In oligo-ovulatory and anovulatory women with polycystic ovary syndrome (PCOS) who wish to conceive, first-line therapy is usually ovulation induction with clomiphene citrate.1,2
The American Society of Reproductive Medicine Practice Guidelines describes the use of progestin to induce a withdrawal bleed before initiation of clomiphene citrate administration in women with PCOS.3 However, there is scant literature on the value or possible detrimental effect of such a practice. Further, if a patient remains anovulatory in response to the initial clomiphene dose, another dose of progestin is recommended by a leading reproductive endocrinology textbook4 to induce a withdrawal bleed before dose escalation. Others have skipped routine withdrawal with progestins after anovulatory cycles and instead proceeded immediately to repeat clomiphene citrate administration in women with PCOS. One group has labeled this a “stair-step protocol” to accelerate treatment progression and time to ovulation with clomiphene. However their report did not describe pregnancy or live birth rates associated with dosage escalation.5
Our original study protocol, titled Pregnancy in Polycystic Ovary Syndrome I,6 used either clomiphene, metformin, or the combination of the two for ovulation induction in women with PCOS. In this report, we have performed a secondary analysis of this study to estimate whether progestin administration to induce a withdrawal bleed immediately before initiation of ovulation induction (with clomiphene citrate alone, metformin alone, or combination clomiphene citrate and metformin) would influence the subsequent likelihood of ovulation, conception, and live birth in the ensuing cycle.
MATERIALS AND METHODS
This study represents an analysis of the database of the National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development Cooperative Reproductive Medicine Network Pregnancy in Polycystic Ovary Syndrome I study. This analysis was approved by the Wayne State University institutional review board; the conduct of the original study was approved by institutional review boards at each participating institution.6 The study protocol has been published previously6–11 and is briefly reviewed here. Six hundred twenty-six women diagnosed with PCOS had oligomenorrhea (history of eight or fewer spontaneous menses per year) or amenorrhea and evidence of hyperandrogenemia (defined as an elevated serum testosterone level). All women were documented to have normal uterine cavities with patency of at least one fallopian tube and a partner who had semen analysis within 1 year with a sperm concentration of at least 20 million sperm/mL.
Patients received in a double-blind double dummy manner both encapsulated clomiphene, 50 mg or placebo (purchased from Teva Pharmaceuticals), and extended-release metformin (Glucophage XR), 500 mg or placebo (provided by Bristol Myers Squibb). Patients were randomized to treatment arms (clomiphene alone, metformin alone, or combination of clomiphene plus metformin) by means of an interactive voice system, with assignment stratified based on the study center and presence or absence of prior exposure to either clomiphene or metformin. Administration of the medications was initiated concurrently and continued for up to six cycles, with the metformin dose increased gradually up to a dose of four tablets per day. Clomiphene was administered as one pill per day for 5 days beginning cycle day 3 in the initial cycle; the dose was increased one tablet per day in subsequent cycles if there was no evidence of ovulation (as assessed by weekly serum progesterone level of 5 ng/mL or higher), up to a maximum dosage of three tablets per day. A negative pregnancy test was confirmed each cycle before clomiphene administration. As previously described, baseline demographic characteristics were comparable in each treatment group.6
Couples were instructed to have intercourse every 2 to 3 days, to record vaginal bleeding in their diaries, and to have serum progesterone measured every 1 to 2 weeks to diagnose ovulation. For women who did not have spontaneous menses, progestin administration to induce withdrawal bleeding was performed at the discretion of each site's principal investigator. Women could receive up to six cycles of treatment if they had failed to conceive. Women with a positive pregnancy test underwent ultrasonography to determine the number of fetuses and their viability, but there were no routine ultrasound examinations of follicular response during the study. Pregnancy outcomes up until the completion of pregnancy were ascertained.
