- Email: [email protected]
Evaluation of ovarian function in 35–40-year-old women with polycystic ovary syndrome
European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Evaluation of ovarian function in 35–40-year-old women with polycystic ovary syndrome Amanda Ladro´n de Guevara a, Nicola´s Crisosto a, Ba´rbara Echiburu´ a, Jessica Preisler a, Natalie Vantman a, Josefina Bollmann a, Francisco Pe´rez-Bravo b, Teresa Sir-Petermann a,* a b
Endocrinology and Metabolism Laboratory, West Division, School of Medicine, University of Chile, Santiago, Chile Laboratory of Nutritional Genomics, Department of Nutrition, Faculty of Medicine, University of Chile, Santiago, Chile
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 October 2012 Received in revised form 16 April 2013 Accepted 7 June 2013
Objective: To assess gonadotrophin secretion, ovarian steroid production and ovarian reserve in PCOS women during the onset of reproductive decline, in order to characterize their ovarian function at this age. Study design: Forty PCOS patients and 35 healthy women (35–40 years of age) were included. Clinical history, anthropometry, transvaginal ultrasound and a leuprolide acetate test (10 mg/kg s.c.) were performed. Gonadotrophins, steroid hormones, SHBG, inhibin B and AMH were determined. Results: Basal and peak LH levels were similar in both groups. Basal and peak FSH levels were significantly higher in the control group. Androgens, peak oestradiol, ovarian volume, antral follicle count and AMH levels were significantly higher in PCOS patients. Conclusion: These observations suggest that during late reproductive age, gonadotrophin secretion in women with PCOS is clearly different from that observed in control women and may also differ from that of younger PCOS patients. New features like normal LH and lower FSH levels associated with a higher ovarian reserve may give a different reproductive profile to these women. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Polycystic ovary syndrome Late reproductive age Gonadal axis Ovarian reserve
1. Introduction Polycystic ovary syndrome (PCOS) is a highly prevalent (6–10%) endocrine-metabolic dysfunction in premenopausal women [1–4]. Many studies have suggested that some features of PCOS may improve with aging [5–7]. Therefore, one of the main difficulties in studying older PCOS women is that they tend to lose some of the features that are usually used to identify PCOS. Moreover, it has been suggested that PCOS patients may exhibit longer reproductive lifespans. This hypothesis is based on the fact that ovarian volume, number of antral follicles and other ovarian reserve markers like AMH are significantly higher in PCOS patients during reproductive age [8–10]. It is possible that during the 35– 40-year-old period, a crucial period in terms of fertility and opportunity for assisted reproduction techniques given their increased follicular mass, PCOS patients may show different reproductive features compared to control women of the same age.
* Corresponding author at: Laboratory of Endocrinology, Department of Medicine, West Division, School of Medicine, Las Palmeras 299, Interior Quinta Normal, Casilla 33052, Correo 33, Zip Code 8320000, Santiago, Chile. Tel.: +56 2 681 46 76; fax: +56 2 681 66 93. E-mail addresses: [email protected], [email protected] (T. Sir-Petermann). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.06.013
Given the limited knowledge on the reproductive characteristics of women with PCOS between 35 and 40 years of age, the aim of the present study was to assess the ovarian function, including the reproductive axis, of a group of PCOS patients during this fiveyear period, and compare them with a group of healthy women. This is the first report of a follow-up study in PCOS patients that will continue through late reproductive age until the menopausal transition. 2. Materials and methods 2.1. Participants The present study is part of a larger ongoing project in which endocrine and metabolic variables are studied in PCOS women born in Santiago de Chile, from the same demographic area and socio-economic level. PCOS women were recruited from patients attending the Unit of Endocrinology and Reproductive Medicine, University of Chile. Although many women were interviewed, we included only those who agreed to participate in this study and fulfilled the inclusion criteria. Forty women with PCOS and 35 controls between 35 and 40 years of age, matched for age, weight and BMI range (20–35 kg/m2),
166
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
participated in this study. PCOS diagnosis was established in our unit according to the NIH consensus criteria [11] when the patients were in the early reproductive age. These patients participated in previous studies and the inclusion criteria are described elsewhere [12,13]. We included only patients who still fulfilled the NIH criteria for PCOS and who had not been under treatment for at least 3 months prior to the evaluation. Control women (Cw) were selected from women attending preventive medical examination at the Department of Obstetrics and Gynecology of our hospital. Women who had similar socioeconomic level, history of regular 28- to 32-day menstrual cycles, absence of hirsutism and other manifestations of hyperandrogenism, and no history of infertility or pregnancy complications were included. The protocol was approved by the Institutional Review Boards of the San Juan de Dios Hospital and the University of Chile. All women signed informed consents before entering the study. 2.2. Study protocol Control and PCOS subjects were studied 3–7 days after menstrual bleeding. In the absence of menstrual bleeding at the time of admittance to the study, the presence of a dominant follicle, recent ovulation or luteal phase was excluded by ultrasound examination and serum progesterone (P) evaluation. Subjects found to be in the preovulatory or luteal phase of a menstrual cycle were not included. On day 3 in both PCOS and control women, we performed a complete physical examination with anthropometric measurements including: weight, height, waist circumference and body mass index (BMI). Additionally a transvaginal ultrasound was performed, with a 3.8–7.5 MHz transvaginal probe, Aloka SSD4000 equipment (Tokyo, Japan). Ovarian volume was calculated using the simplified formula for a prolate ellipsoid. The larger ovary was used to evaluate ovarian size. The total number of 2- to 9-mm follicles from both ovaries was registered. Subsequently on day 4, in all participants a GnRH analogue test with 10 mg/kg leuprolide acetate (Lupron; Abbott Laboratories, North Chicago, IL) was performed as previously described [14,15]. Serum LH and FSH levels were measured before and 3 h after leuprolide injection. Serum testosterone, androstenedione, 17hydroxyprogesterone (17-OHP) and oestradiol concentrations were determined at baseline and 24 h after leuprolide administration (day 5). SHBG, progesterone, AMH and inhibin B were measured in the fasting sample before leuprolide administration (day 4). Basal serum SHBG and testosterone were used to calculate the FAI as the ratio of serum testosterone: SHBG 100. Maximal values after leuprolide testing were defined as the peak value for gonadotrophins at 3 h and for steroids at 24 h. Between days 6 and 7, we performed an oral glucose tolerance test (75 g glucose in 250 ml water) after a 12-h overnight fast. Blood samples (5 ml) were drawn for the measurement of glucose and insulin concentrations before and 30, 60, 90 and 120 min after the glucose load. These measurements were not included in the present study. 2.3. Assays Serum LH, FSH and oestradiol were determined by electrochemiluminescence assays (Roche). Sensitivities for LH, FSH and oestradiol assays were 0.1 U/l, 0.1 U/l and 5.0 pg/ml, respectively. Intra- and inter-assay coefficients of variation were 1.8 and 5.2% for LH, 1.8 and 5.3% for FSH, and 5.7 and 6.2% for oestradiol, respectively. Serum progesterone was assayed by radioimmunoassay (RIA) (Diagnostic Product Corp., Los Angeles, CA). Sensitivity for progesterone was 0.02 ng/ml, and intra- and inter-assay
coefficients of variation were 4.9 and 7.1%, respectively. Serum androstenedione (Diagnostic Systems Laboratories) and 17-OHP (Diagnostic Products Corp., Los Angeles, CA) were assayed by RIA. SHBG was determined by radioimmunometric assay (Diagnostic Products Corp.). Sensitivities for androstenedione, 17-OHP and SHBG assays were 0.1 ng/ml, 0.1 ng/ml and 0.04 nmol/l, respectively, and intra- and inter-assay coefficients of variation were 4.3 and 6.0%, 5.0 and 5.0%, and 3.8 and 7.9%, respectively. Serum testosterone was assayed by RIA (Diagnostic Systems Laboratories) in our laboratory. The limit of detection was 10 ng/dl. Intraand inter-assay coefficients of variation were 7.0 and 11.0%, respectively. Serum AMH was assayed by enzyme immunoassay (Immunotech-Beckman Coulter, Marseille, France) [16]. Analytical sensitivity was 2.1 pmol/l, and intra- and inter-assay coefficients of variation were 5.3 and 8.7%, respectively. Serum inhibin B was assayed by ELISA (Diagnostic Systems Laboratories) with a sensitivity of 7.0 pg/ml and intra- and interassay coefficients of variation of 6.3 and 4.6%, respectively. 2.4. Statistical evaluation Data are expressed as median and range. Data were not normally distributed according to the Shapiro–Wilk test, and non parametric tests were therefore used for data analysis. For comparisons between two groups, a Mann–Whitney test was employed. A chi-square test was used for the comparisons between percentages. Statistical analysis was performed using the STATA 10.0 package. A p-value of less than 0.05 was considered statistically significant. 3. Results Table 1 shows the clinical characteristics of control women (Cw) and PCOS women (PCOSw). By design, age and BMI range were not different between groups. Waist circumference was also comparable. Regular menses and parity were significantly higher in the control group compared to the PCOS group. Clinical hirsutism, evaluated by the Ferriman–Gallwey score, was more prevalent in PCOSw than in Cw. Table 2 shows the hormone levels before and after leuprolide administration. Basal and peak androgen levels, as well as poststimulation oestradiol levels, were significantly higher in PCOSw. SHBG levels were significantly lower in PCOSw compared to Cw. Basal progesterone concentrations were similar in both groups. Regarding ovarian reserve markers, ovarian average volume and total antral follicular count were significantly higher in PCOS patients (p < 0.05). Inhibin B concentrations were similar between
Table 1 Clinical characteristics of control women and PCOS women during the 35–40 year old period.
