University of Udine, Udine, University of Pisa, Pisa, and University of Modena, Modena, Italy; and University of Wien, Wien, Austria

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1 FERTILITY AND STERILITY VOL. 70, NO. 5, NOVEMBER 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Low levels of serum inhibin A and inhibin B in women with hypergonadotropic amenorrhea and evidence of high levels of activin A in women with hypothalamic amenorrhea Felice Petraglia, M.D.,* Beda Hartmann, M.D., Stefano Luisi, M.D., Pasquale Florio, M.D., Silvia Kirchengast, M.D., Massimo Santuz, M.D.,* Alessandro D. Genazzani, M.D., and Andrea R. Genazzani, M.D. University of Udine, Udine, University of Pisa, Pisa, and University of Modena, Modena, Italy; and University of Wien, Wien, Austria Received March 2, 1998; revised and accepted June 18, Reprint requests: Felice Petraglia, M.D., Department of Surgical Sciences, Chair of Obstetrics and Gynecology, University of Udine, Piazzale S. Maria della Misericordia, Udine, Italy (FAX: ; felicepetraglia@dsc.uniud. it.). * Department of Surgical Sciences, Chair of Obstetrics and Gynecology, University of Udine. Department of Obstetrics and Gynecology, University of Wien. Department of Reproductive Medicine and Child Development, Section of Obstetrics and Gynecology, University of Pisa. Department of Pediatric, Obstetric and Gynecological Sciences, University of Modena / 1802/$19.00 PII S (98) Objective: To examine serum levels of inhibin A, inhibin B, and activin A in women with secondary hypergonadotropic or hypothalamic amenorrhea. Design: Retrospective study. Setting: Universities of Udine, Pisa, and Modena in Italy, and of Wien in Austria. Patient(s): Forty women with idiopathic premature ovarian failure (POF), 23 women with hypogonadotropic hypothalamic amenorrhea, 40 healthy postmenopausal women, and 40 age-matched women with normal ovarian function (controls). Intervention(s): Blood samples were collected between 8 and 9 AM. Main Outcome Measure(s): Serum levels of inhibin A, inhibin B, and activin A. Result(s): Women with POF had lower concentrations of serum inhibin A and inhibin B than women with hypothalamic amenorrhea and fertile controls, and the difference between these concentrations was statistically significant. Levels of inhibin A and inhibin B were low in postmenopausal women and were no different than in women with POF. Serum levels of activin A were not significantly different among women with POF, fertile controls, and postmenopausal women. Women with hypogonadotropic hypothalamic amenorrhea had higher activin A values than did controls. No significant correlation was found between the level of inhibin A or inhibin B and the length of amenorrhea or the level of FSH. Conclusion(s): Low levels of circulating inhibins A and B, but not activin A, reflect ovarian failure in women with POF, whereas women with hypogonadotropic hypothalamic amenorrhea have normal levels of inhibins A and B and high levels of activin A. (Fertil Steril 1998;70: by American Society for Reproductive Medicine.) Key Words: Inhibin A, inhibin B, activin A, premature ovarian failure, hypergonadotropic amenorrhea, hypothalamic amenorrhea, menopause The main endocrine change in women with premature ovarian failure (POF) is a decline in circulating levels of ovarian steroid hormones. However, POF is characterized by various degrees of residual ovarian function and an uncertain prognosis. It recently has been shown that the ovary produces growth factors that are involved in the regulation of follicle development. Among these, inhibin A, inhibin B, and activin A are ovarian proteins that are measurable in the systemic circulation (1). In particular, levels of inhibin A are correlated with the luteinization of granulosa cells and represent a marker of luteal function (2), whereas levels of inhibin B reflect follicle maturation, being highest during the follicular phase of the menstrual cycle (3, 4). Both proteins show an equipotent capacity to decrease FSH release from cultured pituitary cells (5) and are suggested to control FSH secretion throughout the menstrual cycle (6, 7). In addition to their effects on FSH secretion, there is evidence supporting a local autocrine/ paracrine regulatory role for these hormones in ovarian follicles (8). Postmenopausal women 907

