Antim ullerian hormone levels decrease in women using combined contraception independently of administration route

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1 Antim ullerian hormone levels decrease in women using combined contraception independently of administration route Sanna Kallio, M.D., a Johanna Puurunen, M.D., a Aimo Ruokonen, M.D., Ph.D., b Tommi Vaskivuo, M.D., Ph.D., b Terhi Piltonen, M.D., Ph.D., a and Juha S. Tapanainen, M.D., Ph.D. a,c a Department of Obstetrics and Gynecology and Clinical Research Center, Oulu University Hospital, Oulu; b Department of Clinical Chemistry, Oulu University Hospital, Oulu; and c Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland Objective: To compare the effects of continuous use of oral (OC), transdermal, and vaginal combined contraceptives on the pituitaryovarian axis and inhibition of follicular development. Design: Spin-off study of a prospective, randomized trial. Setting: University clinic. Patient(s): Forty-two of 54 healthy women completed the study. Intervention(s): Treatment with combined OCs (ethinyl E 2 [EE] and desogestrel), transdermal patches (EE and norelgestromin), or vaginal rings (EE and etonogestrel) for 9 weeks continuously. Blood sampling was performed before and at 5 and 9 weeks of treatment. Main Outcome Measure(s): Changes in serum hormone levels induced by combined contraceptives. Result(s): Serum antim ullerian hormone (AMH), FSH, inhibin B, LH, and E 2 levels had decreased significantly in all study groups after 9 weeks of treatment. Significant declines were already detected after 5 weeks' use of combined contraceptives with regard to all hormone levels apart from those of serum AMH, where the decrease between baseline and 5 weeks was only moderate. Between groups, serum levels of AMH, inhibin B, LH, and E 2 were comparable at baseline and after 5 and 9 weeks of treatment. Conclusion(s): The decrease of serum AMH levels during the use of all combined contraceptives indicates that folliculogenesis is arrested independently of administration route. Clinical Trial Registration Number: NCT (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Antim ullerian hormone, combined contraception, follicle development, inhibin B, FSH Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/kallios-antimullerian-hormone-combined-contraception/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Use of combined contraceptives is a widespread and effective method of reversible contraception (1). The contraceptive effect is established mainly by the suppression of pituitary gonadotropin secretion, which results in reduced ovarian activity (i.e., inhibition of follicular growth and ovulation) (2). The standard 21/7 regimen of combined contraceptives contains a 7-day hormone-free interval with withdrawal bleeding. During this Received July 30, 2012; revised November 2, 2012; accepted November 20, 2012; published online December 20, S.K. has nothing to disclose. J.P. has nothing to disclose. A.R. has nothing to disclose. T.V. has nothing to disclose. T.P. has nothing to disclose. J.S.T. has nothing to disclose. Supported by grants from the National Graduate School of Clinical Investigation (CLIGS), the Academy of Finland, the Sigrid Juselius Foundation, Oulu University Hospital, and the Finnish-Norwegian Medical Foundation. Reprint requests: Juha S. Tapanainen, M.D., Ph.D., Department of Obstetrics and Gynecology, Helsinki University and Helsinki University Central Hospital, P.O. Box 140, HUS, Finland ( juha.tapanainen@helsinki.fi). Fertility and Sterility Vol. 99, No. 5, April /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. hormone-free period, the pituitaryovarian axis resumes its activity and gonadotropin secretion increases (3, 4). This results in follicular development (up to more than 10 mm in diameter) and transient activation of ovarian hormone function (5, 6) reflected by temporary increases in serum levels of E 2, inhibin B, LH, and FSH (7). It has been demonstrated that a shortened hormone-free interval in oral contraceptive (OC) users induces greater pituitary and ovarian suppression compared with the standard regimen (5, 7 9). In addition to oral pills, vaginal rings and transdermal patches VOL. 99 NO. 5 / APRIL

2 are the other alternatives for combined hormonal contraception (10). Variability in circulating steroid levels and profiles has been detected in connection with each route of administration, and exposure to ethinyl E 2 (EE) seems to be lowest in women using vaginal rings and highest in transdermal patch users (11). Antim ullerian hormone (AMH) is a member of the transforming growth factor-b (TGF-b) family. In women, AMH is produced mainly by the granulosa cells (GCs) of early growing follicles up to small antral stage (12) and its serum levels reliably reflect ovarian follicle reserve and changes in follicle count (13 15). Serum levels of AMH are relatively stable throughout the menstrual cycle (16 19), but decrease gradually throughout reproductive life (14). The effect of administration of oral combined contraceptives on serum