Age (years) Weight (kg) Height (m) BMI (kg/m2) Waist circumference (cm) Regular menses (%) Parity (No.) Hirsutism (Ferriman–Gallwey score)
Control women (n = 35)
PCOS women (n = 40)
37.0 (35.0–40.0) 63.5 (50.5–88.5) 1.57 (1.45–1.69) 26.0 (20.1–34.8) 81.0 (66.0–100.0) 100 2 (0–4) 2.0 (0–7)
36.0 (35.0–40.0) 65.5 (48.0–94.0) 1.57 (1.46–1.70) 27.1 (20.5–35.8) 82.5 (68.0–111.0) 22.5* 1 (0–4)* 12 (8–29)*
BMI: body mass index. Results are expressed as median and range. * p < 0.05.
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170 Table 2 Basal and maximal steroid responses to leuprolide acetate, and basal SHBG and progesterone levels in control women and PCOS women during the 35–40 year old period. Control women (n = 35) 17 OH progesterone (ng/ml) Basal 0.9 Peak 1.5 Androstenedione (ng/ml) Basal 1.7 Peak 1.9 Testosterone (ng/ml) Basal 0.5 Peak 0.6 Oestradiol (pg/ml) Basal 40.5 Peak 127.9 SHBG (nmol/l) 68.7 Progesterone (ng/ml) 0.7
PCOS women (n = 40)
(0.2–2.4) (0.6–4.2)
1.2 (0.4–2.8)* 2.7 (1.1–8.5)*
(0.1–3.1) (0.1–4.3)
*
2.6 (1.1–4.9) 2.9 (1.2–8.8)*
(0.1–0.8) (0.1–2.4)
0.7 (0.1–1.8)* 1.0 (0.3–2.1)*
(17.0–123.0) (30.2–520.8) (17.5–123.4) (0.3–2.3)
39.0 199.0 51.0 0.7
(16.2–194.6) (40.3–623.1)* (22.6–128.6)* (0.3–3.0)
SHBG: sex hormone binding globulin. Results are expressed as median and range. Maximal values after leuprolide were defined as the peak value for gonadotropins at 3 h, and for steroids at 24 h after stimulation. * p < 0.05.
groups (Table 3). AMH concentrations were significantly higher in the PCOS group (Fig. 1). The characteristic PCO morphology on ultrasound, according to the Rotterdam criteria, was present in 55.0% of the PCOS women studied [17]. Gonadotrophin secretion and AMH levels are illustrated in Fig. 1. Basal and post-stimulation LH concentrations were similar in the two groups, but basal and post-stimulation FSH levels were significantly lower in the PCOS group. AMH serum levels were significantly higher in PCOSw compared to Cw. 4. Comment In the present study, we evaluated a group of women with PCOS and a group of control women between 35 and 40 years old, a critical reproductive period. Contrary to what has been described in younger PCOS patients, LH levels were similar while basal and peak FSH levels were significantly lower in the PCOS group compared to the control group. Androgen and AMH levels, ovarian volume and follicular count were significantly higher in the PCOS group, while inhibin B levels were similar in both groups. It is known that PCOS patients have an abnormal regulation of steroidogenesis, mainly of androgen secretion by the ovary, an increased follicular mass and higher insulin levels. During reproductive life, this gonadal dysfunction is associated with a classical profile in terms of gonadotrophin secretion, with high LH levels and FSH levels that are usually in the low normal range. In the present study, the GnRH analogue test was employed to evaluate possible changes that may occur in the pituitary gonadal axis during a very specific period in reproductive life, where women still have chances of pregnancy but with much lower fertility rates compared to younger women [18]. The GnRH analogue test is considered an important tool to demonstrate an improvement in the gonadotrophin secretion dysfunction in PCOS women at this age. It has been hypothesized that increased LH levels determine higher AMH levels in PCOS patients during reproductive age, while an inverse correlation between serum AMH and FSH levels has been observed in conditions of abnormal or exhausted follicular development such as the menopause. Thus, high AMH levels associated with increased LH levels represent a pathological condition with decreased fertility, which is what has been reported in PCOS during the 18–35-year-old period. On the other hand, high FSH levels associated with low AMH levels represent decreased
167
Table 3 Ovarian reserve of control women and PCOS women according to different parameters during the 35–40 year old period.
Ovarian volume (cc) Number of follicles Inhibin B (pg/ml)
Control women (n = 35)
PCOS women (n = 40)
5.6 (2.0–10.0) 7 (0–14) 83.1 (2.6–216.4)
10.0 (3.1–25.8)* 14 (2–25)* 92.9 (2.6–676.0)
Results are expressed as median and range. * p < 0.05.
fertility rates during late reproductive life, menopause or infertility conditions [19]. In the present study, we no longer see high LH levels, FSH levels are not yet increased and we still see high AMH levels that are not as high as what has been reported during the main reproductive period [20]. In this regard, recently Carmina et al. showed that lower AMH serum levels predict an improved ovulatory function in PCOS women during the same age period (35–40 years) [21]. Taking all these observations together, we may suggest that this age period may be favorable for reproduction. Thus, we can say that, during the 35–40-year-old period, PCOS patients show a different hormonal profile characterized by higher AMH levels, lower FSH levels and similar LH levels compared to control women. Whether this new profile represents a favorable or an unfavorable reproductive condition in PCOS patients remains to be determined. As previously mentioned, PCOS is characterized by an increased number of pre-antral and small antral follicles due to increased follicular recruitment, which is associated with a disturbed selection process. One of the mechanisms that have been implicated in this phenomenon is enhanced steroidogenesis with higher local and circulating androgens. Hyperinsulinaemia is also implicated in the ovarian steroidogenic dysfunction, resulting in higher production of intraovarian androgens which in turn may aggravate the follicular derangements observed in this syndrome [22,23]. On the other hand, elevated serum LH levels promote ovarian theca interna cell steroidogenesis and offer an additional contribution to follicular arrest. The lack of increased LH levels in 35–40-year-old PCOS patients may release their follicles from one of the factors that perpetuate follicular arrest. In fact, serum testosterone levels decrease in middle-aged women with PCOS [24] and it has been described that 35–49-year-old PCOS patients show regular menses associated with older age and a decreased follicle count [25]. So what we have found here is a transition period in PCOS where androgens are still high, the number of follicles is still increased but the gonadotrophin secretion pattern has changed. Currently in clinical practice, the most employed ovarian reserve markers are basal FSH levels, antral follicle count and AMH levels. Basal FSH levels have been traditionally used to determine ovarian reserve. In women older than 30 years, it has been established that basal FSH levels with a cut-off of 10 UI/ml have high sensitivity, specificity, and positive and negative predictive values for a good response in terms of assisted reproductive cycles [26]. Nevertheless, longitudinal studies have shown that a markedly elevated FSH is a late predictor of menopausal transition, since increased values only occur about 10 years before the menopause [27,28]. Thus, FSH levels cannot be used as an early predictor of reduced fertility [29,30]. It is widely known that FSH concentrations in PCOS are generally in the lower normal range, suggesting an endogenous inhibition of FSH secretion [10,31]. The source of this inhibition is probably ovarian, as loss of ovarian tissue through wedge resection or laparoscopic ovarian diathermy is capable of restoring normal follicular development and ovulation. Moreover, after laparoscopic
168
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
Fig. 1. Basal and peak LH levels (A), basal and peak FSH levels (B) and basal AMH levels (C) in control women and PCOS women during the 35–40 year old period. The box and whisker plots show median and interquartile ranges. *p < 0.05 between PCOS and control groups. The vertical lines protruding from the box extend to the minimum value and the maximum value. The white circles represent outlier values.