2 have low concentrations of circulating immunoreactive inhibin (9), whereas the use of a specific dimeric assay has demonstrated a decrease in inhibin B, but not inhibin A, in older anovulatory women (6). No data are available on women with POF or other forms of hypogonadotropic amenorrhea. Data on activin A are even more intriguing. Activin A is a protein that stimulates FSH release from cultured pituitary cells (5), and some changes in its levels have been demonstrated during the menstrual cycle in healthy women, whereas no changes are seen after menopause (10). Considering the effects of these proteins on follicle maturation and the luteinization of granulosa cells, we performed a retrospective study to evaluate serum levels of inhibin A, inhibin B, and activin A in women with POF or hypothalamic hypogonadotropic amenorrhea and to compare them with levels in healthy postmenopausal women and healthy fertile women. TABLE 1 Serum hormone levels in the four study groups. Study group FSH (miu/ml) Hormone level (mean SEM) LH (miu/ml) E 2 (pg/ml) Fertile HA POF PMP Note: HA hypothalamic hypogonadotropic amenorrhea; PMP healthy postmenopausal women; POF premature ovarian failure. MATERIALS AND METHODS Subjects The study design included four groups of women: group 1 consisted of patients with POF (n 40; age range, years); group 2 consisted of patients with hypothalamic amenorrhea (n 23; age range, years); group 3 consisted of postmenopausal women matched for menopausal age (n 40; age range, years); and group 4 consisted of healthy fertile control women with regular menstrual cycles who were medical students or laboratory staff and who volunteered for the study (n 40; age range, years). All subjects gave their informed consent. The study was approved by the local ethical committee. On the first visit to our outpatient clinic, a complete medical history was obtained and a physical examination was performed for each patient; in addition, appropriate routine tests were performed to exclude internal diseases. Serum FSH, LH, and E 2 levels were measured with the use of specific assays. Patients with pituitary, thyroid, or adrenal disorders were excluded from the study. None of the subjects had taken any hormones for at least 6 months before the study, and most of the participants had never used hormonal preparations. The diagnosis of POF was based on age 35 years, normal menarche, secondary amenorrhea between 6 and 36 months ( months), FSH levels 50 miu/ml, and E 2 levels 20 pg/ml on at least two occasions. Screening for autoimmune diseases and genetic factors was performed by clinical examination, chromosome analysis (leukocyte karyotype), and blood tests for antiovarian as well as antinuclear antibodies. Patients were screened for antiovarian antibodies with the use of an antiovarian antibody latex agglutination test (Privates Institut Bioserv GmbH, Leipzig, Germany). Results showing a titer of 1:100 were considered negative. Analysis of antinuclear antibodies was performed by indirect immunofluorescence on slides coated with Hep-2 cells; a titer of 1:40 was considered negative. None of the patients with POF screened positive for any autoimmune disease. The criteria for the diagnosis of hypogonadotropic hypothalamic amenorrhea were as follows: 1. Disappearance of menses 6 months before the study and no evidence of pregnancy. 2. Low levels of LH, FSH, E 2, and progesterone assayed in at least three different samples collected over a period of 45 days before the study (Table 1). 3. Plasma levels of cortisol, androstenedione, testosterone, dehydroepiandrosterone sulfate, thyroxine, thyroid-stimulating hormone, and prolactin within the normal range. 4. Normal findings on computerized tomographic examination of the sella turcica. 