Methodology for measurement of markers of the androgen milieu (total testosterone, free androgen index, sex hormone–binding globulin and glucose homeostasis (serum glucose, insulin, and proinsulin) have been described previously.6 Similarly, the homeostatic model assessment algorithm to assess insulin sensitivity has been described previously.7,8 Briefly, the homeostatic model assessment index is a measure of insulin action and is derived from fasting glucose and insulin levels based on the following formula: (fasting insulin in micro-international units/mL×fasting glucose in nmol/L)/22.5. These measures were obtained on a monthly basis during the study.
Analysis was conducted on the outcome variables of ovulation, conception, and live birth in cycles in which women had spontaneous menstruation preceding administration of study drug, were anovulatory and underwent a progestin-induced withdrawal bleed before initiating clomiphene study drug, or were anovulatory and received no progestin before receiving study drug. (Menstrual-like flow was termed “spontaneous menses,” but, because of the retrospective nature of this study, it is not possible to be certain whether such bleeding episodes represented endometrial shedding after ovulation or spontaneous shedding in association with anovulation.) This analysis was conducted for all three arms (clomiphene alone, metformin alone, and clomiphene and metformin combined) together, as well as separately. Menstrual cycle status of the preceding cycle was categorized: spontaneous menses, anovulatory with progestin withdrawal, and anovulatory without progestin withdrawal. Data were available in 2,809 of 2,925 cycles of therapy during the study (96%) and are the basis for analysis in this article.
Data were analyzed using SPSS 18.0 for Windows. Statistical analyses were conducted using Fisher exact test and Pearson χ2 test; hormonal and metabolic parameters were analyzed by two-way analysis of variance (ANOVA) and one-way ANOVA for menstrual cycle status and treatment condition with Student-Newman-Keuls post hoc comparisons. Body mass index (BMI, calculated as weight (kg)/[height (m)]2) was analyzed by two-way and one-way ANOVA for menstrual cycle status and either ovulation, conception and live birth status with Student-Newman-Keuls post hoc comparisons. All statistical tests were two-tailed, with a significance level of P<.05 considered statistically significant. Data are expressed as mean±standard deviation.
Ovulation after treatment occurred significantly more often in cycles after a previous spontaneous menses, than in cycles after progestin withdrawal, or cycles without progestin withdrawal (P<.001, Table 1). There was no significant difference in the rate of ovulation between the anovulatory cycles with and without progestin withdrawal. Consistent ovulation rate observations were performed in each of the treatment arms (clomiphene alone: spontaneous menses 280/386 [72.5%], progestin withdrawal 62/178 [34.8%], and no progestin withdrawal 106/336 [31.5%], P<.001; metformin alone: spontaneous menses 202/329 [61.4%], anovulatory with progestin withdrawal 28/201 [13.9%], and anovulatory without progestin withdrawal 60/446 [13.5%], P<.001; and clomiphene plus metformin: spontaneous menses 371/470 [78.9%], anovulatory with progestin withdrawal 76/172 [44.2%], and anovulatory without progestin withdrawal 123/291 [42.3%], P<.001). Comparing the individual treatment arms, there was a significant difference in ovulation rates in cycles preceded by spontaneous menses (P<.001), as well as in prior anovulatory cycles (with [P<.001] or without [P<.001] progestin withdrawal). Ovulation rates were lower in the metformin arm than the clomiphene alone and clomiphene plus metformin arms in cycles with a previous spontaneous bleed (P=.006 and P=.003, respectively) and in anovulatory cycles with (P=.003 and P=.003, respectively) and without (P=.003 and P=.003, respectively) progestin withdrawal. The ovulation rate of the clomiphene arm was significantly less than the combined clomiphene plus metformin arm only in cycles with anovulation without progestin (P=.018).
In the 2,809 cycles, conception in all treatment arms occurred in 131 women, with live births in 118. In the 131 conception cycles, 29.8% followed spontaneous menses, 8.4% followed anovulatory cycles with progestin withdrawal, and 61.8% followed anovulatory cycles without a progestin induced withdrawal bleed (P<.001).
