be helped by the use of the vibrator or the electro-ejaculator. However, if these facilities are not available, or if the man has an anatomical block, then surgical sperm retrieval is required.

SPERM RETRIEVAL FROM THE BLADDER Men with retrograde ejaculation who have not responded to medical therapy (as described above) will need sperm retrieval from the bladder. Sperm may be retrieved from the urine or from medium which has been instilled in the bladder. SPERM RECOVERY FROM URINE Normally, urine is too acidic and concentrated for sperm survival. To improve sperm survival in urine, the urine must be made dilute (reduce osmolality) and

alkaline. Accordingly, to recover sperm in a man having retrograde ejaculation the following procedure is recommended. The man is given an alkalinizing drug (sodium bicarbonate or one of various commercial preparations) four times-a-day for two days prior to the retrieval. On the third day the patient drinks several glasses of water together with the alkalinizing drug. He then passes urine every 20 minutes and the urine is tested for pH (and osmolality if an osmometer is available). Once the pH is over 7.5 (and osmolality < 300 mosmol) then the man is instructed to masturbate. After reaching orgasm he passes urine, as soon as possible, collects it in a sterile beaker and hands it to the laboratory which should immediately process it by centrifuging the urine to separate the sperm. The sperm pellet is re-suspended in medium, centrifuged once more and then processed for IUI. SPERM RECOVERY FROM MEDIUM If the sperm retrieved by the above described method are sufficient in number but show poor motility then the following technique may be effective in improving the motility of the recovered sperm. The patient takes an alkalinizing drug for 48 hours prior to retrieval, as described above. On the morning of retrieval he is kept fasting to achieve mild dehydration and reduce urine formation. Prior to retrieval he takes the alkalinizing drug with a minimum amount of water. He passes urine and is then catheterized using sterile liquid paraffin as a lubricant (do not use anesthetic jelly because petroleum jellies are spermicidal). The bladder is emptied. If there is significant residual urine then the bladder is washed with 100 ml of sodabicarbonate solution. Once the bladder is fully emptied then 30 ml of a buffered sperm-washing medium is instilled in the bladder and the catheter is removed. The man masturbates to orgasm and then micturates; the voided medium is collected and immediately processed by the laboratory to separate the sperm for IUI. Since the medium is far more sperm-friendly than urine, the sperm recovered from medium are often far superior in quality.

Techniques of Non-surgical Sperm Retrieval CONCLUSIONS Laboratory personnel in charge on semen processing should establish rapport with the men who come to give a semen sample and discuss whether they have any difficulty collecting semen. In this way, many collections problems will be anticipated and appropriate corrective measures taken, reducing unpleasant surprises on the day of ovulation or egg pick-up. In many cases, allowing the man to collect semen at home, and if necessary by coitus interruptus or use of a non-spermicidal condom, will help solve many semen collection problems. Those who persist in having anejaculation can be treated with the vibrator or electro-ejaculator. When a problem is known beforehand, training with the vibrator should begin several weeks before the actual treatment cycle, so that the man has sufficient time to learn how to use the vibrator and gain confidence.

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REFERENCES 1. Shah R. Management of anejaculation. In: NP (Ed): Handbook of Andrology. Chennai: TR Publishers, 1999:129-39. 2. Hovav Y, Shotland Y, Yaffe H, et al. Electro-ejaculation and assisted fertility in men with psychogenic anejaculation. Fertil Steril 1996;66:620-3. 3. Nehra A, Werner MA, Bastuba M, et al. Vibratory stimulation and rectal probe electro-ejaculation as therapy for patients with spinal cord injury: Semen parameters and pregnancy rates. J Urol 1996;155:554-9. 4. Bennett C, Seager S, Vasher E, McGuire E. Sexual dysfunction and electro-ejaculation in men with spinal cord injury: Review. BJU 1991;67:191-4. 5. Ohl DA, Wolf LJ, Menge AC, Christman GM. Electroejaculation and assisted reproductive technologies in the treatment of anejaculatory infertility. Fertil Steril 2001; 76(6):1249-55.

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Surgical Methods of Sperm Retrieval Narendra Malhotra, Arun Tewari Jaideep Malhotra, Sudesh A Kamat, Ashok Sharma

INTRODUCTION Today with the availability of intracytoplasmic sperm injection techniques at almost all assisted reproduction techniques centers, it has become possible to achieve fertilization and pregnancies in men who produce only a few spermatozoa or even in men who produce no spermatozoa in the ejaculate. But in men with Aspermia (inability to ejaculate) and in men with Azoospermia (absence of sperms in ejaculate), sperms have to be obtained by some sperm retrieval technique prior to ICSI. Retrieval of sperms may be done by non-surgical or surgical methods.1-3

AZOOSPERMIA

FIGURE 5.1: Normal sperm count

Absence of sperms in the ejaculate is known as azoospermia and this may be obstructive or nonobstructive in etiology (Figures 5.1 and 5.2).

3. Ligation of vas deferens as a complication of hernia, prostate or seminal vesicle surgery. 4. Vasectomy and failed reversal of vasectomy.

Obstructive Azoospermia

Non-obstructive Azoospermia

1. Congenital absence of bilateral vas deferens (CABVD). 2. Epididymovasal occlusion due to infections.

In these cases there is an impairment of the process of spermatogenesis. 1. Hypogonadotropic hypogonadism.

Surgical Methods of Sperm Retrieval

FIGURE 5.3: Small testis FIGURE 5.2: Azoospermia

2. Non-hypogonadotropic azoospermia–primary testicular failure. Clinical Features of Obstructive Azoospermia • • • •

History of infections and previous surgery. Examination of testis and vas. Hormonal levels of anterior pituitary (FSH). Semen analysis showing low fructose in cases of seminal vesicle agenesis or ED obstruction. • Testicular biopsy showing normal spermatogenesis. Non-obstructive

FIGURE 5.4: Cryptorchidism

• History of mumps, trauma, cryptorchidism, chemotherapy radiation. • Examination of small testis and cryptorchidism (Figures 5.3 and 5.4). • FSH is high in testicular failure cases. • FSH is low in hypogonadotropic hypogonadism • Testicular biopsy will show spermatogenic arrest, germinal aplasia, (Sertoli cell only) or testicular fibrosis (Figure 5.5).

SURGICAL METHODS OF SPERM RETRIEVAL Indications 1. Obstructive azoospermia (reconstruction not possible).

FIGURE 5.5: Testicular fibrosis

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3. 4. 5. 6.

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a. Bilateral congenital vas aplasia. b. Iatrogenic obstruction (surgeries). c. Tuberculosis. Obstructive azoospermia failed reconstruction at a. VEA. b. Vaso-vasostomy. Choice of couple in obstructive azoospermia. Non obstructive azoospermia focal spermatogenesis. Situational Anejaculation during an ART cycle. Total astheno/necrozos permia.4

Techniques Surgical techniques may be open procedures or may be percutaneous procedures. Open procedures cannot be repeated on the same side for at least 4 months. FIGURE 5.6: MESA

Epididymal Sperm Retrieval (OPEN) 1. MESA (Microsurgical epididymal sperm aspiration).5 • Scrotum is opened • Epididymis revealed • Tunic incised, expose epididymal duct microsurgically • Aspirate fluid (Figure 5.6). 2. OFNA (Open fine needle aspiration) • Expose epididymis • Directly puncture duct with a 26 G needle • No dissection or suturing involved • Simple procedure • Only few minutes.

Epididymal Sperm Retrieval (Percutaneous) 1. PESA (Percutaneous epididymal sperm aspiration)6 • Puncture through scrotal skin (Figure 5.7) • Insulin syringe with 26 G needle • Quick • Simple • Easily can be repeated • Future VEA jeopardized. Testicular Sperm Retrieval (Percutaneous) (Figure 5.8) 1. TESA (Testicular sperm aspiration)7,8 • Scalp vein set 22 G

FIGURES 5.7A to C: PESA

Surgical Methods of Sperm Retrieval

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3. Needle biopsy • Tissue biopsy needle • TRUCUT needle • Biopty gun10 • Tissue recovered is less than NAB • High chance of hematoma formation 4. RETA (Figure 5.10) (Rete testis aspiration) • Testicular tissue is aspirated from Rete testis area. • Technique similar to needle biopsy.

FIGURE 5.8: TESA

• Suction via 20 ml syringe • Aspirate a tubule • Slight chance of hematoma9 2. NAB (Needle aspiration biopsy)—TESA (Figure 5.9) • 17/18 G scalp vein • Local anesthesia • Suction after needle has entered testis • Rotate needle and push in and out • Clamp scalp vein tubing • With draw carefully • Strand of testicular tissue grasped and pulled out • Tease the tissue and retrieve sperms.

FIGURE 5.10: RETA

5. SPAS (Figure 5.11) • Aspiration of spermato cell fluid

FIGURE 5.11: SPAS

Testicular Sperm Retrieval (Open Methods) FIGURE 5.9: NAB—TESA

1. Conventional biopsy (Figure 5.12)

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FIGURE 5.12: Conventional

FIGURE 5.14: Micro biopsy

2. Testicular biopsy (Figure 5.13) 3. Micro biopsy (Figure 5.14) a. Single seminiferous tubule b. Microsurgical selective biopsy11 3. TESE (Testicular sperm extraction) (Figure 5.15)

CONCLUSIONS FIGURE 5.13: Testicular

Today it is possible to extract sperms by various techniques surgical or non-surgical in men who

FIGURE 5.15: TESE

Surgical Methods of Sperm Retrieval previously had no option other than donor insemination or adoption. It is advisable to use the simplest technique first which is least non-invasive. ART has seen a revolution with ICSI and sperm retrieval techniques and it has now become possible for azoospermic men to have and to father their own genetic offspring.

ACKNOWLEDGEMENTS Sincere thanks to Dr Jatin P Shah and to Dr Rupin Shah for introducing us to the wonderful world of ART.

REFERENCES 1. Shah R. Management of anejaculation. In NP (Ed). Handbook of Andrology. TR Publishers, Chennai 1999; 129-39. 2. Shah R: Ejaculatory and erectile dysfunctions in infertile men. In Jansen R, Mortimer D (Eds): Towards Reproductive certainty. The Partheon Publishing Group, New York 1999. 3. Bennett C, Seager S, Vasher E, et al. Sexual dysfunction and electro ejaculation in men with spinal cord injury: review. Br J Urol 1991;67: 191-4.

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4. Verheyen G, Joris H, Critis K, et al: comparison of different hypo osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Human Reprod update 1997;3: 195-203. 5. Giradi SK, Schlegel P. MESA: Review of techniques, preoperative considerations and results. J Andrology 1996; 17: 5-9. 6. Craft I, Tsirigotis M, Bennett V, et al: Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in management of infertility due to obstructive azoospermia. Fertile Steril 1995;63: 1038-42. 7. Tournaye H, Clasen K, Aytoz A, et al. Fine needle aspiration versus open biopsy for testicular sperm recovery: A controlled study in azoospermic men with normal spermatogenesis. Hum Reprod 1998;13: 901-4. 8. Turek PJ, Givens CR, Schriock ED, et al: Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine needle aspiration mapping in patients with non obstructive azoospermia. Fertil Steril 1999;71: 552-7. 9. Belenky A, Avrech O, Bacher G, et al. Ultrasound guided testicular sperm aspiration in azoospermic patients a new sperm retrieval method for intracytoplasmic sperm injection. J Clin ultrasound 2001;29: 339-43. 10. Money AF, Deshon GE Jr, Rosanski TA, et al. Technique of biopsy gun testis needle. Urology 1993;42: 325-6. 11. Schlegel PN. Testicular sperm extraction: Microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14: 131-5.

The ART Laboratory

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6

The ART Laboratory Hrishikesh Pai, Shilpa Shah Rishma Pai, Nandita Palshetkar

INTRODUCTION Setting up of an high quality ART laboratory is of utmost importance for achieving consistently high pregnancy rates.This is mostly dependant on the location and structuring of the laboratory, high quality of lab equipment, skilled and trained laboratory staff as well as a good quality control (QC) and quality assuarance (QA) program. This chapter discusses the set up of an ART laboratory in all its aspects.

LAB DESIGNING

6. Autoclave room 7. Semen collection room: This should be in a secluded private area. It should have a wash basin with an attached toilet 8. Semen processing laboratory: This room should be near the semen collection room. It should have a sterile laminar flow hood and other equipment used for preparing semen. The lab should maintain good laboratory practice and should take care to properly dispose of the wastes as per government guideline 9. A room for performing IUI.

STRUCTURING

The Sterile Area

As per recent Indian Council of Medical Research (ICMR) guidelines, an ART clinic should a have a sterile and nonsterile area.

The entry to this area must be strictly controlled by an anteroom for changing footwear, area for changing into sterile garments and a scrub station. The sterile area must be airconditioned wherein fresh air filtered through an appropriate hepa filter system is circulated at ambient temperatures of 22 to 25°C. It should have the following three rooms: 1. The operation theater 2. The embryology lab complex 3. The embryotransfer room.

The Nonsterile Area 1. 2. 3. 4. 5.

A reception and a waiting area An examination room A general purpose clinical laboratory A store room A record room

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Atlas of HART noted before selection of the location. Basic air sampling, determination of the volatile organic compounds(VOCs) and ozone measurements should be done both inside and outside of the proposed area. The laboratory should be next to the operating theater. In our personal experience it is preferable to avoid basements and areas which are facing the road. The higher floors are better than lower floors. INTERIORS

FIGURE 6.1: Clean air filtration outflow from lab

FIGURE 6.2: Steel roof painted with epoxy +hepa filter + hanging steel c

As per the ICMR guidelines some of the spaces mentioned above may be combined, as long as they do not compromise on patient care and quality of service. However, the space provisions of the sterile area cannot be combined with space provisions in the nonsterile area and vice versa. LOCATION The choice of the site of the clinic and laboratory is based on various other external factors. The surrounding area, the adjacent buildings, any construction, demolition of adjoining property in foreseeable future, industrial hazards, general pollution, etc. should be

• Over pressured laboratory that uses a high number of fresh air changes. This pressure module creates positive pressure in the laboratory. • All electrical wiring should be concealed. • The lab wall and ceiling should have absolute minimum number of penetrations and airtight utility connections. It is better to avoid false ceiling. • Ducts and equipments must be laid out in such a way that all kind of repairs can be performed outside the lab with minimum lab dysfunction. • Split or central air conditioner is preferable to a window air conditioner. Air conditioning vents have to be fitted with HEPA filters to filter the air. The laboratory should not get contaminated when the AC is being serviced or removed for repairs. Most of the new labs are being equipped with Air Handling Units (AHU) which constantly recycles the air and pass the air through multiple heap filters. In places which have high humidity levels in the atmosphere. one can use dehumidification systems, while passing air through hepa filters. This prevents the moistening of the laboratory walls, if there are temperature fluctuations in the lab. • The floor should be covered with vinyl or ceramic tiles, so that is easy to clean. • The walls should also be covered with tiles. Alternately one can cover the walls with epoxy paint or stainless steel. • Wall junctions should be covered to prevent dust accumulation (Figure 6.3). • The roof should be without a false ceiling. In our new laboratories, we have roofs made of stainless steel and fitted with Hepa filters. The filtered air is conveyed via ducts and enter the laboratory through the Hepa filters (Figure 6.2) placed on the

The ART Laboratory roof. The return air is taken back to the AHU via ducts placed at the floor level (Figure 6.1).

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• Ultraviolet light is necessary to create a sterile area in the laboratory. It is important not to run the UV light when work is going on in the laboratory (Figure 6.5). This can damage the eyes of the workers. One can place the start button of the UV lights outside the lab door. This will enable one to switch on the lights, without getting exposed to them. Normally, one can run the ultraviolet lights on the weekends when there is no work in the lab. Many labs do not like to use UV light, as it can break oxygen into ozone, which may be toxic to the embryo.

FIGURE 6.3: Covering at wall junctions to prevent dust accumulation

• There should be small hatch and a door connecting the lab and operating theater. The hatch is used to pass the tubes filled with follicular fluid into the lab, during the oocyte retrieval. The door is used to transfer the embryotransfer catheter loaded with embryos into the embryotransfer room during ET procedures (Figure 6.4).

FIGURE 6.5: UV lights

• As far as possible one should provide a separate storage closet or space for the liquid nitrogen containers and the CO2 gas cylinders, in the vicinity of the laboratory. One can also keep the cylinder outside the laboratory and bring the CO2 gas to the incubators, in via copper tubing or silicone tubing. This would prevent contamination of the lab, when on is changing the cylinders. EQUIPMENTS Essential Equipments

FIGURE 6.4: Steel door between operation theater and laboratory

• Plain incubator set at 37°C, which can be primarily be used for placing the semen collection jars for liquefaction, prior to sperm processing. Alternately one can also place the jars on digital heaters whose surface temperature is set at 37°C.

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FIGURE 6.6: CO2 cylinders

• Carbon dioxide incubator: This is the heart of the ART lab. In case of CO2 incubators, one can either use water jacketed or non-water jacketed incubators (Figure 6.7). The new incubators have sensors based on infrared technology. It is better to go for non-water jacketed incubators with

infrared control. These are easy to maintain, unlike water jacketed incubators. Nowadays many incubators come with facilities to take the temperature up to 90°C or 180°C. This higher temperature tends to kill the organisms and helps in decontamination measures. It is essential to have at least two incubators in the laboratory. • Sterile laminar flow hood: It should also have thermostatically controlled heating plate. This can be either a vertical or a horizontal flow A vertical flow is less harmful to the lab workers the air flow are not directly aimed at their face. On the other hand, a horizontal flow is more effective in reducing contamination. For IUI, one can order for a smaller one and a half feet breadth laminar flow. It is best to ask for a stainless steel top, as it is durable, easier to clean and maintain as well as it is more aesthetic. One can start the laminar flow 1 hour before preparing samples (Figure 6.8). Nowadays one can order laminar flows with their tops heated digitally by heaters to maintain 37 °C table top temperature. There should be two separate CO2 cylinders for different incubators (Figure 6.6).

FIGURE 6.8: Laminar flow workstation

FIGURE 6.7: Double stacked incubators

• Binocular microscope preferably phases contrast to test the semen for count, motility, morphology and other components such as pus cells. The microscope should have 10x, 20x, 40x and 100x objectives, and a 10x eyepiece.

The ART Laboratory • A stereozoom microscope to scan the follicular fluid, dissect the eggs, inseminate the eggs and load the embryotransfer catheter. • A high resolution inverted microscope with phase contrast or Hoffman modulation contrast optics, preferably with facilities for video recording. • Micromanipulator system for intracytoplasmic sperm injection (Figure 6.9). Preferably each workstations and microscope to be equipped with still camera and/or video camera and a medical monitor.

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or the Makler chamber are commonly used. Although expensive, use of Makler camber is highly recommended as it is very accurate. In recent times disposables sperm counting slides are also available. One slide can be used for analyzing 2 samples. • Refrigerator: This is used to store the various chemicals and culture medias. The fridge should not be used for any other activity. • Liquid nitrogen containers to store sperms,oocytes and embryos. Optional Equipment • Oocyte spindle view machine for the visualization of the internal elements in order to avoid the damage to the chromosomes and to achieve better fertilization rates, during ICSI (Figure 6.10).

FIGURE 6.9: Micromanipulator

• Centrifuge this should have a timer (up to 30 minutes) and a rotor (up to 3000 rpm) whose speed can be controlled. It is better to go for a swing out rotor head. • Digital heater: This can be used especially if HEPES buffered media are used instead of bicarbonate based media. • Cryofreezer equipment for freezing embryos in a programmed manner. • Generators: Even if the electricity supply is reliable, spare generators has to be installed with each unit, in case of emergency. Additional battery “uninterruptible power system” (UPS) must be also installed though its for short period. • Sperm counting chamber: There are various chambers available for analyzing sperms. Nondisposable systems such as the Neubaur chamber

FIGURE 6.10: Spindle view oocyte with laser

• Assited laser Hatching system • Preimplantation genetic diagnosis: fluorescent microscope with FISH software and PCR systems. • Automatic sperm analyzer systems: Systems such as CASA by Hamilton Thorne are being routinely used in labs with a large volume of work. They can also be used when extensive research work on semenology is being carried out in the laboratory. However, it is not required to run a routine IUI laboratory.

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• CODA filters which can be placed in the lab as stand alone units. One can also use inline filters to pass CO2 gas from the cylinder to the incubator. One can also have incubator based filters to filter out the air in the incubator. We change the coda filters every month. STAFFING Depending upon the case load, the staff requirement should be defined. The minimum staffing requirement as per the ICMR guidelines are as follows: 1. Gynecologist 2. Andrologist 3. Program coordinator 4. Clinical embryologist 5. Counsellor. In the lab, besides the senior embryologist one can have a, assistant embryologist and a technician. Their job should be properly assigned for each position for example, the embryologist has to concentrate on gamete and embryo handling and has to also spend quality time on quality control, standardization of the procedure and training. Deviation/loading of work on staff can seriously cause harm to the laboratory outcome. There may be other positions for preimplantation genetics (depending upon facilities to be availed to the patients), research or secretarial work.

GENERAL MEASURES IN THE LAB • It is advisable to change into sterile clothes, slippers, cap and mask when one is working in the lab. This maintains good sterility in the lab area. It is preferable to have clothes made up of terry cotton or silk instead of linen. This will prevent the shedding of small threads from the linen clothes that can contaminate the laboratory environment. However this is not mandatory. • Soap or detergents are not to be used for cleaning. • Bright light can damage eggs and embryo, thus when handling them the light in the laboratory should be as dim as possible.

• The temperature should be maintained at 23 -25°C inside the laboratory. • All tabletops should have laminated or stainless steel tops for easy cleaning. • Alcohol (70% absolute or 70% isopropyl) should be used for cleaning only after the days work is over. • Wet mopping is done regularly so as to free the laboratory from fibers and strands. • Entrance to ART laboratory should be restricted to laboratory staff only. • Washing, changing of clothes wearing a mask, cap and slippers is compulsory before entering the laboratory. Laboratory slippers cannot be taken outside the laboratory. • The CO 2 cylinders should be checked twice everyday. • Keep a constant check on the CO2 levels and the temperature of the incubators continuously. Incubators should be opened as less frequently and as briefly as possible. • The water level in the incubators should be regularly checked and replenished with sterile filtered water when required. • Liquid nitrogen levels to be regularly checked and should be filled on time.

GENERAL RULES FOR LABORATORY CULTURES There are basically two types of culture media used in the laboratory. One is follicle flushing media and the other IVF culture media. Follicle flushing medium has got HEPES which maintains a stable pH in the bicarbonate buffered system which can be used for basic procedures like follicle flushing, sperm preparation, oocyte harvesting, and dissection of oocytes and preparation of ICSI plates. However, since HEPES has been known to alter the ion channel activity in the plasma membrane, it can be embryo toxic. Thus the gametes must be washed in HEPES free medium before overnight culture in IVF culture medium. • Commercially available media is stored in the refrigerator.

The ART Laboratory • When it is to be used, it must be removed from the refrigerator and kept in the laminar flow to come to room temperature. • The bottle is then cleaned with alcohol and kept under the laminar flow hood for a while so that the alcohol fumes evaporate. • The bottles are opened carefully in the laminar flow under aseptic conditions and alliquoted in tubes. Media should not be poured out, but pipetted out. • Ensure heating blocks and stages are pre-warmed to 37°C. Human oocytes are extremely sensitive to temperature and pH conditions and transient cooling can cause irreversible damage to the meiotic spindle. • Culture plates and ICSI plates are overlaid with oil, as the oil acts as a physical barrier to the atmosphere and airborne particles. It prevents evaporation, delays gas diffusion thereby keeping the pH, temperature and osmolarity of the medium stable. It’s thereby protecting the oocytes and embryos form significant fluctuation in their micro environment. • All tissue culture plates, media and oil are incubated in the CO2 incubator overnight before use to equilibrate in 5% CO2 and 37°C to imitate the physiological conditions present in the body. • If, leaving the media in the incubator is not possible CO2 has to be gassed in the media before use.

QUALITY CONTROL IN THE ART LABORATORY Every IVF lab should have a quality management system to establish and maintain a strict Quality control (QC), A good QC/QA system would help ensure an optimal quality of treatment. Although many national registries emphasize pregnancy rate or live birth rates as the sole outcome measure of good QA/QC, this is not good enough. A good QA/QC system should ensure that the laboratory will be successful and guarantee optimal standards of treatment (pregnancy rates) and care (patient satisfaction) for its patients. It contains the following:

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QUALITY CONTROL This consists of establishing quality standards, testing specific parameters within the laboratory, monitoring and taking corrective measures. Staffing This should be adequate. A ratio of 100 to 400 cases per embryologist have been quoted as ideal. An average of 200 cases per embryologist is an acceptable level. Furthermore the embryologist should undergo personal audits of their performance. Procedures Standard operating procedure (SOP) is a must. This is a written document describing each procedure step by step, as it is being carried out in the lab. This also includes details of the relevant equipment, materials and standards used. All staff should read and sign the SOPs. These can be regularly updated when necessary. Laboratory Equipment QC The objective is to ensure that one uses high quality equipment, which is frequently maintained and quality controlled. The following equipment should undergo a regular QC: a. Incubator: Daily recording of temperature (36.5 to 37.5) and CO2 reading(4.5 to 5.5) should be done. These should be verified at regular intervals with independent CO2 monitors(digital or fyrite) and thermocouples(digital thermometers). The pH of culture media in the incubator should be monitored using a microprobe attached to a pH meter. b. Microscopes and heated stages: This can be evaluated by placing the probe into a petridish on the heated stage of the inverted or stereozoom microscope. The temperature should be maintained at 37°C +/- 0.5°C for a fixed time period such as half to one hour. c. Flow hoods/air conditioners and air handling units: These need to be serviced at regular intervals from an independent contractor. We service our hoods once in two months.

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d. Refrigerators: Most culture medias are stored in the fridge. Temperatures are recorded daily, by placing a thermometer in the fridge. e. Sterilizing oven: Most laboratories use disposable ware, However those using ovens to sterilize glassware should monitor the oven temperature of 180° by using a independent thermocouple. f. Liquid nitrogen dewars: The storage dewars must be filled regularly as per schedule and low level alarms should be installed. g. Air quality: Particulate count in the laboratory, the temperatures in the laboratory and bacterial contamination of the table tops in the laboratory can be assessed by new equipments which have been recently released into the market. h. Calibration equipment: These should be standardized by recognized equipment manufacturers. i. Plastic ware and culture media: The batch and company name needs to be recorded. The date on which a particular batch was started also needs to be recorded. Although most products are tested by the manufacturers a standard 48 hours sperm survival test can be used to assess new batches. General Audit In this the performance of certain parameters can be compared with set standards. These parameters can be any or all of the following: a. Oocyte maturity in ICSI b. Fertilization rate c. ICSI damage rate d. Embryo cleavage rate e. Pregnancy rate. Personal Performance Audit These may include number of oocytes collected per

embryologist, the fertilization rates per ICSI oocytes, pregnancy rates per embryologist, etc. QUALITY ASSURANCE (QA) This encompasses all aspects of quality control plus written SOPs. It also encompasses other areas such as quality systems and planning. An important area is to subject the laboratory to scrutiny by external monitoring agencies. TOTAL QUALITY MANAGEMENT (QM OR TQM) This encompasses QC and QA. It is not only restricted to the ART laboratory, but includes all the functions in the ART unit. It involves the patients, suppliers and looks at the entire functional strategy of the ART unit. Although several QM systems are in place worldwide the ISO 9001:2000 series has become most popular. The authors unit is ISO 9001:2000 certified A functioning QM system allows the ART clinic control of its procedures, monitoring of its clinical and non-clinical performance and improvement of its functioning.

