Preventive Powers of Ovulation and Progesterone

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Preventive Powers of Ovulation and Progesterone This series of articles, originally published in the CeMCOR newsletter, illustrate the importance of ovulation throughout women's reproductive life. Scientific evidence shows that ovulation (and therefore an approporiate progesterone level) is necessary for the optimum development and functioning of several physiological systems in women's bodies. The articles explain what ovulation is and address some of the issues and implications of ovulatory disturbances. You will need the free Adobe Acrobat Reader in order to read and print these artcles. If you don't already have this program, you can download it for free here. Choose from the list below: 1. 2. 3. 4. 5. 6. 7.

What is Ovulation? Ovulatory Disturbances Indicators of Ovulation Detection of Ovulation Ovulation and Bone Health (see a related CeMCOR peer-reviewed publication here) Ovulation and Breast Health Ovulation and the Heart (see a related CeMCOR peer-reviewed publication here)

Type: Articles Topic: Cyclic Progesterone Therapy, Ovulation and menstrual cycles, Progesterone therapy Life Phase: Adolescence, Premenopause, Perimenopause Updated Date: October 4, 2014

© 2020 The Centre for Menstrual Cycle and Ovulation Research | Website by Raised Eyebrow Web Studio

Preventive Powers of Ovulation and Progesterone What is Ovulation? by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research. This question is at the heart of the Centre for Menstrual Cycle and Ovulation Research (CeMCOR). It is also what makes CeMCOR unique. Because CeMCOR focuses on ovulation, rather than simply on menstruation, it has the potential to make new discoveries for women. Over the next several issues of the new CeMCOR Newsletter, I will be focusing on different aspects of the question about the importance of ovulation for women’s health. This article will start by asking what we now know about how frequently ovulation occurs or doesn’t in women with menstrual cycles. Before I get into that, however, let me say what I believe: Regular menstrual cycles with consistently normal ovulation during the premenopausal years will prevent osteoporosis, breast cancer and heart disease in women. So, what do we mean by ovulation? Ovulation literally means the release of an egg from the ovary. That is a key event that leads to pregnancy and the birth of a baby. However, ovulation is also important as the process that leads to production of the menstrual cycle’s second important hormone—progesterone. It is the latter meaning of ovulation, having to do with the process of releasing an egg and the production of progesterone that is key, I believe, for women’s health. A complex system must be coordinated to allow release of an egg. As is appropriate, that coordination begins in the brain. It would not be prudent, for example, for a woman who was in severe emotional distress, starving or ill to become pregnant. For that reason, I think that regular ovulation tells us a lot about that woman’s personal environment, social context and health. I called ovulation the “bellwether of women’s well being” (meaning predictor of women’s whole health) when awarded a Research Lectureship by the University of British Columbia Faculty of Medicine in 2002. What might cause disturbances in ovulation? A woman must be in good emotional, nutritional and social health to regularly ovulate. It makes sense that a woman who is under emotional stress is less likely

to ovulate. For example, student nurses in Japan were shown to ovulate less frequently during their school year, that was full of deadlines and exams, than during their summer break (1). Even the worry that what we eat will cause us to gain weight (called cognitive dietary restraint) is a big enough stress in normal weight premenopausal women that their stress hormone level, cortisol, is higher than in other similar women who aren’t restrained (2). Those women with eating restraint also had disturbed ovulation (they didn’t ovulate, called “anovulation” or they ovulated but with too short a time from ovulation to the next flow, called “short luteal phase” cycles) (3;4). It also makes sense to not become pregnant if you are starving! Therefore, someone who is not starving but who is eating fewer calories than she burns will also have a subtle change in her menstrual cycle—she’ll have disturbed ovulation (5). If she is losing a lot of weight, the period may stop all together. Being seriously ill will also disturb women’s (and men’s) reproduction. For example, older menopausal women who were admitted to the hospital or the intensive care unit with pneumonia or acute heart attack showed low levels of the pituitary hormone, follicle stimulating hormone (FSH), when first admitted (6). As their health improved over about two weeks, FSH levels rose to the normal, high levels of a menopausal woman (6). What about exercise? Everyone knows that marathon-training women lose their periods. Right? Wrong! And also wrong to blame the changes that may occur in exercising women on the exercise without considering stress and not eating enough. In fact, I was so angry at the notion that women who ran long distances would inevitably develop “athletic amenorrhea” that I began studying women’s menstrual cycles back in 1980! I did a study with two women who kept track of their weights, periods, ovulation and length of the luteal phase as well as how much they ran over a full year. One was trying to become pregnant. The other was training for her first marathon. Both started with normal menstruation and ovulation and experienced multiple short luteal phase or anovulatory menstrual cycles, but neither lost her period. The woman who wanted to become pregnant did when she gained weight by cutting back her exercise(7). The other woman ran her first marathon and after that decreased her running—her luteal phase length, because she had adapted to her exercise, was normal while running 3-5 miles a day (7). This is the start of a series of articles on this topic. We will discuss how soon after the first period ovulation begins, how scientists decide if a woman is ovulating, and what we know (or don’t know) about ovulation in women in the general population. Eventually this newsletter will cover the important issues like how ovulation is important for bone health, prevention of breast and other women’s cancers, and for

protection against heart disease. Stay tuned! The next installment of this article will appear in the next edition of this newsletter.   Reference List for "Is Ovulation (and are normal Progesterone levels) Important for the Health of Women?" 1. Nagata I, Kato K, Seki K, Furuya K. Ovulatory disturbances. Causative factors among Japanese student nurses in a dormitory. J.Adolesc.Health Care 1986;7:1-5. 2. McLean JA, Barr SI, Prior JC. Cognitive dietary restraint is associated with higher urinary cortisol excretion in healthy premenopausal women. Am.J.Clin.Nutr. 2001;73:7-12. 3. Barr SI, Prior JC, Vigna YM. Restrained eating and ovulatory disturbances: possible implications for bone health. Am.J.Clin.Nutr. 1994;59:92-7. 4. Barr SI, Janelle KC, Prior JC. Vegetarian versus nonvegetarian diets, dietary restraint, and subclinical ovulatory disturbances: prospective six month study. Am.J.Clin.Nutr. 1994;60:887-94. 5. Loucks AB, Thuma JR. Luteinizing hormone pulsatility is disrupted at a threshold of energy availability in regularly menstruating women. J Clin Endocrinol Metab 2003;88(1):297-311. 6. Warren MP, Siris ES, Petrovich C. The influence of severe illness on gonadotropin secretion in the postmenopausal female. J Clin Endocrinol Metab 1977;45:99-104. 7. Prior JC, Ho Yeun B, Clement P, Bowie L, Thomas J. Reversible luteal phase changes and infertility associated with marathon training. Lancet 1982;1:269-70. Originally published March 2008

Preventive Powers of Ovulation and Progesterone Ovulatory Disturbances by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research.

I believe that the answer is Yes! And furthermore, having normal ovulation during most of our menstruating years is the missing link in preventing osteoporosis, heart disease and breast cancer. As I discussed in the first CeMCOR newsletter in March, release of an egg (ovulation) is a carefully coordinated event that normally occurs in the middle of the menstrual cycle. However, many “threats” such as being emotionally upset, not eating enough to cover our body’s energy needs, being ill or over-exercising (almost always along with inadequate caloric intake, +/- stress) can cause ovulation disturbances. By ovulation disturbances I mean two kinds of menstrual cycle variations: 1) Short luteal phase cycles in which an egg is released but the time from ovulation until the next flow is too short; and 2) Anovulatory cycles in which an egg isn’t released and progesterone levels stay low throughout the cycle. Before I go on to describe how disturbances in ovulation and luteal phase length are diagnosed, it is useful to think about the development of ovulation after the first period (menarche). Many of us have thought that getting regular periods means our reproductive system is working perfectly. In fact, most doctors think that too! In other words, if a period comes about once a month, and the time from one period to the next is not too variable, most women and many doctors assume that the cycle is normal. And by “normal”, they mean that ovulation has occurred and the ovary has secreted the right amounts of progesterone as well as estrogen. In fact, although some cycles with anovulation are irregular or too far apart, many cycles which occur perfectly regularly can also be anovulatory. Can I easily tell if I have ovulation disturbances? I first learned about ovulation disturbances from a one-year study we did to try to understand about exercise and menstrual cycles. In order to participate in this study (which was called the Prospective Ovulation Cohort), each woman needed to be healthy, not taking hormonal contraceptives, non-smoking, of normal weight, between the ages of 20 and 42, and she had to have two normally ovulatory cycles in a row in order to join (1). Some of the 66 women were normally active, some were regular recreational runners, and some were training for and ran a marathon (1). At the time, everyone thought that the marathon-training women would lose

their periods, or at least develop irregular or skipped periods. That didn’t happen. Almost all women continued to have periods that were 21 to 36 days apart. And most of the time, they also continued to ovulate. You cannot easily tell if you aren’t ovulating normally. Despite normal cycle lengths, four percent of the cycles in the Prospective Ovulation Cohort were anovulatory. These anovulatory cycles were not perceived by the women to be different from their ovulatory cycles. What surprised most people was that anovulatory cycles did not occur more frequently in women who were training for a marathon than in women who regularly walked the dog or played tennis on the weekend. This study showed that, in well nourished women who started out normally ovulatory, training for and running a marathon did not cause anovulation (1). And women with anovulatory cycles had the same amount of estrogen as women without anovulatory cycles. In fact, if we had not carefully monitored changes in spinal cancellous bone (the honeycomb-like inner part of the vertebrae) bone, we would not have thought these few anovulatory cycles were of any great significance. Short luteal phase cycles are cycles that are ovulatory but with too few days of high progesterone levels. (“luteal phase” refers to the second half of the menstrual cycle that comes following ovulation when progesterone is produced). These, too, are not clearly different from normally ovulatory cycles. They are often perfectly regular, and with normal amounts of flow. The only problem, although an egg has been released, is that the time from ovulation until the next period is shorter than is ideal. The normal or ideal luteal phase length was initially based on potential for pregnancy. We have subsequently learned that a normal luteal phase length is also important to prevent bone loss (1). (More about bone and ovulation in a future newsletter). If you are using a basal temperature method to document ovulation then less than 10 days is too short. If you are using a urinary LH test to assess ovulation, then less than 12 days is too short (2). This is because the LH surge comes before ovulation, while the temperature rise follows ovulation. Short luteal phases were very common in the 66 regularly ovulatory, healthy women in the Prospective Ovulation Cohort. Over 25% of all cycles had short luteal phases. And these short luteal phase cycles, like anovulatory ones, were also as common in the runners than in the non-runners (1). What are menstrual cycles and ovulation like in girls after their first

period? We’ve already discussed that ovulation is carefully coordinated by the brain and is altered in response to things like food and stress and ill health. Therefore it would be surprising if the body learned to do this complex thing overnight. In fact it usually takes a year or more before the first ovulatory cycle following menarche (the first period) (3), and the first of a girl’s ovulatory cycles commonly have short luteal phases. I think it likely that there would be far more teen pregnancies if normal ovulation kicked in at a younger age! What is interesting is that it takes about 12 years before ovulation is at its most regular and predictable. By about age 20-25 approximately 95% of all cycles are ovulatory (3). And even after achieving ovulatory maturity, some menstrual cycles will still have short luteal phases, depending on stress and nutrition. All of this discussion of ovulation and the length of the luteal phase is preparing for the important relationships were are learning between normal ovulation and bone, heart and breast health. In the next newsletter we will discuss how doctors may diagnose ovulation.