5. No psychiatric disease. The postmenopausal women had had spontaneous amenorrhea for at least 1 year (range, months), and they had serum FSH levels of 50 miu/ml and E 2 levels of 20 pg/ml. Patients with climacteric symptoms were excluded. The fertile controls were examined during the early to midfollicular phase, and the occurrence of ovulation was monitored during the current cycle by ultrasound. A blood specimen was collected between 8 and 9 AM from the forearm vein after overnight fasting, bed resting, and 30 minutes of saline infusion. After centrifugation at 3,000 rpm for 10 minutes, the serum was separated and stored at 20 C until it was assayed; it was assyaed in small aliquots so that it was thawed only once. Inhibin A, Inhibin B, and Activin A Assays Inhibin A, inhibin B, and activin A concentrations in the various fluids (culture medium, peritoneal fluid, and serum) were measured with the use of specific two-site enzyme immunoassays as previously described (Serotec, Oxford, United Kingdom). Briefly, standards and samples in each assay were diluted as appropriate and mixed with an equal 908 Petraglia et al. Inhibins, activin, and amenorrhea Vol. 70, No. 5, November 1998

3 FIGURE 1 Mean SEM inhibin A level in women with premature ovarian failure or hypothalamic amenorrhea, postmenopausal women, and healthy fertile controls. P.05. (activin A) or half volume of the sample (inhibin A and inhibin B) of distilled water containing 10% sodium dodecyl sulfate. After 3 minutes at 100 C, the tubes were cooled before adding freshly prepared hydrogen peroxide solution. After additional incubation at room temperature, duplicate aliquots of denatured and oxidized samples and standards were transferred to antibody-coated microtiter plates. The plates were incubated at room temperature for 2 hours (inhibin A) or overnight (inhibin B). In activin A plates, 25 L of biotinylated monoclonal antibody to the A-subunit was added before overnight incubation. After washing with enzyme immunoassay wash buffer (0.1 mol/l [tris(hydroxymethyl)aminomethane] hydrochloride, 0.15 mol/l NaCl, 10% [wt/vol] bovine serum albumin, 5% [vol/ vol] Triton X-100, and 0.1% [wt/vol] sodium azide; ph 7.5), 50 L of alkaline phosphatase conjugated Fab mouse antihuman inhibin -subunit was used in inhibin A and inhibin B assays, whereas streptavidin alkaline phosphatase was used in activin A assays. Then the plates were incubated for 1 (inhibin A), 2 (activin A), or 3 (inhibin B) hours. The plates were washed, and bound alkaline phosphatase was quantitated with the use of a commercially available enzyme immunoassay amplification system (Immuno Select ELISA Amplification System; Dako, Milan, Italy), which was used according to the supplier s instructions. The inhibin A, inhibin B, and activin A plates were read at 490 nm on an automated enzyme immunoassay plate reader (BRIO: Basic Radim Immunoassay Operator; Radim Spa, Pomezia, Italy). The inhibin A detection limit was 20 pg/ml of serum, with intra-assay and interassay coefficients of variation (CVs) for quality control samples of 4% and 8%, respectively. The assay detection limit for inhibin B was 30 pg/ml in serum and 15 pg/ml in peritoneal fluid and culture medium. Within- and between-plate CVs in this case were 5% and 9%, respectively. The limit of detection for activin A was 100 pg/ml, and intra-assay and interassay CVs were 5% and 9%, respectively. Cross-reactions for each assay with the various inhibin-related proteins were 0.5%. Statistical Analysis Statistical analysis of the data was performed with the use of analysis of variance and the Duncan test. Multiple regression analyses were performed to test the correlation among the hormonal parameters and between each hormonal parameter and the duration of amenorrhea. The mean SEM serum levels of inhibin A (Fig. 1) and inhibin B (Fig. 2) in the women with POF and the postmenopausal women were significantly lower than those in the fertile women and the women with hypogonadotropic hypothalamic amenorrhea (P.01). Both the inhibin A and the inhibin B level did not change significantly between the women with hypogonadotropic hypothalamic amenorrhea and the control women. No statistically significant difference in the levels of inhibin A and inhibin B was observed between the postmenopausal women and the women with FERTILITY & STERILITY 909