AMH levels is somewhat contradictory (19 27) and no clear data are available on how the continuous use of combined contraceptives by different routes of administration affects follicle development and the dynamics of serum AMH secretion. As circulating levels of EE in combined contraceptive users vary depending on the route of administration, our hypothesis was that the inhibition of follicular growth may also be divergent during the use of different combined contraceptive preparations and this could be reflected in altered serum AMH and inhibin B levels. To this end, the objective of this study was to compare the effects of continuous use of OC, transdermal, and vaginal combined contraceptives on the pituitary-ovarian axis and inhibition of follicular development. The subjects were randomized to use one of the following preparations continuously for 9 weeks: a combined OC (EE, 20 mg and desogestrel 150 mg [Mercilon; Organon]; n ¼ 18), a transdermal contraceptive patch (EE, 20 mg/day and norelgestromin 150 mg/day [Ortho Evra; Janssen Pharmaceuticals, Inc.]; n ¼ 18), or a contraceptive vaginal ring (EE, 15 mg/d and etonogestrel [active metabolite of desogestrel] 120 mg/day [NuvaRing; Schering-Plough Corp.]; n ¼ 18). The women were advised to take combined contraceptives in the mornings and use phone reminders to avoid omissions. At every follow-up visit compliance was discussed, and two women with nonadherence to the study protocol were excluded from the study. The subjects were advised to use condom contraception during the first week of combined contraceptive use. Transvaginal ultrasonography was performed in all subjects to verify normal ovarian morphology before computer-generated randomization, as described previously (28). Blood samples were taken at baseline on cycle days 2 4 before treatment and thereafter at 5 and 9 weeks of treatment and serum stored at 80 C before analysis. After entering the study, four subjects were excluded because of disturbance in glucose tolerance and two women were excluded as a result of nonadherence to the study protocol (28). In addition, six women dropped out during the first month of the study (three subjects withdrew because of mood swings; other causes were difficulties in using the transdermal patch, and personal reasons). Thus, the study population consisted of 42 women (oral pill group n ¼ 13; transdermal patch group n ¼ 15; vaginal ring group n ¼ 14) who completed the study. MATERIALS AND METHODS Study Design and Subjects The present study was a spin-off study from a recently published randomized trial in which the primary outcome measures were androgen secretion and chronic inflammation (28). The study was a prospective, open-label, single-center study, conducted at Oulu University Hospital between September 2008 and December 2010, and it was approved by the Ethics Committee of Oulu University Hospital (Institutional Review Board) and the Finnish Medicines Agency. All subjects gave written informed consent. The trial was registered in 2007 at the EU Clinical Trials Register ( identifier code ) and later at Clinical Trials ( identifier code NCT ) to ensure international registration. The main outcome measures of the present study were changes in serum hormone levels (AMH, FSH, LH, E 2, and inhibin B) induced by the use of combined contraceptives. A total of 54 healthy white women (aged years, body mass index [BMI] kg/m 2 ) with regular menstrual cycles and no medication participated in the study. Women with previous hormone contraceptive use (28 of 54 subjects) were required to have a washout period of a minimum of 2 months before participation in the study. Exclusion criteria were cigarette smoking, abuse of alcohol, lactation, and general contraindications such as the use of combined contraceptives. Assays Serum levels of LH and FSH were analyzed by chemiluminometric assays (Advia Centaur; Siemens Healthcare Diagnostics), with a sensitivity of 0.07 IU/L for LH and 0.3 IU/L for FSH. The intra-assay and interassay coefficients of variation (CVs) were 2.3% and 2.7%, respectively, for LH at a concentration of 4.2 IU/L, and 2.9% and 3.9% for FSH at a concentration of 6.9 IU/L. Serum levels of E 2 were determined by RIA (Orion Diagnostica), with a sensitivity of 5 pmol/l. The intra-assay and interassay CVs were 2.8% and 5.8%, respectively, at a concentration of 90 pmol/l. Serum AMH levels were analyzed by using an ELISA method (AMH Gen II; Diagnostic Systems Laboratories-Beckman Coulter Inc.). The range of AMH standards used in this assay was ng/ml, the intra-assay CV was 3.3% and the interassay CV was 4.8%. Inhibin B levels were determined by an ELISA method (Inhibin B Gen II ELISA; Beckman Coulter Inc.) with a sensitivity of 2.6 ng/l. The intra-assay and interassay CVs were 2.4% and 4.4%, respectively, at a concentration of 76 ng/l. Statistics Statistical analysis was performed using IBM SPSS Statistics (version for Windows, SPSS Inc.). Variables with skewed distribution were log-transformed before analysis. The limit of statistical significance was set at P<.05. Changes in hormone levels within the groups were analyzed by 1306 VOL. 99 NO. 5 / APRIL 2013