Reference List for "Is Ovulation (and are normal Progesterone levels) Important for the Health of Women?" 1. Prior JC, Vigna YM, Schechter MT, Burgess AE. Spinal bone loss and ovulatory disturbances. N Engl J Med 1990;323:1221-7. 2. Petit MA, Prior JC. Exercise and the hypothalamus: ovulatory adaptions. In: Warren MP, Constantini NW, editors. Sports Endocrinology. Totowa, New Jersey: Humana Press, Inc.; 2000. p. 133-63. 3. Vollman RF. The menstrual cycle. In: Friedman EA, editor. Major Problems in Obstetrics and Gynecology, Vol 7. 1 ed. Toronto: W.B. Saunders Company; 1977. p. 11-193. Originally published October 2008

Preventive Powers of Ovulation and Progesterone Indicators of Ovulation by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research. I believe that ovulation with a normal luteal phase length – and normal amounts of progesterone to counterbalance and complement estrogen – is of key importance for women’s bone, breast and heart health. In the previous two issues we discussed: 1) Regular cycles can be anovulatory (with plenty of estrogen but no progesterone) or have short luteal phase lengths (meaning plenty of estrogen but not enough progesterone). 2) Ovulation disturbances – meaning not ovulating or ovulating with too short a time from ovulation until the period (short luteal phase cycle) – are commonly caused by emotional, social, nutritional or physical stress (like illness or too much exercise for a woman’s adaptation or food intake). Ovulation disturbances are especially common in the 10-12 years after menarche (the first period) because the brain is just learning how to ovulate. Ovulation disturbances are also extremely common in perimenopause. In perimenopause the coordinated system is falling apart. At this time, the ovary’s primary goal is to “get rid of” any remaining estrogen producing cells to avoid a rogue period at 80. (Aging of women’s reproduction is a totally different topic that we will cover in the next series of newsletters.) How can I tell if I am ovulating? That’s a very important question for a woman who wants to be healthy. The simple answer is - we’re still in the process of finding out. When we have completed the analysis of a recent study, we hope to know. We have just completed the Menstruation and Ovulation Study (MOS) in which 610 women participated, answered a question about their cycles, collected urines throughout the cycle and kept the daily Menstrual Cycle Diary© (1) during one menstrual cycle. The purpose of MOS was to determine whether a woman can reliably tell, by the way she feels, that her period is coming. This set of experiences that indicates ovulation is called “Molimina.” What is Molimina? Molimina, from the Greek word meaning the “work” of bringing on the flow, includes all the experiences that are specific for ovulation. This is not a laboratory test but rather is something you can observe. The Molimina Question: “Can you tell, by the way you feel, that your period is coming?” is an important question to ask

yourself every cycle. Before we get to what I currently think can tell us that we are ovulating, we need to realistically discuss the problems with studying this question. One of the first is that, as women, we are taught to ignore, not to pay attention to, our menstrual cycles. Even those of us who pay attention to our periods, don’t likely attend to the changing experiences inside our cycles. Another problem is that there are cultural ideas that tend to surface when we don’t really know the answer to the molimina question. For example, in this culture the only time it is “ok” to be ravenous, irritable or bitchy, is before a period. We expect to have so called “PMS” or premenstrual syndrome. This expectation may override our actual experiences or colour what we perceive. Then, finally, there are more subtle issues that influence our experiences themselves, such as our inherited metabolism of ovarian hormones, or possibly environmental toxins (like phthalates in plastics, or cadmium in oysters) that can interfere with the breakdown of estrogen. Many years ago I was excited by the notion that women could know the important fact of whether or not they were ovulating. I asked the Molimina Question of 61 consecutive regularly menstruating women that I saw in my clinical endocrinology practice. As it turns, although menstruating regularly, all of these women had Anovulatory Androgen Excess (AAE, also called Polycystic Ovary Syndrome or PCOS) (2). Each woman described whether or not she could tell that her period was coming (the majority had no clue). I decided, based on my idea of experiences indicating ovulation, whether or not she had molimina, and then asked each woman to get a progesterone blood test during the week before her next period (within 7 days but not counting the day before flow). What I found was: Not being able to tell that the period was coming was an extremely reliable indicator of anovulation and low progesterone levels. What we are learning from MOS is whether that is also true for women who don’t have AAE. I believe a woman is ovulatory who both: • knows that her period is coming, and, • experiences tenderness in the high side part of her breasts up under her armpits. She may also experience fluid retention. If – however, she only reports moodiness, hunger or front of the breast tenderness – that suggests estrogen is high just before flow and that she is not ovulating. (Estrogen levels should be dropping at that time of the cycle). Using the first morning temperature to tell about ovulation Progesterone, as we have discussed, is produced in high levels by the part of the follicle that has released the egg. This remaining hormone-producing nubbin of tissue, called the “corpus luteum” which means yellow body in Latin, makes all the progesterone for that one cycle. Progesterone works in every tissue of the body

including the brain. One of progesterone’s actions in the brain is to talk to the temperature centre in the hypothalamus and to raise our internal temperature. Although the “basal body temperature” or BBT method to assess ovulation has been used by many generations of women, it is not reliable. By BBT the temperatures are plotted on a graph (difficult for women, or anyone, to do accurately) and then one simply “eyeballs” the graph. Only since the 1970s have we developed scientific, quantitative ways of assessing ovulation by temperature (3). The most reliable and easy way to use Quantitative Basal Temperature (QBT) methods to determine ovulation and luteal phase length is to take your first morning temperature and write it down on the bottom of the daily Menstrual Cycle Diary sheet. At the end of the cycle, get out your calculator, add up all the temperatures and divide by the number of days for which you have a reading. This gives you the average temperature. Now look at your list of temperatures. Where your temperature goes above and stays above the average until at least the day before flow is your luteal phase length. It should be 10-16 days. (See instructions here [PDF]). How about using a “fertility” test with LH to assess ovulation? I prefer the Quantitative Basal Temperature method because we care about progesterone and it can also tell us about the length of luteal phase. The luteinizing hormone (LH) peak occurs over a day or so and triggers the release of the egg. However, even the best of the LH kits with sticks that you stick in urine, will miss ovulation about 20% of the time. And, although rarely, you can have an LH peak and still not release an egg. Finally, these kits are not cheap. In the next newsletter we will discuss the ways that doctors and researchers can determine about ovulation. For next time: How can a doctor tell if I am ovulating? Short of doing an operation and observing the egg actually squirting out of the ovary, as happened recently, there are only indirect ways of telling about ovulation. The three main ways are... To be continued! Reference List for "Is Ovulation (and are normal Progesterone levels) Important for the Health of Women?" 1. Prior JC. Exercise-associated menstrual disturbances. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive Endocrinology, Surgery and Technology. New York: Raven Press, 1996: 1077-1091.

2. Prior JC. Ovulatory disturbances: they do matter. Can J Diagnosis 1997; February:64-80. 3. Prior JC, Vigna YM, Schulzer M, Hall JE, Bonen A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle LH peak. Clin Invest Med 1990; 13:123-131. Originally published October 2008

Preventive Powers of Ovulation and Progesterone Detection of Ovulation by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research. I believe that ovulation with a normal luteal phase length – and normal amounts of progesterone to counterbalance and complement estrogen – is of key importance for women’s bone, breast and heart health (see Ovulatory Disturbances - They Do Matter [PDF]). In the last issue we discussed how you, personally, can tell that you are ovulating using the Molimina Question, recording and analyzing your basal temperature using quantitative methods (called QBT), and possibly using the over-the counter fertility test detecting the mid-cycle luteinizing hormone (LH) peak. We will now look at the medical methods for diagnosis of ovulation. How can a doctor tell if I am ovulating? For some physicians, especially those who specialize in fertility management, all methods for detecting ovulation (short of doing an operation and observing the egg actually squirting out of the ovary-see figure) are indirect and therefore considered inferior. There are three different, indirect medical methods that are commonly used to diagnose ovulation and normal progesterone action: 1) a series of ultrasounds of the ovary across the mid-cycle; 2) a biopsy of the uterine lining about a week after expected ovulation to see if the cells show evidence for progesterone action; and 3) taking one or several measurements of estrogen and progesterone in blood or urine to see if they show the expected ovulatory levels and pattern. We will discuss and critique each of these in turn. Note(for each of the following ovulation methods to be sensitive and specific, a woman must provide accurate information about the date of day one of her tested cycle and the date of the start of the next flow. Therefore, the start of the ovulation detection process using any method requires that you keep track of your own cycle (most easily done by keeping the daily Menstrual Cycle Diary). Detecting ovulation using ovarian ultrasound When sound waves are passed through the lower abdomen and a full bladder to observe the ovary (called abdominal ultrasound) or through the vagina (vaginal