BIBLIOGRAPHY 1. Elder K Elliot T (Eds): Worldwide Conference on Reproductive Biology West Lederville. Australia. Ladybrook Publishing 1998;33-5. 2. ISO 9001: 2000 Quality Management systems Requirements. www.iso.ch. 3. Kastrop P. Quality Management I the ART laboratory Reprod Biomed Online 2003;7:691-4. 4. Keck C. Quality Management in Assisted Reproduction. Prague: KAP Ltd.2003:23-4. 5. Moore LM. High standards ISO 9000 comes to health care Trustee 1999;52:10-4. 6. National Guidelines for Accreditation,supervision and regulation of ART Clinics in India. Indian Council of Medical Research(ICMR). National Academy of Medical Sciences (India) 2004.

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Techniques of Sperm Preparation for ART Sushma Ved, Sonia Malik

INTRODUCTION There is no doubt that semen processing technique used and the care employed are crucial to success of ART. Human spermatozoa vary widely in their characteristics from individual to individual and from sample to sample intra-individual. Even a normal fertile sample contains millions of non-motile sperms. Therefore there are many spermatozoa in a sample that are unlikely to play a role in fertilization and would simply serve to contaminate that culture system if they were not removed. Ejaculated semen is a viscous liquid composed of a mixture of seminal plasma, testicular and epididymal secretions, Polygonal Epithelial cells from urethral tract, spermatogenic cells and leukocytes produced at the time of ejaculation. The seminal plasma contains substances which inhibit capacitation and prevent fertilization.1 With the introduction of ICSI 2 now provides effective IVF treatment for even the most severe cases of male infertility which were previously untreatable. Regardless of the technique used the preparation of semen specimen must be as gentle as possible in order not to affect the fertilizing potential of normal

spermatozoa. During processing, sperm function may be threatened by the secretions of cytotoxic cytokines and the generation of free oxygen radicals (ROS) responsible for the initiation of a deleterious lipid peroxidation cascade in the sperm plasma membrane. A variety of semen processing techniques are described in the literature that is recommended for ART to produce significantly higher percentage of functional spermatozoa. Each of these techniques has its specific advantages and drawback and all methods may be suitable for normal semen samples. Accordingly each laboratory has its own preferred routine methods, subnormal samples indeed present a challenge in such cases it is advantageous to able to draw experience, gathered with different techniques and to use them as circumstances required. Selecting the optimal method for a given sample will depend on the parameters of that sample and procedure for it is intended. Ejaculated as well as epididymal and Testicular spermatozoa are currently frozen as a routine for reasons of convenience or to ensure later fertility. With the development of ICSI (one oocyteone sperm) the storage of sperm whatever source and of whatever quality has become worthwhile.

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SEMEN COLLECTION Semen is collected by masturbation into a 60-100 ml. wide mouth sterile container (glass or nontoxic polypropylene Jar) after an optimal period of 3-5 days of sexual abstinence.4 The container should be labeled with the patients name or Identification code. Special rooms should be available which guarantee a private atmosphere. If the patients is not able to deliver his ejaculate since psychological stress factor on the day of IUI or egg retrieval in case of IVF ICSI patient may collect at home and transported to the hospital within 30-60 minutes. Care must be taken to protect from extreme temperature during transportation, as sperm motility and velocity are dependent on cellular metabolism, are therefore temperature sensitive. Sperm motility should be assessed at standard temp of 37°C. Kenntenich et al 5 recommended sample collection in the presence of the patients wife to reduce stress during ART procedures. For cases of impaired semen quality several modifications of sample collection have been established. One method is the separate collection of the first (two contractions) and second (remaining contractions) semen fractions (split ejaculate). In contrast to split ejaculate the total number of motile spermatozoa can be increased by pooled sequential ejaculates.6 Patients are asked to produce two ejaculates within a period of 2-4 hours. In exceptional cases where the volume of the ejaculate is very less, highly viscous, the sperm concentration is very high or in case of retrograde ejaculates the ejaculate is collected in a container containing sperm preparation medium or culture medium is added to the ejaculate immediately after collection and mixed with medium as there is danger of drying of ejaculate (where the volume of ejaculates very less) or damage to spermatozoa in retrograde ejaculate with different PH of urine. In some patients there are ejaculatory disorder. With regard to ART clinically important disorders are—Retrograde ejaculations and anejaculation. Retrograde ejaculation7—since urine has a deleterious effect on sperm quality. Recovery of sperms from urine is only successful when pH (7.4) and

osmolarity (320 mosm/kg) are adjusted by oral intake of sodium bicarbonate and water. Alternatively culture medium can be injected into the bladder before ejaculation. STEPS 1. Patient is asked to drink 1-2 glass of water with intake of sodium bicarbonate. 2. Empty the bladder—by passing urine. 3. Fill the bladder with culture medium (in case sodium bicarbonate is not taken). 4. Patient is asked to masturbate. 5. Collection of urine in 2 containers: First–Collect 10 ml of urine in first container rest of urine collect in second container. In case of anejaculation and intact reflexes, Penile vibratory stimulation (PVS) to the frenulum may be useful.8 PVS is not indicated for anejaculation due to lesions of the peripheral nerve fibers. Electroejaculation has also been applied in men with spinal cord injury. If conservative treatment for vibratory stimulation and electro ejaculation fails, MESA, TESE are suitable to retrieve spermatozoa for ICSI. In case of when fresh semen samples are not available frozen sperm are used. After collection of semen sample it is left for liquefaction at 37° C which normally occurs within 30- 60 minutes. In cases of impaired liquefaction (often referred to as viscosity) vigorous pipetting or syringing may help to reduce viscosity. The presence of mucous streaks a sign of incomplete liquefaction may interfere with the counting procedure. Normal semen sample may contain Jelly-like grains (gelatinous bodies) which do not liquefy, the significance of this finding is unknown. The semen sample should be thoroughly mixed, examined, processed and inseminated immediately. THE AIMS FOR SPERM PREPARATION 1. To remove seminal plasma (which contains prostaglandins, cytokines as well as possible autigenic or infectious matter which prevents capacitation and fertilization of oocytes.

Techniques of Sperm Preparation for ART 2. To concentrate progressively motile, functional and morphologically normal sperms. 3. To remove defective and non vital sperms as well as cells (polygonal epithelial cells from urethral tract), spermatogenic cells and leukocytes. 4. To retrieve sufficient motile sperms for ICSI in cases of severe oligozoospermia, (presence of few motile sperms) testicular and epididymal sperms. 5. Selection of viable sperms for ICSI in cases of complete Asthenozoospermia by Hos test and by Laser. 6. Freezing (to ensure-later fertility) and sufficient recovery of motile sperms after thawing from frozen semen samples, processed—Frozen testicular biopsy sperm suspension and frozen sperm from MESA (microsurgical epididymal sperm aspiration). As the method of sperm preparation differ according to sperm parameters, semen analysis and their diagnostic criteria with predictive value must be developed in order to decide which form of ART and which method of sperm preparation should be recommended.

CENTRIFUGATION–SWIM UP TECHNIQUE (FIGURE 7.1) The principle of centrifugation is to separate cellular (sperm, round cells etc) and acellular (seminal plasma) from semen component. After centrifugation the progressive motile sperms swim up from the pellet to the fresh media so that they can be selected for insemination. The ejaculate is mixed with culture media and centrifuged. The supernatant containing seminal plasma is rejected, while sperm containing pellet is carefully overlaid with fresh medium, during subsequent period (30-60 minute) the motile sperm migrate into medium, part of supernatant is aspirated with a pipette and used for Insemination. STEPS 1. Determine semen volume, count and motility by measuring 10 μl of liquefied semen into the Makler chamber.

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2. Add sperm prep. medium to the specimen in 1:1 mix well and centrifuge in round bottom 5 ml tube at 550 μg at 2000 rpm for 10 minutes. 3. Discard the supernatant add 2-3 ml of sperm preparation medium to the pellet and mix 4. Centrifuge again at 550 μg for 5-10 minutes. 5. Discard the supernatant. 6. Suspend the pellet with 0.3-0.5 ml of IVF medium very carefully without touching or disturbing the pellet. 7. Keep sperm pellet in CO2 incubator for 30-45 minutes for swim up. 8. Take out from incubator, aspirate the swim up carefully with a 21 gauze needle and transfer in a new 5 ml tube. 9. Look for sperm count motility. 10. Ready for Insemination (IUI, IVF, ICSI).

SWIM UP FROM SEMEN9 (FIGURE 7.2) With this method several aliquots of liquefied semen are placed in round bottom tubes under neath and overlay of culture medium. Preferably small volume (1 ml of semen) and 2 ml of sperm prep. medium is used. This portioning process help to maximize the combined total interface area between semen and culture medium . The tubes may also be prepared by gently layering culture medium over liquefied semen. ADVANTAGE OF SWIM UP FROM SEMEN 1. Centrifugating only once, minimizing the Trauma of centrifugation on sperms 2. Centrifugating only motile sperms (without leukocytes, and immotile sperms, debris) in this way reducing the effect of ROS (produced by leukocytes, immotile sperms) 3. Better recovery of motile sperms in case of viscous semen samples and oligozoospermia. STEPS 1. Pipette 2 ml of sperm prep. medium in 13 ml round bottom tubes. 2. Gently pipette 1 ml of liquefied neat semen underneath the medium (being very careful not to

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FIGURE 7.1: Centrifugation SWIM up technique

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FIGURE 7.2: Continued

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FIGURE 7.2: SWIM up from semen

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3.

4. 5. 6. 7.

disturb the interface formed between the semen and the medium). More than one tube is needed as only 1 ml of ejaculate is overlaid in one tube. Allow to stand for 45-60 minutes at 37° C when the sperm migrating from the semen the overlaid medium, it turns opaque or milky. Carefully aspirate all the supernatant (if more then 1 tube). Centrifuge collectively in a 15 ml conical tube. Aspirate the whole supernatant carefully without disturbing the pellet. Add 0.2-0.5 ml of IVF medium to the pellet mix well ready for insemination.

MINI SWIM UP10 (FIGURE 7.3) The principle of the mini-swim up technique is to concentrate motiles sperms in the smallest possible volume of culture medium in cases of oligo asthenoterato zoospermia. The procedure is as follows: Up to step-2 same as in centrifugation and swim up. 1. Discard the supernatant. 2. Add 0.5-1 ml of sperm prep. medium to the pellet mix well and transfer in an Eppendorf tube. 3. Centrifuge at 550 μg, 2000 rpm for 5-10 minutes. 4. Carefully lift the supernatant. 5. Overlay pellet with 10-20 μl. of IVF medium. 6. Incubate for 30-60 minutes at 37°C. 7. Lift the supernatant and use part or all the supernatant for insemination.

CENTRIFUGATION AND WASHING (FIGURE 7.4) This method is recommended to retrieve the very few motile sperms for ICSI present in a semen sample, must be performed before diagnosing Azoospermia and before patient is subjected to surgical retrieval of sperms. Presence of few motile sperms in an ejaculate can be missed while looking into the Makler chamber as sperms may not be present in that small quantity of

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ejaculate which is examined (20-30 μl). STEPS 1. Add equal quantity of sperm prep. medium in liquefied ejaculate in conical 15 tube and mixed well. 2. Centrifuge at 550 μg at 2000 rpm for 10 minutes. 3. Discard the supernatant by aspiration. 4. Mix the pellet in 20-30 μl of culture medium. 5. Put small quantity (2-3 μl) of mixed pellet into the microdroplets (50 μl) under oil made of culture medium in a culture or ICSI dish. 6. Look for the sperms in microdroplets on a heated stage with forty × objective microscope equipped with micromanipulation device—the motile sperms with good progression swim to the periphery of the microdroplets but motile sperm with poor progression can be seen mixed with debris in the droplets. Once the motile sperms is seen it can be used for ICSI . In order not to miss even a single sperm the whole mixed pellet should be looked for presence of any spermatozoa in several microdroplets.

DENSITY GRADIENT SEPARATION SYSTEM (FIGURE 7.5) Buoyant density gradient centrifugation provides for a substantial separation of progressively motile, high quality spermatozoa by virtue of their enhanced velocity and relatively high density 11 and at the same time produces high quality samples that are essentially free from microbial or other contaminants.12 It has been demonstrated that the density gradient system keep lipid peroxidation of spermatozoa low by separating most of the ROSproducing cells from the normal and functional cells and that spermatozoa prepared by this approach have an enhanced capacity for fertilization. 13 The mechanisms underlying the selection of highly motile spermatozoa are not completely understood. It may be assumed that because of the density gradients, progressively motile sperm yielding to the centrifugal force—tend to actively migrate to the bottom of the

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FIGURE 7.3: Mini SWIM up technique

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FIGURE 7.4: Centrifugation and washing

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FIGURE 7.5: Buoyant density gradient method

centrifuge tube.14 The best known buoyant density gradient substance and most widely used in clinical application has been percoll15. How ever, a few years ago serious concern was expressed because of PVP component and endotoxin level of percoll, a colloidal polyvinylpyrolidone (PVP)—coated silica particle preparation.

This is why percoll should definitely not be used any more for human gametes. Other colloidal gradients which are assumed to be less harmful contain silica particles coated with silane instead of PVP (e.g. Pure sperm, isolate, sil select). There are two type of density gradients—continuous, and discontinuous. While with continuous gradients density increases

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g r a d u a l l y from the top to bottom. Discontinuous gradients are composed of several layers of the different concentration typically in 40-90 range with both methods gradients are overlaid with semen and centrifuged. The discontinuous density gradient, usually consisting of two to three layers is used most widely. DISCONTINUOUS DENSITY GRADIENT 1. A two layered gradient column is prepared by placing for example 1 ml of a 90% solution on the bottom of conical tube and add 1 ml of 45 % solution—gently without disturbing the interface between two concentrations alternatively 90% layer may be pipetted under the 40% layer. 2. A maximum of 2.00 ml of liquefied ejaculate is layered over the gradient. 3. Centrifuge the tube at 550 μg for 10 minutes, 2000 rpm. 4. Remove the supernatant the bottom layer of the gradient, containing the highest percentage of motile is mixed with 2 ml of physiological culture medium. 5. Centrifuge for 10 minutes. 6. Remove the supernatant. 7. Mix the pellet with 0.3-0.5 ml of culture medium ready to use for inseminator. This method is most widely used in sperm preparation for IUI’S

GLASS WOOL FILTRATION (FIGURE 7.6) A glass wool filter, Trapping in its fabric a high percentage of immotile, membrane defective and agglutinated sperms as well as leukocytes, immature germ cells, epithelia and debris, is used to isolate high-quality spermatozoa.16 Ready to use glass-wool filtration kits are commercially available. However a selfmade system can be used. A tuberculin syringe (microfiber code 112: Manville co. Denver colo). The filter fabric should not be too close—meshed or rise more than 3-4 mm from the bottom of the syringe (0.06 ml mark)

FIGURE 7.6: Glass wool filtration

STEPS 1. Just before use, the sterilized filter is rinsed twice with 2 ml of medium to remove any loose glass fiber particles. The rinsing medium is then looked microscopically for glass wool fragments. 2. Normal semen sample is washed and centrifuged once. 3. After centrifugation-supernatant removed. 4. Pellet is mixed with 1 ml of IVF medium, placed upon glass-wool column and allowed passing through the filter solely by gravity. The rate can be increased significantly by employing two filter columns, operating in parallel.17 5. The filter is rinsed with 0.2-0.3 μl of IVF medium to ensure that a little sperm suspension as possible is not left behind. The filtrate is used for insemination. This method is preferred for IUI.

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FIGURE 7.7A: Schematic presentation of typical morphological changes of human spermatozoa subjected to hypo-osomotic stress; a=no change; b-g=various types of tail changes. Tail region showing swelling is indicated by the hatched area. (Jeyendran RS, Van der Ven HH, Perez-Pelaez M. Crabo BG and Zaneveld LJD (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. Journal of Reproduction and Fertility. 70:219-28.)

B

A

C

FIGURE 7.7B: A single laser shot was applied towards the end of the tail of immotile (a,b) or motile (c) spermatozoa. According to the reaction, immotile spermatozoa were classified as presumably nonviable (a) or presumably viable (b). Note that the reaction of the presumably viable immotile sperm is similar to the reaction of motile spermatozoa (c).

Selection of Viable Spermatozoa for ICSI by Hos Test in Case of Complete Astheno-zoospermia (Figure 7.7A) The success of intracytoplasmic sperm injection (ICSI) is poor when only immotile spermatozoa are present in a semen sample. Usually spermatozoa are selected according to motility which is a clear indication of viability. In cases of complete astheno-zoospermia it is impossible to select viable sperm by morphological means. Common staining methods which allow the Identification of viable sperm (e.g. eosin test) are not suitable for ICSI procedure therefore it was proposed the use of hypo-osmotic swelling test to select

spermatozoa for ICSI.19 For selection of spermatozoa by the HOS-test semen is washed and centrifuged, twice in sperm prep. medium. Pellet is resuspended in approximately 20 μl of IVF medium and then 2 μl of sperm suspension is pipetted in 10 μl of hyposmotic swelling medium (Hypo 10.75 mm fructose, 25 mm sodium citrate . 0.06 mg/ml peniculri G) drop made in a ICSI dish. During incubation on a heated microscope stage, spermatozoa are constantly inspected for the beginning of hypoosmotic swelling. After 5-10 minutes of incubation individual HOS-positive spermatozoa can be identified by the occurrence of swollen sperm tails HOS-positive spermatozoa are immediately sucked into an injection

Techniques of Sperm Preparation for ART pipette and washed in several droplets of hepes buffered medium when the sperm tails straightened again. Finally spermatozoa are placed in PVP; the sperm tails are touched by injection pipette and used for ICSI.20 Use of a Laser to Detect Viable but Immobile Spermatozoa (Figure 7.7B) Human ejaculated spermatozoa with complete asthenozoospermia are washed and pellet is resuspended in Hepes buffered culture medium. A noncontact 1.48 μm diode laser system is used. A description of technical concept of this system is described by rink (rink et al. 1994). For laser treatment, sperm preparation are pipetted into 10 ml of hepes buffered culture medium (Hepes buffered medium drops made under oil in a ICSI dish). Laser assessment of sperm viability is performed by a direct laser shot to the tip of the sperm tail using a laser energy of approximately 120 μj applying 1.2 ms of Irradiation time. Spermatozoa that responds to the laser shot by a curling reaction of tail are presumably viable as the reaction is similar to that of motile sperm following laser treatment. The application of a single shot to the tip of the sperm tail results in two possible reaction either the spermatozoa show no reaction at all or the tail of the spermatozoa starts curling within few seconds of the laser shot. The morphological appearance of the laser reaction is similar to reaction of HOS positive spermatozoa (Jeyendran et al, 1984). And can be used for ICSI.

TESTICULAR AND EPIDIDYMAL SPERM RETRIEVAL The development of TESE (testicular sperm extraction) has also changed the diagnostic procedure in andrology. Testicular Biopsies are now performed for diagnostic and therapeutic purposes because testicular spermatozoa can be used for ICSI. TESE test should be performed to determine whether s p e r m a t o z o a from native or cryopreserved testicular tissue can be

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extracted. It is no longer necessary to do TESE/MESA on the same day as the oocyte retrieval. Needle Biopsy or Aspiration of the testes are not accepted because they may damage the testicular tissue and usually do not provide enough material for cryopreservation.21 TESTICULAR SPERM EXTRACTION (TESE) (FIGURE 7.8) Steps 1. Testicular Biopsied tissue should be received in a tube containing prewarmed culture medium and should be processed as soon as possible. 2. Biopsied tissue is taken out of the tube and transferred on a glass slide in a Petri dish which is kept on heated stage tissue crushed with two surgical blades into smallest possible pieces. Attention should be paid that Biopsied tissue does not get dry while mincing and should be kept in medium. 3. After cutting in smallest possible pieces the crushed tissue with medium from the glass slide is transferred into a 13 ml round bottom tube and allow to stand for 10 minutes during this time the fibrous tissue, blood cells, debris settles down at the bottom of the tube. Aspirate the supernatant— Look for the sperm under Makler chamber. 4. Centrifuge the supernatant for 10 minutes at 550 μg 2000 rpm. 5. Discard the supernatant. 6. Mix the pellet in 0.3-0.5 ml of culture medium look for the sperm in makler chamber if no sperm seen in Makler chamber put 2.3 μl of mixed pellet in the microdroplets under oil in a culture or ICSI dish observe for the sperm on a heated sage with 40x objective microscope equipped with micromanipulation device. Motile spermatozoa with good progression come at the periphery of the drop and sperm with decreased motility can be seen in between the blood cells and debris once the motile sperm is seen—“can be used for ICSI part of the sperm suspension is frozen in parts for the future ICSI cycles.

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MICRO-SURGICAL EPIDIDYMAL SPERM ASPIRATION (MESA)

INDICATIONS FOR SPERM CRYOPRESERVATION

Since ICSI was established, an alternative therapeutic approach is available for men presenting with obstructive Azoospermia. MESA is the method of choice in case of surgically untreated obstructive azoospermia. Obstructive Azoospermia may be caused by congenital or acquired reproductive tract occlusion21, Several modified epididymal sperm retrieval technique have been demonstrated. Moreover MESA is superior to PESA (as nonavailability of spermatozoa at times—20% cases22 failed to obtain spermatozoa), because of availability of spermatozoa for cryopreservation, lower frequency of blood cenamination, lower risk of tissue damage and higher no. of better quality of spermatozoa.23 After MESA—sperm collected in a culture tube washed and centrifuged once looked for sperms in the same way as TESE sperm and used for ICSI.

Frozen human sperm can be divided into two main categories— 1. Semen for auto conservation 2. Donar semen

CRYOCONSERVATION OF SPERM In 1776 Spallanzani was the first to report the maintenance of motility of Human spermatozoa after exposure to low temperatures. In 1866, Mantagazza suggested sperm banks for frozen human sperm. Freezing of human spermatozoa was reported shortly afterwards by Sherman and Bunge 1953, who observed that human spermatozoa, after freezing on dry ice followed by thawing, were able to fertilize the egg and to produce normal embryonic development and offspring. The first birth obtained after freezing human spermatozoa with glycerol in liquid nitrogen vapor was described by the same group (Perloff et al., 1964)24. In 1973, Steinberger and Smith 25 reported similar conception rates after cryopreservation and Insemination using ejaculates of very good quality (61%) or fresh semen samples (73%) minimal criteria for cryopreservation were developed before the new methods of assisted reproduction. Since only one living spermatozoa is necessary for ICSI, cut off values are no longer useful.

AUTOCONSERVATION Long-term—cryopreservation to ensure later fertility in men who are to undergo 1. Radio or chemotherapy which might lead to sterility or 2. Surgical sterilization by vasectomy. Prior to the introduction on of ICSI only semen samples of reasonable quality could be stored and successfully used for assisted reproduction. A low sperm yield no longer a obstacle, in theory all sperm suspension whatever the origin or quality may be frozen and used for assisted fertilization once motile (vital) spermatozoa have been observed. OTHER INDICATION 1. Stress with regard to collection of semen on demand on the day of intrauterine and Insemination (IUI), IVF or ICSI 2. Absence of the Husband during the wife’s treatment 3. Large Intraindividual variations in semen quality, improved semen sample can be cryopreserved and later pooled with fresh sample for IUI, IVF ICSI. 4. Surgically obtained spermatozoa26 epididymal or testicular spermatozoa obtained after diagnostic surgery or after sperm retrieval for ICSI must be frozen in order to avoid repeated surgery in following ICSI cycles27 ejaculates obtained after electroejaculation /from spinal cord injured men. DONOR SPERM CONSERVATION For IUI with donor semen, freezing and storage of donor sperm allows repeated testing of the donar for HIV, hepatitis B, other sexually transmitted disease and avoids the risk of infecting the recipient. Another important advantage of using frozen sperm

Techniques of Sperm Preparation for ART is the ready availability of different donar genotypes and phenotypes so as to match as closely as possible the infertile male partner. THE CRYOPROTECTANT AND CRYOMEDIA The commonly used cryoprotectant for human sperm is glycerol, first described by Polga and colleagues in 1949, is used in concentration of 5% to 10% giving minimal toxicity and maximal cryoprotection (Sherman 1973 ) A higher cryosurvival can be obtained by adding various components called extenders, to the cryoprotective agent glycerol, their functions are – a. to optimize osmotic pressure, pH b. to provide an energy source c. to prevent bacterial contamination and d. to stabilize the cell membrane, one of the cryobuffers is egg yolk which protects the sperm membrane against freezing damage by increasing its fluidity.

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6. Place the kryotubes in liquid nitrogen vapors for about 30 minutes. 7. Store the kryotubes in liquid nitrogen. Freezing of Testicular sperm suspension after mincing29 preserves their quality (motility, vitality) better than freezing of the intact biopsies.30 Biopied testicular issue is processed in the same way as in sperm retrieval from testicular tissue. The sperm suspensions may be frozen in straws or cryotubes (same method is applied for freezing MESA sperm). THAWING PROCEDURE Sperm thawing at room temp. or at 37°C preserves motility, vitality31 and fertilizing ability 32 better than slower thawing in ice baths or faster thawing in warm water baths. The chemical toxicity of glycerol requires its removal after thawing, spermatozoa have to be returned to isotonic conditions by dilution with isotonic media. After Thawing

PREFREEZE ADDITION 1. Take out the sperm freeze medium from freeze and keep at room temp for 30 minutes. 2. Let the semen liquefy and do semen analysis. 3. For every 1 ml ejaculate or sperm suspension add 0.7 ml spermfreeze medium drop by drop (while the addition of glycerol is indispensable for cryo survival of spermatozoon, sperm cells are subjected to hyperosmotic stress when exposed to the cryoprotectant, due to its high osmolority. To minimize osmotic injury and prevent severe dehydration the cryoprotectant solution should be added drop by drop or by multistep addition28 spermatozoa show a very high permeability to glycerol that is temperature dependent an osmotic equilibrium is obtained with in two minutes at room temperature or at 37°C, while equilibrium is delayed at lower temperature. 4. Mix well ejaculate or sperm suspension with freeze medium. 5. Fill the kryotube with ejaculate or sperm suspension mixed with freeze medium.

1. Do semen analysis 2. Wash centrifuge with equal volume of sperm prep. medium 3. Discard the supernatant 4. Mix the pellet with IVF medium and immediately used for IUI, or IVF or ICSI.