3 Fertility and Sterility TABLE 1 Clinical characteristics and mean (±SD) hormone levels at baseline, and at 5 and 9 weeks' of use of oral pills, transdermal patches, or vaginal rings. Week Pills P value Patches P value Rings P value No. of women Age (y) BMI (kg/m 2 ) AMH (ng/ml) Inhibin B (ng/l) < < <.001 FSH (IU/L) < < <.001 a LH (IU/L) < < <.001 E 2 (pmol/l) < < <.001 Note: Values of P indicate differences in hormone levels within a group. Differences in serum hormone levels were analyzed by repeated measures analysis of variance (ANOVA). AMH ¼ antim ullerian hormone; BMI ¼ body mass index. a P¼.019 vaginal ring versus transdermal patch group after 9 weeks' use of combined contraceptives (analyzed by one-way ANOVA). repeated measures analysis of variance (ANOVA). To analyze the changes in more detail at 5 and 9 weeks, the paired samples t test was performed as a post hoc test. Correlations between age, BMI, and hormone serum levels between groups were analyzed by means of Pearson's correlation test. Oneway ANOVA was used to explore differences between study groups and Tukey and Scheffe tests were used as post hoc tests. The results are presented as mean SD. RESULTS The baseline characteristics of the three study groups were comparable (Table 1). Compared with baseline there were significant decreases in serum AMH levels after 9 weeks' use of OC pills ( vs ng/ml, P<.001), transdermal patches ( vs ng/ml, P¼.001), and vaginal rings ( vs ng/ml, P¼.001) (Fig. 1). Similar significant declines were detected in levels of serum FSH (P<.001), inhibin B (P<.001), LH (P<.001), and E 2 (P<.001) in all groups as early as at 5 weeks of treatment (Fig. 2, Table 1). However, the moderate decreases in serum AMH levels between baseline and 5 weeks of treatment were not statistically significant in any of the groups. None of the hormonal changes correlated significantly with age or BMI. When comparing the three different routes of administration, similarly to baseline, serum levels of AMH, inhibin B, LH, and E 2 were comparable after 5 and 9 weeks' use of combined contraceptives in all study groups. However, serum FSH levels were higher at 9 weeks in the vaginal ring users compared with the transdermal patch users. No correlation was observed between serum AMH and FSH levels at baseline or after 5 or 9 weeks' use of combined contraceptives in any of the groups. FIGURE 1 Serum antim ullerian hormone (AMH) levels at baseline and after 5 and 9 weeks of administration of combined contraceptives by three different routes (oral [Pill], transdermal [Patch], and vaginal [Ring]). *P¼.001 vs. baseline. VOL. 99 NO. 5 / APRIL

4 FIGURE 2 FIGURE 3 Serum inhibin B levels at baseline and after 5 and 9 weeks of administration of combined contraceptives by three different routes (oral [Pill], transdermal [Patch], and vaginal [Ring]). *P<.001 vs. baseline. DISCUSSION This is the first study in which the effects of continuous use of OC, transdermal, and vaginal combined contraceptives on serum AMH levels have been compared. The data demonstrate that the use of combined contraceptives results in arrest of folliculogenesis independently of the route of administration, as serum AMH levels decreased gradually in all study groups and were approximately 50% lower after 9 weeks' use of contraceptives versus baseline, and significant decreases were also detected in serum inhibin B, E 2, FSH, and LH levels in all three study groups as early as at 5 weeks of treatment. The baseline serum levels of all hormones measured were comparable between the three study groups, but interestingly, after 9 weeks' use of combined contraceptives, serum FSH levels in the ring group were slightly but significantly higher than in transdermal patch group. Exposure to EE has been shown to be lowest in women using vaginal rings when compared with OC pill and transdermal patch users (11), which may lead to less prominent negative feedback on pituitary gonadotropin secretion and thereby higher FSH levels in the ring group. The significant and similar decreases in serum AMH, E 2, and inhibin B levels, however, suggest that a threshold stimulus for inhibition of folliculogenesis was reached with all combined contraceptives studied. Serum AMH levels decreased significantly during the use of all combined contraceptives. Antim ullerian hormone is secreted by GCs of growing follicles, mainly up to 4 6 mm(12). The GC mass of these follicles is higher than in follicles in preceding phases (29) and may contribute significantly to the overall secretion of AMH. Thus, the decrease of serum AMH levels during the use of combined contraceptives probably reflects decreased numbers of small antral follicles, which are already responsive to cyclic changes of FSH (30) and are thereby more sensitive to the reduced serum levels of FSH Effect of FSH on follicular growth and antim ullerian hormone (AMH) secretion in the ovary without (A) and during the use of combined contraception (B). Combined contraceptives reduce the effect of FSH and thus inhibit