ultrasound) the ovaries shows up as small elliptical masses on each side of the pelvis (1). To determine if ovulation is occurring, usually the test is scheduled every day from about cycle day 10 (meaning the 10th day after the first day of flow) until ovulation is detected. What tells about ovulation is that one, of possibly several, small round fluid-filled bodies (called cysts), will gradually grow until it is about the size of a thumbnail (18 mm) (2). This is then called a “dominant follicle” because it is the one that is likely to release an egg. After seeing the growing dominant follicle, one day it will have vanished. This sudden loss of the dominant follicle cyst is presumed evidence that it ruptured thus releasing the egg into the pelvis. The ultrasonographer would declare that the woman ovulated. This method for ovulation detection is expensive (more than $100 for each scan), takes specialized equipment, requires a woman to travel to a medical centre, and is uncomfortable (especially holding a sufficiently full bladder), or the vaginal exam can be embarrassing as well as time consuming. Finally, there are potentially other reasons for “losing” sight of the dominant follicle besides ovulation. Detecting ovulation using tissue from the uterine lining This test (called an endometrial biopsy) involves going to a doctor’s office or centre about three weeks after the last period, having a pelvic exam followed by the insertion of a small probe through the vagina and into the uterus. This probe is equipped to scrape or bite off a small bit of the lining of the uterus. The cells are then prepared, stained and viewed under a microscope. If the uterine lining is proliferative and only under the influence of estrogen it is thick but without small glands. If ovulation has occurred, and the lining is changing, as it should, under the influence of progesterone, the lining has many glands. The endometrium has a fairly typical pattern each successive day of the cycle. The pathologist who is specially trained to read these slides, can compare this standard with the biopsy taken from a given woman. The biopsy is said to be showing proliferation (meaning the effects of estrogen alone), proliferation with atypical changes (a risk factor for endometrial cancer), “secretory” which means evidence for progesterone action and that ovulation has taken place, and finally, whether or not the biopsy is “in phase” or appropriate for the day of the cycle (3). Although this endometrial biopsy is more directly observing the actions of progesterone on the uterine lining, there is a lot of normal variability. It takes several days after ovulation and the rise of progesterone before clear changes occur and ovulation can be diagnosed (just as it takes a few days for the basal temperature to rise after the LH peak) (4). The biopsy is considered abnormal if it is two or more days out of phase. However, the test is only reliable within two to three days. Therefore it is not a very specific test for ovulation.

Besides its lack of specificity, endometrial biopsies are time consuming for the woman (who must be free and get an appointment on the right day of her cycle), difficult for the doctor whose schedule must be flexible, and a very specialized task for the pathologist who reads the slides. Most importantly, an endometrial biopsy hurts – when the probe enters the uterus there is cramping that can be intense. Often, also, there is spotting after the biopsy, which confuses everyone about which day the next flow starts (and this knowledge is an important part of the timing of the test). Test of progesterone levels to decide on ovulation The third and final way that doctors commonly diagnose ovulation or anovulation is to take a single or a series of measurements of progesterone. Progesterone can be measured in blood or urine, although some research and alternate care providers are also measuring it in saliva. Blood tests for progesterone should be obtained about day 21 of the cycle (or three weeks after the first day of flow) for a woman with a regular, approximately 28-day (4-week) cycle (5;6). Often three blood tests are taken approximately every other day during what should be the middle of the luteal phase. The three levels added together should equal at least 65 nmol/L (or 21 ng/ml). If only one blood test is taken, the levels must be over 18 nmol/L (normal range 18 to 90 nmol/L). There is a gray zone between the usual low level of about 2 during the follicular phase and the level of 18 that is diagnostic (6). The ideal progesterone level at the middle of the luteal phase is 45 nmol/L or higher. Blood tests are momentarily uncomfortable, sometimes cause bruising, usually require travel to a laboratory, and must be timed within the menstrual cycle. Each blood test costs from $40-50.00. And you can see from the various normal ranges described above, that coming to a clear conclusion is sometimes not possible. Often doctors will prefer to measure estrogen as well as progesterone and judge ovulation by the changing relationships of the two hormones. This means twice as much blood must be taken and that the cost doubles. It is also not possible to accurately detect the length of the luteal phase unless daily blood tests are performed. Urine tests for ovulation (like we used in the Menstruation and Ovulation Study – MOS) measure pregnanediol glucuronide (PdG), a breakdown product of progesterone that is excreted into the urine. Although there is a close relationship between the blood level of progesterone today and the PdG tomorrow (7), the actual level is highly variable both within and between women. The reason that women’s PdG levels differ is probably because of different genetics and thus differences in the enzyme activity that breaks progesterone down to make PdG. Therefore, for urine, no threshold level can be used to diagnose ovulation. Instead,

the low level of PdG during the first two weeks or so of the cycle must be averaged – to diagnose ovulation, at least two or three levels must be more than three times that follicular phase love value (8). The disadvantages of urine tests for ovulation are that multiple urine samples are needed, and that urine collection can be awkward. Certainly carrying a jug about is difficult/embarrassing. As we used in MOS, “whiz pops” that only require peeing on a sponge in the lid of a small vial, solve most of the urine collection problems. Another disadvantage is that the guidelines for deciding on ovulation require some calculations and the right number of appropriately timed tests. Finally, although this testing is easier for women, many standard labs do not do the urine PdG test. The research cost for a series of tests is at least $60.00 and may be over $100.00. Summary – measurement of ovulation in the real world I believe that ovulation is important for women’s health. This means we need accurate, convenient and inexpensive ways to detect ovulation. Because ovulation is variable from cycle to cycle within a given woman, we need methods that can be used over several cycles. In the last newsletter I described several ways that women, themselves, can determine about whether or not they are ovulating. I continue to believe that the easiest, most accurate, and least expensive method is Quantitative Basal Temperature (QBT) testing. This newsletter’s discussion has shown that the ultrasound, endometrial biopsy and progesterone measurements that physicians use for detecting ovulation are often uncomfortable, expensive or inconclusive. QBT has been shown to be valid related to the midcycle LH peak. Dr. Susan Barr, Jen Bedford PhD student, and Drs. Chris Hitchcock and myself (of CeMCOR) have also studied QBT compared with urine PdG and are expecting that work to be published soon. QBT has the advantage that it tells us about luteal phase length (something we care about because it relates to bone change) which many other methods cannot. Finally, QBT is inexpensive, accessible to all through the CeMCOR website , and is easy for most women to do. In the next newsletter we will start exploring to the key issues for ovulation and women’s health by discussing the importance of ovulation for bone health. Stay tuned!

Reference List for "Is Ovulation (and are normal Progesterone levels) Important for the Health of Women?" 1. Leader A, Wiseman D, Taylor PJ. The prediction of ovulation: a comparison of the basal body temperature graph, cervical mucous score and real-time

pelvic ultra sonography. Fertil.Steril. 1985;43:385-8. 2. Gore MA, Nayudu PL, Vlaisavljevic V. Attaining dominance in vivo: distinguishing dominant from challenger follicles in humans. Hum.Reprod. 1997;12(12):2741-7. 3. Shepard MK, Senturia YD. Comparison of serum progesterone and endometrial biopsy for confirmation of ovulation and evaluation of luteal function. Fertil.Steril. 1977;28(5):541-8. 4. Prior JC, Vigna YM, Schulzer M, Hall JE, Bonen A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle LH peak. Clin.Invest.Med. 1990;13:123-31. 5. Israel R, Mishell DR, Jr., Stone SC, Thorneycroft IH, Moyer DL. Single luteal phase serum progesterone assay as an indicator of ovulation. Am J Obstet Gynecol 1972;112(8):1043-6. 6. Abraham GE, Maroulis GB, Marshall JR. Evaluation of ovulation and corpus luteum functioning using measurements of plasma progesterone. Obstetrics and Gynecology 1974;44:522-5. 7. O'Connor KA, Brindle E, Miller RC, Shofer JB, Ferrell RJ, Klein NA et al. Ovulation detection methods for urinary hormones: precision, daily and intermittent sampling and a combined hierarchical method. Hum.Reprod. 2006;21(6):1442-52. 8. Santoro N, Crawford SL, Allsworth JE, Gold EB, Greendale GA, Korenman S et al. Assessing menstrual cycles with urinary hormone assays. Am J Physiol Endocrinol.Metab 2003;284(3):E521-E530 Originally published October 2008

Preventive Powers of Ovulation and Progesterone Ovulation and Bone Health by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research. I believe that ovulation with a normal luteal phase length – and normal amounts of progesterone to counterbalance and complement estrogen – is of key importance for women’s bone, breast and heart health (see Ovulatory Disturbances: They Do Matter) (1). The four previous issues in this series have discussed what ovulation is, how it is ignored or assumed to be present in regular cycles and that we know little about the prevalence of ovulation from population-based studies. In fact, in the less than 400 women in epidemiology studied for ovulation, the information suggests that in any given regular cycle you may not ovulate once or twice out of a year’s worth of 13 cycles. We also talked about how you can assess your own cycles for ovulation by taking your first morning temperature and analyzing it. Quantitative Basal Temperature(QBT), that you can assess yourself, is much more reliable that oldfashioned BBT method, especially when QBT is coupled with a daily Menstrual Cycle Diary©. In the last issue we discussed how medical doctors assess ovulation using a series of expensive and somewhat embarrassing vaginal ultrasound tests, a painful and invasive endometrial biopsy or an inconvenient and not cheap series of blood tests for progesterone in the latter half of the cycle. With this background, it is now time to begin discussing what consistent, normal ovulation means for specific aspects of our health. The above overview from my 1997 article (1) says that I believe ovulation is important for bone, breast and heart health for women. This article will discuss what we know of ovulation and progesterone for building strong bones and preventing osteoporosis and fractures. How Bone Renovates Itself to Prevent Fracture Before it will be clear why progesterone and normal ovulation are important for bone health and osteoporosis prevention we need to discuss how bone renews itself. To keep the bone strong, old bone must be removed (by cells called osteoclasts) and replaced with new bone (created by cells called osteoblasts). Estrogen slows the action of osteoclasts and thus prevents bone loss. Progesterone directly stimulates osteoblasts to make new bone. Our bone has a natural life cycle. We build bone size and strength starting in utero and continuing until the early teen years. Bone then reaches a high point called “peak bone mass.” After that, women’s bone is ideally kept steady through the

