Vaginally administered estroprogestinic decreases serum inhibin A and inhibin B levels and reduces endometrial thickness

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1 Vaginally administered estroprogestinic decreases serum inhibin A and inhibin B levels and reduces endometrial thickness Stefano Luisi, M.D., Lavinia Estrela Borges, M.D., Lucia Lazzeri, M.D., Ariana dell Anna, M.D., Filiberto Maria Severi, M.D., and Felice Petraglia, M.D. Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy Objective: Serum levels of inhibin A, inhibin B, FSH, and LH were measured in healthy volunteers before and during oral or vaginal estroprogestinic administration. In addition, the effect on endometrial thickness and on follicular growth pattern were also assessed by vaginal ultrasound. Design: Prospective study. Setting: University of Siena. Patient(s): Seventeen healthy fertile women. Intervention(s): This open-label study was performed in 1 healthy volunteers, who were assigned to vaginal ethinylestradiol (15 g) and etonogestrel (12 g), one ring to be used for one cycle, after stratification for the ovulation day in a pretreatment cycle. A similar study on seven women assigned to oral ethinylestradiol (2 g) and levonorgestrel (1 g) was considered, to compare the effects of the two different routes of administration. Blood samples were collected the cycle before (days 8 1) and during (days 8 1) vaginal ring insertion and serum inhibin B, inhibin A, FSH, and LH levels were measured by ELISA. Concomitantly, transvaginal ultrasound was performed in all subjects for endometrial and follicular growth assessment. Main Outcome Measure(s): Inhibin A, inhibin B, FSH, and LH levels. Result(s): Vaginal administration induced a significant decrease of serum inhibin A, inhibin B, FSH, and LH. No significant changes in inhibin B and FSH secretion were observed during oral contraceptive (OC) administration, whereas LH and inhibin A levels significantly decreased. Endometrial thickness and ovarian volume decreased significantly during vaginal ring insertion, but not after OC administration. Conclusion(s): The present findings showed that treatment with vaginal estroprogestinic decreases serum inhibin A and inhibin B levels, the follicular diameter, and endometrial thickness, showing a rapid and significant effect with the vaginal route. (Fertil Steril 26;86: by American Society for Reproductive Medicine.) Key Words: Inhibin, estroprogestinic, endometrial thickness Inhibins are multifunctional ovarian hormones involved in the regulation of pituitary FSH and LH secretion (1, 2). Serum inhibin B levels are related with granulosa cell (GC) function, whereas the corpus luteum (CL) is the major source of circulating inhibin A. Inhibins are also recognized as paracrine ovarian and testicular regulators and have multiple paracrine effects in the uteroplacental unit, representing a promising marker for male and female infertility, gynecological, and gestational diseases (3, 4). Two active molecular forms of inhibin are detected in the circulation: inhibin B is the major form secreted during the follicular phase of the menstrual cycle (marker of follicular growth), and inhibin A levels are low in the early stage of follicular phase and increase from late follicular phase to peak at midluteal phase (marker of luteal activity) (5 1). The availability of vaginal ultrasonography allows the opportunity to study variations and changes on endometrial Received December 13, 25; revised and accepted April 3, 26. Reprint requests: Felice Petraglia, M.D., Department of Obstetrics and Gynecology, University of Siena, Policlinico Le Scotte, viale Bracci, 531 Siena, Italy (FAX: ; petraglia@unisi.it). and follicular growth pattern during the menstrual cycle using a far less invasive method. Although it does not provide information about histological characteristics, it does allow evaluation of the endometrial response to endogenous or exogenous sex steroids in spontaneous or induced menstrual cycles (11). Until the present no clear studies has evaluated the possible ovarian site of effect of estroprogestinic formulations by measuring the changes of inhibin secretion, therefore the present study evaluated the effect of vaginal ring on serum FSH, LH, inhibin A and B secretion, the effect on endometrial thickness and ovarian follicular growth was also assessed. MATERIALS AND METHODS Subjects A group of healthy volunteers (n 1), years of age (25 4 years, mean SD), with regular menstrual cycles were enrolled in this randomized, open label study. All subjects were white. Exclusion criteria consisted of the standard contraindications to hormonal contraceptive use such as pregnancy or lactation, vascular or metabolic diseases, liver or renal dysfunction, obesity, hypertension (systolic blood /6/$32. Fertility and Sterility Vol. 86, No. 5, November 26 doi:1.116/j.fertnstert Copyright 26 American Society for Reproductive Medicine, Published by Elsevier Inc. 1483