CONCLUSIONS There are may variants of each of the semen processing methods. It is not possible to give a single answer. This is why the sperm recovery rate sited above for various methods should be regarded as reference values, which may vary depending on semen quality. For this reason every laboratory should be able to perform several techniques to select—based on their own experience—the most appropriate method for a specific semen sample. We would use the swim up technique primarily for normozoospermias a simple and quick way of producing a purified inseminate containing a high percentage of progressively motile spermatozoa. Given a normal ejaculate volume the sperm loss rate

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to be expected would be acceptable. For the various manifestation of disorders (oligozoospermia) astheno-zoospermia, teratozoospermia) direct swip up from semen, filtration and density gradient separation method came to be superior to swim up technique. How ever this is not the true for every ejaculate and since every separation process may cause damage (for example, to the acrosome or to the spermatozoa’s membrane). It is recommend to prescreen with the method of choice and monitor long term motility, particular attention should be paid however to the presence of abundant leukocytes in ejaculates, leukocytes—especially neutrofils a substantial source of ROS (Reactive oxygen species) However centrifugating high lekocytes ejaculate prior to swip up from semen or Glass Wool filtration will increase the risk of oxygen radical causing of oxidative damage and initiating the lipid peroxidation cascade in the sperm plasma membrane. The oxidative attack of the free radicals is directed against the double bonds of unsaturated fatty acids, resulting in a deleterious accumulation of lipid peroxides in their membranes. As a result of this self propagating mechanism, sperm motility will be impaired and acrosome will be damaged, hence the ability of the sperm fusion will be lost. This is why semen samples with high leukocytes count should be processed using methods which either do not require centrifugation and pelleting (example Swim up from semen, glass wool filtration). In chronic infections, a prolonged antibiotic treatment can effectively reduce leukocyte concentration and enhance sperm motility.33

REFERENCES 1. a) Chang MC. A detrimental effect of seminal plasma on the fertilizing capacity of sperm. Nature (London) 1957; 179: 258-9. b) Bed ford JM, chang MC. Removal of decapacitation factor from seminal plasma by High speed centrifugation. Am J physiol 1962; 202:179-81. c) Reddy JM, stark RA, Zaneveld LJD. A high molecular weight antifertility factor from human semen. J Reprod Fertil 1979; 57: 437-46. d) Van der ven H, Bhattacharya AK, Binor Z, etal. Inhibition of sperm capacitation by a high molecular weight factor

from human seminal plasma fertil steril 1982; 38:753-5. 2. Palermo G, Joris H, Devroey P, et al. Pregnancy after Intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17-18. 3. a. Aitken RJ, Clarson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa J. Reprod Fertil 1987; 81: 459-69. b. Hill JA, Haimovici F, Politch JA, Anderson DJ. Effects of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil steril 1987;47: 460-5. c) Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil steril 1992;57: 40916. d) Weese DL, Peaster ML, Himsl KK, Leach GE, Lad PM Zimmern PE. Stimulated reactive oxygen species generation in the spermatozoa of infertile men. J Urol 1993;149: 64-67. 4. a) Menkveld R. An investigation of Enviornmental influences on spermatogenesis as evidenced in seminal cytology and expirmental production of these deviation dissertation Faculty of medicine, University of Stillenbosch, South Africa, 1987. b) Menkveld R, Kruger TF. Basic semen analysis. In: Acosta AA, Swanson RJ, Ackerman SB, Kruger TF, Van Zyl JA, menkveld R (eds) Human spermatozoa in Assisted reproduction. Williams and Wilkins, Baltimere, 1990; 6884. 5. Kentenich H, schmiady H, Radke E, stief G, Blankau A. The male IVF patient-Psychosomatic considerations. Hum Reprod 1992; 7(Suppl 1): 13. 6. Moghissi KS, Gruber JS, Evans S, Yanez J. Homologous Artificial insemination a reappraisal. Am obstet Gynaecol 1977; 129: 909. 7. Liu DY, Clarke GN, Lopata A, Johnston WIH, Baker HWG. A sperm-zona pillucida binding test and in vitro fertilization. Fertil steril 1989;52: 281 8. a) Beckermann H, Becker J, Lankhorst GJ. The effectivness of vibratory stimulation in anejaculatory men with spinal cord injury. Parapligia 1993;31: 689. b) Wheeler JJ, Walter JS, culkin DJ, Canning JR. Idiopathic anejaculation treated by vibratory stimulation Fertil Steril 1988;50: 377. 9. Cohen J, Edwards RG, Fehilly et al. Invitrofertilization: a treatment for male infertility. Fertil Steril 1985;43: 422-32. 10. AL - Hasani S, Kiipker W, Baschat AA, Sturm R, Bauer O, Diedrich C, Diedrich K. Mini-swim up: A new technique of sperm preparation for intracyto plasmic sperm injection. J Assist Reprod Genet 1995;12: 428. 11.a) Lessley BA, Garner DL. Isolation of motile spermatozoa by density gradient ceutifugation in percoll. Gamete Res 1983;7:49-54. b) Berger T, Marrs RP, moyer DL. Comparison of Techniques for selection of motile spermatozoa. Fertil Steril 1985;43: 268-73.

Techniques of Sperm Preparation for ART c) Pousette A, Akerlof E, Rosenborgl, Fredricsson B. Increase in Progressive motility and Improved morphology of Human spermatozoa following their migration through percoll gradients. Int J Androl 1986;9:1-13. d) Gellert-Mortimer St, Clark GN, Baker HWG, Hyne RV, Johnston WIH. Evaculation of Nycodenz and percoll density gradients for the selection of motile human spermatozoa. Fertil Steril 1988;49: 335-41. e) Tanphaichitr N, millete CF, Agulnick A, Fitzgerald LM. Egg penetration ability and structural properties of Human sperm prepared by percoll gradient centrifugation. Gamete Res 1988;20:67-81. f) Mcclure R, Nunesl., Tomr. Semen manipulation Improved sperm recovery and function with a Two-Layer percoll gradient. Fertil Steril 1989;51: 874. g) Serafini P, Blank W, Tranc, Mansourian M, Tant, Batzofin J. Enhanced penetration of zona-free hamster ova by sperm prepared by Nycodenz and percoll gradient centrifugation. Fertil Steril 1990;53: 551-5. 12.a) Bolton VN, Warren RE, Braude PR. Removal of Bacterial contaminants from semen for in vitro fertilization or Artificial Insemination by the use of buoyant density centrifugation. Fertil Steril 1990;46: 1128-32. b) Punjabi V, Gerris J, Van Bijilen J, Delbekel, Giles M, Buytaert P. Comparison between different pre-treatment techniques for sperm recovery prior to intrauterine insemination, Gift or IVF. Hum Reprod 1990;5: 75-8. 13 a) Aitken RJ, Clarson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988;9: 367-76. b) Serafini et al. Fertil Steril 1990;53: 551-5. c) Jaroudi KA, Carver-Ward JA, Hamilton CJCM, Siek UV, sheth KV. Percoll Semen preparation enhances human ooctye fertilization in male– factor intertility as shown by a randomized crossover study. Human Reprod 1993;8: 1438-42. 14. Rhemrev J, Jeyendran RS, Vermeiden JPW, Zaneveld LTD. Human sperm selection by glass wool filtration and two layer, discontinuous percoll gradient centrifugation. Fertil Steril 1989;51: 685-90. 15.a) Pertoft H, Rubin k, Kjellen L, Laurent TC, Klingborn B. The viability of cells grown or centrifuged in a new density gradient medium, percoll™. Exp Cell Res 1977; 110: 44957. b) Gorus FK, pipeleers DG. A rapid method for the fractionation of human spermatozoa according to their progressive motility. Fertil Steril 1981;35: 662-5. 16.a) Polakoskikll, Paulson JD. Aglass wool column procedure for removing extraneous material from the human ejaculate. Fertil Steril 1977;28: 178-81. b) Jeyendran RS, Perez-Pelaez M, Crabo BG. Concentration of viable spermatozoa for Artificial Insemination. Fertil Steril 1986;45:132-7. 17. Prietl G. Die homologue intrauterine Insemination: klinishe and klinisch-experimentelle untersuchunger Zur optimiering der methode und ihre gesunheitsokonomische Evaluiering, Habilitationsschrift, University

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of Bonn, 1988. 18. Van derven HH, Jeyendran RS, Al-Hasani S, Tünnerhoff A, Hoebbel K, Diedrich K, Krebs D, Perez-Pelaez M. Glass wool column filtration of human semen: relation to swim up procedure and outcome of IVF. Human Reprod 1988;3: 85-8. 19. Bourne H, Richings N. Leu DY, Clarke GN, Harari O, Baker HW. Sperm Preparation for intracytoplasmic injection: Methods and relationship in fertilization results. Reprod Fertil Dev 1995;7: 177-83. 20. S Ved, M Montag, A Schmutzler, G Prietl, G Haidl, H van derv en. Pregnancy following intracytoplasmic sperm injection of inmotile spermatozoa selected by Hypoosmotic swelling = Test a case report. Andrologia 1997; 29:241-2. 21. Girardi SK, Schlegel PN. Microsurgical epididymal sperm aspiration: Review of techniques, preoprative considerations and results. J Androl 1996;17: 5. 22. T sirigotis M, Craft J. Sperm retrieval methods and ICSI for obstructive Azoospermia. Hum Reprod 1995;10: 758. 23. Sheynkin YR, Ye Z, menendezs, Liotta D, Veeck LL, Schlegel PN. Controlled comparison of percutaneous and microsurgical sperm retrieval in men with; obstructive azospermia. Hum Reprod 1998; 13: 3086. 24. Perloff WH, Sterinberger E, Sherman JK. Canception with Human spermatozoa frozen by nitrogen vapour Technique. Fertil Steril 1964; 15: 501. 25. Steinberger E, Smith KD. Artificial insemination with fresh or frozen seman. JAMA 1973;223: 778. 26.a) Schoysman R, Vanderzwalmen P, Nijs M, Segal-Bertin G, Geerts Let et. Pregnancy after fertilization with human testicular spermatozoo. Lancet 1993;342: 1237. b) Tournage H. devroey P Liu J, Nagyz, Lissens W, van steirteghem A. Microsurgical epididynal sperm aspiration and intracytoplasmic sperm injection a new effective approach to infertility as a result of congenital absence of the vas deferens. Fertil Stenil 1994;61:1045-51. c) Devroey P, Liu J, Nagy Z, Tournaye H, Silber, van steirteghem AC. Normal fertilization of Human oocytes after testicular sperm extraction a Intracyto plamic sperm injection. Fertil Steril 1994;62: 639-41. 27. Nagy Z, Liu J, Joris H, Verheyen G, Tournage H. CamusM, Derde M-P, Devroey P, van Steirteghem A. The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod 1995;10:1123-9. 28.a) Mc Laughlin EA, Ford WCL, Hull MGR. A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a semi-programmable freezer. Human Reprod 1990;5: 724-8. b) Gao DY, Mazin P. Kleinhaus FW, Watson PF, Noiles EE, Crister JK. Glycerol permeability of human spermatozoa and its activation ener cryobiology 1992;29: 657-67. 29. Verheyen G, De crooI, Tournage H, Pletincx I, Devroey P, Van steirteghem AC. Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue

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8

The Human Oocyte

Sapna Srinivas

INTRODUCTION Hundreds of oocytes are harvested each month in the IVF lab. The aim of each aspiration being to offer an infertile couple, their dream, a healthy baby with the help of ART. The first exciting and very important stage of the ART procedure is oocyte collection and grading. Though there have been reports2 that oocyte morphology does not effect fertilization rate, embryo quality and implantation rate, many of us in the lab still strongly see a correlation and several studies document the same. Oocyte grading therefore has to be routinely done to assess maturity, quality, plan treatment and prognosticate outcome. Appropriate ovarian stimulation protocols normally provide functional fertilizable mature oocytes. A good embryology laboratory should try to confirm to the threshold limits provided in Table 8.1. The oocyte is a dynamic sensitive cell and among the largest cell in the body. The diameter of a mature oocyte is 110-120 um. It is enveloped by the oolemma. The cytoplasm is homogenous and the main organelles present in it are mitochondria Endoplasmic reticulum and Golgi system, surrounded by a glycoprotein matrix

TABLE 8.1: Desirable threshold limits for various parameters for ART Embryology procedures

Threshold limit

Normal fertilization rates Polyspermic rates ICSI degeneration rates Embryo cleavage rates Cryopreservation survival rates Ongoing pregnancy rate Implantation rates Sperm concentration Sperm morphology Sperm motility

> 60 % < 10 % < 15 % > 80 % > 50 % > 40 % > 20 % +/- 10% of the mean +/- 2 % of the mean +/ - 10 % of the mean

called the Zona pellucida which is approximate 15-20 um wide. Between the oolemma and the ZP is a thin fluid filled perivitteline space. The secondary oocyte is arrested at the MII stage and there is a polar body extruded into the PVS at this stage. Oocytes retrieved in IVF are surrounded by several layers of cells called cumulus cells. Together this is called the Ooocyte cumulus complex (OCC) . The inner most layer of these cells adjacent to the zona are called the Corona. Typically MII oocytes have a sun-burst appearance of the coronal cells and this is called the Corona Radiata.

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FIGURES 8.1A and B: (A) The oocyte seen with radiating coronal cells and expanded cumulus cells, together this is called oocyte cumulus coronal complex (OCC). (B) ICSI on a fully mature oocyte at metaphase II of meiosis o – oolemma, p – perivittelline space, pb – first polar body extruded in to the PVS, z – zona pellucida and c - cytoplasm

MEIOSIS IN THE FEMALE

IDENTIFICATION OF THE OOCYTE

Oogonium (formed in early fetal life)

The goal of the lab during OPU is to pick-up all the oocytes in minimum time and evaluate meiotic maturity and quality of the oocytes in preparation for insemination and injection. Also the follicular fluid and cells must be observed carefully for signs of potential physiological and pathological significance like premature leutinization or endometriosis. As soon as follicular fluid arrives in the IVF lab, the warm fluid is transferred immediately into a 100 mm falcon petri dish and swirled once or twice, so that the entire area is covered. The shiny oocyte cumulus complexes (Figures 8.1A, 8.2A and B)can be easily identified by the naked eye. The oocyte immersed in the mass is confirmed under the stereozoom microscope and assessment of maturity is done later. If there are blood clots or follicular cells attached, the follicular fluid is diluted with media and these clots are teased away gently with the help of a needle.Oocytes are graded for maturity by assessing the cumulus . In ICSI the stripped oocytes are clearly graded under the inverted microscope equipped with Hoffman differential interference contrast optics at 200 × magnification (Figure 8.1B). Oocytes that are scored as MII should be inseminated or injected 3-4 hours after OPU, whereas those that are scored as M I should not be injected until 2-5 hours after 1st polar body extrusion (Figures 8.3A and

Once ovarian differentiation has taken place the female germ cells are called oogonia. Oogonia divide actively by mitosis several times to reach a figure of about half a million by 3-4 months of gestational age. DNA is replicated in preparation for Meiosis I. Oogensis (Begins during embryogenesis) Primary oocyte: Transformation of oogonia to primary oocyte starts from 3-4 months. By birth all germ cells are oocytes. Meiosis I has started and the oocytes are arrested in the Diplotene state (GV stage) (Figures 8.10A, 8.11A and B). This persists for 12 - 45 years until selection and dominance. Secondary oocyte: Formed after puberty. The selected follicle grows with FSH stimulation and the oocyte resumes meiosis I. There is GV breakdown, Diakinesis, Metaphase I (Figures 8.10B and C), Anaphase I, Telophase I. The oocyte enters meiosis II and is ovulated at the metaphase II stage (first PB extruded). This oocyte is viable for < 1 day till fertilization. Pronuclear oocyte or ovulated oocyte: Requires to be fertilized and has to be picked - up by the infundibular fimbria of the fallopian tube. It is generally viable for 12 hours. Sperm penetration activates this oocyte, the 2nd polar body is extruded and PN formation begins.

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FIGURE 8.2: Oocyte – cumulus complexes as seen under the stereozoom microscope x 60

FIGURE 8.3: In ICSI the cumulus cells are treated with hyaluronidase and the oocyte is stripped bare to reveal its structures clearly

B). In view of very poor pregnancy results with GV oocytes, incubated overnight and injected next day, we normally discard GVs.

< 10 mm diameter follicles and have to be matured in vitro before use. Immature /Intermediate OCC

ASSESSMENT OF CUMULUS MATURITY This is based on the appearance of the cumulus and corona cell morphology under the microscope. In IVF it is the only indicator of nuclear maturity of the oocyte housed inside.

The corona radiata is compacted but still distinct from the cumulus and it is usually associated with a MI oocyte with absence of first polar body. This is classified as “Immature oocyte“ (Figures 8.4A and B; 8.5A and B).

Very Immature OCC

Mature OCC

A tightly packed cumulus and corona radiata layer is classified as “very immature” and normally corresponds to an GV oocyte. They are recovered from

A typical and sun burst like corona radiata and nicely expanded fluffy cumulus is generally associated with a pre–ovulatory MII oocyte (Figures 8.6A and B). If the

The Human Oocyte

FIGURE 8.4: Very tightly packed cumulus and corona most likely housing a GV oocyte, generally called “very immature” at OPU shown here under low and high power

FIGURE 8.5: Still compact corona but 2 different layers of corona and cumulus distinct, generally called “Immature” shown here under low and high power

FIGURE 8.6: Sun burst radiating corona cells of a typical MII oocyte shown here under low and high power

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corona is well expanded and is spread within the dish and teased out with 2 needles the first polar body may also be seen. Embryologists can actually feel the stringy/elastic behavior of granulosa and cumulus cells and their typical propensity to attach to the petri dish which is very typical of MII oocytes of good quality. Postmature OCC Post mature oocyte cumulus complex appears like a gelatinous mass or clump (Figure 8.7). There is no clear distinction between the corona radiata and cumulus cells and the oocyte housed inside is generally postmature, has cytoplasmic vacuoles and dark, thick granulosa cells attached to it. These oocytes are seen in cycles with a premature LH surge or a delayed HCG administration. FIGURE 8.8: A Dysmature oocyte, notice the tightly packed coronal cells but a clear polar body visible hence an MII oocyte

Atretic Oocyte/Degenerative OCC Around 5-6% of OCC’s exhibit clear signs of abnormalities in conventional IVF/ICSI cycles. Oocytes with dark and contracted ooplasm, disrupted zona pellucida, empty zona, retracted cumulus. Such oocytes are generally not cultured (Figures 8.9A to D) Various types of normal and abnormal oocytes are shown in Figures 8.12A to F. ASSESSMENT OF NUCLEAR MATURITY FIGURE 8.7: Postmature oocyte cumulus complex

Dysmature OCC In stimulated cycles it is quite common to recover OCC that display a disparity is cumulus expansion and nuclear maturity. For example: you might see nice expanded cumulus and corona but no polar body and an MI stage oocyte housed inside, very often dark compacted cumulus and corona and an MII oocyte housed inside (Figure 8.8). This dysmaturity is well known and is due to a disturbance of cumulus expansion by exogenous gonadotropins.

In conventional IVF maturity of the oocyte has to be estimated only by assessing the OCC and hence has many limitations. Cytoplasmic and nuclear maturity assessment has to wait for 16-18 hours before an oocyte is completely stripped and pronuclei assessment is done. Nuclear maturity assessment is clearly possible only in all cases of ICSI on a routine basis. One indication for ICSI in our lab is to check oocyte quality in long-standing infertility even though the male parameters are normal. There are three categories of maturity: • GV ( Germinal vesicle) (Figures 8.11A and B) • M I ( Metaphase I) (Figures 8.12A) • M II (Metaphase II) (Figures 8.13A to C, 8.14)

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FIGURES 8.9A to D: (A) Contracted cytoplasm of an atretic oocyte. (B) Parthogenetic activation of the oocyte. (C and D) Show the contracted oocyte, a very large PVS, probably due to fracture of the zona during oocyte aspiration

MORPHOLOGICAL CHARACTERISTICS OF THE OOCYTE The morphological assessment of oocytes aspirated for IVF/ICSI is one of the major factors in predicting outcome and must be considered with greatest care, observed patiently and recorded in patient data meticulously. There are several publications suggesting poor outcome in IVF/ICSI with morphologically poor oocytes.9,12,14-16,19 Our personal observations presented in Table 8.2 confirm the same. Our study was done to examine the influence of cytoplasmic morphology on the success

rate of ICSI. A total of 118 patients were included in the study done in the year 2003-2004. After stripping cumulus cells from the oocytes accurate assessment of cytoplasmic morphology was made. 2 morphological abnormalities used in the study were: 1. Increased cytoplasmic granularity – Dark central or uniform granularity all over cytoplasm. 2. Presence of cytoplasmic inclusions – vacuoles of different sizes and shapes, SER and refractile bodies.

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A

B

C

FIGURES 8.10A to C: (A) GV oocyte, (B and C) Metaphase I oocytes

FIGURES 8.11A and B: (A) three oocytes at the GV stage, note the coarse granulation mostly in the center of the oocyte and the clear germinal vesicle. It was difficult to strip these oocytes hence the coronal cells still tightly attached to the zona. (B) An immature oocyte with two germinal vesicles. This oocyte is larger than normal (>250 um) and has central coarse granulation with two peripherally located germinal vesicles

The Human Oocyte

FIGURES 8.12A TO F: (A) A case where 8 oocytes recovered all arrested at the MI stage. Note all 8 oocytes have no polar body and GV has broken down, probably due to meiotic incompetence Patient received HCG 10,000 units 36 hrs pre OPU and her follicular fluid tested positive for HCG. (B) A central metaphase II oocyte, note the thick zona pellucida and normal PVS. The cytoplasm does not exhibit much granulation and has become fine, hence more mature. The oocyte on the left is metaphase I showing transition between oocyte nuclear maturation. (C) An MI oocyte showing a very irregular oolemma and slightly thicker zona. This oolemmal loss of tonicity has led to an irregular shaped oocyte undergoing degeneration. (D) Four oocytes show arrest at MI stage. (E) MI oocyte with finely granular cytoplasm, mild increased PVS at 5 O’ clock position and normal zona. (F) MI oocyte showing an increased granularity on the left side homogenous cytoplasm towards the right. This can be classified as quite immature due to the coarser granularity in the cytoplasm. As the oocyte nears maturity, the cytoplasmic granularity decreases and becomes finely homogenous

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FIGURES 8.13A to C: (A) A fully matured metaphase II (MII) oocyte, note that the cytoplasm is slightly darker than normal and coarsely homogenous. A grade 1 polar body is seen at 6 O’ clock. The PVS and zona are normal. (B) A mature MII oocyte with mild increased cortical granularity at 2 and 3 O’ clock position seen due to clustering of mitochondria. Granular cytoplasmic waves are known to occur in the oocyte and are considered normal. (C) A healthy MII oocyte. Note spherical shape, normal size, normal zona and clear homogenous cytoplasm. This patient had 4 oocytes recovered and fertilized two with very poor sperm profiles with ICSI. Two 8 cell grade I embryos were transferred on day 3. She delivered two healthy female babies

Oocytes were graded as normal if the cytoplasm was clear, fine homogenous and had no inclusions. The results observed are shown in Table 8.2. The difference in pregnancy rates in the two groups is very significant and confirms that results in ICSI are dependant on oocyte quality. Eventhough normal fertilization, cleavage and embryo development occur in poor quality oocytes, the implantation rates and pregnancy rates are not equal to normal oocytes. As shown in our study the clinical pregnancy rate in normal oocytes was 30.7% and in the group in abnormal oocytes was only 2.5%

The possible reasons attributed to poor oocyte morphology are multifactorial • Ovarian stimulation is known to have adverse effect on oocytes. Controlled ovarian hyperstimulation may result in the maturation of abnormal oocytes that would otherwise become atretic in a natural cycle. • Human oocytes recovered from IVF/ICSI cycles have shown > 40-45 % incidence of aneuploidy. (Van Blerkom 92) • Oocyte quality may also be directly affected by the hormonal environment estrogen and progesterone

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TABLE 8.2: Peformance of oocytes undergoing ICSI according to cytoplasmic morphology Group A n = 78 patients Normal cytoplasm

Group B n = 40 patients Granular cytoplasm Cytoplasmic inclusions

No.

Rate%

No.

Rate%

No.

Rate%

Injected oocytes

580

58

221

22

198

20

Fertilized oocytes

394

68

116

53

118

60

Cleaved embryos

315

80

92

79

104

88

Embryo Grade I Grade II Grade III

198 98 19

63 31 6

50 37 5

54 41 5

60 37 7

58 35 7

Mean no. of embryos transferred on day 3

2.5 + 0.5

2.5 + 0.7

Clinical pregnancy rate per transfer

24 (30.7%)

1 ( 2.5%)

are intimately involved in the initiation of cytoplamic maturation and final stage of nuclear maturation. The stimulation regime used and drugs used are therefore very important. • Pure FSH stimulation has demonstrated a high degree of morphological and nuclear anomalies in unfertilized eggs in IVF ( Wojak et al 95) Several studies show that the outcome of IVF/ICSI is dependant on the quality of oocytes retrieved. In our study and several others it has been found that normal fertilization and embryo development can be achieved with dysmorphic oocytes and ICSI but the implantation rate, pregnancy rate and on – going pregnancy rate are as low as 2-3% In view of high rate of aneuploidy and low pregnancy rate probably due to development failure in utero post ET couples should be clearly informed about poor outcome and high rates of abortion even though they will eventually have an embryo transfer. Therefore the importance of morphological evaluation of all oocytes retrieved cannot be understated and until a reliable biochemical assay becomes available it remains the only way to classify oocytes. CYTOPLASM The cytoplasm of an oocyte contains the nucleus, mitochondria, Endoplasmic Reticulum, Golgi complex

and other organelles. It is here that oocyte metabolism occurs. In a normal healthy oocyte it is translucent yellowish colored by phase contrast microscopy, uniformly homogenous and has fine granularity. Very often a polarized granular area can be seen in the cortical region perhaps representing a cluster of mitochondria. An increase in the granularity in the cytoplasm is called Cytoplasmic Granularity. A dysmorphic oocyte shows increased cytoplasmic granularity in two regions. Central Granularity Dark central granularity appearing as a large, dark, spongy granular area in the cytoplasm and its severity is based on the diameter of the granular area and the depth of the lesion. Homogenous Granularity An excessive dark granularity affecting the entire cytoplasm, generally seen in degenerative oocytes. VACUOLE A fluid filled vesicle inside the cytoplasm is called a Vacuole. Vacuoles can be single or multiple of different sizes and shapes (Figures 8.17A to I). Vacuoles may be the result of endocytotic process of cytoplasm repair or abnormal endocytosis due to oolemma instability

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Atlas of HART or atresia in poor quality oocytes. A vacuole appearing close to the PN on day 1 may confuse an embryologist and hence must be observed carefully. It is known that oocytes containing vacuoles result in poor pregnancy outcomes. It is advisable to avoid injecting sperm into a vacuole during ICSI. Various types of granularity encountered are depicted in Figures 8.15A to I and 8.16A to D.

SMOOTH ENDOPLASMIC RETICULUM (SER)

FIGURE 8.14: A metaphase II oocyte with clear homogenous cytoplasm

SER appears as a round, flat disc generally in the center of an oocyte and is due to massive aggregation of saccules of SER. In IVF its presence decreases chances of fertilization which can be overcome by ICSI but

FIGURES 8.15A to I: (A) Dark central granularity occupying nearly three fourth of the cytoplasm, the PVS and zona are normal. (B) Spherical MII oocyte with an increased central granularity. The polar body, PVS and ZP are quite normal. This oocyte fertilized with ICSI and was scored as a Grade I 8 cell embryo on day 3. (C) Dark central granularity in this oocyte which also exhibits a very small polar body. This oocyte could have undergone maturation in culture. (D) A spongy patch of central granularity in this otherwise normal looking MII oocyte. This patient had 8 oocytes recovered all showing same morphology. Patient conceived twins but had a missed abortion at 8 weeks. (E) The area of central granularity in this oocyte is much larger and indicates poorer prognosis, (F) Very dark central granularity in the cytoplasm of this oocyte also exhibiting a fragmented polar body, increased PVS with coarse debris. (G, G, I) Increased central granularity of different sizes and textures

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FIGURES 8.16A to D: (A) This metaphase II oocyte shows a cytoplasm with increased granularity present all over the cytoplasm. Notice the dark color and increased PVS. This patient received very high dose of stimulation as she was a poor responder. (B, C) An increased homogenous granularity in a MII oocyte looks just like the cytoplasm of an immature MI oocyte shown here for comparison. (D) Notice the increased granularity in this oocyte and a glass like appearance. The polar body could not be positioned at 6 O’ clock due to the oval shaped oocyte.

there are several reports claiming a high rate of aneuploidy in these oocytes and no pregnancies (Figures 8.18A to D). REFRACTILE BODIES Small cytoplasmic inclusions about 10 micron diameter are called Refractile bodies. Their presence indicates that these oocytes will have lower fertilization rate and they are known to recur in the same patient in subsequent cycles.

BULL’S EYE Is a very rare occurrence. It signifies lower fertilization rate and high aneuploidy rate of these oocytes (Figures 8.19A to C). PERIVITTELINE SPACE Hassan Ali et al in 19988 did an extensive study of intra perivitteline space granules and found that they may be related to high doses (> 45 ampoules) of HMG. PVS

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FIGURES 8.17A to I: (A) An M1 oocyte showing a large spherical vacuole. (B) A degenerative oocyte showing multiple small and large vacuoles all over the cytoplasm. (C) A single spherical vacuole in this oocyte also has very small inclusions and other refractile bodies in the cytoplasm. This patient had 2 ICSI cycles where the problem repeated in all oocytes in both cycles. (D to I) Oocytes exhibiting different sizes and shapes of vacuoles in their cytoplasm

is a thin fluid filled space between the zona pellucida and the oolemma. The first and second polar bodies are extruded into this space. Normally the PVS is not too wide and the oolemma is almost touching the inner surface of the zona. Sometimes increased PVS are filled with fine or coarse granules and not much is known about these structures (Figures 8.20A to E).