premenopausal years until bone loss occurs when skipped periods start in perimenopause. It is normal to lose bone at about 2% a year beginning when irregular periods start in perimenopause and continue at that high rate until one year after the last period. From menopause for the first four years, we lose bone at a rate of about 1%. Then bone loss is normally slowed to about half a percent a year because osteoblasts can’t keep up with the loss caused by osteoclasts. How Progesterone Works with Bone Cells and Bone Tissue We all know that estrogen is important for bones and prevents bone loss (although it acts indirectly rather than by talking to osteoclasts) What is clear, but most physicians and even some bone experts don’t know is that progesterone sits on specific receptors on osteoblasts and stimulates the formation of new bone. There are numerous papers showing that osteoblast cells cultured in a lab grow rapidly and make bone and bone enzymes when progesterone is added to the culture (24). Ovulation and Building Strong Bones in the Teens and Twenties As we discussed in the second part of this series, it takes a number of years after the first period (called menarche) before the brain, pituitary and ovary learn to have regular ovulation. In fact, the first year after menarche estrogen levels are normal or high and cycles may be regular or irregular but ovulation almost never occurs. In a study of pre-teen and teen growth, bone density, exercise and nutrition performed by UBC nutrition professor, Susan Barr, with assistance from my laboratory (5), we found that the first of these young women to develop an ovulatory cycle was 11 months after menarche and the majority were over one year. When we tracked bone gain, the maximal increase in bone occurred after ovulation first began, rather than with menarche, suggesting that progesterone was important for teen bone growth (Kalyan, J Bone Min Research 2007, abstract). Ovulation and Keeping Strong Premenopausal Bones As I mentioned earlier, my first research studied exercising women and tracked their menstrual cycles. We got funding to study women ages 20-40 for one full year and measure their spine bone density at the beginning and the end of that year. We enrolled 66 healthy, normal weight women who were all proven to not only have regular menstrual cycles but to ovulate normally on two cycles using the QBT method (6). However, when we followed these women’s cycles and ovulation across a year, although all women continued to have regular periods, only 13 women had normal ovulation every cycle, 13 had at least one cycle in which they did not ovulate, and 28 women had more than one short luteal phase cycle. This study showed that the length of the luteal phase (the time of high progesterone

production) explained over 20% of the one-year change in bone (calcium or caloric intake explained only 2%). Said another way, these healthy women with enough estrogen and regular cycles but who didn’t ovulate for even one cycle were losing bone, while those who ovulated every cycle during the year were maintaining bone (7). This was the first study to show that progesterone and ovulation, not just regular cycles and normal estrogen levels were necessary to prevent premenopausal bone loss (7). Since that study, two further investigations of bone in premenopausal women have shown that ovulation disturbances are related to loss of bone (8;9). One of these studies showed that the urinary progesterone peak, and the total amount of progesterone were significantly lower in women from a random sample of the population with the lowest bone density compared with cycles from women in the same study with normal bone density (8). The other study assessed ovulation using progesterone levels in saliva and monitored women over 2 years (9). Progesterone for Preventing Premenopausal Bone Loss Those studies of bone loss in healthy premenopausal women who have regular cycles but don’t ovulate normally raised real concerns about bone loss in premenopausal women who have obviously abnormal menstrual cycles (long cycles as well as skipped periods for months at a time). In addition to recommending regular exercise, stable weight, good intakes of calcium and vitamin D, women with abnormal cycles need something to stimulate new bone to grow (see “ABCs of Premenopausal Osteoporosis Prevention”). We wanted to test the idea that progesterone builds bone by performing a randomized trial of cyclic progesterone[pdf] in premenopausal women with abnormal cycles but who were otherwise well. Because, before 1996 natural oral micronized progesterone called Prometrium® was not available in Canada, we designed a trial using the closest cousin of progesterone, the progestin called medroxyprogesterone acetate (MPA, Provera) for the last 10 days of the menstrual cycle if it was regular, or for the month if it was not (10). We enrolled healthy, normal weight women ages 20-40 with amenorrhea (no periods for six months or longer), women with cycles farther apart than 36 days, those with regular cycles but who were not ovulating, or with regular cycles and ovulation but short luteal phases. Women were randomized to cyclic MPA or placebo. The results showed that women with abnormal cycles given cyclic MPA had a significant gain in bone (23%/year) while those on placebo MPA lost about 2% of their spinal bone—the effect of cyclic progestin was very highly significant (10). This study proves that progestin (which, like progesterone stimulates osteoblasts through the progesterone receptor) not only prevents bone loss but also builds new bone in women with disturbed menstrual cycles or ovulation.

To date no study has given cyclic progesterone to perimenopausal women with abnormal ovulation. It is very clear that perimenopausal women with irregular cycles need increased vitamin D and calcium intakes (see the “ABCs of Midlife Osteoporosis Prevention"). Progesterone’s Role in Osteoporosis Treatment Typically menopausal women who have osteoporosis have had a broken bone with a minor fall or are at high risk for breaking bones. Because such women not only have low bone density but are losing bone, the primary treatments are those medications that slow bone loss (such as estrogen, calcitonin or bisphosphonates). One early clinical study of menopausal women taking estrogen plus 5 mg MPA daily showed a greater gain in bone in these women than menopausal women only taking estrogen therapy (11). In addition, we documented that women with osteoporosis treated with the bisphosphonate, Etidronate, plus 10 mg of MPA daily had a greater gain in bone than women on Etidronate alone (12). Neither of those two clinical studies was randomized or placebo-controlled, however. More convincing evidence that progestins add to the positive effects of estrogen on bone came from a randomized double-blind placebo-controlled study that compared women on standard doses of estrogen with 2.5 mg of MPA daily and on only estrogen (if they had undergone a hysterectomy). These controlled results showed about a one percent greater bone gain on estrogen with MPA than on estrogen alone (13). To date no study has shown that adding progesterone or MPA to a therapy that slows bone loss can prevent fracture more effectively than the boneloss-preventing osteoporosis therapy alone. We are planning a randomized twoyear study of a bisphosphonate plus Prometrium (300 mg/day) compared with the same bisphosphonate plus placebo progesterone. That study will provide the information needed to plan a larger fracture prevention study. All women in any such trial will be treated in the standard way (see “ABCs of Osteoporosis Treatment”) in addition to their bisphosphonate and randomized progesterone therapy. Summary: Progesterone is a Bone-building Hormone To summarize what we’ve covered about bone in relationship to ovulation and progesterone, we can say that progesterone sits on specific receptors on the bonebuilding osteoblast cells. Therefore, women with regular cycles but ovulation disturbances, despite having normal estrogen levels, will continue to lose bone. However treatment with cyclic progestin (and probably progesterone, ideally given for days 14-27 of a 28 day cycle) will significantly increase bone density. Although a few studies in menopausal women have shown that the progestin, MPA, adds to

the benefits of bisphosphonate or estrogen treatment, no study has yet been designed to show that progesterone therapy prevents fractures. In our next newsletter we will cover issues related to progesterone and breast health plus the risk for breast cancer. Stay tuned!   Reference List for "Is Ovulation (and are normal Progesterone levels) Important for the Health of Women?" 1. Prior JC. Ovulatory disturbances: they do matter. Can.J.Diagnosis 1997;February:6480. 2. Tremollieres FA, Strong DD, Baylink D, Mohan S. Progesterone and promogestone stimulate human bone cell proliferation and insulin-like growth factor 2 production. Acta Endocr. 1992;126:329-37. 3. Tertinegg L, Heersche JN. Progesterone stimulates bone nodule formation in rat calvarial cell cultures while estrogen has no effect. J.Bone Min.Res. 1992;7 (Suppl 1):S220. 4. Verhaar HJJ, Damen CA, Duursma SA, Schevens BAA. A comparison of the actions of progestins and estrogens on growth and differentiation of normal adult human osteoblast-like cells in vitro. Bone 1994;15:307-11. 5. Barr SI, Petit MA, Vigna YM, Prior JC. Eating attitudes and habitual calcium intake in peripubertal girls are associated with initial bone mineral content and its change over 2 years. J.Bone Min.Res. 2001;16:940-7. 6. Prior JC, Vigna YM, Schulzer M, Hall JE, Bonen A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle LH peak. Clin.Invest.Med. 1990;13:123-31. 7. Prior JC, Vigna YM, Schechter MT, Burgess AE. Spinal bone loss and ovulatory disturbances. N Engl J Med 1990;323:1221-7. 8. Sowers M, Randolph JF, Crutchfield M, Jannausch ML, Shapiro B, Zhang B et al. Urinary ovarian and gonadotropin hormone levels in premenopausal women with low bone mass. J.Bone Min.Res. 1998;13(7):1191-202. 9. Waugh EJ, Polivy J, Ridout R, Hawker GA. A prospective investigation of the relations among cognitive dietary restraint, subclinical ovulatory disturbances, physical activity, and bone mass in healthy young women. Am.J Clin.Nutr. 2007;86(6):1791801.

10. Prior JC, Vigna YM, Barr SI, Rexworthy C, Lentle BC. Cyclic medroxyprogesterone treatment increases bone density: a controlled trial in active women with menstrual cycle disturbances. Am.J.Med. 1994;96:521-30. 11. Grey A, Cundy T, Evans M, Reid I. Medroxyprogesterone acetate ehnances the spinal bone density response to estrogen in late post-menopausal women. Clin.Endocr. 1996;44:293-6. 12. Prior, J. C. and Hitchcock, C. L. Medroxyprogesterone augments positive bone mineral density effects of cyclic etidronate in menopausal women: pilot data from a random sample of clinical charts of menopausal women with osteoporosis. J Bone Mineral Res 17, S474. 2002. Ref Type: Abstract 13. Lindsay R, Gallagher JC, Kleerekoper M, Pickar JH. Effect of lower doses of conjugated equine estrogens with and without medroxyprogesterone acetate on bone in early postmenopausal women. JAMA 2002;287:2668-76.