ovarian drilling, there is a rapid steep rise in FSH in those who ovulate [32]. Another possibility is a neuroendocrine derangement in the PCOS condition characterized by increased LH pulse amplitude and frequency, and normal to slightly decreased FSH secretion compared to normal ovulatory women. Although abnormal ovarian steroidogenesis probably contributes to the characteristic pattern of gonadotrophin secretion observed in this
syndrome, altered gonadotrophin secretion explains at least in part the characteristic ovarian defects [33]. In the present study, the FSH response in the GnRH analogue test was more pronounced in the control group compared to the PCOS group. Thus, control patients have an increased FSH response compared to PCOS patients, suggesting a higher sensitivity to GnRH pulses, probably related to the endocrine changes associated with ovarian aging [26]. Thus, the magnitude of the response to the GnRH analogue may be an important marker for the assessment of the gonadal axis status. In this regard, Koning et al. showed that the FSH and LH responses to GnRH were higher in patients with imminent ovarian failure compared to controls. In that study the authors concluded that, in women with this condition, the pituitary is more sensitive to GnRH, leading to higher LH and FSH pulse amplitudes, most probably due to lack of a local ovarian factor [34]. Thus, a lower pituitary FSH secretion may mean that there is an increased ovarian reserve or it may represent an abnormal pituitary response in the PCOS group. Regarding ovarian reserve markers, a decrease in inhibin B has been described as a marker of ovarian aging. Inhibin B, however, correlates with age only during a relatively short period before the menopausal transition, and its levels decline to very low or undetectable levels about 4 years prior to the last menstrual period [35]. Therefore it is not an early and sensitive marker. Previous studies have shown conflicting results regarding an elevation of this hormone in PCOS [36,37]. In the present study, we found no significant differences in the levels of inhibin B between the PCOS and control groups. Inhibin A, on the other hand, is secreted by the mature follicle and the corpus luteum, and thus is not directly related to the ovarian reserve [38,39]. Neither of these markers, therefore, is a good candidate to explain the lower FSH levels observed in the PCOS group in the present study. Serum anti-Mullerian hormone (AMH) levels seem to be correlated with the development of preantral and small antral follicles from puberty to the end of reproductive life. It has been shown that AMH levels decrease over time in young normoovulatory women, whereas other markers associated with ovarian aging such as FSH and inhibin B do not change during the same interval [40]. In addition, AMH levels are correlated with age. AMH has been proposed as a very reliable marker of ovarian reserve, having many advantages compared to FSH levels due to its reduced variability during the menstrual cycle, and the facts that it decreases 5 years before the ending of the menstrual period [28,41] and that it reflects the number of growing follicles, independent of gonadotrophins, and therefore the follicular reserve at any point in a woman’s life [42]. In women with PCOS during reproductive life, it has been established that AMH levels are two- to three-fold higher compared with healthy women [43,44], which may be related to the increased number of growing follicles that secrete AMH [8]. In the present study, AMH levels as well as total antral follicle counts were significantly higher in PCOS women than in control women, showing the presence of more small antral follicles and possibly an increased ovarian reserve. In addition, we found significantly higher androgen and peak oestradiol levels compared to the control group. This shows that the steroidogenic capacity of PCOS patients is still higher compared to controls during this age period. Previously we have shown in younger PCOS patients (24.5 5.0 years) that LH, LH/FSH ratio, androgens and AMH were significantly higher than in control women of the same age [45]. Moreover, their LH and AMH levels were clearly higher than the values reported in the present study (LH: 9.3 5.3 UI/l vs. 6.3 4.07 UI/l; p = 0.01 and AMH: 76 36.3 pM vs. 39.7 32.3; p = 0.0003, values are
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
mean SD). Interestingly, FSH levels in those young PCOS patients were very similar to the values observed in the present 35–40-yearold PCOS group (FSH: 5.3 1.7 vs. 5.9 2.3; p = 0.3, values are mean SD). Both groups of PCOS patients were evaluated at the Unit of Endocrinology and Reproductive Medicine of the University of Chile and the samples were measured using the same assays [45]. In summary, our data show that, during the 35–40-year-old period, women with the classical form of PCOS identified during early reproductive age have a reproductive profile that is clearly different from that observed in healthy women. Therefore, the classification of female reproductive aging based on changes in menstrual cycle pattern and FSH levels, as proposed by the Stage of Reproductive Aging Workshop (STRAW) [18], is difficult to apply in PCOS women. New features like normal LH and lower FSH levels associated with a higher ovarian reserve may give a different reproductive profile to these women. Long-term follow-up studies are needed in order to assess the remaining reproductive capacity during this age window in these patients, and appropriately characterize the end of the reproductive stage in women with PCOS. Acknowledgments This work was supported by grants from the National Fund for Scientific and Technological Research (Fondecyt) 1110864, the Chilean Endocrine and Diabetes Society (SOCHED 2009-05) and by the Alexander von Humboldt Foundation. We would like to thank Dr. Gabriel Cavada, Ph.D., Department of Public Health, School of Medicine, University of Chile, for his help with the statistical analysis of this study. This work was presented in part at the 7th Annual Meeting of the Androgen Excess and PCOS Society, Munich, Germany, September 2010; and at the Endocrine Society’s 93th Annual Meeting, Boston, USA, June 2011. References [1] Diamanti-Kandarakis E, Kouli CR, Bergiele AT, et al. A survey of the polycystic ovary syndrome in the Greek island of Lesbos: hormonal and metabolic profile. J Clin Endocrinol Metab 1999;84:4006–11. [2] Asuncio´n M, Calvo RM, San Milla´n JL, Sancho J, Avila S, Escobar-Morreale HF. A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. J Clin Endocrinol Metab 2000;85:2434–8. [3] Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745–9. [4] Azziz R, Carmina E, Dewailly D, et al. Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab 2006;91:4237– 45. [5] Elting MW, Korsen TJ, Rekers-Mombarg LT, Schoemaker J. Women with polycystic ovary syndrome gain regular menstrual cycles when ageing. Hum Reprod 2000;15:24–8. [6] Brown ZA, Louwers YV, Fong SL, et al. The phenotype of polycystic ovary syndrome ameliorates with aging. Fertil Steril 2011;96:1259–65. [7] Carmina E, Campagna AM, Lobo RA. A 20-year follow-up of young women with polycystic ovary syndrome. Obstet Gynecol 2012;119:263–9. [8] Piltonen T, Morin-Papunen L, Koivunen R, Perheentupa A, Ruokonen A, Tapanainen J. Serum anti-Mullerian hormone levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. Hum Reprod 2005;20:1820–6. [9] Mulders AG, Laven JS, Eijkemans MJ, de Jong FH, Themmen AP, Fauser BC. Changes in anti-Mullerian hormone serum concentrations over time suggest delayed ovarian ageing in normogonadotrophic anovulatory infertility. Hum Reprod 2004;19:2036–42. [10] Hudecova M, Holte J, Olovsson M, Sundstrom Poromaa I. Long-term follow-up of patients with polycystic ovary syndrome: reproductive outcome and ovarian reserve. Hum Reprod 2009;24:1176–83. [11] Zawadzky JK, Dunaif A. Diagnosis criteria: towards a rational approach. In: Hershmann JM, editor. Current issues in endocrinology and metabolism. Boston: Blackwell Scientific Publications; 1992. p. 377–84. [12] Sir-Petermann T, Codner E, Pe´rez V, et al. Metabolic and reproductive features before and during puberty in daughters of women with polycystic ovary syndrome. J Clin Endocrinol Metab 2009;94:1923–30.