ZONA PELLUCIDA (FIGURES 8.21A TO F) The zone pellucida is normally 15-20 micron thick, uniform and clear, enveloping the oocyte and the embryo. The functions of the zona are: • It has ZP2 and ZP3 receptors and plays an important role in acrosome reaction, sperm binding and fertilization.

The Human Oocyte

FIGURES 8.18A to D: Oocytes with clustered saccules of smooth endoplasmic reticulum (SER) in the center of the cytoplasm.Notice that they appear like a superficial disk and very smooth

FIGURES 8.19A to C: Bull’s eye ( an aggregation of organelles) in the cytoplasm of these metaphase II oocytes at 10, 1 and 7 O’ clock positions. The homogenous cytoplasm is otherwise totally normal

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FIGURES 8.20A to E: (A,B) Both MII oocytes showing increased PVS with fine granules. (C) This metaphase II oocyte has a very large PVS and fragmented polar body. The cytoplasm is contracted to one side and the entire PVS has very large coarse granules. This oocyte fertilized but arrested at the 4 cell stage. (D, E) Oocytes exhibiting large PVS with coarse granules

• It gives support to the developing blastomeres • It prevents premature implantation of the embryo by hatching in utero. • It protects the embryo from the maternal immune system. Morphological abnormalities in the zona include: • Thick zona > 25 mm • Fragile zona • Compacted zona and septations in the zona • Abnormally shaped zona. Such problems in the zona will impair hatching of the embryo and decrease implantation and pregnancy rates.5 Zona hardening can result in cases of prolonged culture of embryos especially in suboptimal conditions and is also a known cause that prevents hatching of the embryo and implantation.

FIRST POLAR BODY The extrusion of the first polar body indicates the end of meiotic maturation. Edner et al HR 2000 have classified morphology of the polar body into 4 groups: Grade 1. Round or ovoid, intact and with a smooth surface (Figure 8.22). Grade 2. Round or ovoid, intact but with a rough surface (Figure 8.23). Grade 3. Fragmented (Figure 8.24). Grade 4. Large and mostly associated with a large PVS (Figure 8.25). Oocytes with grade 3 and grade 4 first polar bodies have a significantly lowered fertilization rate and can be a valuable tool for predicting oocyte quality. Fragmentation of the first polar body signifies entrapment of mature oocytes in the follicle leading

The Human Oocyte

FIGURES 8.21A TO F: (A) An MII oocyte has a double layered zona at the 4 and 5 O’ clock position. (B) This MII oocyte has a multilayered septate zona and was quite difficult to inject. (C) Oocyte with multilayered septations in the zona at the 6 O’ clock position. The cytoplasm is darker and has several inclusions. (D) An oocyte with a compacted thick zona. (E) This MII oocyte has an elongated zona which is bilayered and appears like a cone. (F) This oocyte developed a crack in the zona, notice the arrow pointing to the crack probably because it is a brittle zona. With brittle zonae oocytes degenerate with ICSI and are better off with IVF if sperm parameters are reasonably ok

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FIGURE 8.22: Grade 1. Round or ovoid, intact and with a smooth surface

FIGURE 8.23: Grade 2. Round or ovoid, intact but with a rough surface

FIGURE 8.24: Grade 3. Fragmented Polar body

to egg aging. This is probably due to a delayed HCG administration. PCO patients who have high concentrations of LH throughout their cycles also exhibit signs of oocyte aging at OPU. OOCYTES OF UNUSUAL SHAPE (FIGURES 8.26A TO D) Occasionally, unusually shaped oocytes may be recovered. Some of these that are elongated or oval

shaped can be fertilized in IVF and ICSI. They are seen to cleave normally and produce healthy offspring. In rare cases binovular oocytes can be seen in IVF. Binovular oocytes and multinuclear oocytes has been described in pre-pubertal stages but rarely persist in adult life and hence rarely ovulate. If found, it is speculated that it could be due to failure of separation of two oocytes in folliculogensis. In nature, if such

The Human Oocyte

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FIGURE 8.25: Grade 4. Large and mostly associated with a large PVS

FIGURES 8.26A to D: (A) A giant binucleate oocyte shows 2 first polar bodies in the cytoplasm. Notice the size in comparison with a normal MII oocyte. (B) A large oocyte with 2 first polar bodies and little fragmentation. The cytoplasm is showing degenerative changes. (C,D) Showing 2 first polar bodies in the PVS.FISH done on such oocytes shows that they are aneuploid and hence should not be used

oocytes ovulate and fertilize they could lead to formation of dizygotic twins. though in IVF no pregnancy has been reported to date.

GIANT AND BINUCLEATE OOCYTES Occasionally in IVF, oocytes that are much larger in diameter than routine size are observed. Such large

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oocytes are presumed to have arisen through failure of cytoplasmic division of oogonia. Most of these large oocytes are binucleate i.e. they possess diploid amount of DNA. Therefore at OPU they have extruded 2 distinct polar bodies into the PVS. When such oocytes are used in IVF/ICSI it is expected that they will develop into triploid embryos. This was confirmed by FISH techniques in several studies. Ideally such oocytes should not be used in ICSI.

CONCLUSION Since ICSI has allowed us to precisely assess all oocytes routinely a lot of information of oocyte dysmorphism on the fertilization potential, embryo cleavage patterns, implantation and pregnancy outcome has been reported in literature. In view of the high incidence of aneuploidy found in such oocytes, prognosticating regarding outcome should be routinely done. More studies will reveal the significance of these various phenotypes of a normal oocyte or a poor quality oocyte. Morphological assessment of human oocytes remains a major factor in predicting IVF/ICSI success and must be considered with greatest care.

ACKNOWLEDGMENTS I sincerely thank Dr. Mamata Deenadayal, Clinical Director, IIRC, for introducing me to the amazing field of ART. Her constant support, encouragement and assistance has always motivated me in my work. I also wish to thank the entire clinical and lab team for their assistance. Special thanks to our lab Director, Prof. G. Sadasivan for being such a wonderful teacher.

BIBLIOGRAPHY 1. A color atlas for Human Assisted Reproduction laboratory and clinical insights – Pasquale Patrizio – Michael J. Tucker, Vanessa Guelman. 2. Balban B, Urman B, Sertac A, et al. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod 1998: 13: 3431-3. 3. Ben-Rafael Z, Mastroianni L Kr, Kopf G. Invitro

4.

5.

6.

7.

8. 9.

10.

11.

12. 13.

14. 15. 16.

fertilization and cleavage of a single egg from a binovular follicle containing two individual eggs surrounded by a single zona pellucida. Fertil Steril 1987;47:707-9. Edner T, Yaman C, Moser M, et al. Progonstic value of the first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection Hum Reprod 2000; 15:427-30. Gabrielsen A, Lindenberg S, Peterson K. The impact of the zona pellucida thickness variation of human embryos on pregnancy outcome in relation to suboptimal embryo development. A prospective randomized controlled study. Human Reprod. 2001;16:2166-70. Hammitt DG, Syrop CH, Van Voorhis BJ. Maturational asynchrony between oocyte cumulus–coronal morphology and nuclear maturity in gonadotropin – releasing hormone agonist stimulation. Fertil Steril 1993;59:375. Hassan Ali H, Hisham-Salesh A, El-Gezeiry D, et al. Perivitelline space granularity: A sign of human menopausal gonadotropin overdose in intracytoplasmic sperm injection. Hum Reprod 1998; 13:3425-30. Hyttel P, Westergaard L, Byskov AG. Ultrastructure of human cumulus-oocyte complexes from healthy and atretic follicles. Human Reprod 1986;1:153-7 Kharamam S, Yakin K, Donmez E, et al. Relationship between granular cytoplasm of the oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum Reprod 2000; 15:2390-3. Laufer N. Tarltzis BC, DeCherney AH. Aysnchrony between human cumulus – corona cell complex and oocyte maturation after human menopausal gonadotropin treatment for in vitro fertilization. Fertil Steril 1984;42:366-9. Quinn P, Kerin JF, Warner GM. Improved pregnancy rate in human invitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-8. Serhal PF, Raniere DM, Kinis A, et al. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod 1997;12:1267b-1270b. Text book of Assisted Reproductive Techniques – laboratory–clinical perspectives – David and Gardner, Ariel Weissman, Colin M Howles and Zeev Shoham 2001. Van Blerkom J, Henry G. Oocyte dysmorphism and aneuploidy in meiotically mature human oocytes after ovarian stimulation. Hum Reprod 1992;7:379-90. Veeck LL. Atlas of the Human Oocyte and Early conceptus. Williams and Wilkins, Baltimore, USA, 1986. Xia P. Intracytoplasmic sperm injection: coorelation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod 1997;12:1750-55.

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In Vitro Fertilization and Blastocyst Culture Alok Teotia

INTRODUCTION

FERTILIZATION

In vitro fertilization became an establish form of therapy for tubal blockage, with the birth of a girl named Louise Joy Brown, in 25 July 1978, in a small place Oldham, near London. The team consisted of Dr. Robert Edwards (The Embryologist) and Dr. Patrick Steptoe (The Clinician). Since then, the technological advancement has resulted in its widespread applications in the treatment of infertility. With the addition of Intracytoplasmic Sperm Injection (ICSI) as a standard technique, even the most severe form of male infertility is treatable. The IVF based techniques utilizing preimplantation genetics diagnosis and production of embryonic stem cell lines, along with in vitro oocyte maturation and cryopreservation has huge therapeutic potentials which may lead to a new era in therapeutics and development of human species. Although true meaning of the term in vitro (invitrous = in glass) has now much changed as plastics are now, globally, the product of choice, because of there better designing and dimensional abilities, optical characters, lack of toxicity, etc.

Fertilization is the event of the union of the male and female gametes. The events involved in fertilization have been studied in detail, both microscopically and ultramicroscopically. Penetration of oocyte by the sperm is followed by activation of the oocyte, which, as a result, is released from the arrested metaphse II stage and resumes its development to form the zygote. More specifically, Fertilization is the union of two different sets 23 chromosomes/set, one set from each genetic parent of chromosomes, to make a new (third) set of chromosomes 23 pairs. This third set is the new genetic blue print, generated for a new individual. The fertilized egg is called an embryo.

IVF IN THE LABORATORY The procedure begins with the procurement of gametes, the oocyte from mother and spermatozoa from father. COLLECTION OF EGGS FROM THE FEMALE’S OVARIES (OOCYTE RETRIEVAL) At present, the most popular technique of oocyte

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B

A

FIGURES 9.1A and B: Oocyte (The female gamete): (A) An oocyte in the Oocyte-Cumulus-Corona Complex (OCC), as seen at the time of oocyte retrieval (100 X). To the necked egg it appears as a non pigmented translucent mucus. (B) A mature Oocyte (M-II), as seen after the complete removal of cumulus – corona cells. Polar body is visible at 2:00 O’ Clock position (300 X). The oocyte is ready to fertilize at this stage.

retrieval consists of aspiration of follicular fluid (containing the oocyte) from each follicle, transvaginally, under ultrasound guidance with a long needle, which can easily reach the ovaries. Follicular fluid is then screened under a microscope for the presence of oocyte-cumulus-complex (OCC) (Figure 9.1A), which are collected and stored in a fluid (culture medium), and transferred to the CO2 incubator as soon as possible the composition of culture medium is designed close to that of the fluid present in the fallopian tube at that phase of menstrual cycle, and is maintained at 37°C and in an atmosphere of 5% CO2 for over 12 hours. Oocyte retrieval is timed approximately 30 to 60 minutes before the expected time of ovulation so that the Oocytes has reached or are approaching the maturity i.e. Metaphase –II (M-II) stage. The M-II stage oocytes (Figure 9.1B) are characterized by the presence of one polar body in the peri-vitelline space. The timing of Oocyte Retrieval is crucial because of the fact that only a mature M-II stage oocyte has the capability to fertilize. Now a days, with the increased coverage of indications, for example, in the patients prone to OHSS, the Oocyte Retrieval can be timed much earlier

and final maturations is allowed in vitro (in vitro Oocyte Maturation). Although the oocytes, immature at the time of collection, can be matured and then fertilized in in vitro culture, but after embryotransfer, the outcomes in terms of pregnancy are not very satisfactory. Efforts are continuing and the field of in vitro oocyte maturation has great potentials, as the number of mature follicles is a major limitations for a large group of patients, and also, more importantly, oocyte banking (cryopreservation) seems much feasible, if used along with in vitro oocyte maturation techniques. OOCYTE HANDLING IN THE LABORATORY: RETRIEVAL TO INSEMINATION The embryology laboratory has to provide a safe and stable environment for oocytes collected from follicular aspirates until they are inseminated or subjected to intracytoplasmic sperm injection. Oocytes, sperms and embryos are to be maintained at a temperature of 37°C. The culture medium used for transitional activities of follicular aspiration, ovum holding and oocyte

In Vitro Fertilization and Blastocyst Culture grading must meet the nutritional requirements, pH stability and osmotic balance and should be compatible with the culture system of insemination, embryo genesis and embryo development. The nutritional requirements for oocyte in transition from the follicular aspirates to fertilization are minimal, or are at least induced to be minimal by methodology. There seen to be no special tonic or amnio-acid requirements for oocytes in this transitional state, and the electrolytic and osmotic needs are met by most balanced salt solutions like Earl’s Balanced Salt Solution (EBSS), Human Tubal Fluid (HTF), Hank’s Balanced Salt Solution (HBSS), Dulbacco’s Phosphate Buffered Saline (D-PBS) etc. Functions of Basal Salt Solution 1. Provide water and certain bulk ions for osmotic balance and cell metabolism. 2. Provide a buffering system to maintain the medium within the physiological pH range.

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both mono and diabasic phosphate as Na or K salts. The most widely used phosphate buffered saline (PBS) was described by Dulbecco and Vogt. Although physiological, but concentration of phosphate needed to provide physiological buffering is quite high and therefore may be toxic. Drawbacks: A Poor buffering capacity above pH 7.5. and tendency to precipitate most polyvalent cations. 3. HEPES {2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid}: • Provide 35% more buffering capacity than PBS • Does not impair buffering capacity of bicarbonate when used together. HEPES increases the buffer capacity and stabilizes the pH within the range of 7.2 to 7.6. Addition of HEPES allows the media to better resist fluctuations of the pH during out of incubator manipulations. OOCYTE GRADING (OG)

Bulk Ions • Sodium: maintains osmotic pressure in the medium. • Potassium: maintains osmotic pressure in the cell. • Calcium and Magnesium: Essential for intracellular enzymes. Participate cell attachment and spreading. Buffers Three most popular buffers used in ART culture medium are – 1. Bicarbonate: When bicarbonate is added to the media an equilibrium is reached at pH 7.4 in the presence of elevated CO2 (5 to 6%). Advantages: Being physiological it is less toxic. It ensures that metabolic requirements for CO2 are met (Participate in basic biochemical processes). Disadvantages: To maintain the desired (physiological) pH, it needs increased CO2 environments (5 to 6% CO2). 2. Phosphoric acid: An appropriate concentraion of phosphoric acid that provide buffering capacity in the physiological range is obtained by including

To shorten the retrieval process, Oocyte Grading is performed at the end of retrieval process. Oocyte Grading can be best performed at 100-200x magnifications using a suitable contrast enhancing optical system. A highly dispersed cumulus and radiating coronal layers signify arrival of M-II stage (Figures 9.1C and D). A tightly packed cumulus with dense coronal layers indicates meiotic immaturity (Figure 9.1B). In this way maturity of OCCs is scored not the meiotic stage of the oocyte. Knowledge of meiotic state of the oocytes allows determining the timing of insemination or ICSI in a more precise manner. It has been suggested that oocyte in M-II (Figure 9.1D) at scoring should be inseminated or injected 3-5 hrs after collection. A more detailed grading system for human oocytes as described by Bongso et al (1999) incorporates a GV stage (grade-I), a M-I stage (grade II), 2 M-II stages (grade III – mature; grade IV: very mature) and a post mature stage.

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D

C

FIGURES 9.1C and D: Oocyte Grading: (C) A mature OCC, (corona cells seen at the periphery are not compact and do not form complete layers), as seen after disection of excess cumulus mass. (OCC grading). (200 X). (D) A slightly immature OCC, (compact corona cells can be seen at the periphery, 4-6 layers), as seen after dissection of excess cumulus mass. (OCC grading). (200 X)

SEMEN Semen is a combination of sperm cells (20 million/ml in a normal semen) and the secretions of accessory glands. The evaluation of the male is based upon the semen analysis. Although, the overall prognosis for a successful pregnancy is dependent on the complex combination of variables in semen quality coupled with the multiple factors inherent on the female partner (Baker et al 2002 – textbook). Sperm morphology has been reported to be directly related to its fertilization potential and can be used to predict a patient’s fertility. Normal Sperm Morphology A mature sperm consists of three regions: 1. Head—Contains the Nucleus (23 Chromosomes). At the tip of the head is the acrosome, a bag of digestive enzymes that help the sperm to penetrate the protective layers that surround and egg cell. Size = 7-9 μ × 3-5 μ (Figures 9.2A to E). 2. Mid piece or neck—Packed with Mitochondria (Energy Source). To supply energy that is required for sperm to reach an egg. Length = 10-15 μ (Figures 9.2F and G).

3. Tail – Consists of a Single, Powerful Flagellum that propels the Sperm. Normal length = 30 to 45 mm (Figure 9.2H). THE SEMINOGRAM (EVALUATION OF THE SEMEN) The fertilization potential of sperm can be reasonably predicted on the basis of their motility, particularly, the forward progression and their morphology, but because of the involvement of the oocyte (as variable) in the process of fertilization, the fertilization can never be predicted. There are chances of a complete fertilization failure, even in the gametes of a previously proven fertile couple. However, with the successful advent and practice of ICSI, the risks has been reduced, and with most of the couples opting for ICSI on at least few of the oocytes to save the risk of a complete fertilization failure, has considerably reduced the incidences of a cancellation of embryotransfers because of unavailability of embryos for transfer, even if there is a complete fertilization failure in standard IVF. ICSI as an ultimate technique ensures that the sperm has been deposited inside the oocytes. Sperm entry in the oocytes, triggers the initiation of fertilization events, or activates the oocyte for fertilization to occur.

In Vitro Fertilization and Blastocyst Culture

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FIGURES 9.2A to D: Normal Sperm Morphology: (A) Normal sperm head: Sperm head is like an elliptical disc.(400 x). Dimension: 7-9 μ x 3-5μ. (B) Normal sperm head: When seen from (thinner) side, it appears much smaller in width. (2-3 μ). (C) Normal sperm head: Intermediate orientation can appear in between the two extremes i.e. width and thickness. (400 x). (D) Acrosome: The anterior light colored area represents. Acrosome, which in a normal sperm should constitute 40-60% of the total sperm head size.

The commonly accepted standard for defining the normal semen analysis is defined by the WHO. As per the WHO (1992) norms, values for a normal semen analysis are: Liq.

– Complete within 60 minutes at RT

Appearance

– Homogeneous, gray and opaque

Odor

– Fresh and characteristic

Consistency

– Leaves pipette as discrete droplets

Volume

– >2 ml

pH

– 7.2 – 8.0

Concentration – > 20 million sperm / ml semen

Motility

– > 50% with forward progression or > 25% with rapid progression both within 60 minutes of collection Morphology – > 30% with normal forms Viability – > 75% Leukocytes – < 1 million/ml Immunobead – < 20% with adherent particles test MAR test – < 30% with normal forms Head abnormality: round, pyriform, pin, double and amorphous. Dimensions of sperm (Kruger’s criteria)

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E

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G

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FIGURES 9.2E to H: Abnormal sperm morphology: (E) Abnormal Sperm: Mid-piece defect -Bend Neck (400x). (F) Abnormal sperm: Head/Midpiece defect - Pin Head (400 x). (G) Spermatozoa showing a Cytoplasmic droplet: It represents relative immaturity, cytoplasmic droplets ½ the size of sperm head are considered normal. (400 x). (H) Spermatozoa showing Tail Curling. Test of vitality: Hypoosmotic swelling Test (Host): Tail curling is induced only in the live sperm when placed in the hypoosmotic solution (50% the molarity).

OBTAINING SPERM FROM THE MALE (SEMEN COLLECTION)

Sperm Preparation Techniques

If possible semen is normally collected by masturbation. The semen is then processed to collect the best quality sperm from the sample. Sperm characteristics important for fertilization with standard IVF include: Normal morphology, Normal (intact) Acrosome, Ability to bind and penetrate zona pellucida, fuse with oolemma, activate the oocyte and form a male pronucleus.

Preparation is aimed towards obtaining the spermatozoa with the highest potential for normal fertilization. It is important to remove seminal plasma as it contains certain decapacitation factor that prevents the acrosome reaction, which is an important step in the fertilization process. In addition, round cells and abnormal sperm responsible for increased oxidative stress should also be separated out.

In Vitro Fertilization and Blastocyst Culture In general oocytes are inseminated 4-6 hrs. after the collection and sperm can be prepared during this time. During preparation, damage to the sperm due to dilution, temperature change, centrifugation and exposure to the potentially toxic materials, must be minimized. Swim up is the most commonly used technique, but can be successfully performed only in normal or slightly subnormal semen samples. Prolonged incubation time may result in a reduced yield of motile sperm from gravitational effect. For discontinuous gradient preparation, media containing silane coated silica are available for clinical use. Some studies suggest that the gradient materials may damage the sperm. Scanning electron microscopy of human sperm after preparation of semen for IVF. Combination of density gradient and swim up may produce higher yields of good quality sperm. Ideally a tailored approach is required for individual samples and should be aimed towards minimizing the centrifugation and other stress. Various agents have been reported to enhance sperm motility (Pentoxifyline (POF) 0.3 to 0.6 m mal/ liter and Caffeine). INSEMINATION To achieve fertilization, the eggs are exposed to a specific number of motile spermatozoa (50–100,000 sperm/egg) for 1 to 18 hrs in controlled conditions. There is increased risk of polyspermy with the higher concentration. If good quality sperms are collected, then reduced concentrations are equally or more effective. The oocytes can be Inseminated conventionally in one of several configurations. Multiple or single oocytes in one well of a four well culture dish containing 0.5 to 1.0 ml insemination medium, with, or without oil. Multiple or single oocytes in an organ culture dish, (single well, 60 x 15 mm) containing 0.8 to 1.0 ml insemination medium, with, or without oil.

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Individual or multiple oocytes in 30-50 μl drops (micro drop insemination) of insemination medium in various dishes for eg., 60 × 15 mm, 35 × 10 mm, 90 × 20 mm, 4 well dish, six/twelve well dish etc. In order to prevent the osmolarity changes, the microdrop cultures are invariably covered with liquid paraffin or mineral oil provided by the media suppliers. In micro drop insemination, because of the fact that all the sperm remain in close proximity of the oocyte, a lower sperm concentration is generally used. In the patients with borderline seminogram, microdrop insemination has been advocated by a number of researchers. Time of insemination: Insemination is generally performed 4-6 hrs. after the oocyte collection, unless immaturity is noticed during scoring. Duration of sperm exposure: Oocytes are generally exposed to sperm for 1:00 to 16 hrs. Fertilization check (PN check): the oocytes are observed at 16-18 hours after the sperm exposure to look for the observable signs of fertilization which includes: • Expulsion of 2nd polar body in the perivitelline space (Figure 9.3A) • Formation of two pronuclei (2 PN), one containing the male and another the female chromosomes (Figures 9.3A and B). Both the pronuclei migrate towards the center where they unite to form a new (third) set of chromosomes.

ANALYSIS OF FERTILIZATION Sperm entry has been claimed to initiate the fertilization process in a mature oocyte. For fertilization to occur, the oocyte has to get activated, and should have the ability to extrude the 2nd polar body, decondense the sperm head, form both the pronuclei (the male and the female pronuclei) and fuse both the pronuclei in order to form a set of 23 pairs of chromosomes. Both, the spermatozoa and the oocyte, providing 23 chromosomes each.

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B

A

FIGURES 9.3A AND B: Normally Fertilized Oocytes-2PN (18 hrs. post insemination). (A) A good quality – 2 PN oocyte. (5-7 Nucleoli in each aggregating towards point of union). (B) A poor quality – 2 PN oocyte. (Less no of nucleoli (3 and 4), scattered)

Fertilization is an oocyte driven process that starts with the sperm entry or we can say that, as soon as the oocyte gets its lacking chromosome set (sperm providing 23 chromosomes). Certain factors released by the spermatozoa are supposed to play important part in the oocyte activation, however parthenogenic activation also is not very uncommon.

Third cleavage start at 60 hours, giving rise to an eight cell embryo, after which, it become difficult to count the cells. The embryo is now referred to as morula. In a normally growing embryo, blastocyst stage is achieved at 5-6 days after sperm exposure (Figures 9.5G to J and 9.6A to C). Figure 9.6D shows extruded inner cell mass and Figure 9.6E shows break in the zona.

ABNORMAL FERTILIZATION

TRANSFERRING THE RESULTING EMBRYOS INTO THE UTERUS

As explained in the Figures 9.4A and B the various abnormalities in the fertilization process can be observed.

Embryos are usually transferred in the uterus either on day 2 or 3 after the egg retrieval (cleavage stage embryotransfer, 4–8 cell stage), or on day 5, 6 or 7 after the egg retrieval (blastocyst transfer).

CLEAVAGE (CELL DIVISION) The embryos are examined carefully at intervals to ensure that the division is continuing at the right pace. First cleavage starts at about 26 hrs after initial sperm exposure and give rise to a 2-cell embryo (Figures 9.5A and B). 2nd cleavage starts at 40 hours after sperm exposure and involves two divisions giving rise to a 4-cell embryo (Figures 9.5C to F). A 3 PN embryo at 3 celled stage.

EMBRYO SCORING Before transfer, embryos are evaluated (embryo grading) to select the most competent embryos from the entire lot. Here, it is important to note that, the reference time should be the time of insemination or ICSI. The course of embryo development is supposed to start with the oocyte activation which is a result of sperm entry in the oocyte. Although in ICSI it is the time of injection, in IVF.

In Vitro Fertilization and Blastocyst Culture

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B

FIGURES 9.4A and B: Abnormally Fertilized Oocytes - (18 hrs. post insemination). (A) A 3 PN oocyte. Absence of 2nd polar body indicates that 3rd (extra) PN could be of maternal origin. (Retained Polar body), Signs of activation are not very prominent. On cleavage, these oocytes very frequently result in a 3 celled stage with equal sized blastomeres. (B) A 1 PN Oocyte (presence of 2nd polar body and the signs of activation are suggestive of either the parthenogenic oocyte activation, or, failure to decondense the sperm head. One possibility of early Pronuclear abuttal cannot be ruled out, but in that situation this stage will last for a very short time, unless, its development is blocked (arrested) at this stage.

B

A

FIGURES 9.5A and B: (A) Initiation of cleavage. (B) A cleaved 2 cell embryo

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D

C

FIGURES 9.5C and D: (C) Initiation of 2nd cleavage. (D) A normal embryo at 3 cell stage

E

F

FIGURES 9.5E and F: (E) A 3 PN. (F) 3 cell embryo

In Vitro Fertilization and Blastocyst Culture

H

G

FIGURES 9.5G and H: (G) A good quality -4 cell embryo showing crossing over of blastomeres. (H) A 6 cell embryo

I

J

FIGURES 9.5I and J: (I) An eight celled embryo. (J) A Morula (> 8 cell stage)

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A

B

FIGURES 9.6A and B: An early Blastocyst. Formation of blastocele is visible at various locations within the blastocysts

FIGURE 9.6C: Expanded blastocyst, peripheral single cellular layer is visible along with the blastocele extended in the entire space, because of the increase in size, zona pellucida has stretched (thinning of zona pellucida)

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E

FIGURES 9.6D AND E: (D) Extruded inner cell mass (E) break in the zona

During transfer, the embryos are loaded in the catheter in a tiny drop (5 to 20 μl) of culture media, which is then very gently introduced into the uterus through vagina, often under ultrasound guidance. Once the tip of the catheter reaches at the desired location, the embryos are gently expelled with the small amount of culture media in which they are suspended. The selection of embryotransfer catheter is crucial and should be aimed towards a non-traumatic yet precisely controlled embryo deposition at the preferred site.