4 FIGURE 2 Mean SEM inhibin B level in women with premature ovarian failure or hypothalamic amenorrhea, postmenopausal women, and healthy fertile controls. P.05. POF, resulting in all women having concentrations below the range of normal values. In regard to activin A levels, no statistically significant difference was observed between the fertile controls and the women with POF or the postmenopausal women, whereas the mean SEM values of activin A for the women with hypogonadotropic hypothalamic amenorrhea were significantly higher than those for the other study groups (P.01) (Fig. 3). The results of the multiple regression analysis showed that in women with POF or hypogonadotropic hypothalamic amenorrhea and in postmenopausal women, serum levels of inhibins A or B or of activin A did not correlate with patient age, length of amenorrhea, or serum LH, FSH, or E 2 levels. DISCUSSION The present study showed that premature or physiologic ovarian failure is associated with decreased serum inhibin A and inhibin B levels, whereas the temporary and reversible failure of ovarian function in women with hypogonadotropic hypothalamic amenorrhea is not associated with any changes in inhibin A or inhibin B levels. Whereas the finding in women with POF is the first reported in literature, the data obtained for postmenopausal women confirm the findings of previous studies that showed low levels of immunoreactive inhibin in postmenopausal women (9). Therefore, in association with high FSH and low E 2 levels, the definitive lack of ovarian function may be predicted by low levels of inhibins, independent of patient age and the duration of amenorrhea. Levels of activin A do not change in women with POF or in postmenopausal women, whereas they increase in women with hypogonadotropic hypothalamic amenorrhea, a finding that supports the growing concept that the source of activin A is different from that of inhibin A and inhibin B. The ovary is the major source of inhibin A and inhibin B in healthy fertile women. Human granulosa cells and corpus luteum (CL) express inhibin -, activin A-, and activin B-subunit messenger RNAs (11, 12), and ovarian follicular fluid contains large amounts of inhibin A, inhibin B, and activin A (10). The present findings support the current hypothesis that changes in circulating inhibin levels reflect the secretory activity of ovarian granulosa/luteal cells, and when their function is exhausted, no inhibins are released in the blood. In particular, whereas inhibin A reflects CL secretion (2), inhibin B is the physiologically relevant form of inhibin during the follicular phase of the cycle (7). In fact, serum levels of inhibin A in patients with luteal dysfunction are significantly lower than those in controls (3), and a poorer response to ovulation induction has been demonstrated in women with low day 3 serum inhibin B concentrations (13). 910 Petraglia et al. Inhibins, activin, and amenorrhea Vol. 70, No. 5, November 1998

5 FIGURE 3 Mean SEM activin A level in women with premature ovarian failure or hypothalamic amenorrhea, postmenopausal women, and healthy fertile controls. P.05. An in vitro study showed that inhibin A and inhibin B may play a role in the paracrine regulation of the follicle and CL (1). Decreased levels of inhibin B in older ovulatory women have been indicated to reflect a diminished follicular pool, typical of ovarian aging (6). Thus, on the basis of previous reports and our data, it appears that low levels of circulating inhibin A and inhibin B correlate well with the premature suppression of ovarian follicle development in young women. Because of their feedback effect on pituitary FSH release, the reduced secretion of inhibin A and inhibin B may explain the reported increase in FSH levels in women with POF or postmenopausal women. However, there is no statistical correlation between inhibin A or inhibin B levels and FSH levels in these two groups of patients. In contrast, because FSH stimulates inhibin secretion in vitro from cultured granulosa cells (14, 15), our present data suggest that women with POF and postmenopausal women have impaired ovarian responsiveness. The unchanged serum levels of activin A in women with POF and postmenopausal women suggest that the ovary is not the major source of circulating activin A. Follicular fluid contains activin A (8), but the mean concentration of activin A in follicular fluid changes little in follicles of any size (8), suggesting a constant, not dynamic, contribution from the ovary. Small changes in circulating activin A levels are described during the menstrual cycle; they decline in the early follicular phase, reach a nadir at the midfollicular phase, and then