Originally published April 2009

Preventive Powers of Ovulation and Progesterone Ovulation and Breast Health by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research. I believe that ovulation with a normal luteal phase length – and normal amounts of progesterone to counterbalance and complement estrogen – is of key importance for women’s bone, breast and heart health (1). The five previous issues in this series have discussed what ovulation is, how it is ignored or assumed in regular cycles and that we have little solid evidence about how frequently or not ovulation occurs among menstruating women in the whole population. The little epidemiology evidence we have suggests that 17% of the time women in the population, often despite regular cycles, do not ovulate. We also have talked about how you can assess your own cycles for ovulation by taking your first morning temperature and analyzing it. This “quantitative basal temperature” (2) is much more reliable than the old fashioned BBT methods, especially when coupled with a daily Menstrual Cycle Diary (3). Finally, we discussed the many problems with how ovulation and luteal phase lengths are assessed by physicians. In the last issue we discussed the importance of ovulation and normal progesterone for building and maintaining strong bones. Estrogen levels rise and fall during the normal menstrual cycle. When estrogen levels fall, there is a small amount of bone loss—this loss needs to be offset by an increased bone formation, caused by normal luteal phase lengths and normal progesterone in order to prevent bone loss (4;5). Progesterone’s job is to stimulate bone formation. I believe progesterone could be added to a bone-loss-slowing medication to form improved fracture-preventing osteoporosis treatment for women. Now it is time to examine the complex relationships among ovulation, progesterone and breast health. We will start by discussing the hormonal influences on breast growth and development during adolescence. Then we will look at the roles estrogen and progesterone play in normal breast cell function. Finally, and of crucial importance, we will review the new evidence that ovarian hormone therapy (OHT, menopausal hormone therapy) with estrogen plus progesterone is not associated with an increased risk for breast cancer. As every woman knows and fears, treatment of menopausal hot flushes with estrogen alone or with estrogen plus medroxyprogesterone (a cousin of natural progesterone) increases the risk for breast cancer. When we understand more we may eventually have evidence that normal ovulation and progesterone levels prevent breast cancer.

Grown up breasts As you recall from our earlier discussion, at menarche (the first period), our ovaries are making plenty of estrogen. However, at that time our bodies have not yet learned the complex process of ovulating. There is a natural clue in how breasts look that reflects whether or not ovulation has been established (or the breasts have been exposed to the progestins in contraceptives or the standard Pill). There are a series of reasonably orderly steps that the breasts go through in the process of becoming “grown up.” These steps are called Tanner Stages, after the doctor who took pictures of girls’ breast and pubic hair changes yearly from before to several years after menarche. A young child will have breasts that are Tanner Stage I (small circle of skin called the “areola,” around the small nipple and both are lying flat against the chest). Around age 8-10, a small lump, made up of the glands that will eventually form the mature breast, begins to be present beneath the small areola and nipple—this gives us Tanner Stage II. At the first period we have usually reached Tanner Stage III, meaning that the breast is round and somewhat full but the areola (the now darker bull’s-eye circle of skin surrounding the nipple) is still small—about the size of a nickel or quarter. You can see all the stages of breast development in this figure. Starting about a year or so after the first period, around the time that the first cycles are ovulatory, the areola starts to balloon out and get bigger around. This awkward phase is called Tanner IV and occurs because the ducts are starting to mature under the influence of progesterone. Once ovulation is established, the areola then flattens but is now bigger (about the size of a Canadian two-dollar coin) and is now fully grown up, Tanner V. By this time the breasts will be able to make milk for nursing a baby, their fundamental purpose. Unfortunately, doctors tend to consider breasts based only on their size—which continues to increase across adolescence, increases during pregnancy and may increase in perimenopause—while ignoring the key issue, how big around the areola is. That means that Tanner Stage III and Tanner Stage V can be mixed up. The drawback is that medicine has missed an important clue—a clear sign that tells us whether or not women have established normal ovulation (or breasts have been exposed to progesterone/ progestins). Breasts changes with estrogen and progesterone Have you ever noticed that your breasts get bigger or swollen before your period? Maybe you found that it was sore if you accidentally bumped your nipple at the middle of your cycle. Or perhaps you’ve had the experience of knowing that you were pregnant even before you’d missed a period—the clue, breasts that were too

sore to touch! All of these experiences are telling us that estrogen and progesterone are working in our breasts. What is important to understand is what changes each hormone causes and the necessity that estrogen and progesterone be in balance. Many hundreds of studies have examined breast cells grown in dishes in the laboratory and noted the changes that occur when estrogen or progesterone are added to the cultured cells. In general these studies observe that both estrogen and progesterone stimulate breast cells to grow. Cell growth—or proliferation as it is commonly called—is associated with more chance for a mistake and overgrowth of abnormal cells leading to breast cancer. Therefore, for years it has been assumed that both estrogen and progesterone play roles in the risk for breast cancer. However, cell culture studies often use breast tumour cell lines, or cells that are not natural (because they can continue to grow in culture). In addition, most investigators only observe the cells for a day or two. It turns out that in every tissue studied in many different animals, estrogen initially and continuously causes proliferation (6). However, although progesterone initially causes cell proliferation, it then begins and continues to stimulate cells to differentiate(7). Because more differentiated or more mature cells are less likely to be cancerous, progesterone, according to this measure, should have anti-cancer effects. The best way to understand what estrogen and progesterone do in breast cells is to study these cells in living, breathing women. This turned out to be possible because women were scheduled for a small surgery to remove a lump that, in each case, turned out to not be cancer. Two excellent randomized, double blind controlled studies have done just that—in each trial women applied onto the breast that was scheduled for a surgical biopsy a gel containing one of four things: estrogen, progesterone, estrogen and progesterone or just the alcohol base. The first study was in menstruating women who were scheduled for the biopsy on day 11 of their cycle (8). They began applying the gel on the first day of their menstrual period and continued through day 11 when they had surgery. The second study was in menopausal women who were randomized to apply the gel for 14 days with the biopsy scheduled on the 14th day (9). Both of these studies showed that the estrogen and progesterone got into cells and in amounts that were natural; they also showed that breast cell proliferation changed in response to hormones compared with placebo. In both studies, estrogen caused proliferation, and progesterone decreased proliferation. This suggests that estrogen’s job is to make breasts grow and progesterone’s job is to stop the growth and allow breast cell maturation. The next time your breasts become sore or enlarged, ask yourself if estrogen is stimulating your breast cells to proliferate. Also, you might wonder whether your

body is making enough progesterone to counterbalance estrogen. What about history of ovulation problems and breast cancer? On this question, we have few good answers. Remember that we said it is commonly and wrongly understood that regular cycles mean normal ovulation? We know that women with more years of menstrual cycles (earlier first period, later menopause) have a higher risk for breast cancer (10). But we also know that regular cycles can be anovulatory and without progesterone. Two moderate sized long cohort studies from the 1980s in women with medical diagnoses associated with ovulation problems—infertility and anovulatory androgen excess (AAE, also known as polycystic ovary syndrome, PCOS)—have documented inadequate progesterone and then followed women to observe how many of these women compared with controls, developed breast cancer. The first was a study of 1083 women who were documented between 1945 and 1965 to have infertility that was presumably caused by ovulation disturbances because blocked tubes or problems with their husbands’ sperm were excluded (11). Compared with controls who had other reasons for infertility all of whom were followed through 1978, women with ovulation disturbances showed 5.4 times increased risk for premenopausal breast cancer (11). The second study observed all 1270 women hospitalized for AAE/PCOS at the Mayo Clinic beginning in the 1930s (12) and compared their risk until the late 1970s with women hospitalized for other reasons. The women with chronic anovulation and androgen excess (AAE/PCOS) had a risk for menopausal breast cancer that was 3.6 times higher than their controls (12). Both of these studies suggest that chronic ovulation disturbance (especially if estrogen levels are normal or high as they are in AAE/PCOS) is an important risk for breast cancer. Risk for breast cancer with ovarian hormone therapy Multiple large, long observational studies from the 1940s through the 1990s showed that menopausal women who took ovarian hormone therapy, meaning estrogen or estrogen with progestins, appeared to have fewer heart attacks, to be less likely to develop dementia and more likely to live longer. There were concerns, however, that this treatment might cause breast cancer. Plus it was never clear if these women were healthier in the first place, or better at taking pills, or more likely to see their doctors regularly. To test whether menopausal ovarian hormone therapy (OHT) was causing benefit or harm, women scientists pushed the National Institutes of Health in the USA to do randomized controlled trials—the Women’s Health Initiative (WHI) hormone trials. In July 2002 the almost 17,000 women in the estrogen plus progestin (E + P) arm

of the WHI were told to suddenly stop their study drug—this was four years earlier than planned—because more women taking hormones than taking the placebo developed breast cancer and heart attacks (13). Although the estrogen only (E only) WHI trial in women who had undergone a hysterectomy continued for another two years, it, too, was stopped early. In this case, the reasons for stopping were lack of heart disease prevention and more strokes in those taking estrogen (14). Surprisingly, the E only WHI trial did not show an increase in breast cancer. Although many had blamed the low dose medroxyprogesterone (2.5 mg/d) in the E + P arm for the increased breast cancer that arm of the WHI showed, it has been known for many years that women who have had pelvic surgery (hysterectomy, even tubal ligation) have a lower risk for breast cancer (15). It is likely that the WHI E only trial didn’t have enough women in it to show a risk for breast cancer, given the lower risk in women with hysterectomy and especially since that trial had only about 11,000 women enrolled. Our best guess about why hysterectomy decreases breast cancer risk is that, for reasons not yet quite clear, the ovaries are getting less blood flow and hence make lower levels of testosterone, which, because it gets made into estrogen by our bodies, causes lower levels of estrogen itself (16). How can it be that a cousin of progesterone, medroxyprogesterone, that, like progesterone also causes less breast cell growth/proliferation, should cause breast cancer when taken plus estrogen? Given what we know about estrogen causing proliferation and progesterone or progestins stopping that growth and causing maturation, I initially wondered if the reason for breast cancer increase with E + P might be that it was a full dose of estrogen (Premarin® 0.625 mg/d) but only a quarter luteal phase equivalent dose of medroxyprogesterone (MPA, Provera® 2.5 mg/d). The dose imbalance in the WHI E + P trial is still a probable reason, however, a recent randomized trial in monkeys without their ovaries of hormone therapy with estrogen plus MPA (2.5 mg/d) or estrogen plus progesterone (200 mg/d) for two months showed that estrogen with MPA caused markedly increased breast cell growth but that estrogen with progesterone did not (17). The MPA dose was lower in relative terms than the progesterone dose but the difference in effect was marked. In addition, although there are many guesses, none of which can be proved at the moment, one of the most recent ones is that medroxy-progesterone activates quiet or hiding breast cancer stem cells that estrogen then stimulates to grow (18). Of course, the reason everyone will understand is that breast cancer increase is just one further evidence that estrogen is good and progestins or progesterone are somehow bad. Although observational studies and randomized ones differ (like in heart disease risk with hormone therapy), in the case of breast cancer, multiple studies have show a greater risk for cancer from estrogen with MPA than with estrogen