Among all the cycles with ovulation, conceptions occurred in 4.5% and 6.6% of the cycles that were preceded by spontaneous menses and anovulatory cycles with progestin withdrawal, respectively; in contrast, the conception rate was dramatically increased (27.7%) in the cycles with anovulation without progestin withdrawal (P<.001; see Table 1). These relationships persisted when examined by each of the individual treatment arms (clomiphene alone, metformin alone, and clomiphene plus metformin) separately (each P<.001) (data not shown).
Similarly, when evaluating live births (Table 1) as a function of the preceding cycle menstrual status (spontaneous menses, anovulatory with progestin withdrawal, and anovulatory without progestin withdrawal), the rates were 3.0%, 5.4%, and 19.7%, respectively (P<.001). Again, these significant relationships were all manifested when examining each of the clomiphene alone, metformin alone, and clomiphene plus metformin arms separately (each P<.001) (data not shown).
To reduce the effect of including different numbers of cycles from each patient and to assess the effect of correlated data from the same patient, we repeated the analysis by including only the initial treatment cycle in the 626 women and identified similar results. Thus, the use of data from the multiple cycles of the same patients does not alter our conclusion. Specifically, among women who ovulated who had a preceding spontaneous menses, conceptions occurred in 8 of 70 cycles (11.4%); previously anovulatory women who did or did not undergo progestin withdrawal conceived in 11 of 159 (6.9%) and 3 of 10 (30%) of cycles, respectively (P=.037). Examining live births in the initial cycles, a total of 17 occurred. Among women with previous spontaneous menses or anovulation with or without progestin withdrawal, the rate of live births among those who ovulated was 5 of 70 (7.1%), 9 of 159 (5.7%), and 3 of 10 (30%), respectively (P=.015).
We examined the effects of several possible confounding variables. As previously noted,6 a significant relationship of BMI to ovulation, conception, and live birth was observed, with women with lower BMI being more likely to have each of these outcomes (Table 2). This relationship persisted within each of the three groups when considering the menstrual status of the preceding cycle. However, there was no significant interaction between the menstrual status of the preceding cycle with respect to BMI and either ovulation, conception, or live birth. Testosterone and sex hormone–binding globulin were both significantly different as a function of menstrual groupings of the preceding cycles; those with spontaneous menses had significantly lower total testosterone levels, as well as higher sex hormone–binding globulin levels (P<.001 and P<.008, respectively). Levels in the women with anovulatory cycles with or without progestin were comparable. Assessing markers of glucose homeostasis, the preceding menstrual status was not associated with any difference in plasma glucose. However plasma insulin and proinsulin were both significantly different (P=.011 and P<.001, respectively) (Table 2). Post hoc analysis revealed that cycles in women with a preceding spontaneous menses had lower insulin and proinsulin levels as compared with cycles preceded by anovulation (with or without progestin induced menses), and had greater insulin sensitivity, as assessed using the homeostatic model assessment formula (Table 2).
Our secondary analysis of the Reproductive Medicine Network Pregnancy in Polycystic Ovary Syndrome I study data, looking at the administration of progestin in an anovulatory cycle before ovulation induction, has found that a menstrual bleed immediately before the cycle, whether induced or not, is associated with a reduced chance of conception and live birth in the subsequent cycle of ovulation induction. We noted this association both in the initial cycle as well as all cycles combined, and identified it as independent of ovulation induction agent. Further, we noted that this effect was unrelated to ovulation in the previous cycle, which was significantly more likely in cycles with spontaneous menses compared with anovulatory cycles. Importantly, these findings challenge the widespread clinical practice tradition of routinely administering progestin to induce a withdrawal bleed in the context of anovulatory women undergoing ovulation induction.
Administration of fertility-promoting medication to anovulatory of oligo-ovulatory women with PCOS is often successful in helping couples conceive. However, multiple cycles of therapy are often required, both to find the proper dose of ovulation inducing medication and to allow for sufficient opportunity for conception when ovulatory. A recent publication has highlighted the time “lost” in successive cycles of attempted ovulation induction when the generally recognized approach is to administer progestin to induce endometrial shedding after each nonresponsive (anovulatory) cycle.5 Compared with historical controls,3,4 that report suggested that use of a stair-step protocol increasing the dose of clomiphene citrate reduced the time to ovulation by 32–53 days, and was associated with a three-fold higher overall rate of ovulation (64% at the 100 mg/d dosage) as compared with the progestin withdrawal group (where the ovulation rate was 22%). That report did not assess conception or live birth rates.