return to early follicular phase concentrations by midcycle (10). Our data support the hypothesis that serum levels of activin A do not reflect ovarian activity and that the ovary is not the major source of activin A. The finding of augmented levels of activin A in patients with hypogonadotropic hypothalamic amenorrhea is interesting but difficult to explain. The lack of any correlation with FSH levels does not support a possible pituitary origin and reduces the putative role as a reproductive hormone. Neuroendocrine and metabolic derrangements are typical of women with hypogonadotropic hypothalamic amenorrhea, and the high levels of activin A may reflect a homeostatic imbalance as occurs in patients with cancer or patients with liver or kidney disease (16). Another source in women with hypogonadotropic hypothalamic amenorrhea could be the adrenal gland, in which both A- and B-subunits of activin have been detected (17). A role for activin in the secretion of adrenal hormones already has been suggested (18), and in women with hypogonadotropic hypothalamic amenorrhea, increased activity of adrenal glands has been shown. In conclusion, low circulating levels of inhibin A and inhibin B in women with POF reflect ovarian failure, FERTILITY & STERILITY 911

6 whereas normal levels of the inhibins and high levels of activin A characterize hypothalamic amenorrhea. Acknowledgments: The present study was conducted in collaboration with the Associazione Sviluppo Studi Ormoni e Donna Atenei Toscani (ASSO- DAT) Italy. References 1. Judd HL, Judd GE, Lucas WE, Yen SS. Endocrine function of the postmenopausal ovary: concentration of androgens and estrogens in ovarian peripheral vein blood. J Clin Endocrinol Metab 1974;35: Eramma M, Ritvos O. Prostaglandin E 2 induces inhibin- and Asubunit mrna and secretion of dimeric inhibin A in cultured human granulosa-luteal cells. Mol Hum Reprod 1996;2: Yamoto M, Imai M, Otani H, Nakano R. Serum levels of inhibin A and inhibin B in women with normal and abnormal luteal function. Obstet Gynecol 1997;89: Groome NP, Illingworth PJ, O Brien M, Cooke I, Ganesan TS, Baird DT, et al. Detection of dimeric inhibin throughout the human menstrual cycle by two-site immunoassay. Clin Endocrinol (Oxf) 1994;40: Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsack T, et al. Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res 1988;44: Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia DE, Soules MR. Decreased inhibin B secretion is associated with monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J Clin Endocrinol Metab 1996;81: 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: Magoffin DA, Jakimiuk AJ. Inhibin A, inhibin B and activin A in the follicular fluid of regulatory cycling women. Hum Reprod 1997;12: Hee J, MacNaughton J, Bangah M, Burger H. Perimenopausal patterns of gonadotropins immunoreactive inhibin, oestradiol and progesterone. Maturitas 1993;18: Muttukrishna S, Fowler PA, George L, Groome NP, Knight PG. Changes in peripheral serum levels of total activin A during the human menstrual cycle and pregnancy. J Clin Endocrinol Metab 1996;81: Miro F, Hillier SG. Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells. J Clin Endocrinol Metab 1992;75: 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: Seifer DB, Lambert-Messerlian G, Hogan JW, Gardiner AC, Blazar AS, Berk CA. Day 3 serum inhibin-b is predictive of assisted reproductive technologies outcome. Fertil Steril 1997;67: Turner IM, Saunders PTK, Shimasaki S, Millier SG. Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells. Endocrinology 1989;125: Seifer DB, Gardiner AC, Lambert-Messerlian G, Schieyer AL. Differential secretion of dimeric inhibin in cultured luteinized granulosa cells as a function of ovarian reserve. J Clin Endocrinol Metab 1996;81: Harada K, Shintani Y, Sakamoto Y, Wakatsuki M, Shitsukawa K, Saito S. Serum immunoreactive activin A levels in normal subjects and patients with various diseases. J Clin Endocrinol Metab 1996;81: Spencer SJ, Rabinovici J, Mesiano S, Goldsmith PC, Jaffe RB. Activin and inhibin in the human adrenal gland. J Clin Invest 1992;90: Voutilainem R. What is the function of adrenal inhibins? Eur J Endocrinol 1995;132: Petraglia et al. Inhibins, activin, and amenorrhea Vol. 70, No. 5, November 1998