the growth of follicles, mainly in the antral stage. Reduced granulosa cell (GC) mass of the follicles results in a significant decrease in AMH secretion, mainly in antral follicles, but perhaps to some extent also in follicles at preceding stages. brought about by combined contraceptives (Fig. 3). Antral follicle counts during the treatment could have provided additional support for this hypothesis, but for practical reasons ultrasonography was performed only at baseline. In previous studies, intraovarian androgens have been shown to promote preantral follicle growth and consequently to increase the number of growing small antral follicles (31, 32). In addition, increased serum levels of AMH have been observed in women with polycystic ovary syndrome (PCOS) (33, 34). Thus, ovarian androgen suppression caused by continuous use of combined contraceptives, as shown in our primary study (28), may also be connected to the decreased serum AMH and inhibin B levels. In addition, a direct effect of E 2 on AMH secretion cannot be excluded, as some animal and in vitro studies have demonstrated that E 2 has an influence on AMH expression (35 38). However, Liberty et al. (39) reported that the effect of E 2 on AMH secretion is likely to be indirect in cycling infertile women 1308 VOL. 99 NO. 5 / APRIL 2013

5 Fertility and Sterility and thus the role of E 2 as a direct regulator of AMH secretion is somewhat unclear and needs further investigation. It is thought that early growing follicles are FSH independent, as in the absence of bioactive FSH or functional FSH receptors follicles grow to preantral/small antral stage (40 42). However, studies in hypogonadal or hypophysectomized rodents, as well as a case study on a patient with idiopathic hypogonadotropic hypogonadism, have shown that early follicular growth is impaired but not abolished, and exogenous administration of FSH restores the growth and number of follicles (43 45). Thus, preantral follicles may be responsive to, but not dependent on, FSH (46) and therefore the gradual decrease of serum AMH levels observed during the use of combined contraceptives may also at least partially be a result of protracted growth of these follicles (Fig. 3). As the results of some previous studies have suggested that a shortened hormone-free interval during the use of OC combined contraceptives suppresses pituitary-ovarian function more effectively than a standard 21/7 regimen (5, 7 9), continuous administration for 9 weeks, as used in our study, may theoretically inhibit follicular growth even more significantly, as reflected by substantially decreased serum AMH levels. The results of previous studies concerning serum AMH concentrations and the use of cyclic combined contraceptives have been controversial. Decreased (22, 26, 47) or unchanged (19, 21) serum AMH concentrations during the hormone-free interval compared with natural cycles or with days when using pills (19, 48) have been reported. Fewer larger antral follicles (>6 mm) have been found in OC combined contraceptive users compared with nonusers, but the numbers of small antral follicles (2 6 mm) and serum AMH levels were similar in a study by Deb et al. (24). In a population-based study (25), however, combined contraceptive users were shown to have slightly decreased AMH levels. The more pronounced decrease of serum AMH levels observed in our study may be theoretically explained by continuous administration of the combined contraceptives, as prolonged suppression caused by combined contraceptives may more effectively inhibit the growth of smaller antral follicles, which produce AMH. However, this has to be confirmed in future studies. Inhibin B is secreted mainly by GCs of preantral and small antral follicles and its serum concentrations are highest at the midfollicular phase (49). In a previous study, serum inhibin B concentrations decreased rapidly 8 10 days after initiation of vaginal combined contraceptive administration, whereas no decline was observed when combined contraceptives were administered orally (50). In addition, in some previous reports, levels of inhibin B have been shown to increase during the hormone-free interval of cyclic use of OC combined contraceptives (4, 51, 52). However, in the present study, serum concentrations of inhibin B decreased significantly in all study groups. This difference may be explained by the fact that in our study the first blood samples were collected 5 weeks after the initiation of treatment. In conclusion, the present results demonstrate that the continuous use of combined contraceptives inhibits follicular development significantly, independently of administration route. This was reflected in significantly decreased serum AMH levels in all study groups after 9 weeks' use of combined contraceptives. In addition, the marked decline in serum AMH levels suggests that the continuous use of combined contraceptives inhibits the early phase of FSH-dependent follicular development and perhaps also to some extent follicular growth in preceding stages. 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