alone (19). A recent large, observational study from France called the E3N study of women (mostly teachers) in an insurance programme, has provided important information about the progesterone and breast cancer question. (Note that this is not a randomized, double blind placebo-controlled trial like the WHI, therefore it may well have biases we don’t yet understand.) However, E3N was performed in France, a country that has had oral micronized progesterone therapy (called Prometrium® in North America or Utrogestan® in France) since the 1980s—in Canada it has been available only since the mid-1990s. The E3N study examined risk for breast cancer in about 80,000 menopausal women followed for about eight years by whether they didn’t take ovarian hormone therapy (the control group), or used estrogen alone, estrogen with progesterone (about a third of those taking combined therapy), or estrogen with MPA (20). They found that estrogen alone increased the risk 29%, estrogen with MPA increased the risk for breast cancer by 79% but that estrogen with progesterone showed no increased risk (20). This study suggests that MPA differs significantly from its parent hormone, progesterone, especially in relationship to breast cancer risks. Making sense out of progesterone and breasts All of the evidence we have right now suggests to me that the currently unknown major risk factor for breast cancer is being exposed to enough or too much estrogen without enough progesterone. One day we may know that having normal ovulatory cycles throughout our reproductive lives is a way to prevent breast cancer. It is clear that estrogen needs to be counterbalanced by progesterone in the breast to prevent tenderness, overgrowth or cancerous growth in breast cells. We have reviewed the evidence that our breasts need progesterone as well as estrogen to mature into organs with large areolae that have the ability to make milk. Two good randomized studies show that progesterone causes breast cells in women to become more mature and less likely to cause cancer. Yet the Women’s Health Initiative randomized placebo-controlled trial of estrogen with the progestin, MPA, showed an increased risk for breast cancer not shown in the estrogen only trial, in women who had their uterus and possibly ovaries removed. Finally, a large observational study indicated that estrogen with progesterone therapy did not cause breast cancer although estrogen alone or estrogen with MPA did. Estrogen causes important cell growth that progesterone must transform into mature cells for milk production, normal soft and non-tender breasts and, most importantly, to avoid an increased risk for breast cancer. In the next newsletter we will discuss ovulation and progesterone related to women’s blood vessels, cholesterol and risks for heart disease. Stay tuned!

Reference List for "Is Ovulation (and are normal Progesterone levels) Important for the Health of Women?" 1. Prior JC. Ovulatory disturbances: they do matter. Can.J.Diagnosis 1997;February:64-80. 2. Prior JC, Vigna YM, Schulzer M, Hall JE, Bonen A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle LH peak. Clin.Invest.Med. 1990;13:123-31. 3. Prior JC. Exercise-associated menstrual disturbances. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive Endocrinology, Surgery and Technology. New York: Raven Press; 1996. p. 1077-91. 4. Prior JC, Vigna YM, Schechter MT, Burgess AE. Spinal bone loss and ovulatory disturbances. N Engl J Med 1990;323:1221-7. 5. Sowers M, Randolph JF, Crutchfield M, Jannausch ML, Shapiro B, Zhang B et al. Urinary ovarian and gonadotropin hormone levels in premenopausal women with low bone mass. J.Bone Min.Res. 1998;13(7):1191-202. 6. Clarke CL, Sutherland RL. Progestin regulation of cellular proliferation. Endocr.Rev. 1990;11:266-301. 7. Graham JD, Clarke CL. Physiological action of progesterone in target tissue. Endocr.Rev. 1997;18:592-19. 8. Chang KJ, Lee TTY, Linares-Cruz G, Fournier S, de Lignieres B. Influence of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil.Steril. 1995;63:785-91. 9. Foidart J, Collin C, Denoo X, Desreux J, Belliard A, Fournier S et al. Estradiol and progesterone regulate the proliferation of human breast epithelial cells. Fertil.Steril. 1998;5:963-9. 10.Titus-Ernstoff L, Longnecker MP, Newcomb PA, Dain B, Greenberg ER, Mittendorf R et al. Menstrual factors in relation to breast cancer risk. Cancer Epidemiol.Biomarkers Prev. 1998;7(9):783-9. 11.Cowan LD, Gordis L, Tonascia JA, Jones GS. Breast cancer incidence in women with a history of progesterone deficiency. Am J Epidemiol. 1981;114(2):209-17. 12.Coulam CB, Annegers JF, Kranz JS. Chronic anovulation syndrome and associated neoplasia. Obstetrics and Gynecology 1983;61:403-7.

13.Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in health postmenopausal women: prinicpal results from the Women's Health Initiative Randomized Control trial. JAMA 2002;288:321-33. 14.Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H et al. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial. JAMA 2004;291(14):1701-12. 15.Kreiger N, Sloan M, Cotterchio M, Kirsh V. The risk of breast cancer following reproductive surgery. Eur.J.Cancer. 1999;35:97-101. 16.Laughlin GA, Barrett-Connor E, Kritz-Silverstein D, von Muhlen D. Hysterectomy, oophorectomy, and endogenous sex hormone levels in older women: the Rancho Bernardo Study. J Clin Endocrinol Metab 2000;85:64551. 17.Wood CE, Register TC, Lees CJ, Chen H, Kimrey S, Cline JM. Effects of estradiol with micronized progesterone or medroxyprogesterone acetate on risk markers for breast cancer in postmenopausal monkeys. Breast Cancer Res Treat. 2007;101(2):125-34. 18.Horwitz KB, Sartorius CA. Progestins in hormone replacement therapies reactivate cancer stem cells in women with preexisting breast cancers: a hypothesis. J Clin Endocrinol.Metab 2008;93(9):3295-8. 19.Beral V. Breast cancer and hormone-replacement therapy in the Million Women Study. Lancet 2003;362(9382):419-27. 20.Fournier A, Berrino F, Clavel-Chapelon F. Unequal risks for breast cancer associated with different hormone replacement therapies: results from the E3N cohort study. Breast Cancer Res Treat. 2008;107(1):103-11. Originally published July 2009

Preventive Powers of Ovulation and Progesterone Ovulation and the Heart by Dr. Jerilynn C. Prior, Scientific Director, Centre for Menstrual Cycle and Ovulation Research. We have been asking, through a series of newsletters, what we know about women’s health related to normal progesterone levels and ovulation. We have discussed the fact that ovulatory disturbances (meaning anovulation and short luteal phase cycles) are common and hidden within menstrual cycles that seem perfectly normal. I speculate that at least a third of all cycles - in regularly menstruating, healthy women 10-30 years since menarche (their first period) produce too little progesterone. (We don’t really know the percentage of cycles with ovulatory disturbances in the general population because no one has studied it. CeMCOR in joint study with Norwegian scientists and funded by Canadian Institutes of Health Research is currently trying to learn what proportion of women’s cycles is anovulatory.) In previous newsletters we described how difficult it is to know if we have ovulatory disturbances. Most of the time, unless we are working to become pregnant, we think everything’s fine. Thus, doctors would call ovulatory disturbances “subclinical” because they don’t come to medical attention. The majority of ovulatory disturbances occur within cycles of normal length, normal flow and even with perfectly normal estrogen levels (1). However, they are lacking any (anovulation) or have too little progesterone production (short luteal phase). Thus, ovulatory disturbances provide an “experiment of nature” that allows us the opportunity to understand how progesterone alone - not just in combination with estrogen, its essential partner ovarian hormone - contributes to women’s health. Earlier we discussed that estrogen’s job is to stimulate the growth of cells (i.e. proliferation) but that progesterone’s role is to decrease that proliferation and induce maturation and differentiation of tissues (2). Although much of our research and treatment has focused on estrogen, which is considered the primary “woman’s hormone”, I believe that progesterone is an essential partner hormone to estrogen. These two important ovarian hormones are meant to work together, complementing or counter-balancing each other in every tissue and every cell of women’s bodies and across our life cycles (3). I’ll say again what I believe, and what we are making progress in proving: Regular menstrual cycles with consistently normal ovulation during the premenopausal years will prevent osteoporosis, breast cancer and heart disease in women.

The purpose of this article is to describe new and suggestive evidence that progesterone is important for preventing women’s cardiovascular diseases (CVD, heart attacks, strokes and blood clots). However, before we can make sense of any information about progesterone and CVD, we have to put what we currently believe and know into a context. We are now ready to wrap up this review of progesterone and women’s health. This final section concerns women’s risk for heart disease, stroke and diseases of the blood vessels and the relationships of these vascular problems to estrogen and progesterone. Let’s start with what “we” believe—by this I mean the two main cultural myths that surround women’s heart disease. Myths about women’s heart disease 1. The first myth--women’s heart disease is the same as men’s Obviously it is simpler for doctors, media and organizations to give one consistent message that applies to young and old, woman and man alike. It is also advantageous to pharmaceutical interests and will expand the potential customers to have a one-size fits all marketing campaign. However, these messages are fundamentally untrue. Women’s heart disease first occurs at an older age than in men. Furthermore, contrary to advertising, women’s heart disease rates never becomes as high as in men, and the population-adjusted death rate in women remains lower than in men (4). In addition, in a population follow-up study, men whose cholesterol levels were in the lowest quarter of the population level had higher heart attack rates than women with cholesterol levels in the highest quarter of the population level(5). Furthermore, low dose aspirin (a single 325 mg tablet a week, or 81 mg every day) doesn’t prevent heart disease in women although it has been shown to prevent heart attacks in men (6). And, as opposed to men, there is no credible evidence that the popular lipid lowering drugs (such as statins) are effective at preventing as opposed to treating heart disease in women (7). Despite this myth of women and men having similar risks for heart disease, there are sex-related disparities in the health care for women and men with acute heart attacks. According to a recent report from Ontario, women are more likely to be taken care of by a family doctor without a specialist than are men of similar income bracket and age. Women are also likely to wait longer before they get infusion of the clot-busting intravenous drugs (thrombolytic) when they come to the emergency room with a heart attack. And finally, women are less likely to have the diagnostic angiogram testing that tells physicians the extent of the cardiovascular risk. These facts are ironic given the messaging that says women and men are at the same risk for heart disease.