2 pressure 14 mm Hg or diastolic blood pressure 9 mm Hg), and use of any medication that could affect the efficacy of the vaginal or oral contraceptive (OC). A complete medical, gynecological, and obstetrical history was obtained from each patient. Physical and gynecological examinations were performed at the pretreatment visit. Average body mass index was kg/m 2 (range kg/m 2 ). Subjects were assigned to vaginal ethinylestradiol (15 g) and etonogestrel (12 g), one ring to be used for one cycle of 28 days, each cycle having a 21-day treatment period followed by a 7-day ring-free period. A similar study on seven women assigned to oral ethinylestradiol (2 g) and levonorgestrel (1 g) was considered, to compare the effects of the two different routes of administration. Blood samples were collected (every 1 minutes for 2 hours) twice: [1] control, on days 8 1 of the last menstrual period and [2] during, days 8 1, vaginal application. A single blood sample was collected 18 2 days after vaginal estroprogestinic application. Blood samples were centrifuged and serum was stored at 2 C until inhibin A, inhibin B, FSH, and LH levels were measured. The study was approved by the Institutional Review Board of the Academic Health Center of Siena, and informed consent forms were signed by all subjects before enrollment. Inhibins Assay Dimeric inhibin A and inhibin B were measured by ELISA (Diagnostic System Laboratories [DSL], Oxford, UK) levels. The inhibin A detection limit was 4 pg/ml in serum, with intraassay and interassay coefficient of variation (CV) for quality control sample of 4.% and 8.%, respectively. The inhibin B detection limit was 5 pg/ml in serum, with intraassay and interassay CV for quality control sample of 6.% and 8.%, respectively. Cross-reactions for the assay with the various proteins of the inhibin-related family were.1%. Esaote SpA (Genova, Italy) ultrasound machine, equipped with a multifrequency microconvex vaginal probe ( mhz). The transvaginal approach was performed by following classic transvaginal section planes, using high magnification of images to provide a good definition of endometrial lines and accurate measurement of three ovarian diameters. The endometrium was measured as total thickness in the sagittal plane. The echolucent layer surrounding the endometrium was not included. In the presence of intracavitary fluid, both single layers were measured and the sum was recorded. Both ovaries were evaluated for follicular growth identification and volume measurement. Statistical Analysis The pulsatile secretion of serum inhibin A, inhibin B, FSH, and LH before and after the vaginal ring insertion was calculated as area under curve (AUC) and data were compared by using the two-sided Student s t-test for paired groups. When appropriate, 95% confidence intervals were calculated for the observed differences. Probability values.5 were considered statistically significant. RESULTS Inhibin Levels A significant decrease of serum inhibin A and inhibin B AUC (mean SD) during vaginal hormonal treatment was observed (P.1) (Fig. 1). Serum inhibin A, but not inhibin B, slightly decreased during oral estroprogestinic treatment (Fig. 2). Gonadotropins Levels vaginal ring insertion serum LH and FSH secretion evaluated on AUC (mean SD) significantly decreased (P.1) (Fig. 1). Serum LH levels significantly decreased during oral estroprogestinic administration (P.1), whereas no change in serum FSH levels was observed (Fig. 2). FSH and LH Assay The FSH and LH serum concentrations were measured using commercially available immunometric kits (Diagnostic Products Corporation, Los Angeles, CA). The analytical sensitivity of both the LH and FSH assays was.1 mlu/ml. The intraassay CVs of FSH and LH were 5.4% and 4.8%, respectively, and the interassay CVs were 8.1% and 9.9%, respectively. For the quantitative measurement of FSH and LH in serum, the Immulite analyzer (Siena, Italy) was used. Transvaginal