TECHNIQUES ASSOCIATED WITH IVF ASSISTED HATCHING Hatching: For implantation, the growing embryo has to break open its outer covering (the Zona pellucida). This process is called Hatching and is an important event in the embryonic growth. It is believed that in some females, the Zona pellucida is so hard that, the embryos are unable to break it and therefore, are unable to implant. The zona hardening can also occur in a variety of

laboratory conditions, thus making it difficult to implant. This problem can be rectified by, either by cutting a small part of zona pellucida(mechanical), or, by, dissolving some part of zona pellucida (chemical) in a solution designed particularly, for this purpose. Both these procedures promote hatching and come under – Assisted Hatching. EMBRYO, EGG AND SPERM FREEZING Both, the gametes (sperm and egg), and the embryos can be frozen under liquid nitrogen (at -196°C), in frozen state, they can be kept for years, after which they can then be used, if required, to produce a child. PREIMPLANTATION GENETIC DIAGNOSIS (PGD) A single cell (blastomere) is taken out (biopsy) of the embryo when it is at > 8 cell stage, this cell can then be analyzed genetically to check for any inheritable disorders of the baby may have. Embryonic cells are remarkable in their capacity for different into all of the body tissue as well as the germ-line. ES cells also can express their pluripotency

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in vitro, where they are capable of differentiating spontaneously onto numerous cell types, including cardiac myocytes, blood cells and neuronal cells. The future developments in this field of research, including potential clinical applicants. Will require an efficient blastocyst culture from which a healthier ICM can be obtained.

BIBLIOGRAPHY 1. Baker, Harald Bowine, David H Edgar: Sperm Preparation Techniques: In Textbook Of Assisted Reproductive Technologies, Laboratory And Clinical Perspectives Ed. D K Gardner, A Weissman, Colin M Howles, Zeev Shoham, Page 77, Martin Dunitz Ltd Publication. 2. Bongso A, Trouson AO, Gardner DK: IVF: In Trouson AO, Gardner DK (Eds): Handbook of IVF; 2nd ed Boca Raton: CRC Press LLC, 1999; 127-43. 3. Bourne H, Richings N, Liv DY, Clarke GN, Harari O, Baker HW. Sperm preparation for ICSI: methods and relationship to fertilization results. Reprod Fertil Dev 1995;7:177-83. 4. Dozortsev D, Rybouchin A, De Sulter P, Qian C, Dhont M. Human oocyte activation following ICSI: the role of the sperm cell. Hum Reprod 1995;10: 403-7.

5. Gordeon Baker, Harald Bourne, David H Edgear. Sperm preparation techniques: In Textbook of ARTs Laboratory and Clinical Perspectives Ed. DK Gardner, A Weissman, Colin M Howles, Zeev Shoham. Page 77, Martin Dunitz Ltd. Publication. 6. Grab D, Thirauf S, Rosenbusch B, Sterzic KK. Arch Gynaecol Obstet 1993; 252:137-4, 7. Kaylen M Silverberg, Tom Turner: Text Book. 8. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in-vitro fertilization. Fertil Steril 1988;49: 112-7. 9. Kruger TF, Menkuld R, Stander FS, et al. sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986;46:1118-23. 10. Nagy Z . Special applications of ICSI , Hum Reprod 1998; 13(Suppl 1): 143-54. 11. Nagy Z. Sterilization AC: Using ejaculated sperm for ICSI. Fertil Steril (1995) 63:808-15. 12. Ng F l, Liu D Y, Baker H W. Comparison of percoll, minipercoll and swim up methods for sperm preparation from abnormal semen samples. Hum Reproduction 1992;7: 261-6. 13. Veek LL, An atlas of human gametes and conceptures NY: Parthenon Publishing group, 1999. 14. Wor Heal Org. WHO Laboratory manual for the examination of human sperm and sperm-cervical mucus interaction. 3rd ed. New York; Cambridge University Press 1992;3-27.

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Intracytoplasmic Sperm Injection (ICSI) Sudesh A Kamat

INTRODUCTION One of the most significant advances in the field of reproductive medicine is the successful introduction of ICSI for the alleviation of male factor infertility. In Intracytoplasmic sperm injection (ICSI) a single spermatozoon is injected directly into the cytoplasm of the oocyte in order to achieve fertilization. The first pregnancies in humans following ICSI were reported in 1992.1 Although ICSI is now considered a routine technique, it remains one of the most demanding techniques to master, due partly to its inherent technical difficulty and partly to the heterogeneity of the cases encountered for treatment.2

INDICATIONS FOR ICSI 1. Male factor infertility which includes oligospermia, cryptozoospermia, asthenozoospermia, teratozoospermia and globozoospermia. 2. Azoospermia: Surgical sperm retrieval techniques like percutaneous epididymal sperm aspiration (PESA) and testicular sperm extraction (TESE) can be used in conjunction with ICSI for patients with

3. 4. 5. 6. 7. 8. 9.

obstructive or non-obstructive azoospermia (hypospermatogenesis, Sertoli cell-only syndrome and spermatogenic arrest). Patients with high titre of anti-sperm antibodies. Poor cryopreserved sperm motility upon thawing. Immotile cilia syndrome. Ejaculatory dysfunction (anejaculation, retrograde ejaculation). Unexplained infertility. Fertilization failure with standard IVF (rescue ICSI). Female factor infertility where previous cycles of standard IVF has resulted in either poor fertilization or repeated high incidence of polyspermy.

BASIC STEPS IN ICSI 1. 2. 3. 4. 5. 6.

Setting up the micromanipulator. Preparation of dishes for ICSI. Sperm preparation. Scanning, identification and grading of the oocytes. Denuding the oocytes. The actual ICSI procedure.

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FIGURE 10.1: ICSI micromanipulator

SETTING UP THE MICROMANIPULATOR The micromanipulator (Figure 10.1) consists of an inverted microscope supported on a solid vibration free table. The hydraulic manipulators are mounted on to the microscope pillars and bear micro-pipette holders that are connected to the micro-injector syringes. The coarse manipulators are motorized whereas the fine manipulators consists of hanging joysticks capable of moving in x, y and z axis. Hoffman modulation contrast optics is employed and the temperature controlled microscope stage is maintained at 37°C to help maintain cell viability. The microscope is fitted with video camera for viewing and recording the ICSI procedure. The micro-injector syringes are filled with oil, taking care to remove any air bubbles from the system. The holding pipette is usually fitted on the left-hand side holder and the microinjection pipette on the right hand side. The pipettes are aligned, first under low power and then high power, parallel to the stage and facing each other so that under the microscope it appears like a straight line (Figure 10.2). The holding pipette has an outer diameter of 80 μm and an inner diameter of 20 μm whereas the microinjecting pipette has a tip diameter of 5 μm. The microinjection pipette has a sharp beveled end with or without a spike, to facilitate easy penetration of the egg membrane. The micropipettes are angled at about 30 to 35 degrees to ensure smooth manipulation without interference

FIGURE 10.2: Alignment of micropipettes. The holding pipette is on the left hand side and the microinjection pipette on the right hand side

from the bottom surface of the petri dish. The pipettes can be aligned just prior to the ICSI procedure. PREPARATION OF DISHES For manipulation of gametes outside the CO2 incubator, commercially available HEPES buffered flushing medium is widely used, as it maintains the physiological pH of the media between 7.2 and 7.4. Oocytes and embryos are cultured in bicarbonate based IVF media inside the CO2 incubator (5% CO2, 37°C). All media and oil is equilibrated overnight in CO2 incubator. The following dishes are prepared and properly labeled: 1. Oocyte scanning: center well dish (falcon 3037) with 1 ml. HEPES buffered medium in the inside well and 3 ml. medium in the outside well. 2. Oocyte culture: center well dish with 1 ml. IVF culture medium, overlaid with paraffin oil. 3. Oocyte denuding: center well dish with 0.5 ml hyaluronidase (80 IU/ml). 4. ICSI dish: 2 to 3 droplets (10 μl each) of 10% PVP (polyvinyl pyrollidone) are placed in the center of the ICSI dish (Falcon 1006). 7 to 8 droplets (10 μl each) of HEPES buffered flushing medium are then placed in a circle around the PVP droplets and overlaid with oil. The droplets are numbered on the back-side of the dish (Figure 10.3).

Intracytoplasmic Sperm Injection

FIGURE 10.3: ICSI dish: 3 central PVP droplets and 7 droplets of HEPES buffered flushing medium which are numbered and overlaid with paraffin oil

SPERM PREPARATION FOR ICSI Semen sample is collected in a wide mouth sterile container and allowed to liquefy for 20 minutes. The sperm parameters like count, motility, morphology are recorded. Add 2 to 3 ml of flushing medium to the semen sample, mix and transfer into 6 ml. round bottom tubes. The tubes are labeled and centrifuged at 1500 rpm. for 7 minutes. The supernatant is discarded; the pellet is gently layered with 0.3 to 0.5 ml of culture medium and placed in the incubator. Density gradient centrifugation method can also be used for sperm preparation. In case of TESE, the seminiferous tubules are mechanically teased under the stereo-zoom microscope using two needles. The contents of the tubules are washed, centrifuged and observed for spermatozoa. In case of viable immotile spermatozoa, the viable spermatozoa may be identified using the hypo-osmotic swelling test. SCANNING, IDENTIFICATION AND GRADING OF OOCYTES The contents of the follicular fluid is poured out into a plain petridish (falcon 3652) and scanned under the stereo-zoom microscope. Once an oocyte is identified, it is picked up with a sterile transfer pipette (falcon 7575), washed in HEPES buffered medium and

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FIGURE 10.4: The oocyte cumulus complexes in culture medium

transferred into the dish with IVF culture media and placed back into the incubator. Based on the appearance of cumulus and corona cells the oocytes are graded as immature, pre-ovulatory and hypermature. DENUDING THE OOCYTES The oocyte cumulus complexes (Figure 10.4) are transferred from the culture medium and placed into the dish containing enzyme hyaluronidase. The denudation of oocytes relies upon the mechanical dispersal of the cumulus cells that comprise the corona radiate. The oocytes are gently aspirated in and out, using a 300 um denuding pipette attached to a mouth tubing aspiration assembly, so as to loosen the cumulus cells. The oocytes should not remain in hyaluronidase for more than 1 min. The partially denuded oocytes, with its corona radiate intact (Figure 10.5), are then transferred into droplets of flushing medium and denuded further using flexipets of reducing diameters (170 um and 140 um). The oocytes are simultaneously washed repeatedly in different droplets of flushing medium till all the coronal cells are removed (Figure 10.6). The oocytes are then placed in the HEPES buffered medium in the ICSI dish and observed for different stages of oocyte maturity. The

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Atlas of HART contain a haploid set of chromosomes, so that a normal diploid karyotype should be carried by the zygote following injection of another haploid set of chromosomes from the spermatozoa. THE ICSI TECHNIQUE

FIGURE 10.5: The partially denuded oocytes, with its corona radiate intact

ICSI involves the injection of a single spermatozoon into the cytoplasm of a mature metaphase II oocyte. The entire process is carried out in the ICSI dish. The holding and injection pipettes are properly aligned onto the micromanipulator. The ICSI dish containing the oocytes is removed from the incubator and the washed sperm suspension is added to one of the droplets of PVP. The viscous nature of PVP slows down the sperm movement making us easy to manipulate them. PVP enhances fine control aspiration and injection and also lubricates the inner wall of the microinjecting pipette thereby preventing sperm from sticking inside. The prepared ICSI dish is then placed on the heated stage of the micromanipulator. The actual procedure of ICSI can be divided into two parts: Sperm Selection and Immobilization

FIGURE 10.6: Denuded oocytes with all the coronal cells removed

dish with the oocytes is placed back into the incubator. The denuded oocytes are classified as follows: 1. Germinal vesicle: presence of a large nucleus inside the cytoplasm, absence of polar body. 2. Metaphase I: absence of germinal vesicle, absence of polar body. 3. Metaphase II: presence of a polar body, absence of germinal vesicle. Metaphase II oocyte is a prerequisite for successful fertilization through ICSI. This ensures that they

It is absolutely vital to damage the plasmalemma of the spermatozoon and so immobilize it, to prevent undue disruption of the ooplasm and ensure oocyte activation. Oocyte activation is a prerequisite for decondensation of the sperm nucleus and formation of the male pronucleus. The aligned microinjection needle is gently lowered into the droplet of plain PVP. PVP will be sucked in by capillary action. The microinjection pipette is raised and then lowered into another droplet of PVP containing the sperm. A morphologically normal motile sperm is selected. The spermatozoon is first focused and then the microinjection needle is adjusted so as to make sure they are in the same plane (Figure 10.7). The sperm is immobilized by hitting the sperm tail with the tip of the microinjection pipette (Figure 10.8). The immobilized sperm is then aspirated tail in first into the microinjection pipette (Figures 10.9 and 10.10). The pipette is then raised out of the PVP droplet.

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FIGURE 10.7: A morphologically normal motile spermatozoon is selected and the microinjection needle is adjusted so as to make sure they are in the same plane

FIGURE 10.9: The immobilized sperm is aspirated tail in first into the microinjection pipette

FIGURE 10.8: The selected sperm is immobilized by hitting the sperm tail with the tip of the microinjection pipette

FIGURE 10.10: The immobilized sperm is clearly visible at some distance away from the tip of the microinjection pipette

Ooplasmic Penetration and Sperm Deposition A mature metaphase II oocyte is brought into focus using the moving stage (Figure 10.11). The holding pipette is next lowered into the droplet containing the mature egg. The egg is then rotated using the holding pipette, so as to bring the polar body either at 6 O’clock or 12 O’clock position. Placement of the polar body at 6 or 12 O’clock position ensures that the injection pipette does not penetrate the oocyte closer to where the spindle is presumed to lie. The oocyte is fixed to

the holding pipette, at 9 O’ clock position, by aspirating or applying negative pressure (Figure 10.12). The oocyte is again focused so that the plasma membrane is visible as a distinct line. The injection pipette is then gently lowered into the oocyte droplet and focused in the same plane as the oocyte. The sperm is then gently advanced forward towards the pipette tip, so that minimum amount of PVP (1-2 pl) would be pushed into the ooplasm. The microinjection pipette is then aligned close to the egg at the 3 O’clock position and

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FIGURE 10.11: A mature metaphase II oocyte with the polar body visible at 12 O’clock position

FIGURE 10.14: The sperm is gently advanced forward towards the pipette tip and the pipette is slowly and steadily inserted through the zona pellucida

FIGURE 10.12: The oocyte is fixed to the holding pipette, at 9 O’clock position, by aspirating or applying negative pressure

FIGURE 10.15: The microinjection pipette has penetrated the zona pellucida and is gently advanced forward towards the oolemma

FIGURE 10.13: The microinjection pipette is aligned close to the egg at the 3 O’ clock position

FIGURE 10.16: The intact oolemma apparent by the invagination around the micropipette

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FIGURE 10.17: A small quantity of ooplasm is then aspirated in the microinjection pipette

FIGURE 10.20: The sperm can be seen deposited in the center of the oocyte

FIGURE 10.18: The oolemma has ruptured which is confirmed by the sudden free flow of the ooplasm into the microinjection pipette

FIGURE 10.21: The microinjection pipette is slowly withdrawn out of the egg as gently as possible making sure that the sperm remains inside

FIGURE 10.19: The sperm is slowly released inside the oocyte

FIGURE 10.22: The microinjection pipette has been completely withdrawn from the oocyte. The ICSI procedure is over

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slowly and steadily inserted through the zona pellucida and oolemma (Figures 10.13 to 10.15). The oolemma usually remains intact due to its inherent strength and elasticity which is apparent by the fact that the oolemma remains invaginated around the micropipette (Figure 10.16). A small quantity of ooplasm is then aspirated in the microinjection pipette (Figure 10.17). Initially, the flow of ooplasm into the injection pipette will be slow as it is impeded by the intact oolemma. Eventually, however, the oolemma will rupture which is confirmed by the sudden free flow of the ooplasm into the microinjection pipette (Figure 10.18). Once the oolemma is ruptured and penetrated, the sperm is then slowly released and deposited in the center of the oocyte (Figures 10.19 and 10.20). The microinjection pipette is slowly

withdrawn out of the egg as gently as possible making sure that the sperm remains inside (Figures 10.21 and 10.22). The oocyte is then released from the holding pipette. The procedure is repeated until all oocytes have been injected in a similar fashion. The injected oocytes are then transferred from the ICSI dish into IVF culture media dishes and placed back into the CO2 incubator. The micropipettes are removed from the tool holder and discarded. Oocytes are assessed for evidence of fertilization 16-18 hours after ICSI.

REFERENCES 1. Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17-8. 2. Fleming S, King R. Micromanipulation in assisted conception. UK Cambridge University Press, 2003.

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Cryopreservation: Gametes, Oocytes and Ovarian Tissue Vijay Mangoli, Ranjana Mangoli

INTRODUCTION Cryopreservation of human gametes, embryos and fertility related tissues are of great significance in Assisted Reproductive Technology. The concept of cryopreservation of human gametes and embryos is challenging and exiting mainly due to unpredictable results. Over past two decades, freezing techniques in human IVF have improved due to increased knowledge about biochemistry and membrane structure of human germ cells. However, still the pregnancy outcome in humans is quite low as compared to rodents and cattle.1 Due to limitations in experiments with human embryos for ethical reasons, the protocols applied are mainly based on animals. The species specificity plays a crucial role in selection of cryoprotectants and cooling- thawing rates for gametes and embryos at different stages. Freezing of an embryo is a complex physiochemical process of heat and water transport between the embryo and the surrounding medium.

CRYOBIOLOGICAL PRINCIPLES Primarily, cryopreservation is a method of preserving

live cells at subzero temperatures without loosing its regaining capacity of biochemical activities after thawing. The whole procedure can be divided into five phases. Pre-freeze, freezing, storage, thawing, and post thaw (Figure 11.1). During pre-freeze, the embryos are exposed and equilibrated to the cryoprotectants, which are absolutely necessary for a successful procedure. There are two types of cryoprotectants - permeating or intracellular and non-permeating or extracellular. The membranes around the ooplasm i.e. oolemma and zona pellucida have selective permeability for different cryoprotectants e.g. glycerol, propanediol, dimethylsulphoxide can enter the oolemma, whereas, solutions of high molecular weight compounds like sucrose act as extracellular cryoprotectant. The main aim of the permeating cryoprotectants is to minimize intracellular water content by replacing it. Extracellular cryoprotectants help them by increasing osmotic pressure in the medium surrounding the cell. As the cells are more permeable to water than cryoprotectants, they shrink and loose water to achieve equilibrium between intracellular and extracellular solutions. The complete process depends

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FIGURE 11.1: Schematic diagram showing steps of embryo freezing and thawing

upon extent of membrane permeability, type of cryoprotectant, and volume of the cell to be cryopreserved. The rate of cryoprotectant influx and that of water efflux, in turn depends upon permeability coefficient, temperature coefficient, and the reflexion coefficient. The choice of cryoprotectant depends upon type of cell to be frozen.2 Freezing phase begins at room temperature and ends at –196°C. This can be divided into four steps. Initial cooling to seeding temperature, the seeding process, slow freezing to –30/–50°C and rapid freezing to liquid nitrogen temperature. Cells remain unharmed as the temperature in the surrounding medium lowers to 0°C. The constituents of the cryoprotective solution make it undergo ‘supercooling’ i.e. maintaining liquid state below 0°C which reduces the temperature of ice formation. Culture medium freezes at approximately –5°C or lower and one of its constituents- sodium chloride

has a eutectic temperature of –21.3°C. As ice formation takes place in the medium, the water is lost and the solute concentration rises. Cryodamage is caused by increased salt fractionation in the remaining unfrozen aqueous solution before sodium chloride separates at its eutectic temperature, and the resulting high internal salt concentrations.3 Prior to seeding there is a drop in the temperature by about 30°C. To avoid extensive super cooling and nucleation at temperatures below –10°C, crystallization is induced 2°C below the freezing point of the solution by seeding the medium column containing oocytes and embryos, which acts as an ice nucleator. Seeding is an important step during cryopreservation that induces crystallization of the super cooled intracellular solution at appropriate temperature rather than allowing it to initiate in an uncontrolled manner that may be harmful for delicate cell organelles including spindle. Slow freezing is

Cryopreservation: Gametes, Oocytes and Ovarian Tissue preferred over rapid cooling for mammalian embryos mainly because of their comparatively low water permeability and low surface to volume ratio. Most common drawback of cryopreservation is freezing injury, mainly caused due to intracellular crystallization. Storage below –120°C maintains cell integration without alterations. Generally cells are frozen at 196°C, which is the boiling point of liquid nitrogen, because the only possible change that, can occur is DNA breakage caused by background radiation. However, there is experimental evidence to prove that gamma radiation equivalent to 2000 years of background radiation does not produce any detectable genetic change or reduction in embryo viability.4 Thawing rate depends upon freezing regime used. The procedure involving slow cooling below – 60°C from seeding temperature till immersing in LN2, requires slow thawing at the rates of below 40°C/ min. whereas, the protocol where slow cooling is done till -30°C before free fall of temperature of LN2 needs rapid thawing. Otherwise osmotic shock caused due to slow efflux of solute from the cell to its surrounding causes cell damage. As thawing is reverse procedure of freezing, it requires stepwise removal of cryoprotectant along with simultaneous refilling of water content. If freezing and thawing procedures are not coordinated properly, rapidly changing gradients of solutes develop causing membrane damage of the egg cell or embryo. Postthaw phase begins once the equilibrium is reached between the embryo and suspending medium. As the rate of water entering the cell is higher than cryoprotectant leaving the cell, the cell tends to increase in its volume. However, there is a limit of any cell to stretch itself. Embryos can withstand an increase in volume only by a factor of 2 without loosing their developmental capacity. 5 Thawed embryos swell osmotically when they are returned to isotonic solutions. The ability of the cell to expand is probably due to microvilli on the surface. Zona pellucida also plays a role in limiting its expansion. To reduce damage due to osmotic stress

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the cryoprotectant must be gradually removed from embryos. This can be achieved by lowering the extracellular cryoprotectant concentration in a stepwise manner. Sucrose acts as an osmotic counterforce to restrict water movement across the membranes. As a result the embryo shrinks progressively as the cryoprotectant leaves the cells. The embryo then regains its normal volume when it is transferred into sucrose free isotonic solution.

CRYOPRESERVATION IN ART Preservation of sperm and embryos has its own significance in ART. In some situations semen freezing becomes absolute necessity e.g. male partner is not available at the time of Ovum Pick up or female partner alone is coming from out of country for the treatment, males where sperm count is very low or having ejaculatory dysfunction or those undergoing chemotherapy in near future etc. in such cases their semen can be effectively frozen and used for IVF. During IVF, excess eggs are obtained as a result of controlled ovarian stimulation protocol. This may result in getting more than 3 embryos. As, transferring more embryos can result in multiple pregnancies; it becomes necessary to cryopreserve these excess embryos. In case if patient doesn’t become pregnant in fresh cycle, she can take her frozen embryos in subsequent cycle without undergoing ovum pick up. Cryostorage is an effective method of preservation. As mentioned earlier, the only damage to the stored tissue is ionizing radiation, yet the total dose received by a sample over some hundreds of years would hardly impair embryonic growth. There are no apparent mutagenic effects of cryopreservation per se. All tissue damage accumulated in storage is manifested on thawing because DNA repair enzymes do not function at such low temperatures.6 Human pronucleate eggs and embryos were frozen successfully using DMSO at a freezing rate of –0.3°C/minute.7 However, propanediol is generally preferred for freezing early stage human embryos.

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CRYOPRESERVATION OF MALE GAMETE – THE SPERM Human spermatozoa were among the first living cells to be studied for the effect of freezing - thawing. The recent history of cryopreserved human semen began in 1953 with the demonstration that thawed semen was capable of fertilization and the induction of normal embryonic development 8. The successful cryopreservation of cells is affected by the rates of freezing both above and below the freezing point, and the composition of the solution in which the cells are frozen. Human spermatozoa are resistant to cold shocks with respect to motility and oxygen consumption. 9 The most significant injuries to spermatozoa appear to be plasma membrane swelling and acrosomal leakage and breakdown.10 To prevent injury to sperm, even at controlled rates of freezing, cryoprotectants and extenders are added to the freezing medium. Glycerol is one of the cryoprotectants used for its protective action due to its ability to depress the freezing point and to reduce the electrolyte concentration to which the cells are exposed during freezing procedure. Glycerol is also beneficial in maintaining the pH and changing the temperature at which the liquid to solid phase change is reached. Various media, termed extenders, assists glycerol during cryopreservation by (a) Optimizing the osmotic pressure and pH: (b) Providing an energy source to prevent the sperms undesirable use of its own intracellular phospholipid; (c) Preventing bacterial contamination when an antibiotic is included; and (d) Allowing for dilution of the semen. The choice of an extender for human spermatozoa should be based on its ability to maintain cellular integrity and function during cryopreservation. FREEZING AND THAWING PROTOCOL Cryopreservation is cell specific. Cells may differ both in the amount of intracellular water and the permeability of the cell membrane. Sherman introduced the standard technique of vapor freezing.11 The optimal freezing rates of mammalian spermatozoa – with their small volume, large surface, and small amount of

intracellular water are usually in the range of 10 ° to 100°C per minute. Semen samples can be frozen using ultra rapid freezing and slow cooling (using programmable freezer) freezing method. For ultra rapid freezingto the liquefied semen sample, the mixture of cryoprotectant and the extender is added drop by drop in 1:1 proportion. Aliquots of this mixture are made into 1 ml Cryovials. These Cryovials are then placed 20 cm above liquid nitrogen (approximately 2°C) for 25 minutes. The samples are then plunged directly into liquid nitrogen.12 In slow cooling method, samples are frozen using a programmable freezer. The mixture of semen and

FIGURE 11.2: Semen freezing program

cryoprotectant will be loaded in the 0.25/0.5 ml plastic straws for freezing. The straws are sealed with a heat sealer. The sealed straws are placed in the chamber of the electronically controlled biological freezer. When the final temperature is reached, the straws are plunged directly into liquid nitrogen for storage at –196°C (Figure 11.2) Thawing is done by single stage method whereby vials/straws are brought to room temperature by exposing them to 30°C warm water for 3 minutes. These thawed semen samples are processed using semen preparation techniques that can separate dead sperm, debris, leukocytes, and pus cells from morphologically normal and motile spermatozoa. These techniques vary depending upon the quality

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FIGURE 11.3: Embryo freezing program

of thawed semen sample.