2. The second myth—women’s heart disease is caused by estrogen deficiency The reasoning behind this notion goes like this—young women have lots of estrogen and don’t get heart attacks. Older menopausal women are “estrogen deficient” and get heart attacks. Therefore, lack of estrogen causes women’s heart disease. That is like saying that headache is an aspirin-deficiency disease! Ten years before the first Women’s Health Initiative (WHI) proved me correct in suspecting that cardiovascular disease would not be prevented by estrogen treatment (8), I could “see” that this myth about estrogen therapy preventing heart disease was wrong (9). This myth has now, finally, been repeatedly tested in randomized double-blind placebo-controlled trials in both women (8;10) and men (11). In every scientific (randomized, placebo-controlled) test, this estrogentreatment-heart-disease-prevention myth has failed—and yet the myth persists (12;13). The only possible reason for such a nonsensical idea to persist is because it serves some purpose. I can guess that its purpose is to re-enforce the “woman problem.” As a culture, we fundamentally believe women to be somehow lacking (the anatomy and physiology of men—thank goodness) or that women are weak or ill. Pharmaceutical companies, some specialist physicians and those dominant in our culture appear to gain power by treating women’s “deficiencies,” often with estrogen. What’s the evidence for the Estrogen-Heart Disease Prevention Myth? Large observational studies, including some of the data from longitudinal population-based studies like the Framingham Heart Study, have shown that women taking estrogen had fewer heart attacks than did women not taking socalled hormone “replacement” therapy (14). The reasons estrogen (here read pill estrogen as in conjugated equine estrogen, CEE, or Premarin) was proposed to prevent heart disease were that it increased the apparently preventative, good high-density cholesterol (HDL) level. Estradiol is also undoubtedly active in the complex nitric-oxide system through which the microcirculation (small blood vessels) is controlled (15). But we knew, even many years ago, that the women who take estrogen treatment differ in heart-protective ways from the women who don’t—they are more likely to have a personal physician, to be well educated, to exercise regularly, to be non-smokers, to be of normal weight and without diabetes or high blood pressure (16). Oral estrogen treatment increases HDL cholesterol and makes blood flow better in

small and medium sized blood vessels—this is called endothelial function because it is controlled by complex changes in endothelial lining of vessels. Estrogen treatment also doesn’t appear to cause high blood pressure, diabetes or obesity. Although estrogen treatment, in general, has no effect on blood pressure, insulin resistance or obesity in randomized controlled trials—in some women it does appear to contribute to individual-specific increases in blood pressure, blood sugar and weight gain. How could estrogen or estrogen-progestin treatment cause the increase in risk for heart disease shown in multiple randomized controlled trials (8;17)? One possible way is through its increased levels of C-reactive protein, a strong marker of inflammation, which is now considered a common pathway to many diseases including heart disease (18). CEE also increases triglycerides that are now known to be more strongly associated with causing women’s heart disease than HDL levels are at preventing it. Most important of all, oral estrogen increases women’s risk for blood clots(19). I believe that the formation of clots within arteries is estrogen’s main negative cardiovascular effect (both on heart attacks and strokes)—we used to call heart attacks “coronary thrombosis” (meaning heart artery blood clots). The good news about estrogen and blood clots is that estrogen applied through the skin (transdermal estrogen—as a patch or gel or cream), doesn’t go from the stomach through the liver first and thus increase levels of blood clotting proteins— transdermal estrogen does not cause blood clots (20;21). I believe that no one, who needs estrogen treatment, should ever be treated with oral estrogen, given that safer transdermal bio-identical estradiol is available as a patch, gel or cream. Progesterone, Blood Vessels and Heart Disease I believe that ovulatory disturbances in young menstruating women cause an increased risk for heart disease in older menopausal women. This postulate is a very hard one to test—large numbers of menstruating women with frequent ovulatory disturbances would need to be given a placebo or cyclic progesterone for years and then followed for at least 10 years following the last menstrual flow. This is because ovulatory disturbances occur in young, menstruating women whereas heart disease is largely a disease of the very elderly. And heart disease takes years to develop. Despite the difficulty in doing a definitive experiment about progesterone and heart disease, there are many heart disease risk factors that progesterone appears to decrease. We’ll begin with these CVD markers, and then discuss the two experiments that strongly suggest that ovulatory disturbances cause subsequent heart disease.

Cardiovascular Risk Markers and Progestin or Progesterone We have known since a controlled trial in 1985 that oral micronized progesterone decreased blood pressure in both menopausal women and in men (22). This means progesterone should decrease women’s risk for strokes for which high blood pressure and blood clotting are major risks. Here it is worth recalling that both the Estrogen-Progestin and the Estrogen-only arms of the Women’s Health Initiative trials showed higher risks for stroke with hormone treatment compared with controls (8;23). We have repeated the study of progesterone and blood pressure in a randomized double-blind trial in of progesterone for hot flushes in healthy menopausal women and expect to know the results in the next year. Potential CVD reducing mechanisms have also been shown for medroxyprogesterone MPA (a progestin most closely related to oral micronized progesterone) although MPA is often blamed for heart disease (24). MPA may decrease CVD risk by lowering triglycerides and C-reactive protein levels (Kalyan Pharmacotherapy 2010). These data are from a randomized blinded one-year comparison of CEE and MPA that showed an important difference between estrogen and MPA. Women randomized to MPA had lower triglyceride and C-reactive protein levels at the end of the trial. Although the women on MPA also had lower HDL levels than did those on CEE, their HDL levels remained within the normal range (Kalyan Pharmacotherapy 2010). In that randomized comparison study of estrogen and MPA (the only one that has been published) there were no differences in blood pressure. We have these data about MPA, however, it is difficult to know about the effect of progesterone on cholesterol, other lipids and inflammatory markers because, to our knowledge, no study has compared placebo with oral micronized progesterone without estrogen. Again, we have collected this information in a controlled trial of progesterone for hot flushes and hope to publish the results within a year. Another observation in the randomized blinded comparison of CEE and MPA is that women on CEE gained more weight (almost five versus about 2.5 kg) (25), and tended to increase their truncal fat more than did those women on MPA (Kalyan Pharmacotherapy 2010). It is abdominal fat that is associated with insulin resistance, diabetes and an increased risk for heart disease. We do know that most women exposed to progesterone can eat this imperceptible amount more and not gain weight. From studies in which young normal-weight women kept a three-day diet diary about a week after flow started and a week before the next flow, we discovered that the women who ovulated were eating about 300 calories more during the luteal than the follicular phase yet kept their weights steady (26). This occurs because progesterone raises our core temperature about 0.2 degrees C. and increased temperature requires increased energy. This fact makes it likely that

progesterone aids women in avoiding obesity, insulin resistance and potentially diabetes mellitus, a very important women’s heart disease risk factor. Endothelial function is another cardiovascular marker for which there are positive progesterone data. Abnormalities in the control of blood flow by the endothelium of arteries are associated with an increased risk for heart attack. Control of blood flow is a complex process through which nitric oxide is released in the endothelial lining of blood vessels. Some years ago we did a randomized study in which healthy menopausal women came once a week for the study of blood flow. During each session, blood flow in the forearm was measured following standardized stimulations when (a week apart) estrogen, progesterone, estrogen plus progesterone or just the base solution (control) were infused into the local artery (15). This study showed that progesterone was as effective or better at increasing blood flow as estrogen(15). We have repeated this study in women randomized to oral micronized progesterone or placebo and will soon be able to present our results. Primate and Human Studies of Ovulation and Risk for Heart Disease The most convincing studies are always those with disease outcomes—like measured blood vessel abnormalities or better yet heart attacks. There are two such studies of the potential association of ovulatory disturbances and risk for CVD—one is in colonies of female monkeys fed a high cholesterol diet, and the other a study of ovulation over three cycles in a large number of women who were followed for heart attacks in a population-based Dutch study. The monkey study has the advantage that the animals were captive, could be studied closely and at the end their arteries could be carefully examined for the plaques that indicate a risk for heart attack (27). The scientists first observed that some of the female monkeys were groomed more, got to the food first and were dominant over other female monkeys. They then observed that, although the dominant monkeys tended to weigh more, their menstrual cycles were the same lengths but the stressed, isolated subordinate monkeys were more likely to have ovulatory disturbances. After three years of this monitoring, when they looked at the arteries they found that, although the male monkeys had the most abnormal arteries the subordinate females had similar artery disease. However, the regularly ovulatory, non-stressed dominant female monkeys had little or no artery plaque (27). Although, in this study they did not measure estrogen levels which were likely similar between groups, progesterone levels were lower and cortisol stress hormone levels were higher. Therefore, the lower progesterone levels, the higher cortisol levels or both appear to cause female monkeys serious blood vessel disease.