169
[13] Crisosto N, Echiburu´ B, Maliqueo M, et al. Improvement of hyperandrogenism and hyperinsulinemia during pregnancy in women with polycystic ovary syndrome: possible effect in the ovarian follicular mass of their daughters. Fertil Steril 2012;97:218–24. ˜ ez L, Potau N, Zampolli M, Street ME, Carrascosa A. Girls diagnosed with [14] Iba´n premature pubarche show an exaggerated ovarian androgen synthesis from the early stages of puberty: evidence from gonadotropin-releasing hormone agonist testing. Fertil Steril 1997;67:849–55. [15] Sir-Petermann T, Hitchsfeld C, Codner E, et al. Gonadal function in low birth weight infants: a pilot study. J Pediatr Endocrinol Metab 2007;20:405–14. ˜ ı´guez G, et al. Low risk of impaired testicular Sertoli and [16] Rey RA, Codner E, In Leydig cell functions in boys with isolated hypospadias. J Clin Endocrinol Metab 2005;90:6035–40. [17] Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19–25. [18] Soules MR, Sherman S, Parrott E, et al. Stages of reproductive aging workshop (STRAW). J Womens Health Gender-Based Med 2001;10:843–8. [19] Panidis D, Katsikis I, Karkanaki A, Piouka A, Armeni A, Georgopoulos A. Serum anti-Mu¨llerian hormone (AMH) levels are differentially modulated by both serum gonadotropins and not only by serum Follicle Stimulating Hormone (FSH) levels. Med Hypotheses 2011;77:649–53. [20] Skalva P, Cygal A, Madej P, et al. Is the plasma anti-Mu¨llerian hormone (AMH) level associated with body weight and metabolic, and hormonal disturbance in women with and without polycystic ovary syndrome? Eur J Obstet Gynecol Reprod Biol 2011;158:254–9. [21] Carmina E, Campagna AM, Mansuet P, Vitale G, Kort D, Lobo R. Does the level of serum antiMu¨llerian hormone predict ovulatory function in women with polycystic ovary syndrome with aging? Fertil Steril 2012;98:1043–6. [22] Homburg R. Androgen circle of polycystic ovary syndrome. Hum Reprod 2009;24:1548–55. [23] Crisosto N, Sir-Petermann T, Greiner M, et al. Testosterone-induced downregulation of anti-Mu¨llerian hormone expression in granulosa cells from small bovine follicles. Endocrine 2009;36:339–45. [24] Winters SJ, Talbott E, Guzick DS, Zborowski J, McHugh KP. Serum testosterone levels decrease in middle age in women with the polycystic ovary syndrome. Fertil Steril 2000;73:724–9. [25] Elting MW, Kwee J, Korsen TJ, Rekers-Mombarg LT, Schoemaker J. Aging women with polycystic ovary syndrome who achieve regular menstrual cycles have a smaller follicle cohort than those who continue to have irregular cycles. Fertil Steril 2003;79:1154–60. [26] de Carvalho BR, Rosa e Silva AC, Rosa e Silva JC, dos Reis RM, Ferriani RA, Silva de Sa´ MF. Ovarian reserve evaluation: state of the art. J Assist Reprod Genet 2008;25:311–22. [27] van Rooij IA, Tonkelaar Id, Broekmans FJ, et al. Anti-Mullerian hormone is a promising predictor for the occurrence of the menopausal transition. Menopause 2004;11:601–6. [28] van Rooij IA, Broekmans FJ, Scheffer GJ, et al. Serum antiMullerian hormone levels best reflect the reproductive decline with age in normal women with proven fertility: a longitudinal study. Fertil Steril 2005;83:979–87. [29] van Montfrans JM, Hoek A, van Hooff MH, de Koning CH, Tonch N, Lambalk CB. Predictive value of basal follicle-stimulating hormone concentrations in a general subfertility population. Fertil Steril 2000;74:97–103. [30] Sowers MR, Eyvazzadeh AD, McConnell D, et al. Anti-Mullerian hormone and inhibin B in the definition of ovarian aging and the menopause transition. J Clin Endocrinol Metab 2008;93:3478–83. [31] Nardo LG, Yates AP, Roberts SA, Pemberton P, Laing I. The relationships between AMH, androgens, insulin resistance and basal ovarian follicular status in non-obese subfertile women with and without polycystic ovary syndrome. Hum Reprod 2009;24:2917–23. [32] Pasquali R, Stener-Victorin E, Yildiz B, et al. PCOS Forum: research in polycystic ovary syndrome today and tomorrow. Clin Endocrinol (Oxf) 2011;74:424–33. [33] Kazer RR, Kessel B, Yen SS. Circulating luteinizing hormone pulse frequency in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1987;65:233–6. [34] de Koning CH, Popp-Snijders C, Schoemaker J, Lambalk CB. Elevated FSH concentrations in imminent ovarian failure are associated with higher FSH and LH pulse amplitude and response to GnRH. Hum Reprod 2000;15:1452–6. [35] Lambalk CB, van Disseldorp J, de Koning CH, Broekmans FJ. Testing ovarian reserve to predict age at menopause. Maturitas 2009;63:280–91. [36] Welt CK, Taylor AE, Martin KA, Hall JE. Serum inhibin B in polycystic ovary syndrome: regulation by insulin and luteinizing hormone. J Clin Endocrinol Metab 2002;87:5559–65. [37] Laven JS, Imani B, Eijkemans MJ, de Jong FH, Fauser BC. Absent biologically relevant associations between serum inhibin B concentrations and characteristics of polycystic ovary syndrome in normogonadotrophic anovulatory infertility. Hum Reprod 2001;16:1359–64. [38] Roberts VJ, Barth S, el-Roeiy A, Yen SS. Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle. J Clin Endocrinol Metab 1993;77:1402–10. [39] Groome NP, Illingworth PJ, O’Brien M, et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996;81:1401–5.
170
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
[40] de Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC. AntiMu¨llerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002;77:357–62. [41] Sowers M, Eyvazzadeh A, McConnell D, et al. Anti-Mullerian hormone and inhibin B in the definition of ovarian aging and the menopause transition. J Clin Endocrinol Metab 2008;93:3478–83. [42] Hehenkamp WJ, Looman CW, Themmen AP, de Jong FH, Te Velde ER, Broekmans FJ. Anti-Mu¨llerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. J Clin Endocrinol Metab 2006;91: 4057–63.
[43] Pigny P, Merlen E, Robert Y, et al. Elevated serum level of anti-Mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 2003;88:5957–62. [44] Laven JS, Mulders AG, Visser JA, Themmen AP, De Jong FH, Fauser BC. AntiMu¨llerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 2004;89:318–23. ˜ ı´guez G, Villarroel C, et al. Hormonal profile in women with [45] Codner E, In polycystic ovarian syndrome with or without type 1 diabetes mellitus. J Clin Endocrinol Metab 2007;92:4742–6.