Ultrasound Transvaginal ultrasound examination was performed to evaluate the endometrium and ovarian follicle growth in women receiving the vaginal ring. All ultrasound examinations were performed by the same operator using a Technos MPX Transvaginal Ultrasound Ovarian volume and endometrial thickness (Fig. 3) significantly decreased during the period of vaginal hormone administration in comparison with the cycle before vaginal ring insertion (P.4). DISCUSSION The present data showed for the first time that ovarian inhibin secretion is strongly inhibited during vaginal estroprogestinic administration and confirmed a potent effect on pituitary LH and FSH secretion. A previous study has already shown a gradual decline of gonadotropins in the first week after estroprogestinic administration, and suggested a rapid effect on the suppression of development of nondominant follicles (11) Luisi et al. Vaginal estroprogestinic and inhibins Vol. 86, No. 5, November 26

3 FIGURE 1 Serum inhibin A (A), inhibin B (B), FSH (C), and LH (D) levels 8 1 days after the last spontaneous menstrual period (before estroprogestinic administration) and 8 1 days during vaginal ring insertion. Inhibin A (pg/ml) A *p<.1 Inhibin B (pg/ml) B *p<.1 C D FSH (ng/ml) 5. *p<.1 LH (ng/ml) 5. *p<.1.. Luisi. Vaginal estroprogestinic and inhibins. Fertil Steril 26. According to Timmer et al. (12), the maximum estroprogestinic serum concentrations were achieved at the first week after vaginal ring insertion and were approximately 3% and 4% of those reported with low-dose OCs containing levonorgestrel and ethinylestradiol, respectively. The absolute progestinic bioavailability was higher in patients using the vaginal ring, whereas systemic exposure to ethinylestradiol was half of that observed with the oral administration (12). The vaginal route promotes a constant hormonal absorption with prolonged activity and greatest bioavailability in contrast to the oral route. In addition, vaginal administration of sex steroids has been associated with localized effects, including increased efficacy and tolerability (13, 14). Because inhibins are very sensitive biochemical markers of follicular activity, the reduced secretion of both inhibins during the administration of estroprogestinic reflects a rapid and concomitant blockade of the preovulatory follicle development. Inhibin A and LH levels were significantly suppressed during oral estroprogestinic administration, whereas no effect on inhibin B and FSH levels was noted. Mulders et al. (15) showed that the hypothalamic pituitary ovarian axis is completely suppressed 3 days after the vaginal ring insertion, in contrast to the generally accepted requirement of 7 days with combined OCs. In addition, during the pill-free interval, a significant increase of inhibin B levels and a slight but nonsignificant increase of inhibin A levels in patients using low-dose OCs for 3 months, were observed, whereas P levels were completely suppressed (16). Furthermore, the oral administration of estroprogestinic for a short interval of time does not reduce local ovarian inhibin secretion and, despite their high level of efficacy, oral estroprogestinics do not completely suppress ovarian activity. Nevertheless, follicular development up to the stage of ovulation is never reported (17 19). Ovarian volume and endometrial thickness were significantly reduced after vaginal ring insertion, suggesting an immediate effect on the ovary and endometrium. This suppressive effect on endometrial thickness and on ovarian follicles, observed at the first week after the vaginal ring insertion, has been confirmed previously (2, 21). In addition, prolonging the use of the vaginal ring for an additional 2 weeks did not compromise ovarian suppression and inhibition of ovulation was maintained (2). Oral hormone administration also causes suppression of both ovarian volume and endometrial thickness, but the onset of suppression commences later when compared with vaginal administration (22, 23). Fertility and Sterility 1485