In our report, the rate of ovulation after anovulatory cycles with or without progestin withdrawal in women with PCOS was significantly reduced when compared with cycles with a preceding menses. Thus these findings are inconsistent with the calculations of effect on the rate of ovulation in the report of Hurst et al5 where use of progestin withdrawal in the historical control group was associated with a reduced rate of ovulation. In our study, we showed that in cycles not preceded by spontaneous menses, ovulation rates were comparable whether or not treatment was preceded by progestin-induced endometrial shedding. However, we found that cycles preceded by endometrial shedding (either spontaneously or after progestin withdrawal) were associated with several fold lower rates of conception and live birth. Thus, our data does not support “conventional wisdom” that progestin induced endometrial shedding in anovulatory women improves the preparation of the endometrium for pregnancy initiation in the subsequent cycle.
This observation of an apparent detrimental effect of endometrial shedding was observed whether evaluating all cycles that women underwent in the study, or just the initial cycle of each patient. This relationship was also manifest in each of the three treatment arms (clomiphene citrate alone, metformin alone, or clomiphene citrate plus metformin). Additionally, although BMI was associated with the likelihood of each of the endpoints (ovulation, conception, and live birth),6,12,13 there was no significant effect of the menstrual status of the preceding cycle with respect to BMI for any of these outcomes.
These observations raise the question of a mechanism by which lack of endometrial shedding could exert a positive effect on subsequent conception and live births. Possibilities include an indirect consequence of the effect of progestin on the hypothalamic-pituitary-ovarian axis, as well as direct or indirect effect of the differences in the androgenic and metabolic milieus. Progestin administration has direct effects on the endometrium that may be deleterious to subsequent conception. Alternatively, it is generally well-accepted that women with PCOS have reduced sensitivity to suppression of luteinizing hormone (LH) pulse frequency by progesterone and estradiol compared with women without PCOS.14–17 Thus, the increased LH pulse frequency observed in women with PCOS may be due to impairment of sensitivity of the gonadotropin-releasing hormone pulse generator to these hormones. Administration of estradiol and progesterone for 7 days to women with PCOS reduced serum LH, follicle-stimulating hormone, and testosterone levels as well as the LH pulse frequency.14 Thus progestin exposure (either endogenous or exogenous) may have altered the hypothalamic-pituitary-ovarian axis sufficiently to alter effects of sex hormones and protein factors directly on the endometrium in women receiving clomiphene citrate, metformin or a combination of these two agents, and thereby indirectly affecting the endometrium.
There are multiple other possible mediators of the effect of the status of the preceding menstrual cycle. For example, endometrial androgen receptor expression fluctuates throughout the menstrual cycle, with a gradual reduction from the proliferative phase to the mid-secretory phase, and further diminution in the late secretory phase.18,19 The potential significance of this variation to conception and live birth is highlighted by the observation that expression of androgen receptors in endometrium is elevated in women with PCOS,20 an observation consistent with reports that androgen receptor expression is androgen dependent.21
Interestingly, expression of sex hormone–binding globulin, which usually is attributed to the liver, is also found in human uterine endometrium.22 In women with PCOS, sex hormone–binding globulin expression in endometrial stroma is reduced as compared with fertile women during the proliferative phase of the menstrual cycle.23 Similarly, women with PCOS have been reported to have reduced expression of insulin-signaling molecules (such as insulin-receptor substrates-1 and peroxisome proliferator-activated receptor gamma) as compared with women in a control group.24,25 Lack of endometrial shedding in the preceding cycle may influence expression of factors considered to be important to endometrial receptivity, such as integrins, osteopontin, leukemia inhibitory factor, glycodelin, tumor necrosis factor-α, transforming growth factor-β, and vascular endothelial growth factor.20,26–31 Importantly, these observations are consistent with evaluation of androgen receptor expression in pig endometrium during early pregnancy, which indicated a role for the androgen receptor in defining the window of implantation and early pregnancy endometrium, thereby affecting the potential for early pregnancy loss.32
Although the Reproductive Medicine Network Pregnancy in Polycystic Ovary Syndrome I study was well designed to examine efficacy of clomiphene citrate, metformin or a combination of these agents for ovarian stimulation and live birth rates in women with PCOS, this post hoc evaluation has many limitations that should lead to a cautious interpretation of the finding. The investigator(s) at each site made the decision of whether, and when, to administer progestin in each patient, as well as the type and duration of progestin therapy. Additionally, although inclusion criteria mandated a normal uterine cavity, at least one patient fallopian tube, and a male partner having at least 20 million sperm/mL, variability existed in parameters related to fertility prognosis, including age of the female partner.