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Evaluation of ovarian function in 35–40-year-old women with polycystic ovary syndrome Amanda Ladro´n de Guevara a, Nicola´s Crisosto a, Ba´rbara Echiburu´ a, Jessica Preisler a, Natalie Vantman a, Josefina Bollmann a, Francisco Pe´rez-Bravo b, Teresa Sir-Petermann a,* a b
Endocrinology and Metabolism Laboratory, West Division, School of Medicine, University of Chile, Santiago, Chile Laboratory of Nutritional Genomics, Department of Nutrition, Faculty of Medicine, University of Chile, Santiago, Chile
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 October 2012 Received in revised form 16 April 2013 Accepted 7 June 2013
Objective: To assess gonadotrophin secretion, ovarian steroid production and ovarian reserve in PCOS women during the onset of reproductive decline, in order to characterize their ovarian function at this age. Study design: Forty PCOS patients and 35 healthy women (35–40 years of age) were included. Clinical history, anthropometry, transvaginal ultrasound and a leuprolide acetate test (10 mg/kg s.c.) were performed. Gonadotrophins, steroid hormones, SHBG, inhibin B and AMH were determined. Results: Basal and peak LH levels were similar in both groups. Basal and peak FSH levels were significantly higher in the control group. Androgens, peak oestradiol, ovarian volume, antral follicle count and AMH levels were significantly higher in PCOS patients. Conclusion: These observations suggest that during late reproductive age, gonadotrophin secretion in women with PCOS is clearly different from that observed in control women and may also differ from that of younger PCOS patients. New features like normal LH and lower FSH levels associated with a higher ovarian reserve may give a different reproductive profile to these women. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Polycystic ovary syndrome Late reproductive age Gonadal axis Ovarian reserve
1. Introduction Polycystic ovary syndrome (PCOS) is a highly prevalent (6–10%) endocrine-metabolic dysfunction in premenopausal women [1–4]. Many studies have suggested that some features of PCOS may improve with aging [5–7]. Therefore, one of the main difficulties in studying older PCOS women is that they tend to lose some of the features that are usually used to identify PCOS. Moreover, it has been suggested that PCOS patients may exhibit longer reproductive lifespans. This hypothesis is based on the fact that ovarian volume, number of antral follicles and other ovarian reserve markers like AMH are significantly higher in PCOS patients during reproductive age [8–10]. It is possible that during the 35– 40-year-old period, a crucial period in terms of fertility and opportunity for assisted reproduction techniques given their increased follicular mass, PCOS patients may show different reproductive features compared to control women of the same age.
* Corresponding author at: Laboratory of Endocrinology, Department of Medicine, West Division, School of Medicine, Las Palmeras 299, Interior Quinta Normal, Casilla 33052, Correo 33, Zip Code 8320000, Santiago, Chile. Tel.: +56 2 681 46 76; fax: +56 2 681 66 93. E-mail addresses: [email protected], [email protected] (T. Sir-Petermann). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.06.013
Given the limited knowledge on the reproductive characteristics of women with PCOS between 35 and 40 years of age, the aim of the present study was to assess the ovarian function, including the reproductive axis, of a group of PCOS patients during this fiveyear period, and compare them with a group of healthy women. This is the first report of a follow-up study in PCOS patients that will continue through late reproductive age until the menopausal transition. 2. Materials and methods 2.1. Participants The present study is part of a larger ongoing project in which endocrine and metabolic variables are studied in PCOS women born in Santiago de Chile, from the same demographic area and socio-economic level. PCOS women were recruited from patients attending the Unit of Endocrinology and Reproductive Medicine, University of Chile. Although many women were interviewed, we included only those who agreed to participate in this study and fulfilled the inclusion criteria. Forty women with PCOS and 35 controls between 35 and 40 years of age, matched for age, weight and BMI range (20–35 kg/m2),
166
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
participated in this study. PCOS diagnosis was established in our unit according to the NIH consensus criteria [11] when the patients were in the early reproductive age. These patients participated in previous studies and the inclusion criteria are described elsewhere [12,13]. We included only patients who still fulfilled the NIH criteria for PCOS and who had not been under treatment for at least 3 months prior to the evaluation. Control women (Cw) were selected from women attending preventive medical examination at the Department of Obstetrics and Gynecology of our hospital. Women who had similar socioeconomic level, history of regular 28- to 32-day menstrual cycles, absence of hirsutism and other manifestations of hyperandrogenism, and no history of infertility or pregnancy complications were included. The protocol was approved by the Institutional Review Boards of the San Juan de Dios Hospital and the University of Chile. All women signed informed consents before entering the study. 2.2. Study protocol Control and PCOS subjects were studied 3–7 days after menstrual bleeding. In the absence of menstrual bleeding at the time of admittance to the study, the presence of a dominant follicle, recent ovulation or luteal phase was excluded by ultrasound examination and serum progesterone (P) evaluation. Subjects found to be in the preovulatory or luteal phase of a menstrual cycle were not included. On day 3 in both PCOS and control women, we performed a complete physical examination with anthropometric measurements including: weight, height, waist circumference and body mass index (BMI). Additionally a transvaginal ultrasound was performed, with a 3.8–7.5 MHz transvaginal probe, Aloka SSD4000 equipment (Tokyo, Japan). Ovarian volume was calculated using the simplified formula for a prolate ellipsoid. The larger ovary was used to evaluate ovarian size. The total number of 2- to 9-mm follicles from both ovaries was registered. Subsequently on day 4, in all participants a GnRH analogue test with 10 mg/kg leuprolide acetate (Lupron; Abbott Laboratories, North Chicago, IL) was performed as previously described [14,15]. Serum LH and FSH levels were measured before and 3 h after leuprolide injection. Serum testosterone, androstenedione, 17hydroxyprogesterone (17-OHP) and oestradiol concentrations were determined at baseline and 24 h after leuprolide administration (day 5). SHBG, progesterone, AMH and inhibin B were measured in the fasting sample before leuprolide administration (day 4). Basal serum SHBG and testosterone were used to calculate the FAI as the ratio of serum testosterone: SHBG 100. Maximal values after leuprolide testing were defined as the peak value for gonadotrophins at 3 h and for steroids at 24 h. Between days 6 and 7, we performed an oral glucose tolerance test (75 g glucose in 250 ml water) after a 12-h overnight fast. Blood samples (5 ml) were drawn for the measurement of glucose and insulin concentrations before and 30, 60, 90 and 120 min after the glucose load. These measurements were not included in the present study. 2.3. Assays Serum LH, FSH and oestradiol were determined by electrochemiluminescence assays (Roche). Sensitivities for LH, FSH and oestradiol assays were 0.1 U/l, 0.1 U/l and 5.0 pg/ml, respectively. Intra- and inter-assay coefficients of variation were 1.8 and 5.2% for LH, 1.8 and 5.3% for FSH, and 5.7 and 6.2% for oestradiol, respectively. Serum progesterone was assayed by radioimmunoassay (RIA) (Diagnostic Product Corp., Los Angeles, CA). Sensitivity for progesterone was 0.02 ng/ml, and intra- and inter-assay
coefficients of variation were 4.9 and 7.1%, respectively. Serum androstenedione (Diagnostic Systems Laboratories) and 17-OHP (Diagnostic Products Corp., Los Angeles, CA) were assayed by RIA. SHBG was determined by radioimmunometric assay (Diagnostic Products Corp.). Sensitivities for androstenedione, 17-OHP and SHBG assays were 0.1 ng/ml, 0.1 ng/ml and 0.04 nmol/l, respectively, and intra- and inter-assay coefficients of variation were 4.3 and 6.0%, 5.0 and 5.0%, and 3.8 and 7.9%, respectively. Serum testosterone was assayed by RIA (Diagnostic Systems Laboratories) in our laboratory. The limit of detection was 10 ng/dl. Intraand inter-assay coefficients of variation were 7.0 and 11.0%, respectively. Serum AMH was assayed by enzyme immunoassay (Immunotech-Beckman Coulter, Marseille, France) [16]. Analytical sensitivity was 2.1 pmol/l, and intra- and inter-assay coefficients of variation were 5.3 and 8.7%, respectively. Serum inhibin B was assayed by ELISA (Diagnostic Systems Laboratories) with a sensitivity of 7.0 pg/ml and intra- and interassay coefficients of variation of 6.3 and 4.6%, respectively. 2.4. Statistical evaluation Data are expressed as median and range. Data were not normally distributed according to the Shapiro–Wilk test, and non parametric tests were therefore used for data analysis. For comparisons between two groups, a Mann–Whitney test was employed. A chi-square test was used for the comparisons between percentages. Statistical analysis was performed using the STATA 10.0 package. A p-value of less than 0.05 was considered statistically significant. 3. Results Table 1 shows the clinical characteristics of control women (Cw) and PCOS women (PCOSw). By design, age and BMI range were not different between groups. Waist circumference was also comparable. Regular menses and parity were significantly higher in the control group compared to the PCOS group. Clinical hirsutism, evaluated by the Ferriman–Gallwey score, was more prevalent in PCOSw than in Cw. Table 2 shows the hormone levels before and after leuprolide administration. Basal and peak androgen levels, as well as poststimulation oestradiol levels, were significantly higher in PCOSw. SHBG levels were significantly lower in PCOSw compared to Cw. Basal progesterone concentrations were similar in both groups. Regarding ovarian reserve markers, ovarian average volume and total antral follicular count were significantly higher in PCOS patients (p < 0.05). Inhibin B concentrations were similar between
Table 1 Clinical characteristics of control women and PCOS women during the 35–40 year old period.