FIGURE 11.4A: Zygote before freezing

CRYOPRESERVATION OF OOCYTES AND EMBRYOS Initially, Dimethylsulphoxide (DMSO) was used in 1.5M to 3M of final concentration as a cryoprotectant for ultrarapid freezing. When it was observed that DMSO exerts toxic effects, 1-2 propanediol (PROH) in 1.5M concentrations is used as an intracellular cryoprotectant along with 0.1M sucrose as extracellular cryoprotectant for slow cooling technique13 Cryoprotectants react with proteins and lipids present in the outer protective layers – the zona pellucida and the oolemma. At higher concentration, they can damage oocytes and embryos. So they must be added and removed carefully at the lowest concentration compatible with successful cryopreservation (Figure 11.3). During thawing, the cryopreservatives are removed quickly and smoothly in 3-4 steps by exposing the embryos to progressively lower concentration of cryoprotectant in each step. Sucrose levels are raised slightly and solutions are made with declining concentrations of propanediol. Propanediol is used in 1M and 0.5M concentration and sucrose in 0.2M concentrations. Human embryos can be frozen at different stages of preimplantation development from pronucleate zygotes to expanded blastocyst. Cleaved embryos with intact zona and 50% or more of their original blastomeres intact are considered as surviving

FIGURE 11.4B: Same zygote post thaw

FIGURE 11.5A: 8- cell embryo in freezing medium

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FIGURE 11.5B: Same embryo in thawing medium

FIGURE 11.7: Few blastocysts post thaw

freezing and thawing. After thawing these embryos are cultured for 24 hours before replacing back into the uterus (Figures 11.4 to 11.7). BLASTOCYST CRYOPRESERVATION

FIGURE 11.5C: Same embryo at blastocyst stage

Glycerol is generally used for blastocyst freezing. Glycerol penetrates tissue slowly, so embryos must be exposed to it gradually Glycerol is used in dilutions of 2%, 4%, 6%, and 8%. Embryos are transferred through these increasing concentrations of glycerol at room temperature for 10 minutes in each concentration. Freezing rate is set at 4°C/min to 0°C, then 1°C to –7°C, which is held for 5 min when the seeding is done, then 3°C/min to –37°C and then plunge into liquid nitrogen. Thawing must be done very carefully, using more steps to remove glycerol using 5%, 4%, 3%, 2%, and 1% concentration of glycerol and then culture medium, exposing the embryos for 10 minutes in each dilution. A minimum of 6 hours in culture is usually required because blastocyst should be expanding when ready to replace into the uterus. 14 VITRIFICATION

FIGURE 11.6: Post thawed embryos at different stages

The main concern with ultrarapid or slow cooling cryopreservation techniques is formation of intracellular ice crystals that may damage the cells. Vitrification is the solidification of a solution at low

Cryopreservation: Gametes, Oocytes and Ovarian Tissue temperature without crystallization. Unlike slow freezing protocols, the entire solution remains unchanged during vitrification and the water does not precipitate, so no ice crystals are formed 15. Though this technique has been successful in other animals, only recently researchers have reported acceptable results in humans, and are still trying to improve results for different embryonic growth stages. Vitrification of water inside the cells can be achieved either by increasing the speed of temperature conduction or by increasing the concentration of cryoprotectant. Vitrification solutions are aqueous cryoprotectants that do not freeze when cooled at high cooling rates to very low temperature. Buffered medium base is used for vitrification (Phosphate buffer saline or Hepes buffered culture medium). High concentration of cryoprotectants (1.5 to 3 M) is used for Vitrification to minimize the cryoinjury. Ethylene glycol (EG) is the most commonly used cryoprotectant for vitrification process. It shows rapid diffusion and quick equilibration into the cell through zona pellucida and the cell membrane. Vitrification process has been successfully used blastocyst16 for the cryopreservation of oocytes, zygotes, cleaving embryos and OVARIAN TISSUE FREEZING Cryopreservation of ovarian tissue is useful for banking reproductive potential of young females for the future. It may be helpful for cancer patients who undergo chemotherapy or radiation treatment inducing sterility. Chemotherapy, especially with alkylating agents, is known to be gonadotoxic. Radiation is also toxic to the ovaries, with the combination of chemotherapy and radiation being particularly devastating. This new technology may have application in preventing natural menopause by protecting oocytes from chemotherapy, radiation or reproductive aging and preserving them for later use. Embryos were not only obtained, but the birth of a healthy girl was recently reported after cryopreservation and transplantation17. Pregnancies and births

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have been reported in different animal species after ovarian tissue and autologous grafts such as mice. One ovary or the portion of an ovary is removed surgically. The ovarian tissue, which contains immature primordial follicles, is frozen. Once the patient is cured of her diseases; her ovarian tissue could be thawed and used. The oocytes in the ovarian tissue are immature and must undergo further maturation in vitro before the eggs can be fertilized. Ovarian tissue freezing and autografting is still experimental, but there have been successes with its use to date. Resumption of endocrine function is reported for limited periods of time in women who had re-ovarian tissue implanted after freezing 18 . Embryos have also been derived from transplanted, frozen ovarian tissue. In near future, it will be possible to stop the ‘Biological clock’ of women by preserving their fertility and utilizing it at a later stage.

REFERENCES 1. Polge C, Willadsen SM. Freezing eggs and embryos of farm animals. Cryobiology 1978; 15: 370-3. 2. Mazur P, Rigopoulos N, et al. Preliminary estimates of the permeability of mouse ova and early embryos to glycerol. Biophys J 1976; 16:232a. 3. Mazur P. freezing of living cells: Mechanism and implications. Am J Physiol (Cell Physiol 16) 1984; 247: C125-C142. 4. Glenister PH, Whittingham DG, Lyon MF: Further studies on the effect of radiation during storage of frozen 8-cell mouse embryos at –196 C. J Reprod Fert 1984; 70:229-234 5. Mazur P, Schneider U. Osmotic responses of preimplantation mouse and bovine embryos and their Crobiological implications. 6. Ashwood-Smith MJ. The low temperature preservation of fetal cells. In Edwards RG (Ed). Fetal Tissue Transplants in Medicine. Cambridge, Cambridge University Press, 1992, P299. 7. Edwards RG, Steptoe PC. A Matter of Life. London, Hutchinsons, 1980. 8. Mahadevan M, Trounson AO. Effect of cooling, freezing, and thawing rates and storage conditions on preservation of human spermatozoa. 1984 Andrologica 16-52. 9. Bunge RG, Keetal WC, Sherman JK. Clinical use of frozen semen. Fertil Steril 1954;5: 520. 10. Weidel L, Prins GS. Cryosurvival of human spermatozoa frozen in eight different buffer systems. J Androl 1987;8: 41.

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11. Sherman JK. Temperature shock in human spermatozoa. Proc Soc Exp Biol Med 1987;88:6. 12. Weidel L, Prins GS. Cryosurvival of human spermatozoa frozen in eight different buffer systems. J Androl 1987;8:41. 13. Emilini S, Van den Bergh M et al: Comparison of ethylene glycol, 1,2 propanediol, and glycerol for cryopreservation of slow cooled mouse zygote 4 cell embryos and

blastocysts. Hum Reprod 2000;15, 905-10. 14. Etienne Van den Abbeel, Michel Camus, et al. Slow controlled-rate freezing of sequentially cultured human blastocysts: An evaluation of two freezing strategies. Human Reproduction 2005;20(10):2939-45. 15. Fahy G M. Vitrification: A new approach to organ cryopreservation. In: Meryman H T (Ed): Transplantation: Approaches to graft rejection. New York: Alan R. Liss; 1986;305-35. 16. Chen SU, et al. Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertil Steril 2000;74: 804. 17. Donnez J, Dolmans MN, Demylle D, et al. Live birth after Orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364: 1405-10. 18. Gunasena KT, Villines PM, et al. Live births after analogous transplant of cryopreserved mouse ovaries. Hum Reprod 1997;12:101-6.

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Laser in Assisted Reproduction Technologies Col. RK Sharma

INTRODUCTION As early as 1917, Albert Einstein had developed the theoretical basis for lasers. The term L-A-S-E-R is an acronym for Light Amplification by Stimulated Emission of Radiation (radiation here meaning light radiation). However, it was only in 1960, following much work by Soviet and US scientists, that an American team of researchers finally overcame the technological hurdles and assembled the first rudimentary but workable laser. Since then, lasers have undergone a rapid advance. Surgeons were quick to recognize the potential for a highly focussed cutting beam. By 1965, the first laser was in use for treating certain eye problems. Although the early machines were cumbersome and impractical, by the early 1970s argon lasers were being used experimentally for several medical procedures, including the treatment of diabetic eye problems and removal of small skin growths. Today the instruments are ubiquitous - contributing to areas as diverse as Beatlemania light shows, removing hemorrhoids and eradicating birthmarks. In producing light beams with different properties, laser

technology takes advantage of the special characteristics of a whole range of gases, solids and liquids, some of them exotic elements - including argon, krypton, neodymium, titanium and holmium. Depending on the substance used, the laser beam has a longer or shorter wavelength. The wavelength and pulse time determine the laser’s properties - its color and what it can do. Recently, diode laser system has been introduced in IVF fulfilling all safety requirements, while achieving a high standard of reproducibility in terms of ablation diameter. This 1.48-μm wavelength indium-galliumarsenic-phosphorus (InGaAsP) semiconductor laser offers a variety of laser applications to the embryologist, providing a promising tool for the microdissection of subcellular targets. The diode laser stands out due to the rapidity, the simplicity and the safety of the procedure, which is supported by healthy offspring after laser application. The laser assisted hatching software is designed for easy positioning, focus and measurement of embryos and simple alignment of the laser. The laser has three preset energy intensities of low (35 mW), medium (45 mW) and high

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FIGURE 12.1: 1480 Nm diode laser integrated in the inverted microscope incorporated

(55 mW) that can be delivered in a single 25 ms pulse with a single click of the mouse controller. Low power is used for perforating very thin (15 μm) or hard zona pellucida. Figure 12.1 shows the Nm Diode Laser assembly.

APPLICATIONS – Assisted hatching. Zona drilling and zona thinning. – Laser Assisted ICSI. – Embryo biopsy (Polar body, blastomere, blastocyst). – Sperm immobilization prior to ICSI. – Laser assisted microsurgical fertilization. – Removal of fragments by optical tweezer. – Separations of corona radiata. – Discriminate between viable and dead immotile spermatozoa. – LASER hemi dissection of zona pellucida. – Inner cell mass (ICM) excision. – Somatic cell nuclear transfer. – Transgenic applications. – Detecting zona pellucida hardness. – Cleaning of micro tools.

ASSISTED HATCHING Zona hardening after zona reaction subsequent to

fertilization occurs and is evidenced by an increased resistance to dissolution by different chemical agents. A loss of elasticity is also observed. This physiological phenomenon is essential for polyspermy block and for embryo protection during transport through the reproductive tract. It has been postulated that additional zona pellucida hardening may occur as a consequence of in vitro culture. Hatching could be inhibited in some in vitro cultured human embryos owing to the inability of the blastocysts to escape from thick or hardened ZP. The first report on the use of assisted hatching (AH) in human embryos was published by Cohen et al in 1990. These authors reported an important increase of implantation rates with mechanical AH in embryos from unselected IVF patients. WHY PERFORM ASSISTED HATCHING ? The ratio of lysine production to ZP thickness could determine whether the embryo will lyse the zona and perform the hatching. Embryos with thick zonae or those that present extensive fragmentation or cell death after freezing and thawing may benefit from assisted hatching. Both quantitative and qualitative deficiencies in lysine secretion could result in hatching impairment. Suboptimal culture conditions may cause such deficiencies. The trophectoderm of some embryos may not be able to secrete the “hatching factor” and lysine production could be influenced by a patient’s age. Uterine lysins action could also be impaired in some patients or cycles. Khalifa et al have shown that ZP thinning significantly increases the complete hatching of mouse embryos. Gordon et al demonstrated the usefulness of ZP thinning with acid Tyrode’s to improve hatching in hatching defective mouse embryos created by the destruction of one quarter of the blastomeres. They reported normal implantation rates after the transfer of assisted hatched embryos that had cell numbers reduced in pseudopregnant female mice. In a randomised study Liu et al demonstrated that

Laser in Assisted Reproduction Technologies implantation occurred significantly earlier in patients whose embryos were submitted to AH when compared to the control group, possibly by allowing earlier embryo endometrium contact. SELECTION CRITERIA FOR ASSISTED HATCHING 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

High basal FSH. Previous implantation failure. Thick zona pellucida. (More that 17 micron). Embryo with slow cleavage rate. Excessive fragmentation (more that 20%). Elderly patient (more than 38 years). Severe endometriosis. Post thawed embryos. In vitro maturation cycle. Poor responders. Unexplained infertility. Patients with three or less embryos.

METHODS When breaches are made in the ZP of early cleavage IVF embryos, embryonic cell loss may occur through the zona, as a result of uterine contractions after replacement of the embryos. It is advisable to manipulate the embryos for AH after the adherence between blastomeres has increased, just before compaction. Embryos at the 6 to 8 cell stage, at day three after insemination, can be manipulated with different methods for the performance of AH. It is very important to minimize the time the embryo is out from the incubator and to optimise the methodologies to reduce pH and temperature varia-tions that can be detrimental for embryo development. To reduce environment variations, Assisted Hatching is performed in microdrops of HEPES buffered medium covered with oil, under an inverted microscope with Nomarski or Hoffman optics, on a heated microscope stage, at 37°C. Proper alignment of the embryo is important to achieve the best results. Figures 12.2A and B show good and bad alignment. It is important that the size of the hole created in the zona is large enough to avoid trapping of the

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embryo during hatching but not so large that permits blastomere loss. Monozygotic twinning has been described as a consequence of Assisted Hatching. The adequate size of the hole seems to be 30-40 mm. Instead of making a big single hole it is better to make two or three holes of smaller diameter, which are located very close to each other (Figures 12.2C and D). Different protocols have been described, but a minimum of 30 minutes culture period seems to be sufficient before transfer of the manipulated embryos. Embryos transfer to the uterus has to be performed as atraumatically as possible to avoid damage of ZP manipulated embryos. Treatment during four days starting on the day of oocytes retrieval with broad-spectrum antibiotics and corticosteroids (methylprednisolone, 16 mg daily) had been postulated. Cohen et al suggested that such treatment might be useful to avoid infection and immune cell invasion of the embryos. Laser hatching has been proposed as more reproducible, controlled, and technically easier compared with mechanical and chemical means, which were used earlier. LASER ASSISTED HATCHING The use of laser techniques in the field of assisted reproduction for application in gametes or embryos was first described by Tadir et al. For fast and efficient clinical use of lasers systems in assisted hatching it is important that the laser is accurately controlled and produces precise ZP openings without thermal or mutagenic effects. The application of laser on the ZP for Assisted Hatching results in photoablation of the zona pellucida. Different Laser Systems Contact type: Erbium-yttrium-aluminium garnet (Er:YAG) laser system - Operates in the infrared region of the light spectrum. Non-contact type:

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Holmium: yttrium scandium gallium garnet (Ho:YSGG) laser system. UV laser 337.1 nm (Liow et al, 1996). 1480 nm diode laser (Germond et al, 1996; Rink et al, 1996; Montag et al, 1998; Blake et al, 2001).

thinning with the use of an Er: YAG laser, Five to eight pulses were needed to ablate 50% of the ZP in a length of 20 mm. The necessity of sterile micropipettes and optical fibers to deliver the laser beam to the target are the main disadvantages of contact mode lasers.

Contact Lasers The procedure is performed on a microscope slide, and the embryo is placed in a drop of medium covered with paraffin oil. The embryo is held with a holding pipette, and the laser is delivered through a microscope laser glass fiber, fitted to the manipulator by a pipette holder, in direct contact with the ZP. Several pulses are necessary to penetrate the ZP. Because each laser pulse removes only small portions of the ZP, the fiber tip has to be continuously readjusted to guarantee close contact with the remaining zona. Antinori et al described the method for ZP Good alignment

Non-contact Lasers Non-contact laser systems allow microscope objective delivered accessibility of laser light to the target,. Laser propagation is made through water and as it avoids the UV absorption peak of DNA, no mutagenic effect on the oocytes or embryo is expected. Blanchet et al first reported the use of a noncontact laser system (248 mm KrF excimer) for mouse ZP drilling. Neev et al described the use of a non-contact laser Ho:YSGG (2.1 mm wavelength) for assisted hatching Bad alignment

A

B

C

D

FIGURE 12.2: Good and bad alignment of embryo before laser is applied with laser beam

Laser in Assisted Reproduction Technologies in mice. The study shows the lack of embryo toxic effects as well as improved blastocyst hatching. Similar results were reported by Schiewe et al. Rink et al recently designed and introduced a noncontact infrared diode laser (1.48 mm) that delivers laser light through the microscope objective. The drilling mechanism is explained by a thermal effect induced at the focal point by the absorption to the laser energy by water and /or ZP macromolecules, leading to a thermolysis of the ZP matrix. Laser absorption by the culture dish and medium is minimal. The LASER beam has to be aligned properly before use to get a clean hole at a desired place. Pilot beam is helpful for the same. The red pilot beam to align the laser perfectly so that no flaring can occur. The effect on the ZP is greatly localized, and the holes are cylindrical and precise as seen on the electron microscope picture. Exposure time (10-40 ms) can be minimized. The safety and usefulness of the system was demonstrated in mice and humans. Its use for PB as well as blastomere and blastocyst biopsy has also been reported. The system is compact and easily adapted to all kinds of microscopes. The size of the hole is related to the laser exposure time, and thus the system is simple and easy to use. Two pulses of 15-20 ms are usually needed to drill a 30-403 μm hole in 15-17 μm thick zona. Assisted hatching may be clinically useful and individual assisted reproduction programmes should evaluate their own patient populations in order to determine which subgroups may benefit from the procedure. The routine or universal performance of assisted hatching in the treatment of all IVF patients appears, at this point, to be unwarranted (American Society of Reproductive Medicine, 2000).

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non-contact diode laser for assisted hatching of in vitro matured/in vitro fertilized/in vitro cultured (IVM/IVF/IVC) blastocysts. Short irradiation exposure times (3-5 ms) were applied and a significant increase in the hatching rate was observed. Even though no statistically significant differences were observed, a trend towards higher pregnancy and implantation rates was obtained when laser drilled assisted hatching blastocysts were replaced compared with non-drilled blastocysts (44.4 v 23.8% and 30.6 v 11.6%). LASER ZONA THINNING During this procedure embryo is stabilized with the holding pipette at 9 O’ clock and laser thinning is done from 3 O’ clock to 6 O’ clock position. Advantages • Zona thinning is more efficient than zona drilling and hatching after zona thinning is more similar to the natural one. • Loss of blastomeres is prevented. • Toxic chemicals, bacteria or white blood cells do not come in contact with embryo.

BLASTOCYST ASSISTED HATCHING Even though Assisted hatching is usually performed on early cleavage embryos (day 3, 6-8 cell stage), it can also be applied to blastocysts to increase implantation rates. Park et al recently reported the use of 1.48 mm

FIGURE 12.3: Electron microscopic picture of a hole made by laser in the zona pellucida

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Atlas of HART (amniocentesis/CVS) and it is performed on preimplantation embryos and only unaffected embryos are transferred. Polar body or blastomere from cleavage/Blastocyst stage are taken after making a hole with the LASER. First pregnancy after PGD for X-linked disorder was reported by Handyside et al in 1990. SPERM IMMOBILIZATION PRIOR TO ICSI The application of a non-contact diode laser for sperm immobilization prior to ICSI is a potentially useful alternative to the conventional mechanical approach and reduces time of ICSI procedure. FIGURE 12.4: Laser assisted ICSI

Figure 12.3 shows electron microscope pictures of hole in the zona pellucida. IVM AND LASER The aspirated oocytes in the early follicular phase for IVM are immature with the result the zona pellucida is affected and LASER hatching helps in increasing implantation.

LASER ASSISTED ICSI Performing ICSI through a laser-drilled hole in the zona pellucida reduces the risk of oocyte damage related to deformation during the initial phase of the microinjection procedure. LASER assisted ICSI is suitable for patients whose oocyte show inherent fragility or with difficult ZP/ oolemma penetration and high degeneration rates after the standard ICSI procedure (Rienzi L et al, Center for Reproductive Medicine, European Hospital, Rome, Italy) (Figure 12.4). Laser-assisted zona pellucida thinning prior to routine ICSI increases the hatching rate in vitro, which may explain the increase in pregnancy rate, at least in day 3 transfers (M Moser et al). EMBRYO BIOPSY Preimplantation genetic diagnosis (PGD) was developed in 1980’s as an early method of genetic diagnosis and as an alternative to PND

LASER ASSISTED MICRO SURGICAL FERTILIZATION Human sperm have been successfully manipulated in an optical trap and these optically trapped directed single sperm cell can be moved through a precise LASER created hole in the zona pellucida and sperm fused with oocytes. APPLICATIONS Fertilization using immotile and acrosome defective sperms. Removal of Fragments of Optical Tweezer Optical tweezers use a focused laser beam to trap a microscopic object and work by means of the gradient force of light. A sharp focal point is created using a laser, and when a small particle comes into contact with the focus it remains trapped. The particle is pulled towards the more intense light because it has a greater index of refraction than the surrounding medium, and momentum must be conserved. Optical tweezers have many uses, and the list is growing. Objects ranging from tens of nanometers to many micrometers in size can be manipulated by optical tweezers with laser powers ranging from several milliwatts up to several watts depending on the size of the object and the amount of radiation it can withstand. These can be used to remove the fragments

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a curling of the sperm tail only in viable sperm, similar to the reaction observed in the hypo-osmotic swelling test. Laser Dissection of Zona Pellucida Dissection of the zona pellucida into two halves with equal diameter for the hemizona assay can be done by giving LASER shots across the center of the oocytes at 6 and 12 O’ clock (Figures 12.5A and B). Inner Cell Mass (ICM) Excision

A

LASER assisted ICM dissection is useful to establish ESC lines in a controlled and xenogenic by-product free environment (T Takeuchi et al) (Figure 12.6). Somatic Cell Nuclear Transfer LASER suitably makes a very small, neat slit in the zona pellucida which facilitates the DNA removal and thus improves enucleating efficiency, reduces the lysis rates of the eggs and improves ability to create human cloned embryos for the purposes of deriving patient specific stem cell lines. Transgenic Applications Insertion of cells into the blastocysts cavity is easier with the LASER and causes less damage to the

B

FIGURES 12.5A and B: Hemizona dissection with laser at 6 and 12 O’clock position

from the embryo without lateral damage. Separation of Corona Radiata LASER shots over the corona radiata can loosen the cells and makes passage for the sperms easier to the ZP and can be useful in cases with oligospermia. Discriminate between Viable and Dead Immotile Spermatozoa Single laser shot to the far end of the sperm tail causes

FIGURE 12.6: Inner cell mass excision

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blastocysts than with their old micropipette method. Detecting Zona Pellucida Hardness Use of a LASER to evaluate zona pellucida hardness at different stages of embryonic development in vitro and in vivo can help in study of the ZP properties. Cleaning of Micro Tools During micromanipulation some times the cellular elements or debris get stuck with the micro tools and are difficult to remove mechanically. Few LASER shots can help in cleaning the tools. To conclude LASER is a solution looking for a problem and ART is a fertile field for its application. Imagination and dexterity of the embryologist would determine its future.

BIBLIOGRAPHY 1. Alikani M, Cohen J. Micromanipulation of cleaved embryos cultured in protein-free medium: A mousemodel for Assisted hatching. J Exp Zoology 1992; 263:458. 2. Bleil JD, Wassarman PM. Structure and function of the Zona pellucida: Identification and characterization of mouse oocyte’s zona pellucida. Dev Biol 1980; 76:185. 3. Check JH, Hoover I, Nazari A, O’Shaughnessy A, Summers D. The effect of Assisted hatching on pregnancy rates after frozen embryotransfer. Fertil Steril 1996;65: 254-7. 4. Chao KH, Chen SU, Chen HF, Wu MY, Yang YS, Ho HN. Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)-embryotransfer (ET), but not that of IVF-tubal ET in patients with repeated IVF Failures. Fertil Steril 1997; 67: 904-8. 5. Cohen J. Assisted hatching of human embryos. J In Vitro Fertil and Embryotransfer. 1991; 8:179-90. 6. Cohen J, Elsner C, Kort H, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisted hatching using micromanipulation. Hum Reprod (1990) 5:7-13. 7. Cohen J, Ahkani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992; 12:2-7. 8. Cohen J, Feldberg D. Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol Reprod Dev 1991;30:70-8. 9. Cohen J, et al. Selective Assisted hatching of human embryos, Ann Med Sing (in press).

10. Cohen J, Feldberg D. consequences of size and number of zona pellucida opening on hatching and trophoblast outgrowth in the mouse. Mol Reprod Dev 1991;30:70. 11. Cieslak J, Ivakhnenko V, Wolf G Sheleg S, Verlisky Y. Three dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching. Fertil Steril 1999; 71:308-13. 12. Dean J. Biology of mammalian fertilization the role of the zona pellucida. J Clin Investig 1992; 89:1055-9. 13. Drobnis EZ, Andrew JB, Katz DF. Biophysical properties of the zona pellucida measured by capillary suction: Is Zona hardening a mechanical phenomenon? J Exp Zool 1988; 245:206. 14. Hellebaut S, De Sutter P, Dozortsev D, Onghena A, Qian C, Dhont M. Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? A prospective randomized study. J Assist Reprod Genet 1996; 13:19:22. 15. Khalifa EAM, Tucker MJ. Partial thinning of the zona pellucida for more successful enhancement of Blastocyst hatching in the mouse. Hum Reprod 1992; 7:532-6. 16. Khalifa EAM, Tucker M J, Hunt P. Cruciate thinning of the Zona pellucida for more successful enhancement of Blastocyst hatching in the mouse. Hum Reprod 1992; 7:5326. 17. Neev J, Schiewe M, Sung, et al. Assisted hatching in mouse embryos using a non-contact Ho: YSGG laser system. J Assisted Reprod Genet 1995; 12:288-93. 18. Obruca A, Strohmer H, Sakkas D, et al. Use of lasers in Assisted fertilization and hatching. Hum Reprod 1994; 9:1723-6. 19. Palanker D, Ohad S, Lewis A, et al. Technique for cellular microsurgery using the 193 nm Excimer laser. Laser Surg Med 1991; 11:589-6. 20. Rink K, Delacreaz G, Saalthe RP et al. Non-contact micro drilling of mouse zone pellucida with an objective delivered 1.48pm diode laser. Laser Surg Med 1996; 18:5262. 21. Schoolcraft W, Schenker T, Gee M, Jones GS, Jones HW. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994; 62:551-4. 22. Strohmer H, Feichtigner W. Successful clinical application of laser for micromanipulation in an in vitro fertilization program. Fertil Steril 1992; 58:212-4. 23. Tadir Y, Wright WH, Vafa O et al. Micromanipulation of sperm by a laser generated optical trap. Fertil Steril 1989; 52:870-3. 24. Tadir Y, et al. Micromanipulation of gametes using laser microbeams. Hum Reprod 1991; 6:1011. 25. Trounson AO, Moore NW. The survival and development of sheep eggs following complete or partial removal of the zona pellucida. Repro Ferti 1974; 41:97-105. 26. Veiga A, Sadalinas M, Benkhalifa M, et al. Laser blastocyst biopsy for perimplantation diagnosis in the human Zygote 1997; 5:351-4.