4 FIGURE 2 Serum inhibin A (A), inhibin B (B), FSH (C), and LH (D) levels 8 1 days after the last spontaneous menstrual period (before estroprogestinic administration) and 8 1 days of oral contraceptive administration. 75 A 2 B Inhibin A (pg/ml) 5 25 Inhibin B (pg/ml) 1 C D FSH (ng/ml) 5. LH (ng/ml) 5. *p<,1.. Luisi. Vaginal estroprogestinic and inhibins. Fertil Steril 26. In conclusion, the present findings show that a short treatment with vaginal ring estroprogestinic decreases ovarian inhibin A and inhibin B secretion, follicular and endometrial thickness, hereby suggesting an immediate effect of the vaginal route. FIGURE 3 Endometrial thickness measurement 8 1 days after the last spontaneous menstrual period before vaginal ring insertion and 8 1 days after vaginal ring insertion. Luisi. Vaginal estroprogestinic and inhibins. Fertil Steril 26. REFERENCES 1. Kingsley DM. The TGF- superfamily: new members, new receptors and new genic tests of function in different organism. Genes & Dev 1994;8: Cobellis S, Luisi S, Pezzani I, Reis FM, De Leo V, Petraglia F. Serum inhibin A, inhibin B, and pro-alpha C levels are altered after surgically or pharmacologically induced menopause. Fertil Steril 22;77: Petraglia F, Zanin E, Faletti A, Reis FM. Inhibins: paracrine and endocrine effects in female reproductive function. Curr Opin Obstet Gynecol 1999;11: Welt CK. Regulation and function of inhibins in the normal menstrual cycle. Semin Reprod Med 24;22: Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T, et al. Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res 1988;44: Groome NP, Illingworth PJ, O Brien M, Pai R, Rodger FE, Mather JP, et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996;81: Schneyer AL, Fujiwara T, Fox J, Welt CK, Adams J, Messerlian GM, et al. Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentrations. J Clin Endocrinol Metab 2;85: Sehested A, Juul A, Andersson AM, Petersen JH, Jensen TK, Muller J, et al. Serum inhibin A and inhibin B in healthy prepubertal, pubertal, and adolescent girls and adult women: relation to age, stage of puberty, menstrual cycle, follicle-stimulating hormone, luteinizing hormone, and estradiol levels. J Clin Endocrinol Metab 2;85: Luisi S, Florio P, Reis FM, Petraglia F. Inhibins in female and male reproductive physiology: role in gametogenesis, conception, implantation and early pregnancy. Hum Reprod Update 25;11: Kline J, Kinney A, Kelly A, Reuss ML, Levin B. Predictors of antral follicle count during the reproductive years. Hum Reprod 25;2: Luisi et al. Vaginal estroprogestinic and inhibins Vol. 86, No. 5, November 26

5 11. Heusden AM, Fauser BC. Residual ovarian activity during oral steroid contraception. Hum Reprod Update 22;8: Timmer CJ, Mulders TM. Pharmacokinetics of etonogestrel and ethinylestradiol released from a combined contraceptive vaginal ring. Clinical Pharmacokinetics 2;39: van den Heuvel MW, van Bragt AJ, Alnabawy AK, Kaptein MC. Comparison of ethinylestradiol pharmacokinetics in three hormonal contraceptive formulations: the vaginal ring, the transdermal patch and an oral contraceptive. Contraception 25;72: Roumen FJ, Dieben TO. Comparison of uterine concentrations of ethinyl estradiol and etonogestrel after use of a contraceptive vaginal ring and an oral contraceptive. Fertil Steril 26;85: Mulders TM, Dieben TO, Bennink HG. Ovarian function with a novel combined contraceptive vaginal ring. Hum Reprod 22;17: Renier MA, Vereecken A, Van Herck E, Straetmans D, Ramaekers P, Vanderheiden J, et al. Dimeric inhibin serum values as markers of ovarian activity in pill-free intervals. Contraception 1998;57: Heusden AM, Fauser BCJM. Residual ovarian activity during oral steroid contraception. Hum Rep 22;8: Oddsson K, Leifels-Fischer B, de Melo NR, Wiel-Masson D, Benedetto C, Verhoeven CH, et al. Efficacy and safety of a contraceptive vaginal ring (NuvaRing) compared with a combined oral contraceptive: a 1-year randomized trial. Contraception 25;71: Bjarnadottir RI, Tuppurainen M, Killick SR. Comparison of cycle control with a combined contraceptive vaginal ring and oral levonorgestrel/ethinyl estradiol. Am J Obstet Gynecol 22;186: Mulders TM, Dieben TO. Use of the novel combined contraceptive vaginal ring NuvaRing for ovulation inhibition. Fertil Steril 21;75: Duijkers IJ, Klipping C, Verhoeven CH, Dieben TO. Ovarian function with the contraceptive vaginal ring or an oral contraceptive: a randomized study. Hum Reprod 24;19: Baerwald AR, Olatunbosun OA, Pierson RA. Ovarian follicular development is initiated during the hormone-free interval of oral contraceptive use. Contraception 24;7: Schlaff WD, Lynch AM, Hughes HD, Cedars MI, Smith DL. Manipulation of the pill-free interval in oral contraceptive pill users: the effect on follicular suppression. Am J Obstet Gynecol 24;19: Fertility and Sterility 1487

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