Age (years) Weight (kg) Height (m) BMI (kg/m2) Waist circumference (cm) Regular menses (%) Parity (No.) Hirsutism (Ferriman–Gallwey score)
Control women (n = 35)
PCOS women (n = 40)
37.0 (35.0–40.0) 63.5 (50.5–88.5) 1.57 (1.45–1.69) 26.0 (20.1–34.8) 81.0 (66.0–100.0) 100 2 (0–4) 2.0 (0–7)
36.0 (35.0–40.0) 65.5 (48.0–94.0) 1.57 (1.46–1.70) 27.1 (20.5–35.8) 82.5 (68.0–111.0) 22.5* 1 (0–4)* 12 (8–29)*
BMI: body mass index. Results are expressed as median and range. * p < 0.05.
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170 Table 2 Basal and maximal steroid responses to leuprolide acetate, and basal SHBG and progesterone levels in control women and PCOS women during the 35–40 year old period. Control women (n = 35) 17 OH progesterone (ng/ml) Basal 0.9 Peak 1.5 Androstenedione (ng/ml) Basal 1.7 Peak 1.9 Testosterone (ng/ml) Basal 0.5 Peak 0.6 Oestradiol (pg/ml) Basal 40.5 Peak 127.9 SHBG (nmol/l) 68.7 Progesterone (ng/ml) 0.7
PCOS women (n = 40)
(0.2–2.4) (0.6–4.2)
1.2 (0.4–2.8)* 2.7 (1.1–8.5)*
(0.1–3.1) (0.1–4.3)
*
2.6 (1.1–4.9) 2.9 (1.2–8.8)*
(0.1–0.8) (0.1–2.4)
0.7 (0.1–1.8)* 1.0 (0.3–2.1)*
(17.0–123.0) (30.2–520.8) (17.5–123.4) (0.3–2.3)
39.0 199.0 51.0 0.7
(16.2–194.6) (40.3–623.1)* (22.6–128.6)* (0.3–3.0)
SHBG: sex hormone binding globulin. Results are expressed as median and range. Maximal values after leuprolide were defined as the peak value for gonadotropins at 3 h, and for steroids at 24 h after stimulation. * p < 0.05.
groups (Table 3). AMH concentrations were significantly higher in the PCOS group (Fig. 1). The characteristic PCO morphology on ultrasound, according to the Rotterdam criteria, was present in 55.0% of the PCOS women studied [17]. Gonadotrophin secretion and AMH levels are illustrated in Fig. 1. Basal and post-stimulation LH concentrations were similar in the two groups, but basal and post-stimulation FSH levels were significantly lower in the PCOS group. AMH serum levels were significantly higher in PCOSw compared to Cw. 4. Comment In the present study, we evaluated a group of women with PCOS and a group of control women between 35 and 40 years old, a critical reproductive period. Contrary to what has been described in younger PCOS patients, LH levels were similar while basal and peak FSH levels were significantly lower in the PCOS group compared to the control group. Androgen and AMH levels, ovarian volume and follicular count were significantly higher in the PCOS group, while inhibin B levels were similar in both groups. It is known that PCOS patients have an abnormal regulation of steroidogenesis, mainly of androgen secretion by the ovary, an increased follicular mass and higher insulin levels. During reproductive life, this gonadal dysfunction is associated with a classical profile in terms of gonadotrophin secretion, with high LH levels and FSH levels that are usually in the low normal range. In the present study, the GnRH analogue test was employed to evaluate possible changes that may occur in the pituitary gonadal axis during a very specific period in reproductive life, where women still have chances of pregnancy but with much lower fertility rates compared to younger women [18]. The GnRH analogue test is considered an important tool to demonstrate an improvement in the gonadotrophin secretion dysfunction in PCOS women at this age. It has been hypothesized that increased LH levels determine higher AMH levels in PCOS patients during reproductive age, while an inverse correlation between serum AMH and FSH levels has been observed in conditions of abnormal or exhausted follicular development such as the menopause. Thus, high AMH levels associated with increased LH levels represent a pathological condition with decreased fertility, which is what has been reported in PCOS during the 18–35-year-old period. On the other hand, high FSH levels associated with low AMH levels represent decreased
167
Table 3 Ovarian reserve of control women and PCOS women according to different parameters during the 35–40 year old period.
Ovarian volume (cc) Number of follicles Inhibin B (pg/ml)
Control women (n = 35)
PCOS women (n = 40)
5.6 (2.0–10.0) 7 (0–14) 83.1 (2.6–216.4)
10.0 (3.1–25.8)* 14 (2–25)* 92.9 (2.6–676.0)
Results are expressed as median and range. * p < 0.05.
fertility rates during late reproductive life, menopause or infertility conditions [19]. In the present study, we no longer see high LH levels, FSH levels are not yet increased and we still see high AMH levels that are not as high as what has been reported during the main reproductive period [20]. In this regard, recently Carmina et al. showed that lower AMH serum levels predict an improved ovulatory function in PCOS women during the same age period (35–40 years) [21]. Taking all these observations together, we may suggest that this age period may be favorable for reproduction. Thus, we can say that, during the 35–40-year-old period, PCOS patients show a different hormonal profile characterized by higher AMH levels, lower FSH levels and similar LH levels compared to control women. Whether this new profile represents a favorable or an unfavorable reproductive condition in PCOS patients remains to be determined. As previously mentioned, PCOS is characterized by an increased number of pre-antral and small antral follicles due to increased follicular recruitment, which is associated with a disturbed selection process. One of the mechanisms that have been implicated in this phenomenon is enhanced steroidogenesis with higher local and circulating androgens. Hyperinsulinaemia is also implicated in the ovarian steroidogenic dysfunction, resulting in higher production of intraovarian androgens which in turn may aggravate the follicular derangements observed in this syndrome [22,23]. On the other hand, elevated serum LH levels promote ovarian theca interna cell steroidogenesis and offer an additional contribution to follicular arrest. The lack of increased LH levels in 35–40-year-old PCOS patients may release their follicles from one of the factors that perpetuate follicular arrest. In fact, serum testosterone levels decrease in middle-aged women with PCOS [24] and it has been described that 35–49-year-old PCOS patients show regular menses associated with older age and a decreased follicle count [25]. So what we have found here is a transition period in PCOS where androgens are still high, the number of follicles is still increased but the gonadotrophin secretion pattern has changed. Currently in clinical practice, the most employed ovarian reserve markers are basal FSH levels, antral follicle count and AMH levels. Basal FSH levels have been traditionally used to determine ovarian reserve. In women older than 30 years, it has been established that basal FSH levels with a cut-off of 10 UI/ml have high sensitivity, specificity, and positive and negative predictive values for a good response in terms of assisted reproductive cycles [26]. Nevertheless, longitudinal studies have shown that a markedly elevated FSH is a late predictor of menopausal transition, since increased values only occur about 10 years before the menopause [27,28]. Thus, FSH levels cannot be used as an early predictor of reduced fertility [29,30]. It is widely known that FSH concentrations in PCOS are generally in the lower normal range, suggesting an endogenous inhibition of FSH secretion [10,31]. The source of this inhibition is probably ovarian, as loss of ovarian tissue through wedge resection or laparoscopic ovarian diathermy is capable of restoring normal follicular development and ovulation. Moreover, after laparoscopic
168
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
Fig. 1. Basal and peak LH levels (A), basal and peak FSH levels (B) and basal AMH levels (C) in control women and PCOS women during the 35–40 year old period. The box and whisker plots show median and interquartile ranges. *p < 0.05 between PCOS and control groups. The vertical lines protruding from the box extend to the minimum value and the maximum value. The white circles represent outlier values.