In summary, this secondary analysis has suggested that endometrial shedding whether spontaneous or progestin-induced before ovarian stimulation results in a reduction in the subsequent rate of conception and live birth. Taken together with another recent report that estimated a shorter time to ovulation if progestin therapy was withheld, these reports suggest that there may be an advantage with respect to conception and live birth in not initiating progestin withdrawal after an anovulatory cycle in women with PCOS who are about to undergo ovarian stimulation.
1. Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Consensus on infertility treatment related to polycystic ovary syndrome [published erratum appears in Hum Reprod 2008;23:1474]. Hum Reprod 2008;23:462–77.
2. Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Consensus on infertility treatment related to polycystic ovary syndrome. Fertil Steril 2008;89:505–22.
3. Practice Committee of the American Society for Reproductive Medicine. The use of clomiphene citrate in women. Fertil Steril 2006;86:S187–93.
4. Speroff L, Fritz MA. Clinical gynecologic endocrinology and infertility. 7th ed. Philadelphia (PA): Lippincott Williams & Wilkins; 2004.
5. Hurst BS, Hickman JM, Matthews ML, Usadi RS, Marshburn PB. Novel clomiphene “stair-step” protocol reduces time to ovulation in women with polycystic ovarian syndrome. Am J Obstet Gynecol 2009;200:510.e1–4.
6. Legro RS, Huiman X, Barnhart HX, Schlaff WD, Carr BR, Diamond MP, et al.. Clomiphene, metformin, or both for infertility in the polycystic ovary syndrome. N Engl J Med 2007;356:551–66.
7. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and cell function from fasting plasma glucose and β-cell function from fasting plasma glucose and insulin concentration in man. Diabetologia 1985;28:412–9.
8. Haffner SM, Miettinen H, Stern MP. The homeostasis model in the San Antonio Heart study. Diabetes Care 1997;20:1087–92.
9. Legro RS, Myers ER. Surrogate end-points or primary outcomes in clinical trials in women with polycystic ovary syndrome? Hum Reprod 2004;19:1697–704.
10. Myers ER, Silva SG, Hafley G, Kunselman AR, Nestler JE, Legro RS. Estimating live birth rates after ovulation induction in polycystic ovary syndrome: sample size calculations for the Pregnancy in Polycystic Ovary Syndrome trial. Contemp Clin Trials 2005;26:271–80.
11. Legro RS, Myers ER, Barnhart HX, Carson SA, Diamond MP, Carr BR, et al.. The Pregnancy in Polycystic Ovary Syndrome Study: baseline characteristics of the randomized cohort including racial effects. Fertil Steril 2006;86:914–33.
12. Rausch ME, Legro RS, Barnhart HX, Schlaff WD, Carr BR, Diamond MP, et al.. Predictors of pregnancy in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2010;94:3458–66.
13. Zhang H, Legro RS, Zhang J, Zhang L, Chen X, Casson PR, et al.. Decision trees for identifying predictors of treatment effectiveness in clinical trials and its application to ovulation in a study of women with polycystic ovary syndrome. Hum Reprod 2010;25:2612–21.
14. Pastor CL, Griffin-Korf ML, Aloi JA, Evans WS, Marshall JC. Polycystic ovary syndrome: evidence for reduced sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab 1998;83:582–90.