The study of pre-/perimenopausal women was part of a population-based breast screening programme of over 11,000 women ages 44-49 in 1986-8 who initially completed an extensive questionnaire and brought three consecutive cycle day 22 overnight urine samples to the laboratory (28). About eight years later, local hospital registers were systematically searched for women who had participated looking for those with either acute heart attack or chest pain (angina) plus at least a 50% blockage in a coronary artery on angiography (29). Women with heart disease were matched by age, screening and other variables with three women without CVD—those with heart attacks (cases) and those without (controls) were compared for things that differed. Researchers found that those with heart attacks were more likely to smoke (60%!), to have treated high blood pressure and to have diabetes. Also, although there were no differences in actual levels of estrogen, progesterone or testosterone in their urine, more of those with heart disease had low levels of progesterone designated as anovulatory levels than did controls. This suggests that those with major heart risk factors (smoking, diabetes, high blood pressure) were more likely to have a heart attack in their mid-50s if they also had been anovulatory earlier. Although this study did not take into account the great differences between ovulatory women in their metabolism and excretion of progesterone, it is suggestive that those without adequate progesterone in perimenopause have higher rates of heart attack later. Thus both cardiovascular risk factors (like blood pressure, inflammation, triglycerides, less weight gain and improved endothelial function) and two studies of ovulation in female monkeys and women all suggest that ovulation and normal progesterone levels with normal estrogen may be protective for heart disease in women. Summary - Progesterone Prevents Osteoporosis, Breast Cancer and Heart Disease In this series of newsletter articles we have discussed the difficulties in making a clinical diagnosis of ovulatory disturbances (multiple blood, urine or saliva tests or serial ultrasound studies). We also assert that a motivated woman, with little equipment or cost, can know her own cycle using the Menstrual Cycle Diary and measurements of her first morning temperature analyzed scientifically and accurately using a quantitative method (30;31). We have estimated that approximately 10-20% of women’s cycles are anovulatory and about a third have short luteal phases thus ovulatory disturbances occur in a high percentage of seemingly normal menstrual cycles. Thus we know that ovulatory disturbances with their normal estrogen but lower progesterone levels are both common and silent. In this series of articles we have already shown that progesterone is important for women’s bone health. Progesterone, acting through the bone-forming osteoblast

cells, is important for the increased bone gain that occurs in the first years after menarche as cycles are “growing up” to become ovulatory (32). We also know that young, healthy and regularly menstruating women with more ovulatory disturbed cycles are silently losing bone (1;33). It may be that, eventually, progesterone will be used as part of the treatment for osteoporosis and used to prevent fractures. We have also made a strong case that progesterone may prevent breast soreness, lumpiness (sometimes called “fibrocystic disease”) and breast cancer risk. We showed that progesterone is necessary for the breast to mature to its grown up, Tanner Stage V form that has a Canadian two-dollar sized darker areola surrounding the nipple (34). That progesterone can stop the excessive cell growth caused by estrogen is also shown in two randomized human trials of hormones applied daily to one breast before a breast biopsy (35;36). Finally, the latest evidence from a large prospective observational study is that progesterone (but not progestins) with estrogen decreases the risk for breast cancer caused by the estrogen alone (37). This final article suggests that, although women’s heart disease is under an unscientific cloud of myths and disadvantages in clinical care, that there are evidences that progesterone is positive for heart disease risk factors and some clinical studies suggesting normal ovulation prevents later heart attacks. All of these ideas need testing in well documented prospective studies and randomized controlled trials before they will be proven. The data to date confirm CeMCOR’s postulate that normal ovulatory cycles during the premenopausal years prevent later, menopausal osteoporosis, breast cancer and heart disease, the three major health issues for women in industrialized countries who live to become older women. Reference List 1. Prior JC, Vigna YM, Schechter MT, Burgess AE. Spinal bone loss and ovulatory disturbances. N Engl J Med 1990;1221-7. 2. Clarke CL, Sutherland RL. Progestin regulation of cellular proliferation. Endocr.Rev. 1990;266-301. 3. Baxter S, Prior JC. The Estrogen Errors: Why Progesterone is Better For Women's Health. Westport: Praeger Publishers, 2009:1. 4. Tunstall-Pedoe H. Myth and paradox of coronary risk and the menopause. Lancet 1998;1425-7.

5. Isles C, Hole DJ, Hawthorne VM, Lever AF. Relation between coronary risk and coronary mortality in women of the Renfew and Paisley survey: comparison with men. Lancet 1994;702-6. 6. Ridker PM, Cook NR, Lee IM, Gordon D, Gaziano JM, Manson JE, Hennekens CH, Buring JE. A randomized trial of low-dose aspirin in the primary prevention of cardiovascular disease in women. N.Engl.J Med. 2005;1293304. 7. Rosenberg H, Allard D. Evidence for Caution: Women and statin use. 1-36. 2007. Winnipeg, Canadian Women's Health Network. Women and Health Protection. Ref Type: Report 8. Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in health postmenopausal women: prinicpal results from the Women's Health Initiative Randomized Control trial. JAMA 2002;321-33. 9. Prior JC. Postmenopausal estrogen therapy and cardiovascular disease (letter). N Engl J Med 1992;705-6. 10.Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ, Lane D, Langer RD, Lasser N, Lewis CE, Manson J, Margolis K, Ockene J, O'Sullivan MJ, Phillips L, Prentice RL, Ritenbaugh C, Robbins J, Rossouw JE, Sarto G, Stefanick ML, Van Horn L, Wactawski-Wende J, Wallace R, Wassertheil-Smoller S. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial. JAMA 2004;1701-12. 11.Coronary Drug Project Research Group. Coronary drug project: findings leading to the discontinuation of the 2.5 mg/day estrogen group. Journal of the American Medical Association 1973;652-7. 12.Derry PS. Hormones, menopause, and heart disease: making sense of the Women's Health Initiative. Womens Health Issues 2004;212-9. 13.Barrett-Connor E. Hormones and heart disease in women: the timing hypothesis. Am.J.Epidemiol. 2007;506-10. 14.Castelli WP, Anderson K, Wilson PW, Levy D. Lipids and risk of coronary heart disease. The Framingham Study. Ann.Epidemiol. 1992;23-8.

15.Mather KJ, Norman EG, Prior JC, Elliott TG. Preserved forearm endothelial responses with acute exposure to progesterone: a randomized cross-over trial of 17-b estradiol, progesterone, and 17-b estradiol with progesterone in healthy menopausal women. J Clin Endocrinol Metab 2000;4644-9. 16.Barrett-Connor E, Bush T. Estrogen and coronary heart disease in women. Journal of the American Medical Association 1991;1861-7. 17.Beral V, Banks E, Reeves G. Evidence from randomised trials on the longterm effects of hormone replacement therapy. Lancet 2002;942-4. 18.Frohlich M, Muhlberger N, Hanke H, Imhof A, Doring A, Pepys MB, Koenig W. Markers of inflammation in women on different hormone replacement therapies. Ann.Med. 2003;353-61. 19.Brosnan JF, Sheppard BL, Norris LA. Haemostatic activation in postmenopausal women taking low-dose hormone therapy: less effect with transdermal administration? Thromb.Haemost. 2007;558-65. 20.Brosnan JF, Sheppard BL, Norris LA. Haemostatic activation in postmenopausal women taking low-dose hormone therapy: less effect with transdermal administration? Thromb.Haemost. 2007;558-65. 21.Canonico M, Oger E, Plu-Bureau, Conard J, Meyer G, Levesque H, Trillot N, Barrellier MT, Wahl D, Emmerich J, Scarabin PY. Hormone therapy and venous thromboembolism among postmenopausal women: impact of the route of estrogen administration and progestogens: the ESTHER study. Circulation 2007;840-5. 22.Rylance PB, Brincat M, Lafferty K, De Trafford JC, Brincat S, Parsons V, Studd JW. Natural progesterone and antihypertensive action. Br.Med.J. 1985;13-4. 23.Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ, Lane D, Langer RD, Lasser N, Lewis CE, Manson J, Margolis K, Ockene J, O'Sullivan MJ, Phillips L, Prentice RL, Ritenbaugh C, Robbins J, Rossouw JE, Sarto G, Stefanick ML, Van Horn L, Wactawski-Wende J, Wallace R, Wassertheil-Smoller S. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial. JAMA 2004;1701-12.

24.Koh KK, Sakuma I. Should progestins be blamed for the failure of hormone replacement therapy to reduce cardiovascular events in randomized controlled trials? Arterioscler.Thromb.Vasc.Biol. 2004;1171-9. 25.Prior JC, Vigna YM, Wark JD, Eyre DR, Lentle BC, Li DKB, Ebeling PR, Atley LM. Premenopausal ovariectomy-related bone loss: a randomized, doubleblind one year trial of conjugated estrogen or medroxyprogesterone acetate. J.Bone Min.Res. 1997;1851-63. 26.Barr SI, Janelle KC, Prior JC. Energy Intakes Are Higher During the LutealPhase of Ovulatory Menstrual Cycles. American Journal of Clinical Nutrition 1995;39-43. 27.Kaplan JR, Adams MR, Clarkson TB, Koritnik DR. Psychological influences on female 'protection' among cynomolgus macaques. Atherosclerosis 1984;28395. 28.Gorgels WJ, Graaf Y, Blankenstein MA, Collette HJ, Erkelens DW, Banga JD. Urinary sex hormone excretions in premenopausal women and coronary heart disease risk: a nested case-referent study in the DOM-cohort. J Clin Epidemiol. 1997;275-81. 29.Gorgels WJ, Graaf Y, Blankenstein MA, Collette HJ, Erkelens DW, Banga JD. Urinary sex hormone excretions in premenopausal women and coronary heart disease risk: a nested case-referent study in the DOM-cohort. J Clin Epidemiol. 1997;275-81. 30.Bedford JL, Prior JC, Hitchcock CL, Barr SI. Detecting evidence of luteal activity by least-squares quantitative basal temperature analysis against urinary progesterone metabolites and the effect of wake-time variability. Eur.J Obstet Gynecol Reprod Biol. 2009;76-80. 31.Prior JC, Vigna YM, Schulzer M, Hall JE, Bonen A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle LH peak. Clin.Invest.Med. 1990;123-31. 32.Kalyan S, Barr SI, Alamoudi R, Prior JC. Is Development of Ovulatory Cycles in Adolescence Important for Peak Bone Mass? J Bone Miner.Res 22, S494 W511. 2007. Ref Type: Abstract 33.Waugh EJ, Polivy J, Ridout R, Hawker GA. A prospective investigation of the relations among cognitive dietary restraint, subclinical ovulatory disturbances, physical activity, and bone mass in healthy young women. Am.J Clin.Nutr. 2007;1791-801.

34.Prior JC, Vigna YM, Watson D. Spironolactone with physiological female gonadal steroids in the presurgical therapy of male to female transexuals: a new observation. Arch.Sex.Beh. 1989;49-57. 35.Chang KJ, Lee TTY, Linares-Cruz G, Fournier S, de Lignieres B. Influence of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil.Steril. 1995;785-91. 36.Foidart J, Collin C, Denoo X, Desreux J, Belliard A, Fournier S, de Lignieres B. Estradiol and progesterone regulate the proliferation of human breast epithelial cells. Fertil.Steril. 1998;963-9. 37.Fournier A, Berrino F, Clavel-Chapelon F. Unequal risks for breast cancer associated with different hormone replacement therapies: results from the E3N cohort study. Breast Cancer Res Treat. 2008;103-11. Originally published February 2010