ovarian drilling, there is a rapid steep rise in FSH in those who ovulate [32]. Another possibility is a neuroendocrine derangement in the PCOS condition characterized by increased LH pulse amplitude and frequency, and normal to slightly decreased FSH secretion compared to normal ovulatory women. Although abnormal ovarian steroidogenesis probably contributes to the characteristic pattern of gonadotrophin secretion observed in this
syndrome, altered gonadotrophin secretion explains at least in part the characteristic ovarian defects [33]. In the present study, the FSH response in the GnRH analogue test was more pronounced in the control group compared to the PCOS group. Thus, control patients have an increased FSH response compared to PCOS patients, suggesting a higher sensitivity to GnRH pulses, probably related to the endocrine changes associated with ovarian aging [26]. Thus, the magnitude of the response to the GnRH analogue may be an important marker for the assessment of the gonadal axis status. In this regard, Koning et al. showed that the FSH and LH responses to GnRH were higher in patients with imminent ovarian failure compared to controls. In that study the authors concluded that, in women with this condition, the pituitary is more sensitive to GnRH, leading to higher LH and FSH pulse amplitudes, most probably due to lack of a local ovarian factor [34]. Thus, a lower pituitary FSH secretion may mean that there is an increased ovarian reserve or it may represent an abnormal pituitary response in the PCOS group. Regarding ovarian reserve markers, a decrease in inhibin B has been described as a marker of ovarian aging. Inhibin B, however, correlates with age only during a relatively short period before the menopausal transition, and its levels decline to very low or undetectable levels about 4 years prior to the last menstrual period [35]. Therefore it is not an early and sensitive marker. Previous studies have shown conflicting results regarding an elevation of this hormone in PCOS [36,37]. In the present study, we found no significant differences in the levels of inhibin B between the PCOS and control groups. Inhibin A, on the other hand, is secreted by the mature follicle and the corpus luteum, and thus is not directly related to the ovarian reserve [38,39]. Neither of these markers, therefore, is a good candidate to explain the lower FSH levels observed in the PCOS group in the present study. Serum anti-Mullerian hormone (AMH) levels seem to be correlated with the development of preantral and small antral follicles from puberty to the end of reproductive life. It has been shown that AMH levels decrease over time in young normoovulatory women, whereas other markers associated with ovarian aging such as FSH and inhibin B do not change during the same interval [40]. In addition, AMH levels are correlated with age. AMH has been proposed as a very reliable marker of ovarian reserve, having many advantages compared to FSH levels due to its reduced variability during the menstrual cycle, and the facts that it decreases 5 years before the ending of the menstrual period [28,41] and that it reflects the number of growing follicles, independent of gonadotrophins, and therefore the follicular reserve at any point in a woman’s life [42]. In women with PCOS during reproductive life, it has been established that AMH levels are two- to three-fold higher compared with healthy women [43,44], which may be related to the increased number of growing follicles that secrete AMH [8]. In the present study, AMH levels as well as total antral follicle counts were significantly higher in PCOS women than in control women, showing the presence of more small antral follicles and possibly an increased ovarian reserve. In addition, we found significantly higher androgen and peak oestradiol levels compared to the control group. This shows that the steroidogenic capacity of PCOS patients is still higher compared to controls during this age period. Previously we have shown in younger PCOS patients (24.5 5.0 years) that LH, LH/FSH ratio, androgens and AMH were significantly higher than in control women of the same age [45]. Moreover, their LH and AMH levels were clearly higher than the values reported in the present study (LH: 9.3 5.3 UI/l vs. 6.3 4.07 UI/l; p = 0.01 and AMH: 76 36.3 pM vs. 39.7 32.3; p = 0.0003, values are
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
mean SD). Interestingly, FSH levels in those young PCOS patients were very similar to the values observed in the present 35–40-yearold PCOS group (FSH: 5.3 1.7 vs. 5.9 2.3; p = 0.3, values are mean SD). Both groups of PCOS patients were evaluated at the Unit of Endocrinology and Reproductive Medicine of the University of Chile and the samples were measured using the same assays [45]. In summary, our data show that, during the 35–40-year-old period, women with the classical form of PCOS identified during early reproductive age have a reproductive profile that is clearly different from that observed in healthy women. Therefore, the classification of female reproductive aging based on changes in menstrual cycle pattern and FSH levels, as proposed by the Stage of Reproductive Aging Workshop (STRAW) [18], is difficult to apply in PCOS women. New features like normal LH and lower FSH levels associated with a higher ovarian reserve may give a different reproductive profile to these women. Long-term follow-up studies are needed in order to assess the remaining reproductive capacity during this age window in these patients, and appropriately characterize the end of the reproductive stage in women with PCOS. Acknowledgments This work was supported by grants from the National Fund for Scientific and Technological Research (Fondecyt) 1110864, the Chilean Endocrine and Diabetes Society (SOCHED 2009-05) and by the Alexander von Humboldt Foundation. We would like to thank Dr. Gabriel Cavada, Ph.D., Department of Public Health, School of Medicine, University of Chile, for his help with the statistical analysis of this study. This work was presented in part at the 7th Annual Meeting of the Androgen Excess and PCOS Society, Munich, Germany, September 2010; and at the Endocrine Society’s 93th Annual Meeting, Boston, USA, June 2011. References [1] Diamanti-Kandarakis E, Kouli CR, Bergiele AT, et al. A survey of the polycystic ovary syndrome in the Greek island of Lesbos: hormonal and metabolic profile. J Clin Endocrinol Metab 1999;84:4006–11. [2] Asuncio´n M, Calvo RM, San Milla´n JL, Sancho J, Avila S, Escobar-Morreale HF. A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. J Clin Endocrinol Metab 2000;85:2434–8. [3] Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745–9. [4] Azziz R, Carmina E, Dewailly D, et al. Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab 2006;91:4237– 45. [5] Elting MW, Korsen TJ, Rekers-Mombarg LT, Schoemaker J. Women with polycystic ovary syndrome gain regular menstrual cycles when ageing. Hum Reprod 2000;15:24–8. [6] Brown ZA, Louwers YV, Fong SL, et al. The phenotype of polycystic ovary syndrome ameliorates with aging. Fertil Steril 2011;96:1259–65. [7] Carmina E, Campagna AM, Lobo RA. A 20-year follow-up of young women with polycystic ovary syndrome. Obstet Gynecol 2012;119:263–9. [8] Piltonen T, Morin-Papunen L, Koivunen R, Perheentupa A, Ruokonen A, Tapanainen J. Serum anti-Mullerian hormone levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. Hum Reprod 2005;20:1820–6. [9] Mulders AG, Laven JS, Eijkemans MJ, de Jong FH, Themmen AP, Fauser BC. Changes in anti-Mullerian hormone serum concentrations over time suggest delayed ovarian ageing in normogonadotrophic anovulatory infertility. Hum Reprod 2004;19:2036–42. [10] Hudecova M, Holte J, Olovsson M, Sundstrom Poromaa I. Long-term follow-up of patients with polycystic ovary syndrome: reproductive outcome and ovarian reserve. Hum Reprod 2009;24:1176–83. [11] Zawadzky JK, Dunaif A. Diagnosis criteria: towards a rational approach. In: Hershmann JM, editor. Current issues in endocrinology and metabolism. Boston: Blackwell Scientific Publications; 1992. p. 377–84. [12] Sir-Petermann T, Codner E, Pe´rez V, et al. Metabolic and reproductive features before and during puberty in daughters of women with polycystic ovary syndrome. J Clin Endocrinol Metab 2009;94:1923–30.