15. Daniels TL, Berga SL. Resistance of gonadotropin releasing hormone drive to sex steroid induced suppression in hyperandrogenemic anovulation. J Clin Endocrinol Metab 1997;82:4179–83.
16. Eagleson CA, Gingrich MB, Pastor CL, Arora TK, Burt CM, Evans WS, et al.. Polycystic ovarian syndrome: evidence that flutamide restores sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab 2000;85:4047–52.
17. Blank SK, McCartney CR, Chhabra S, Helm KD, Eagleson CA, Chang RJ, et al.. Modulation of gonadotropin-releasing hormone pulse generator sensitivity to progesterone inhibition in hyperandrogenic adolescent girls–implications for regulation of pubertal maturation. J Clin Endocrinol Metab 2009;94:2360–6.
18. Mertens HJ, Heineman MJ, Koudstaal J, Theunissen P, Evers JL. Androgen receptor content in human endometrium. Eur J Obstet Gynecol Reprod Biol 1996;70:11–3.
19. Mertens HJ, Heineman MJ, Theunissen PH, de Jong FH, Evers JL. Androgen, estrogen and progesterone receptor expression in the human uterus during the menstrual cycle. Eur J Obstet Gynecol Reprod Biol 2001;98:58–65.
20. Apparao KB, Lovely LP, Gui Y, Lininger RA, Lessey BA. Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome. Biol Reprod 2002;66:297–304.
21. Ruizeveld de Winter JA, Trapman J, Vermey M, Mulder E, Zegers ND, van der Kwast TH. Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem 1991;39:927–36.
22. Misao R, Fujimoto J, Nakanishi Y, Tamaya T. Expression of sex hormone-binding globulin exon VII splicing variant mRNA in human uterine endometrium. J Steroid Biochem Mol Biol 1997;62:385–90.
23. Maliqueo M, Bacallao K, Quezada S, Clementi M, Gabler F, Johnson MC, et al.. Sex hormone-binding globulin expression in the endometria of women with polycystic ovary syndrome. Fertil Steril 2007;87:321–8.
24. Fornes R, Ormazabal P, Rosas C, Gabler F, Vantman D, Romero C, et al.. Changes in the expression of insulin signaling pathway molecules in endometria from polycystic ovary syndrome women with or without hyperinsulinemia. Mol Med 2010;16:129–36.
25. Kohan K, Carvajal R, Gabler F, Vantman D, Romero C, Vega M. Role of the transcriptional factors FOXO1 and PPARG on gene expression of SLC2A4 in endometrial tissue from women with polycystic ovary syndrome. Reproduction 2010;140:123–31.
26. Quezada S, Avellaira C, Johnson MC, Gabler F, Fuentes A, Vega M. Evaluation of steroid receptors, coregulators, and molecules associated with uterine receptivity in secretory endometria from untreated women with polycystic ovary syndrome. Fertil Steril 85:1017–26.
27. Tabibzadeh S. Molecular control of the implantation window. Hum Reprod Update 1998;4:465–71.
28. Von Wolff M, Thaler CJ, Strowitzki T, Broome J, Stolz W, Tabibzadeh S. Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion. Mol Hum Reprod 2000;6:627–34.
29. Haider S, Knöfler M. Human tumour necrosis factor: physiological and pathological roles in placenta and endometrium. Placenta 2009;30:111–23.
30. Giudice LC. Endometrium in PCOS: implantation and predisposition to endocrine CA. Best Pract Res Clin Endocrinol Metab 2006;20:235–44.
31. Leach RE, Jessmon P, Coutifaris C, Kruger M, Myers ER, Ali-Fehri R, et al.. High throughput, cell type-specific analysis of key proteins in human endometrial biopsies of women from fertile and infertile couples. Hum Reprod 2012;27:814–28.
32. Kowalski AA, Vale-Cruz DS, Simmen FA, Simmen RC. Uterine androgen receptors: roles in estrogen-mediated gene expression and DNA synthesis. Biol Reprod 2004;70:1349–57.