Preimplantation Genetic Diagnosis

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Preimplantation Genetic Diagnosis Prochi Madon, Arundhati Athalye Nandkishor Naik, Firuza Parikh

INTRODUCTION Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis. PGD is done during the IVF procedure, mainly by biopsy of a single cell or blastomere from a cleavage stage embryo. Genetic diagnosis of the embryo is done on the nucleus of this cell by fluorescence in situ hybridization (FISH) to check for common aneuploidies, or inherited translocations. Molecular analysis by single cell PCR can also be carried out for single gene disorders such as beta-thalassemia, though at present, only PGD by FISH is carried out in India. PGD is a very specialized technique and needs a lot of expertise. Before embryo biopsy, the embryo is placed in a medium free of calcium and magnesium, to prevent adhesion of cells, so that a single cell can be aspirated easily. The embryo is held with the holding pipette on the left, under a micromanipulator in such a way that a cell with a prominent nucleus, suitable for biopsy is on the right. A hole is drilled in the zona adjoining this cell with a non-contact diode laser, or with acid tyrode (Figure 13.1). A single cell is then gently aspirated with the suction pipette, without

damaging the embryo (Figures 13.2A to G). The cell is then placed in a labeled drop of medium under oil in a petri dish. One cell is similarly aspirated from each embryo for genetic testing, and taken to the genetics laboratory for fixation and FISH. During fixation, the aim is to fix the cell to a microscope slide in such a way that the cytoplasm is removed, but the nucleus is intact and is not lost in the

FIGURE 13.1: Inverted microscope with micromanipulator and LASER system required for blastomere biopsy

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A

B

C

D

E

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G

FIGURES 13.2A to G: Cleavage stage blastomere biopsy. (A) A 30 µm hole is drilled with a diode LASER beam in the zona pellucida of an 8-celled embryo near the blastomere to be biopsied. (B) A pipette is introduced through the opening with the help of micromanipulator. (C,D,E) The blastomere is slowly aspirated into the pipette and pulled out. (F) Intact embryo after complete removal of blastomere. (G) Intact blastomere after biopsy

FIGURE 13.3A: Fixed blastomere (good fixation). An intact nucleus (40X) without cytoplasm

Preimplantation Genetic Diagnosis

FIGURE 13.3B: Fixed blastomeres (fair fixation)

FIGURE 13.3C: Fixed blastomeres (Bad or poor fixation). Cytoplasm not removed

FIGURE 13.3D: Binucleated fixed blastomere

FIGURE 13.3E: Fixation of entire arrested embryo

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FIGURES 13.4A and B: (A) FISH on blastomere showing normal diploid signals for chromosomes 13 (2 green) and 21 (2 orange) using Vysis Aneuvysion LSI probe mix. (Counterstained with DAPI). (B) FISH on blastomere showing normal diploid signals for chromosomes 18 (2 Aqua) and sex chromosomes—X (1 green) and Y (1 orange) using Vysis Aneuvysion CEP probe mix. (Counterstained with DAPI)

FIGURES 13.5A and B: (A) Multicolor FISH on a blastomere showing 2 signals each for chromosomes 13 (red), 16 (2 aqua-sky blue), 18 (dark blue), 21 (green) and 22 (gold-yellow) indicating no aneuploidy for these chromosomes, using the Vysis Multivysion -PB probe mix (No counterstain used). (B) FISH on same blastomere in the 2nd step showing 2 signals each for chromosomes 18 (Aquablue) and X (green) indicating no aneuploidy of chromosome 18 and sex chromosomes (female) using Vysis Aneuvysion CEP probe mix (Counterstained with DAPI). A total of 7 chromosomes analyzed in 2 steps

Preimplantation Genetic Diagnosis

FIGURES 13.6A and B: (A) FISH on a blastomere of an arrested embryo showing 3 signals each for chromosomes 13 (green) and 21 (orange) indicating triploidy. (B) FISH on a blastomere showing 3 signals each for chromosome 18 (aqua) and sex chromosomes (XXY – 2 green, 1 orange) indicating triploidy

FIGURE 13.7: FISH on blastomere showing trisomy 13 (3 green and 2 orange signals) using the Vysis Aneuvysion LSI probe mix

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FIGURES 13.8A and B: FISH on a blastomere in 2 steps enables aneuploidy detection of 5 chromosomes in a cost effective manner using the Vysis Aneuvysion probe kit. (A) shows 2 signals each for chromosomes 13 (green) and 21 (orange) using LSI probes. (B) FISH on the same blastomere (step 2) using CEP probes shows Klinefelter syndrome (XXY) indicated by the presence of 2 green and 1 orange signal. Two aqua signals indicate a normal diploid status for chromosome 18

FIGURE 13.9: A balanced translocation t(1;3)(q24;q25) in a woman with repeated spontaneous abortions. PGD in such cases, using special probes can help to identify embryos with an unbalanced translocation, which are not transferred

A normal cell, or one with a balanced translocation, will show 2 signals for each probe used. A combination of 2 centromere and 1 telomere, or 2 telomere and 1 centromere probe of the chromosomes involved is required.

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FIGURE 13.10: Difficulties in analysis of the signals due to poor fixation and cross-hybridization

FIGURES 13.11A and B: (A) Regular split signals are 1 or 2 diameters apart. (B) Wide split signals (green) showing a bridge-like link. These 2 are counted as 1 split signal

process (Figures 13.3A to E). The cell from each embryo is properly labeled, so that the normal embryos can be transferred later. Each biopsied cell is first placed in hypotonic solution for a minute, and then transferred to a circled area on a slide, with a very small drop of hypotonic, under the stereo microscope. Just before the hypotonic dries, a small drop of Carnoys fixative is added, and air is gently blown onto it. One or two additional drops of fixative are added when the nucleus is fixed to the slide. The cell is then relocated under

phase contrast, on the fluorescent microscope which will be used for FISH. We use a Zeis fluorescent microscope and Metasystems image analysis software. Cells from the other embryos are similarly fixed. Difficulties in analysis of signals due to poor fixation and cross hybridization is seen in Figure 13.10. Split signals are shown in Figures 13.11A and B. For FISH, the slides are dehydrated in grades of ethanol, and co-denatured with the appropriate FISH probes for 5 minutes. We use a combination of Vysis PB

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probes and Aneuvysion probes, to check for aneuploidy of chromosomes 13, 16, 18, 21, 22, X and Y (Figures 13.5A and B). The slides are hybridized in an incubator for at least 3 hours. The extra probe is then washed off in hot buffered detergent, and the slides are mounted with an antifade and a counterstain (DAPI). The cell is relocated under 40X, and then examined under the oil immersion lens, with the appropriate filters. Two signals for each autosome indicate a normal diploid pattern (Figures 13.4A and B). Three signals of a particular chromosome indicate Trisomy (Figure 13.7) whereas three signals of each autosome indicate Triploidy (Figures 13.6A and B). Sex chromosome abnormalities can also be detected. It is possible to check for additional chromosomes, by stripping off the probes and then hybridizing the same cell with other probes. On the other hand, testing for only 5 common aneuploidies helps to reduce the cost and make the test more affordable (Figures 13.8A and B). Mosaicism cannot be detected by testing only one cell, hence prenatal diagnosis is necessary by amniocentesis in the second trimester in all cases undergoing PGD.

For the detection of inherited reciprocal translocations, a combination of 3 centromere/telomere probes of the chromosomes involved needs to be checked. Two signals of each probe tested indicate a normal embryo, or one with a balanced translocation (Figure 13.9). Embryos with unbalanced translocations are not transferred. As the required probes have to be specially obtained for the patient in advance, it increases the cost, compared to PGD of common aneuploidies. In the absence of availability of single cell PCR for PGD of molecular genetic disorders in India, X-linked disorders such as hemophilia and DMD can be avoided by transferring only female embryos, which may either be normal, or carriers. The male embryos may either be affected or normal, and are not transferred, to eliminate the risk of carrying an affected child. PGD is an option when parents cannot face the trauma of repeatedly aborting an affected fetus in high risk pregnancies, when the abnormality is detected at a later stage by prenatal diagnosis.

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14

In Vitro Maturation of Human Oocytes

H Ingolf Nielsen, Anne Lis Mikkelsen

INTRODUCTION In vitro fertilization (IVF) and embryotransfer (ET) was first carried out by Steptoe and Edwards (1978) during a natural cycle. In such cycles there are obvious problems associated with the fact that usually just one oocyte is obtained. Failures in obtaining this oocyte at all during oocyte retrieval, and the possibility of no fertilization and no or suboptimal preimplantation embryo development and implantation led to techniques, where gonadotropin administration secured multiple follicle development. This made the retrieval of several oocytes possible and thereby increased the probability of success. IVF and ET have since become an established and successful treatment of subfertility, and severable improvements have been added over the years, most notably ICSI. A relatively recent addition is in vitro maturation (IVM). In spite of the success there are several problems associated with hyperstimulated standard IVF with or without ICSI, most notably OHSS in PCOS patients. Therefore in vitro maturation of human oocytes has in recent years gained increasing importance. Oocyte maturation is generally defined as the reinitiation of the first meiotic division leading to

metaphase II (M II), combined with the appropriate cytoplasmic processes, which are necessary for proper fertilization and early embryo development. Using in vitro maturation techniques, oocytes are harvested in the GV stage (germinal vesicle, prophase I) (Figure 14.1) without or with minimal gonadotropin

FIGURE 14.1: Immature (GV) oocyte

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Atlas of HART Cha et al (1991) reported the first pregnancy and triplet delivery following in vitro maturation of immature human oocytes obtained surgically from unstimulated ovaries and donated to another patient. Since then, experience using in vitro maturation has been obtained mainly from two groups of patients. One group are women suffering from PCOS. These women are extremely sensitive to stimulation with follicle stimulating hormone (FSH) and have a significant risk of developing ovarian hyperstimulation syndrome (OHSS). The other group are regularly cycling women with normal ovaries referred for IVF and ICSI due to severe male infertility problems.

IVM TREATMENT OF WOMEN WITH PCOS

FIGURE 14.2: Mature (MII) oocyte

administration, and matured in special maturation media. When mature (M II) (Figure 14.2), the oocytes are either cryopreserved or inseminated using ICSI or standard IVF methods.

SELECTION OF PATIENTS FOR IVM Not infrequently immature oocytes are collected in normal IVF cycles using ovarian hyperstimulation. At the time of hCG administration the oocyte population may be heterogeneous. This leads to retrieval of oocytes at different stages of maturation. Often around 15% of oocytes will remain in prophase I of meiosis. Some patients have almost 100% immature oocytes one time after the other. These oocytes can mature in vitro and develop into viable embryos. The first delivery recorded following in vitro maturation resulted from such a case (Veeck et al, 1983), and other groups have reported on similar cases (Prins et al, 1987, Nagy et al, 1996, Liu et al, 1997). These oocytes, however, may represent an inferior population as they failed to mature although the follicles were exposed to supra-physiological concentrations. Generally, therefore, “oocyte rescue” in stimulated cycles are unsuccessful using present IVM techniques.

In 1994 Trounson et al described the first pregnancy and delivery after in vitro maturation of oocytes obtained in a patient with PCOS. The following year Barnes reported a similar case (Barnes et al, 1995). During the following years several reports on successful IVM procedures appeared. In unstimulated cycles Cha et al, (2000) achieved a pregnancy rate of 27.1%. This pregnancy rate was obtained after transfer of an average of 6.3 embryos per patient, and the implantation rate was quite low (6.9%). To compensate for this, endogenous priming with FSH (Suikkari et al, 2000, Mikkelsen and Lindenberg, 2001) or hCG (Chian et al, 2000) was suggested before oocyte pick-up and IVM. Suikkari et al, (2000) proposed low-dose (37.5 IU) recombinant FSH from previous lutheal phase until the leading follicle reached 10 mm. This resulted in maturation and fertilization rates in women with PCOS comparable with those in regularly cycling women, however, no pregnancies were achieved. A beneficial effect of FSH priming has been found in a prospective randomized study (Mikkelsen and Lindenberg, 2001). Oocytes obtained after priming with rFSH for 3 days followed by deprivation for 2-3 days before aspiration was compared with oocytes obtained in unstimulated PCOS patients. FSH priming improved the pregnancy rate (29% versus 0%) and implantation rate (21.6% versus 0%) compared with

In Vitro Maturation of Human Oocytes the non-primed group. In 1999 Chian et al reported, that giving 10.000 IU hCG 36 hours before oocyte retrieval improved maturation rate of immature oocytes from PCOS women (Chian et al, 1999). In a prospective randomised study this group later demonstrated, that hCG priming not only improved the maturation rate, but also hastened the maturation process (Chian et al, 2000). Pregnancy rates of 30-35% have been obtained in a multicenter study including 1000 cycles with hCG priming before immature oocyte retrieval (Chian, 2004). The implantation rate was, however, still low (10-15%). Lin et al (2003) using hCG priming have reported similar pregnancy rate of 33.8% and implantation rate of 10.5%. Additional FSH priming (75 IU per day for 6 days initiated on day 3) did not make any difference to oocyte recovery, maturational and developmental potential, fertilization rate and pregnancy rate. The potential mechanism of the action of hCG on these small follicles is unclear.

IVM TREATMENT OF REGULAR CYCLING WOMEN WITH NORMAL OVARIES Control of the menstrual cycle is a complex process involving both the hypothalamic-pituitary axis as well as local (paracrine and endocrine) factors (Baker and Spears, 1999). The follicles destined to ovulate will be selected from a cohort of follicles, which enter the follicular phase of the menstrual cycles with a diameter of 2-6 mm. The selected follicle will grow to a diameter

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of 20-25 mm at the time of ovulation. Circulating levels of FSH and LH regulate the follicular growth and development. The rise in serum FSH levels during the early follicular phase causes a cohort of follicles, responsive to FSH stimulation to grow. The dominant follicle can be distinguished from other cohort follicles by size (>10 mm diameter) (Pache et al, 1990). Synthesis of estradiol is closely linked to development of the preovulatory follicle. The concentration of estradiol in the follicle and serum correlates significantly with the size of the follicle, and is the principal factor for establishment of dominance. It has a negative feedback on the hypothalamus axis with subsequent decrease in the level of FSH. The dominant follicle withstands this decline, while subordinate follicles are susceptible to a decline in gonadotropins and undergo atresia. The subordinate follicles, however, can be rescued and thereby avoid atresia by stimulatory treatments with FSH (Macklon and Fauser, 2000) or by retrieval of immature oocyte followed by in vitro maturation. The first deliveries following IVM of immature oocytes in unstimulated cycles resulted from oocytes being retrieved at different times during the menstrual cycle (Barnes et al, 1996, Russell et al, 1997,Thorton et al, 1998, Cobo et al, 1999). Mikkelsen et al (2000) aimed at having the oocyte pick-up coincide with selection of the dominant follicle. Oocytes were aspirated after a leading follicle of 10 mm and an endometrial

FIGURES 14.3A and B: Ultrasound image prior to OPU in an IVM cycle. (A) Leading follicle 12 mm. (B) Cohort follicles 5-7 mm

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thickness of at least 5 mm were observed during ultrasound scanning (Figure 14.3). In 87 cycles a pregnancy rate of 18% per transfer was obtained. Oocytes originating from the ipsilateral ovary did not show to have an impaired competence to mature and cleave compared to oocytes originating from the contralateral ovary (Mikkelsen et al, 2001d). Few studies have examined the effect of priming with FSH before aspiration of immature oocytes in regularly menstruating women (Trounson et al, 1998, Suikkari et al, 2000, Wynn et al, 1998, Mikkelsen et al, 1999, Mikkelsen et al, 2003). The series are small, a variety of stimulation regimens have been used and only few conclusions can be drawn. An unstimulated IVM cycle is shown schematically in Figure 14.4. In two studies (Wynn et al, 1998; Suikkari et al, 2000) an improvement in the harvest of oocytes has

been reported and perhaps oocyte quality can be improved by mild ovarian stimulation with FSH prior to oocyte collection. However, Wynn et al, (1998) did not attempt fertilization of the oocytes and no conclusion concerning developmental capacity can be drawn from that experiment. Mikkelsen et al (1999) found no beneficial effect of FSH priming in a prospective randomized study.

REASONS FOR CHOOSING IN VITRO MATURATION Table 14.1 gives an overview of the differences in standard IVF treatment and IVM. As seen, there are multiple reasons for doing IVM. 1. The safety aspect: PCOS patients are susceptible to ovarian hyperstimulation syndrome (OHSS) following administration of the relatively large amounts of fertility drugs necessary for superovulation in standard IVF treatment.

FIGURE 14.4: An unstimulated IVM cycle

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TABLE 14.1: Differences in traditional IVF treatment and IVM treatment Traditional IVF • Relatively many oocytes/embryos • ‘High’ pregnancy rate per OPU • Down regulation • Daily hormone injections • hCG injection • Emotional stress • Long treatment time - 4-6 weeks • Potential side effects (e.g. OHSS)

2. The ethical aspect: If the fertility problems of the couple are due to male factor, would it not make sense to avoid a stressful treatment of the female partner? 3. The economical aspect: If the patients are paying for the treatment themselves, it is obviously very attractive to save the costs of fertility hormones. If the public or private insurances—are paying, money could be saved or used to treat more patients. The IVF center has the advantage of shorter cycle length and fewer consultations and examinations, leaving more time to increase the number of patients, to improve patient care or to generally reduce the stress level of staff involved. 4. Another treatment–another chance: Poor or slow responders are often given massive amounts of FSH over a prolonged period of time – and with poor results. The usually very few oocytes present may well be damaged and rendered unable to fertilize or to develop into good quality embryos. Also repeated poor embryo quality in general may be a result of incorrect gonadotropin stimulation. And obviously, repeated failure of ovulation induction would justify an attempt to do in vitro maturation. 5. Oocyte donation: Worldwide there is a general shortage of altruistic oocyte donors. Some possible donors may well consider donation, if it were made less troublesome and ovarian hormone stimulation were not involved. 6. Preservation / extension of fertility: Men and boys can have their sperm cryopreserved prior to

IVM® • Fewer oocytes and embryos • Lower pregnancy rate per OPU • No down regulation, no manipulation of hormone balance • No hormone injections - or • Minimal hormone injections (PCOS) • No hCG injections (? – see text) • Reduced psychological impact • Reduced treatment time – 2 weeks • Reduced interference with daily life • No known side effects

chemotherapy or other types of cancer treatment– and then thawed and used later in life to obtain a pregnancy with their partner. Using IVM, the same is the case for women. Very often a cancer treatment has to be initiated quickly, leaving too short time for a stimulated cycle to be carried out prior to oocyte pick-up. Even if there were time, the hormone administration may induce complications during cancer treatment. Subsequently, oocytes retrieved can be frozen – preferably after IVM. For some patients career or life style considerations are important: Fewer visits to the fertility center during treatment and no or very few injections. Another option is to have a relatively trouble-free oocyte collection early in life, followed by IVM and freezing of the oocytes, and ultimately embryotransfer and pregnancy at a more suitable time later in life. This is also an obvious solution for single women, who have not yet found a partner, and who therefore do not have the possibility of freezing embryos.

OOCYTE PICK-UP TECHNIQUE Ultrasound guided transvaginal aspiration of immature oocytes was described by Trounson and co-workers, 1994. They introduced two major modifications compared to conventional IVF ultrasound guided oocyte pick-up: A new and more rigid aspiration needle with a shorter bevel at the tip (Cook, Australia Ltd) and the use of a reduced vacuum of 80-100 mm Hg. The reduced vacuum seemed to be far the most significant change. Although the above mentioned needle and standard double lumen needles may be used with no

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FIGURE 14.5: Oocytes are collected from small antral follicle of 2-10 mm and allowed to mature in vitro

difference in recovery rate (Wynn et al, 1998), most reports have described the use of a single lumen needle for aspiration of follicles of 2-10 mm without flushing and under ultrasound guidance (Figure 14.5) (Russell et al, 1997; Barnes et al, 1996; Chian et al, 2000; Cha et al, 2000; Wynn et al, 1998; Cobo et al, 1999). Transvaginal oocyte aspiration has been performed under general anesthesia (Wynn et al, 1998) or spinal anesthesia (Chian et al, 2000). The use of paracervical block was described by Mikkelsen et al, (1999).

LABORATORY PROCEDURE DAY -1 (OOCYTE PICK-UP) In order to use a time scale similar to standard IVF, the day of oocyte pick-up is called Day -1. Immature oocyte collection is generally carried out using a medium similar or identical to the one used for standard pick-up of mature oocytes in a stimulated

cycle. Usually the follicles are not flushed. As in standard IVF oocyte pick-up, it essential to work under sterile conditions and to maintain the correct temperature and pH in the media used (Figure 14.6A). The follicular fluid containing possible oocytes is examined in the laboratory (Figure 14.6B) and the oocytes identified using a stereomicroscope (Figures 14.7 and 14.8). This may be done by simply pouring the fluid into a petri-dish for examination as in standard IVF. However, due to the small size of the oocyte cumulus complexes (OCCs) a better way is to use a strainer for filtering the aspirate and subsequently wash the oocytes and transfer them into the proper medium (Figures 14.9A to F), called LAG Medium (MediCult, Denmark). The oocytes obtained are usually GV-oocytes, which may be classified into those with a complete cumulus (Figure 14.10), those with sparse cumulus (Figure 14.11), and the nude oocytes with no or almost no cumulus cells attached (Figure 14.12). The more complete the oocyte cumulus complex, the higher probability of a successful maturation. The oocyte pick-up is in fact carried out short time before the degeneration of the small follicles and their oocytes would normally start. Therefore it is not uncommon to find atretic oocytes (Figure 14.13). The oocytes stay in the LAG medium for 3-4 hours. This medium is a fairly simple medium, which mainly functions as a holding medium for the oocytes until the preparation and equilibration of the maturation

FIGURES 14.6A and B: (A) Ovum pick up (B) Laboratory set up

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FIGURES 14.7A and B: (A) Follicular fluid containing two immature GV-oocytes. (B) One of these oocytes in greater magnification

FIGURES 14.8A and B: (A) Follicular fluid containing an immature GV-oocyte. (B) Greater magnification

medium has been completed. The oocytes are subsequently incubated in the latter medium for 2832 hours. The maturation medium consists of IVM® Medium (MediCult, Denmark) with addition of FSH (final conc. 0.075 IU/ml), hCG (final conc. 0.1 IU/ml), and autologous serum (10%) drawn on the day of oocyte pick-up (Table 14.2). DAY 0 (CHECK FOR MATURATION; INSEMINATION) Day 0 is the day when the oocytes are checked for maturation (Figure 14.14), and when insemination of

Table 14.2: Preparation of maturation medium on Day -1. Preparation of the Maturation Medium • MediCult IVM® Medium • FSH solution (final conc. 0.075 IU/ml) • hCG solution (final conc. 0.1 IU/ml) • Patient serum (10%) • Sterile filtration of mixture through 0.22 ìm filter • Equilibration for 2-3 hours at 37°C and 5-6% CO2

the mature oocytes takes place. In most centers this is done by ICSI (Figure 14.15), although standard IVF can be used, if the sperm quality allows it. It should be noted that in order to do standard IVF, the oocytes

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FIGURES 14.9A to F: Use of a strainer to identify oocyte cumulus complexes

cannot be denuded, which means that immature oocytes may be transferred into the sperm suspension, as well. This obviously reduces the fertilization rate, and maturation cannot be safely determined on day 0. From a statistical viewpoint, therefore, maturation rate and fertilization rate from a center doing IVF

cannot be compared with those from a center doing ICSI. From a clinical viewpoint, however, there seems to be no difference in pregnancy rates. Following insemination the oocytes are transferred into a standard IVF culture medium and treated exactly as in normal IVF and ICSI procedures.

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FIGURE 14.10: Immature GV oocytes completely surrounded by cumulus

FIGURE 14.11: Immature GV oocytes with sparse cumulus

DAY 1 (CHECK FOR FERTILIZATION)

DAY 2 (CHECK FOR CLEAVAGE; POSSIBLY EMBRYOTRANSFER)

On day 1 the cultures are examined for signs of fertilization, as judged by the transformation of the oocytes into pre-zygotes (Nielsen et al, 2001) containing pronuclei (Figure 14.16).

As in standard IVF, the appearance of cleaved embryos is determined on day 2 (Figure 14.17). Some centers prefer to do the embryotransfer on the same day, while other wait till day 3 (Figure 14.18). Although still rarely

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FIGURE 14.12: Immature GV oocytes with hardly any cumulus cells attached

FIGURE 14.13: Atretic oocytes

In Vitro Maturation of Human Oocytes

FIGURES 14.14A TO D: Human oocyte before (A and C) and after (B and D) maturation in vitro

FIGURE 14.15: ICSI of a mature (MII) denuded oocyte

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FIGURE 14.16: Human pre-zygote with two pronuclei on day 1

FIGURE 14.17: Human 2-cell embryo on day 2

used, it would also be possible to carry out blastocyst culture and transfer on day 5 (Figure 14.19).

ACKNOWLEDGEMENTS We would like to thank Dr Mariabeatrice Dal Canto, Biogenesi Reproductive Medicine Center, Monza, Italy, and Dr Daniela Nogueira, Women and Infants’

FIGURE 14.18: Human 8-cell embryo on day 3

FIGURE 14.19: Human expanded blastocyst on day 5

Hospital, Boston, USA., for kindly providing part of the illustrations.

BIBLIOGRAPHY 1. Baker SJ, Spears N. The role of intra-ovarian interactions in the regulation of follicle dominance. Human Reproduction Update 1999;5:153-65.

In Vitro Maturation of Human Oocytes 2. Barnes FL, Crombie A, Gardner DK, et al. Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Human Reproduction 1995;10:3243-7. 3. Barnes FL, Kausche A, Tiglias J, et al. Production of embryos from in vitro-matured primary human oocytes. Fertility and Sterility 1996;65:1151-6. 4. Cha KY, Koo JJ, Ko JJ, et al. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertility and Sterility 1991;55:109-13. 5. Cha KY, Chian RC. Maturation in vitro of immature human oocytes for clinical use. Human Reprodution Update 1998;4:103-20. 6. Cha KY, Han SY, Chung HM. Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrom. Fertility and Sterility 2000;73:978-83. 7. Chian RC, Gulekli B, Buckett WM, Tan TL. Priming with human chorionic gonadotropin before retrieval of immature oocytesin women with infertility due to polycystic ovary syndrome. New England Journal of Medicine 1999;341:1624-26. 8. Chian RC, Buckett WM, Tulandi T, et al. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Human Reproduction 2000;15:165-70. 9. Chian RC. In vitro maturation of immature oocytes for infertile women with PCOS. Reproductive BioMedicine Online 2004;8:547-52. 10. Cobo AC, Requena A, Neuspiller F, et al. Maturation in vitro of human oocytes from unstimulated cycles: selection of the optimal day for ovum retrieval based on follicular size. Human Reproduction 1999;14:1864-8. 11. Lin YH, Hwang JL, Huang LW, Mu SC, Seow KM, Chung J, Hsieh BC, Huang SC, Chen CY, Chen PH. Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes. Human Reproduction 2003;18:1632-6. 12. Liu J, Katz E, Garcia JE, et al. Successful in vitro maturation of human oocytes not exposed to human chorionic gonadotropin during ovulation induction, resulting in pregnancy. Fertility and Sterility 1997;67:566-8. 13. Macklon NS, Fauser BCJM. Regulation of follicle development and novel approaches to ovarian stimulation for IVF. Human Reproduction Update 2000;6: 307-12. 14. Mikkelsen Al, Smith SD, Lindenberg S. In vitro maturation of human oocytes from regular menstruating women may be successful without FSH priming. Human Reproduction 1999;14:1847-51. 15. Mikkelsen AL, Smith S, Lindenberg S. Impact of oestradiol and inhibin A concentrations on pregnancy

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26. 27.

28. 29.

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rate in in-vitro oocyte maturaturation. Human Reproduction 2000;15:1685-90. Mikkelsen AL, Andersson AM, Skakkebæk NE, et al. Basal concentrations of oestradiol may predict the outcome of IVM in regular menstruating women. Human Reproduction 2001a;16:862-7. Mikkelsen AL, Lindenberg S. Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome. A randomizxed prospective study. Reproduction 2001b;122:587-92. Mikkelsen AL, Høst E, Blåbjerg J, et al. Maternal serum supplementation in culture medium benefits maturation of immature human oocytes. Reproductive Biomedicine Online 2001c;2:112-6. Mikkelsen Al, Lindenberg S. Influence of the dominant follicle on in vitro maturation of human oocytes. Reproductive Biomedicine Online 2001d;3:199-204. Mikkelsen AL, Host E, Blaabjerg J, Lindenberg S. Time interval between FSH priming and aspiration of imature human oocytes for in-vitro maturation: a prospective randomized study. Reproductive Biomedicine Online 2003;4:416-20. Nagy ZP, Cecile J, Liu J, et al. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal vesicle stage oocytes:case report. Fertility and Sterility 1996;65:1047-50. Nielsen HI, Bahadur G, Hinrichsen MJ, Mortimer D, Tesarik J. Definitions of human fertilization and preimplantation growth revisited. Reproductive Biomedicine Online 2001;3:90-3. Pache TD, Wladimiroff JW, de Jong FH, Hop WC, Fauser BCJM. Growth patterns of nondominant ovarian follicles during the normal menstrual period. Fertility and Sterility 1990;54:638-42. Prins GS, Wagner C, Weidel L et al. Gonadotropins augment maturation and fertilization of human immature oocytes cultured in vitro. Fertility and Sterility 1987;47: 1035-7. Russell JB, Knezevich KM, Fabian K, et al. Unstimulated immature oocyte retrival: early versus midfollicular endometrial priming. Fertility and Sterility 1997;67:61620. Steptoe JD, Edwards RG. Successful birth after IVF. Lancet 1978;ii:366. Suikkari AM, Tulppala M, Tuuri T, et al. Lutheal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes. Human Reproduction 2000;15: 747-51. Thornton MH, Francis MM, Paulson RJ. Immature oocyte retrieval: lessons from unstimulated IVF cycles. Fertility and Sterility 1998;70:647-50. Trounson A, Wood C, Kausche A. In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertility and Sterility 1994;62:353-62.