169
[13] Crisosto N, Echiburu´ B, Maliqueo M, et al. Improvement of hyperandrogenism and hyperinsulinemia during pregnancy in women with polycystic ovary syndrome: possible effect in the ovarian follicular mass of their daughters. Fertil Steril 2012;97:218–24. ˜ ez L, Potau N, Zampolli M, Street ME, Carrascosa A. Girls diagnosed with [14] Iba´n premature pubarche show an exaggerated ovarian androgen synthesis from the early stages of puberty: evidence from gonadotropin-releasing hormone agonist testing. Fertil Steril 1997;67:849–55. [15] Sir-Petermann T, Hitchsfeld C, Codner E, et al. Gonadal function in low birth weight infants: a pilot study. J Pediatr Endocrinol Metab 2007;20:405–14. ˜ ı´guez G, et al. Low risk of impaired testicular Sertoli and [16] Rey RA, Codner E, In Leydig cell functions in boys with isolated hypospadias. J Clin Endocrinol Metab 2005;90:6035–40. [17] Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19–25. [18] Soules MR, Sherman S, Parrott E, et al. Stages of reproductive aging workshop (STRAW). J Womens Health Gender-Based Med 2001;10:843–8. [19] Panidis D, Katsikis I, Karkanaki A, Piouka A, Armeni A, Georgopoulos A. Serum anti-Mu¨llerian hormone (AMH) levels are differentially modulated by both serum gonadotropins and not only by serum Follicle Stimulating Hormone (FSH) levels. Med Hypotheses 2011;77:649–53. [20] Skalva P, Cygal A, Madej P, et al. Is the plasma anti-Mu¨llerian hormone (AMH) level associated with body weight and metabolic, and hormonal disturbance in women with and without polycystic ovary syndrome? Eur J Obstet Gynecol Reprod Biol 2011;158:254–9. [21] Carmina E, Campagna AM, Mansuet P, Vitale G, Kort D, Lobo R. Does the level of serum antiMu¨llerian hormone predict ovulatory function in women with polycystic ovary syndrome with aging? Fertil Steril 2012;98:1043–6. [22] Homburg R. Androgen circle of polycystic ovary syndrome. Hum Reprod 2009;24:1548–55. [23] Crisosto N, Sir-Petermann T, Greiner M, et al. Testosterone-induced downregulation of anti-Mu¨llerian hormone expression in granulosa cells from small bovine follicles. Endocrine 2009;36:339–45. [24] Winters SJ, Talbott E, Guzick DS, Zborowski J, McHugh KP. Serum testosterone levels decrease in middle age in women with the polycystic ovary syndrome. Fertil Steril 2000;73:724–9. [25] Elting MW, Kwee J, Korsen TJ, Rekers-Mombarg LT, Schoemaker J. Aging women with polycystic ovary syndrome who achieve regular menstrual cycles have a smaller follicle cohort than those who continue to have irregular cycles. Fertil Steril 2003;79:1154–60. [26] de Carvalho BR, Rosa e Silva AC, Rosa e Silva JC, dos Reis RM, Ferriani RA, Silva de Sa´ MF. Ovarian reserve evaluation: state of the art. J Assist Reprod Genet 2008;25:311–22. [27] van Rooij IA, Tonkelaar Id, Broekmans FJ, et al. Anti-Mullerian hormone is a promising predictor for the occurrence of the menopausal transition. Menopause 2004;11:601–6. [28] van Rooij IA, Broekmans FJ, Scheffer GJ, et al. Serum antiMullerian hormone levels best reflect the reproductive decline with age in normal women with proven fertility: a longitudinal study. Fertil Steril 2005;83:979–87. [29] van Montfrans JM, Hoek A, van Hooff MH, de Koning CH, Tonch N, Lambalk CB. Predictive value of basal follicle-stimulating hormone concentrations in a general subfertility population. Fertil Steril 2000;74:97–103. [30] Sowers MR, Eyvazzadeh AD, McConnell D, et al. Anti-Mullerian hormone and inhibin B in the definition of ovarian aging and the menopause transition. J Clin Endocrinol Metab 2008;93:3478–83. [31] Nardo LG, Yates AP, Roberts SA, Pemberton P, Laing I. The relationships between AMH, androgens, insulin resistance and basal ovarian follicular status in non-obese subfertile women with and without polycystic ovary syndrome. Hum Reprod 2009;24:2917–23. [32] Pasquali R, Stener-Victorin E, Yildiz B, et al. PCOS Forum: research in polycystic ovary syndrome today and tomorrow. Clin Endocrinol (Oxf) 2011;74:424–33. [33] Kazer RR, Kessel B, Yen SS. Circulating luteinizing hormone pulse frequency in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1987;65:233–6. [34] de Koning CH, Popp-Snijders C, Schoemaker J, Lambalk CB. Elevated FSH concentrations in imminent ovarian failure are associated with higher FSH and LH pulse amplitude and response to GnRH. Hum Reprod 2000;15:1452–6. [35] Lambalk CB, van Disseldorp J, de Koning CH, Broekmans FJ. Testing ovarian reserve to predict age at menopause. Maturitas 2009;63:280–91. [36] Welt CK, Taylor AE, Martin KA, Hall JE. Serum inhibin B in polycystic ovary syndrome: regulation by insulin and luteinizing hormone. J Clin Endocrinol Metab 2002;87:5559–65. [37] Laven JS, Imani B, Eijkemans MJ, de Jong FH, Fauser BC. Absent biologically relevant associations between serum inhibin B concentrations and characteristics of polycystic ovary syndrome in normogonadotrophic anovulatory infertility. Hum Reprod 2001;16:1359–64. [38] Roberts VJ, Barth S, el-Roeiy A, Yen SS. Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle. J Clin Endocrinol Metab 1993;77:1402–10. [39] Groome NP, Illingworth PJ, O’Brien M, et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996;81:1401–5.
170
A.L. de Guevara et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 165–170
[40] de Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC. AntiMu¨llerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002;77:357–62. [41] Sowers M, Eyvazzadeh A, McConnell D, et al. Anti-Mullerian hormone and inhibin B in the definition of ovarian aging and the menopause transition. J Clin Endocrinol Metab 2008;93:3478–83. [42] Hehenkamp WJ, Looman CW, Themmen AP, de Jong FH, Te Velde ER, Broekmans FJ. Anti-Mu¨llerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. J Clin Endocrinol Metab 2006;91: 4057–63.
[43] Pigny P, Merlen E, Robert Y, et al. Elevated serum level of anti-Mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 2003;88:5957–62. [44] Laven JS, Mulders AG, Visser JA, Themmen AP, De Jong FH, Fauser BC. AntiMu¨llerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 2004;89:318–23. ˜ ı´guez G, Villarroel C, et al. Hormonal profile in women with [45] Codner E, In polycystic ovarian syndrome with or without type 1 diabetes mellitus. J Clin Endocrinol Metab 2007;92:4742–6.