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30. Trounson A, Anderiesz C, Jones GM et al. Oocyte maturation. Human Reproduction 1998;13S:52-62. 31. Veeck LL, Wortham JW Jr, Witmeyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW Jr. Maturation and fertilization of morphological immature oocytes in a program of in vitro fertilization. Fertility and Sterility 1983;39:594-602.

32. Wynn P, Picton HM, Krapez J, et al. Pretreatment with follicle stimulating hormone promotes the number of human oocytes reaching metaphase II by in vitro maturation. Human Reproduction 1998;13:3132-8.

15

Stem Cells: A Brief Overview

NS Moorthy

INTRODUCTION Stem cells are the primordial cells produced in the human body, with the capability to differentiate along any line or into any type of cell (totipotent). They divide and multiply within the human body. The first stem cells originate within the developing embryo (blastocyst). These embryonic stem cells have to ultimately create the entire human body. Umbilical stem cells are harvested from the umbilical cord of a full term live birth, not from an aborted fetus. This type of stem cell therapy involves the introduction of healthy new stem cells into the body to repair and replace damaged or lost cells.

FACTS ABOUT STEM CELLS (FIGURE 15.1) Stem cells have two important characteristics that distinguish them from other types of cells. • First, they are unspecialized cells that renew themselves for long periods through cell division. • The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulinproducing cells of the pancreas.

FIGURE 15.1: Stem cells

WHAT MAKES THIS CELL SPECIAL? Stem cells differ from the other cells in the body. All stem cells, regardless of their source, have three general properties • Capacity for self-renewal • Immunological Immaturity • Plasticity or ability to differentiate

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SOURCES OF STEM CELLS

TYPES OF STEM CELLS

• • • • • • •

EMBRYONIC STEM CELLS

Adipose tissue Bone Marrow Embryo Olfactory Ensheathing cells Peripheral blood Skin Umbilical Cord Blood

MILESTONES IN STEM CELL RESEARCH • In 1988, Elaine Gluckman replaced allogenic cord blood for a bone marrow transplant in order to treat Fanconi’s Anemia, a rare recessive blood disorder. The child remains completely disease free. • In 1998, at the University of Wisconsin, James Thompson isolated the first embryonic stem cells from a blastocyst of a five day old in vitro fertilized egg. • In 2001 treatment protocols were developed which permitted the removal of white blood cells from the cord, making the treatment safe with no risk of Graft verse Host Disease. This treatment also proved there is no need for HLA matching. • In 2002, Catherine Verfaillie at the University of Minnesota proved that CD34+ stem cells from bone marrow could repopulate every single cell in a developing mouse. It became proven there are great potentials for adult stem cells to treat a wide range a blood diseases, cancers, degenerative diseases and injuries. • In 2004, Duke University published data from a human study confirming the Verfaillie study. The study featured the heart treatment of a boy who received CD34+ stem cells derived from donated cord blood. Not only did the investigation show differentiation to neurons and other cell types, but also proved that cord blood stem cells: • Migrate to the site of disease • Had the ability to differentiate into specialized heart cells • Engraft yielding clinical benefits.

Embryonic stem cells as the name suggests, are derived from embryos. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called blastocysts. ADULT STEM CELLS An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. UMBILICAL CORD BLOOD STEM CELLS Blood collected from the Umbilical cord of the neonate is rich in stem cells that can be used to generate red blood cells and cell of immune system. PROMISE BEHIND UMBILICAL CORD BLOOD BANKING Umbilical cord blood banking consists of the collection, processing and cryo preservation of cord blood stem cells. Cord blood is the remaining blood within the Umbilical and Placental circulation following the birth of a child and typically prior to placental delivery. This “left-over” blood is traditionally discarded with the placenta as medical waste. In recent times, it has been discovered that cord blood is a rich source of Hematopoietic stem cells. Promise of cord blood banking is the fact that cord blood stem cells are progenitor cells that can reconstitute blood and immune systems. These cells are present in cord blood with concentrations nearly 10 times greater than that in bone marrow and they are more proliferative. Unlike the risks inherent in bone marrow transplants, they can be collected safely without any maternal or neonatal risk following delivery.

Stem Cells: A Brief Overview The cord blood collection is performed by gravity method using blood bag method and it takes 2 to 5 minutes. Stem cells are isolated from the collected samples, frozen and stored cryogenically in liquid phase of Nitrogen. Advantages of Umbilical Cord Blood Stem Cells over Embryonic Stem Cells (Table 15.1) Ethical Issues: Use of Cord Blood stem cells in Cellbased therapies is less controversial as it does not involve destruction of embryos. Immune Tolerance: Patient’s own cord blood stem cells can be used to generate tissue for transplantation, thus avoiding problems with immune rejection.

FREQUENT APPLICATIONS OF STEM CELLS • Acute and Chronic Leukemias • Myelo-dysplastic Syndromes

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Table 15.1: Advantages of cord blood over bone marrow as a source of stem cells Bone marrow

Cord blood

• • • • • • • •

Harvest quantum less Engraftment takes longer GVHR Low Less contamination Simple collection Donor search time –halved HLA match –3/6 or 4/6 Limitless supply potential

• • • • • • • •

Quantity of harvest larger Engraftment faster GVHR High Contamination more Tedious collection Longer time to find donors HLA match –5/6 or 6/6 Limited supply of donors

Stem cell disorders Myelo-proliferative disorders Lympho-proliferative disorders Phagocyte disorders Inherited platelet abnormalities Inherited metabolic disorders Histiocytic disorders Inherited RBC abnormalities

FIGURE 15.2: Promise of stem cell research (Courtesy: Stem Cell Report, NIH)

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Inherited immune system disorders Plasma cell disorders Solid tumors Other inherited disorders

PROMISE OF STEM CELL RESEARCH See Figure 15.2. THE FUTURE LOOKS PROMISING The promise of stem cell therapies is an exciting one. The Potential uses of Stem cells include; 1. Neurology • Parkinson’s disease • Demyelinating disorders • Alzheimer’s disease • Stroke • Spinal injury • Cerebral palsy • ALS 2. Cardiology • Myocardial Infarction • Congestive cardiac failure • Angiogenesis

3. Plastic Surgery • Breast implants • Reconstruction of bone, cartilage • Skin graft—in burns and other skin diseases • Baldness 4. Diabetes mellitus

CONCLUSION Stem cells are one of the most fascinating areas of life sciences today. Research on stem cells is taking place in every possible area. Human stem cells could also be used to discover new drugs. Future looks bright, but only time will tell how many diseases can be cured by stem cells.

BIBLIOGRAPHY 1. 2. 3. 4. 5.

BMJ 2000;321:433-7. N Engl J Med 1996; 335: 157-66. National Bone Marrow Donor Program www.marrow.org. Proc Natl Acad Sci 1989;86: 3828-32. Stem Cell Information – National Institute of Health http;// stemcells.nih.gov/info/basics. 6. Stroke 2004;35:2390-95. 7. The Journal of Clinical Investigation 2005;115:1395.

16

Vitrification of Embryos

Rama Raju GA, Murali Krishna K, Jaya Prakash G, Madan K

INTRODUCTION The extensive use of gonadotropins in ART programmes frequently results in the production of multiple embryos. Transfer of more than two embryos may result in multiple pregnancies causing pre- and postnatal complications. Hence the advantages of cryopreservation of embryos can be summarized as: 1. It is possible to limit the number of embryos to be transferred there by reducing the incidence of multiple pregnancies. 2. Embryos stored in an earlier attempt of IVF can be transferred later avoiding a fresh IVF cycle. 3. Cryopreservation allows the parents to have an additional child if they wish to, at a later date. 4. Cryopreservation provides an option for donation of embryos. 5. Embryo transfer can be postponed in the patients with OHSS with cryopreservation of embryos.

PRINCIPLES OF CRYOPRESERVATION When cells are cooled to below -5°C, the cells and the surrounding medium remain unfrozen and supercooled (supercooling is the ability of an aqueous

solution, to lower it’s normal freezing point, without changing it’s state; i.e. from liquid to solid (ice)). Between -5°C and -15°C ice forms in the external medium (either spontaneously or as result of seeding), whereas cell contents remain unfrozen and supercooled, and this is because the plasma membrane blocks the growth of ice crystals into the cytoplasm. The supercooled water inside the cells has a higher chemical potential than that of water present in the partially frozen extracellular solution resulting in flow of water to outside the cell and freezes externally. If the rate of cooling is slow, then the cell will be able to lose water rapidly enough to concentrate the intracellular solutes sufficiently to prevent freezing. As a result the cell dehydrates and does not freeze intracellularly. However if the cell is cooled at rapid rate, then it is not able to lose water fast enough to maintain equilibrium; the cell reaches equilibrium by freezing intracellularly with the resultant intracellular ice formation being lethal to cell survival. If the cells are cooled too slowly, then there will be a severe volume shrinkage and long-term exposure to highsolute concentrations. Both volume shrinkage and

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FIGURE 16.1: Principles of cryobiology

long-term exposure to high solute concentration may cause cell injury (Figure 16.1). Cell freezing requires the use of a cryoprotectant and cells cannot survive the freeze-thaw process without these special additives. Cryoprotectants are divided into two major groups, permeating and nonpermeating. The most widely used permeating cryoprotectants are ethylene glycol, dimethylsulfoxide (DMSO), and glycerol. Sucrose is the most widely used nonpermeating cryoprotectant, and mostly serves for osmotic control. Cryoprotectants facilitate the freezing process by: 1. Lowering the freezing point, and therefore allow the embryos and freezing medium to be supercooled to a specific sub-zero temperature before seeding,

2. Protecting the cell membrane from freeze-related injury , 3. Decreasing the deleterious effects of high salt concentrations as cells dehydrate during the freezing process. Successful cryopreservation of mouse embryos was reported in 1972 by Whittingham et al. In 1983, Trounson and Mohr reported a pregnancy following freezing and thawing of 8-cell embryo in humans. Slow-freezing protocols are usually employed for freezing of different cleavage stage human embryos. During slow-cooling, cells dehydrate, shrink and the concentration of solutes increases as water freezes in the extracellular medium. Intracellular crystallization is thought to be the major cause of cryoinjury, because it damages both membranes and organelles. However,

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197

there is a delicate balance when removing the water from the cell, as too much dehydration can result in an increase in the intracellular solute concentration to toxic levels and too slow dehydration may prolong the exposure of the embryos to cryoprotectant solutions. Furthermore, with slow-freezing procedures, care must also be taken in the thawing procedure, as ice crystals can form during warming. Although slow freezing method was proven effective for embryos of several mammalian species, the disadvantage of this method is that it requires long time for cooling and an expensive programmed freezer to control the cooling rate.

straws, micro drops, cryotops and cryoloops with a small quantity of cryoprotectant and plunged into liquid nitrogen directly, thereby achieving a very rapid temperature drop of 15,000 to 30,000°C/min. Applying this method, critical temperatures during which the cells are damaged by chilling injury can be quickly bypassed and intracellular ice formation can be avoided by forming a glassy state and minimizing the cryoprotectant toxicity by a very short exposure to cryoprotectant.

VITRIFICATION

The buffered media base most commonly employed for vitrification is either phosphate buffered saline (PBS) of HEPES buffered culture medium.

Rall and Fahy in 1985 developed a vitrification protocol for freezing mouse embryos. Vitrification is the solidification of a solution (water is rapidly cooled and formed into a glassy, vitrified state from the liquid phase) at low temperature, not by ice crystallization, but by extreme elevation in viscosity during cooling. Vitrification involves a short equilibration of embryos in a vitrification solution containing high concentration of cryoprotectants and a rapid cooling in liquid nitrogen. Introduction of vitrification has changed many existing concepts cryopreservation of embryos. Vitrification involves exposure to high concentration of cryoprotectants and is carried out at super fast cooling rates. During vitrification samples containing embryos reach low temperatures in a glassy state which has the molecular structure of a viscous liquid and is not crystalline. However consistently high survival rates have not been achieved with either by slow cooling or vitrification. The reasons could be due to sensitivity of embryos to chilling, lower permeability of cell membrane and due to toxicity of cryoprotectant. More recently the success rate of vitrification has been increased by ultra rapid vitrification, in which the cooling and warming rate is markedly increased by minimizing the volume of the cryoprotectant solution and thereby increasing the rate of cooling further. This method employs loading of embryos in carriers like electron microscopic grids, open pulled straws, hemi

COMPOSITION OF VITRIFICATION MEDIUM (TABLE 16.1) Base Medium

Cryoprotectants Cryoprotective agents are essential for the successful cryopreservation of embryos. They are classified into two categories: 1. Permeating cryoprotectants (Ethylene glycol, DMSO (dimethyl sulfoxide), Glycerol) 2. Non-Permeating cryoprotectants (Saccharides, proteins, polymers) 3. Clevage media 4. Blastocyst media Table 16.1: Composition of Vitrification media S.no Composition of vitrification solutions

Availability

1 2

Hepes buffered media Cryoprotectants

3 4 5

Saccharides Cleavage media Blastocyst media

Gamete (vitrolife) Ethylene glycol, DMSO (dimethyl sulfoxide), Glycerol Sucrose, raffinose Commercially available media Commercially available media

PREP ARA TION OF VITRIFICA TION SOLUTION ((ADAPTED ADAPTED FROM PREPARA ARATION VITRIFICATION DANASOURI EETT AL) Solution ‘1’ Ethylene Glycol

-

10 %

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Solution ‘2’ Ethylene Glycol 40% Sucrose 0.6M Micro filter both the solutions and store in refrigerator. PREPARATION OF WARMING SOLUTION Solution ‘1’ Sucrose

-

1M

Solution ‘2’ Sucrose

-

0.5M

Solution ‘3’ Sucrose

-

0.25M

Solution ‘4’ Sucrose 0.125M Microfilter the solutions and store in refrigerator.

FIGURE 16.3: Vitrification of embryos

Embryos are then transferred to 40% ethylene glycol in 0.6 mol/l sucrose solution for 30 s. The embryos are then immediately loaded onto a nylon loop with a thin film of vitrification solution (3–5 μl), and directly plunged into a cryovial (Figure 16.2) containing liquid nitrogen. These cryovials are closed tightly and placed in liquid nitrogen storage tanks. WARMING

FIGURE 16.2: Cryovial (Inhouse fabricated and also available from other commercial sources)

VITRIFICATION OF EMBRYOS During the vitrification embryos are rinsed in HEPESbuffered medium (Figure 16.3). Then embryos are placed in 10% ethylene glycol solution using a Pasteur pipette, and incubated for 5 min at 37°C. During this period embryos undergo transient volume shrinkage and then return to near-normal volume as the cryoprotectant permeates the embryos (Figure 16.4).

During warming vitrified embryos are thawed by placing the cryoloop directly into 1 mol/l sucrose solution for 2.5 min at 37°C (Figure 16.3). Embryos are then transferred through different concentrations of sucrose solution i.e. 0.5 mol/l, 0.25 mol/l, and 0.125 mol/l for 2.5 min in each step at 37°C. The embryos were finally placed in blastocyst media for culturing in the CO2 incubator for 3-4 h before embryo transfer (Figure 16.5). COMAPRISON BETWEEN SLOW FREEZING AND VITRIFICATION See Table 16.2 and Figure 16.6.

Vitrification of Embryos

FIGURE 16.4: Morphological changes of 8 cell embryo during vitrification

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Atlas of HART Table 16.2: Following are the differences between slow freezing and vitrification S.no Accessibility and regulation 1 2 3 4 5 6 7

Time consumed Instruments Mechanical damage Intracellular ice formation Chemical damage Concentration of cryoprotectant Accessibility and regulation i Can be observed ii Can be analysed iii Interaction with the oocyte or embryo iv Control of solute penetration v Control of dehydration rate vi Maintenance of physiological temperature during equilibration procedure vii Duration out of incubator viii Prolonged temperature shock ix Interference with oocyte or embryo x Fracture of zona pellucida xi Capture by growing ice crystals

Vitrification

Slow freezing

Less (10 mins) Inexpensive Less or none Less More High

More (3 hrs) Expensive More More Less Low

Yes Yes Yes Yes Yes

No No No No No

Yes ~10 mins No Low No No

No ~3 hrs Yes High Possible Possible

(Adapted from Kuleshova LL)

FIGURE 16.5: 8-cell embryo after thawing

LIMITATIONS One aspect which needs to be focused on during the vitrification procedure is viral contamination from liquid nitrogen. Recent studies by Bielanski et al. (2000, 2003) reported the possible transmission of pathogens to bovine embryos vitrified and stored in liquid nitrogen (Bielanski et al, 2000, 2003). However proper

sealing of straws and cryovials containing embryos has been considered an effective measure against contamination during storage. An alternative preventive step described by Vajta et al (1998) against contamination is liquid nitrogen filtration and the application of accessory protective storage containers (Vajta et al, 1998). A straw in straw method for

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201

FIGURE 16.6: Schematic diagram of an embryo before cooling, during cooling and in liquid nitrogen in slow freezing, conventional straw vitrification, and ultra rapid vitrification (Adapted from: Dr. Kasai M)

successful storage of vitrified blastocysts, reported by Lieberman et al. (2002) and Vanderzwalmen et al (2003) is also an additional measure to reduce the viral contamination from liquid nitrogen (Lieberman et al,

2002; Vanderzwalmen et al, 2003). Cross-contamination may be prevented by storing embryos from known infectious patients in separate liquid nitrogen tanks. Though liquid nitrogen contamination has

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not been reported in human embryology, preventive measures should be taken as suggested by Vajta et al (1998) and further studies are required in this aspect.

CONCLUSIONS Cryopreservation by a vitrification method is a simple, inexpensive and efficient method of freezing human embryos because it greatly simplifies the freezing process and eliminates ice crystal formation. Further optimization and modification of vitrification protocols may improve the survival rates, establishing vitrification as the procedure of choice for human embryo cryopreservation.

4. 5. 6.

7. 8. 9.

BIBLIOGRAPHY 1. Bielanski A, Bergeron H, Lau PCK, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146-52. 2. Bielanski A, Nadin Davis S, Sapp T, Lutze Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000;40:110–6. 3. Danasouri DVM, Selman H. 2001 Successful pregnancies

10.

11.

and deliveries after a simple vitrification protocol for day 3 human embryos. Fertility and sterility 76, 400-2. Kasai M, Mukida T. Cryopreservation of animal and human embryos by vitrification. Reproductive Biomedicine Online 2004;9:164-70. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002 ;78(3):44954. Lieberman J, Tucker M, Gardner DK, et al. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reproductive BioMedicine Online 2002;4:146-50. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos by vitrifi cation. Nature 1985;313;573–5. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing, and transfer of an eight-cell embryo. Nature 1983;305:707–9. Vajta, G, Lewis IM, Kuwayama M, et al. Sterile application of the open pulled straws (OPS) vitrification method. Cryo Letters 1998;19:389–92. Vanderzwalmen P, Bertin G, Debauche C, et al. Vitrification of human blastocysts with the Hemi Straw carrier: Application of assisted hatching after thawing. Human Reproduction 2003;18:1504-11. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to -196ºC and -269ºC. Science 1972;178: 411–4.

Index A Analysis of fertilization 135 abnormal fertilization 136 cleavage [cell division] 136 embryo scoring 136 transferring the resulting embryos into the uterus 136 Anejaculation 71 situational 71 total 71 ART laboratory 85 Assisted hatching 160 blastocyst assisted hatching 163 IVM and laser 163 laser assisted hatching 161 contact lasers 162 different laser systems 161 non-contact lasers 162 laser zona thinning 163 methods 161 selection criteria for assisted hatching 161 Azoospermia 76 non-obstructive azoospermia 76 obstructive azoospermia 76 clinical features 77

B Basic steps in ICSI 143 denuding the oocytes 145 ICSI technique 14 ooplasmic penetration and sperm deposition 147 sperm selection and immobilization 146 preparation of dishes 144 ICSI dish 144 oocyte culture 144 oocyte denuding 144 oocyte scanning 144 scanning, identification and grading of oocytes 145 setting up the micromanipulator 144 sperm preparation for ICSI 145 Binucleated fixed blastomere 169

C Centrifugation and washing 99 steps 99 Centrifugation-swim up technique 95 steps 95 clinical features 77

Controlled ovarian hyperstimulation 21 assessment of ovarian reserve 21 Anti-mullerian hormone 21 estradiol levels 21 dynamic tests for ovarian function 22 ovulation induction protocols 23 fixed protocol 25 flexible protocol 25 GnRH antagonists 25 long protocol 23 short protocols 23 ultrashort protocol 23 protocols for ovarian stimulation 22 Corona radiata 110 Cryobiological principles 151 Cryoconservation of sperm 106 autoconservation 106 cryoprotectant and cryomedia 106 donor sperm conservation 106 indications 106 other indication 106 prefreeze addition 107 thawing procedure 107 after thawing 107 Cryopreservation in ART 153 blastocyst cryopreservation 155 cryopreservation of male gamete-the sperm 153 cryopreservation of oocytes and embryos 154 freezing and thawing protocol 154 ovarian tissue freezing 157 vitrification 156

D Delineation of pelvic pathology 49 Density gradient separation system 99 discontinuous density gradient 103

E Electro-ejaculation 73 Evaluation of the female partner 3 assessment of the uterus and fallopian tubes 5 mullerian defects 5 gynecological and obstetrical history 4 age 4 menstrual history 4 obstetrical history 4 medical history 3 adrenal disorders 4

diabetes 4 pelvic inflammation 4 pituitary disorders 4 thyroid disorders 4 tuberculosis 4 surgical history 4

F Facts about stem cells 191 Fixation of entire arrested embryo 169 Fixed blastomeres 169 Frequent applications of stem cells 193

G General measures in the lab 90 General rules for laboratory cultures 90 Glass wool filtration 103 steps 103 selection of viable spermatozoa for ICSI by Hos test in case of complete astheno-zoospermia 103 use of laser to detect viable but immobile spermatozoa 105

H Human oocyte 110 meiosis in the female 111 oogensis 111 oogonium 111 Hyperprolactinemia 15 hyperprolactinemia and infertility 16 investigations 16 medical management 17 pituitary gland involvement 16 pituitary tumors 16 acromegaly 16 empty sella syndrome 16 macroadenoma 16 microadenoma 16 systemic disorders 16 clinical features 16 Hysteroscopy, laparoscopy and evaluation of pelvis 7 endometriosis 7 fibroids 13 medical treatment 15 hydrosalpinx 12 polycystic ovarian disease 8

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Atlas of HART I

Identification of the oocyte 111 assessment of cumulus maturity 112 atretic oocyte/degenerative OCC 114 dysmature OCC 114 immature/intermediate OCC 112 mature OCC 112 post mature OCC 114 very immature OCC 112 assessment of nuclear maturity 114 In vitro fertilization and blastocyst culture 129 In vitro maturation of human oocytes 177 Indications for ICSI 143 Infection of the reproductive system 17 infections in male partner 17 infections in the female partner 18 chlamydia trachomatis 18 gonorrhea 18 mycoplasma 19 trichomoniasis 18 tuberculosis 19 diagnosis 20 history 20 immunohystochemistry 20 laboratory tests 20 Interventional procedures 68 Intracytoplasmic sperm injection 129, 143 IVF in the laboratory 129 collection of eggs from the female’s ovaries [oocyte retrieval] 129 insemination 135 obtaining sperm from the male [semen collection] 134 sperm preparation techniques 134 oocyte grading 131 oocyte handling in the laboratory: retrieval to insemination 130 buffers 131 bulk lons 131 functions of basal salt solution 131 semen 132 normal sperm morphology 132 seminogram 132 IVM treatment of regular cycling women with normal ovaries 179 IVM treatment of women with PCOS 178

L Lab designing 85 equipments 87 essential 87 optional 89 interiors 86 location 86 staffing 90 structuring 85 nonsterile area 85 sterile area 85 Laser assisted ICSI 164 embryo biopsy 164 sperm immobilization prior to ICSI 164

Laser assisted microsurgical fertilization 164 applications 164 cleaning of micro tools 165 detecting zona pellucida hardness 165 discriminate between viable and dead immotile spermatozoa 165 inner cell mass (ICM) excision 165 Laser dissection of zona pellucida 165 removal of fragments of optical tweezer 164 somatic cell nuclear transfer 165 separation of corona radiata 164 transgenic applications 165 Laser in assisted reproduction technologies 159 Luteal phase support and adjuvants 33 clinical significance of various semen parameters 43 examination 41 genital examination 41 scrotal assessment 41 testicular assessment 41 history taking 38 adult history 41 childhood history 38 family history 38 history of drug intake 41 history of illness 41 sexual history 41 specific instructions 42 hormonal 42 prostate examination 42 semen analysis 42 yoga and acupuncture 34

M Micro-surgical epididymal sperm aspiration 105 Milestones in stem cell research 192 Mini swim up 99 Morphological characteristics of the oocyte 115 cytoplasm 119 central granularity 119 homogenous granularity 119 vacuole 119

O Oocyte pick-up technique 181 Oocyte cumulus complex 110 Organic anejaculation 72 Ovulation monitoring 65 Ovum pick-up and embryotransfer 26 embryotransfer 28 catheters 29 loading the catheter 29 time of embryotransfer 30 ovum pick-up technique 26 anesthesia 27 empty follicle syndrome 28 needles 26 procedure 27

P Prediction of ART outcomes 57 Preimplantation genetic diagnosis 167 Principles of cryopreservation 195 Promise of stem cell research 194 futurelooks promising 194

Q Quality control in the ART laboratory 91 quality assurance 92 quality control 91 general audit 92 laboratory equipment QC 91 procedures 91 staffing 91 total quality management 92

R Reasons for choosing in vitro maturation 180 another treatment-another chance 181 economical aspect 181 ethical aspect 181 oocyte donation 181 preservation/extension of fertility 181 safety aspect 180 Recurrent implantation failure 32

S Selection of patients for IVM 178 Semen collection 94 aims for sperm preparation 94 steps 94 Smooth endoplasmic reticulum 120 bull’s eye 121 first polar body 124 giant and binucleate oocytes 126 oocytes of unusual shape 124 perivitteline space 121 refractile bodies 121 zona pellucida 122 Sources of stem cells 192 Sperm retrieval from the bladder 74 sperm recovery from medium 74 sperm recovery from urine 74 Surgical methods of sperm retrieval 77 indications 77 techniques 78 epididymal sperm retrieval (percutaneous) 78 testicular sperm retrieval (open methods) 78 Swim up from semen 95 advantage 95 steps 95

T Techniques associated with IVF 141 assisted hatching 141

Index embryo, egg and sperm freezing 141 preimplantation genetic diagnosis 141 Techniques of non-surgical sperm retrieval 71 Techniques of sperm preparation for ART 93 Technological advancement 129 Testicular and epididymal sperm retrieval 105 testicular sperm extraction 105 steps 105 Third party reproduction 30 ovum donation 30 donor profile 31 indications for oocyte donation 31 preparation of recipient 32 screening tests for oocyte donors 31 surrogate pregnancy 32

205

Total anejaculation 71 non-organic total anejaculation 71 organic total anejaculation 71 situational anejaculation 71 Types of stem cells 192 adult stem cells 192 embryonic stem cells 192 promise behind umbilical cord blood banking 192 advantages of umbilical cord blood stem cells over embryonic stem cells 193 umbilical cord blood stem cells 192

Vitrification 197 comaprison between slow freezing and vitrification 198 composition of vitrification medium 197 base medium 197 cryoprotectants 197 limitations 200 preparation of vitrification of vitrification solution (adapted from Danasouri et al) 197 vitrification of embryos 198 warming 198 Vitrification of embryos 195

V

Z

Vibrator therapy 72

Zona pellucida 110