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Serum inhibin A, inhibin B, pro-αC, and activin A levels in women with idiopathic premature ovarian failure

2004; Elsevier BV; Volume: 82; Issue: 3 Linguagem: Inglês

10.1016/j.fertnstert.2004.05.065

1556-5653

W. Munz, Mohamed E. Hammadeh, R. Seufert, Michael Schaffrath, Werner Schmidt, K. Pollow,

Ovarian cancer diagnosis and treatment

Serum inhibin A, inhibin B, pro-αC, and activin A levels in 30 women with idiopathic premature ovarian failure (POF), 30 postmenopausal women, and 30 age-matched fertile women were determined. Women with POF showed low levels of inhibin A and inhibin B, but not of activin A, whereas the levels of pro-αC were significantly higher than in postmenopausal women. Thus, the circulating level of pro-αC could be a marker for assessing residual ovarian function in women with POF. Serum inhibin A, inhibin B, pro-αC, and activin A levels in 30 women with idiopathic premature ovarian failure (POF), 30 postmenopausal women, and 30 age-matched fertile women were determined. Women with POF showed low levels of inhibin A and inhibin B, but not of activin A, whereas the levels of pro-αC were significantly higher than in postmenopausal women. Thus, the circulating level of pro-αC could be a marker for assessing residual ovarian function in women with POF. Premature ovarian failure (POF) is defined as a syndrome causing amenorrhea, anovulation, hypoestrogenism, and increased gonadotropin levels in women aged <40 years (1Aiman J. Smentek C. Premature ovarian failure.Obstet Gynecol. 1985; 66: 9-14PubMed Google Scholar, 2Rebar R.W. Connolly H.V. Clinical features of young women with hypergonadotropic amenorrhea.Fertil Steril. 1990; 53: 804-810Abstract Full Text PDF PubMed Google Scholar). Inhibin A, inhibin B, and activin A are glycoproteins produced by granulosa cells that have been determined to play a role in the nonsteroidal regulation of pituitary FSH secretion. These compounds are members of the transforming growth factor β superfamily (3Woodruff T.K. Besecke L.M. Groome N. Draper L.B. Schwartz N.B. Weiss J. Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner.Endocrinology. 1996; 137: 5463-5467Crossref PubMed Google Scholar, 4Pangas S.A. Woodruff T.K. Activin signal transduction pathways.Trends Endocrinol Metab. 2000; 11: 309-314Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Inhibins are heterodimeric glycoproteins consisting of an α subunit linked by two disulphide bonds to one of the two related β subunits (βA, βB), resulting in inhibin A (αβA) and inhibin B (αβB). A homodimer of the βA subunit is the glycoprotein activin A, which has a function opposite to that of inhibin.In the circulation, inhibin A, inhibin B, and activin A are detectable, as well as, named a precursor of the α chain pro-αC. Pro-αC is a monomeric pro-α subunit with no biologic activity. Inhibins control FSH secretion throughout the menstrual cycle. In particular, inhibin A is synthesized by the dominant follicle and the corpus luteum, whereas inhibin B is secreted by granulosa cells of developing antral follicles (5Petraglia F. Zanin E. Faletti A. Reis F.M. Inhibins: paracrine and endocrine effects in female reproductive function.Curr Opin Obstet Gynecol. 1999; 11: 241-247Crossref PubMed Scopus (27) Google Scholar). There are age-related changes concerning inhibin release. Low levels of both inhibin A and inhibin B are typical in older fertile and postmenopausal women. Indeed, women with POF have very low levels of inhibins. Activin A stimulates FSH synthesis and secretion and some changes in its level have been shown during the menstrual cycle in fertile women, whereas no changes are seen in postmenopausal women (6Burger H.G. Dudley E.C. Hopper J.L. Shelly J.M. Green A. Smith A et al.The endocrinology of the menopausal transition: a cross-sectional study of a population-based sample.J Clin Endocrinol Metab. 1995; 80: 3537-3545Crossref PubMed Google Scholar, 7Muttukrishna S. Child T. Lockwood G.M. Groome N.P. Barlow D.H. Ledger W.L. Serum concentrations of dimeric inhibins, activin A, gonadotrophins and ovarian steroids during the menstrual cycle in older women.Hum Reprod. 2000; 15: 549-556Crossref PubMed Scopus (75) Google Scholar, 8Petraglia F. Hartmann B. Luisi S. Florio P. Kirchengast S. Santuz M et al.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.Fertil Steril. 1998; 70: 907-912Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).Although the pathogenesis of POF is still unknown, cellular mechanisms can be suggested to be responsible for the degree of residual ovarian function, including a reduced primordial follicle pool and an alteration of follicular recruitment and maturation (9Christin-Maitre S. Vasseur C. Portnoi M.F. Bouchard P. Genes and premature ovarian failure.Mol Cell Endocrinol. 1998; 145: 75-80Crossref PubMed Scopus (64) Google Scholar). Recently, a longitudinal study has demonstrated that women with amenorrhea induced by GnRH-analogue treatment or by chemotherapy still produce inhibin A and pro-αC, thus indicating residual ovarian activity despite granulosa cell suppression (10Cobellis L. Luisi S. Pezzani I. Reis F.M. De Leo V. Petraglia F. Serum inhibin A, inhibin B, and pro-alphaC levels are altered after surgically or pharmacologically induced menopause.Fertil Steril. 2002; 77: 745-749Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The current study was carried out to evaluate the serum levels of inhibin A, inhibin B, activin A, and pro-αC in women with POF and postmenopausal women and to compare them with serum levels in healthy fertile women.The study design included three groups of women. Group 1 comprised 30 women with POF (median age 32 years; range, 28–39 years). The POF status was defined as follows. Age <40 years, age of menarche between 10 and 12 years, secondary amenorrhea between 6 and 38 months (14.4 ± 8.2 months), E2 concentration 40 mIU/mL detected on two different occasions. Screening tests for autoimmune disease and genetic factors were carried out by clinical examination, leucocyte karyotype, and serum tests for antinuclear and antiovarian antibodies (11Wheatcroft N.J. Salt C. Ward-Milford A. Cooke I.D. Weetman A.P. Identification of ovarian antibodies by immunofluorescence, enzyme-linked immunosorbent assay or immunoblotting in premature ovarian failure.Hum Reprod. 1997; 12: 2617-2622Crossref PubMed Scopus (65) Google Scholar, 12Hoek A. Schoemaker J. Drexhage H.A. Premature ovarian failure and ovarian autoimmunity.Endocr Rev. 1997; 18: 107-134Crossref PubMed Scopus (390) Google Scholar, 13Anasti J.N. Kalantaridou S.N. Kimzey L.M. Defensor R.A. Nelson L.M. Bone loss in young women with karyotypically normal spontaneous premature ovarian failure.Obstet Gynecol. 1998; 91: 12-15Crossref PubMed Scopus (120) Google Scholar). None of the women with POF screened positive for any autoimmune disease.Group 2 comprised 30 postmenopausal women (median age 55 years; range, 50–61 years) who had experienced a natural menopause. The time between decreased menstruation and final menstruation was 2–11 years. All women had serum E2 concentrations of 40 mIU/mL. Women with climacteric symptoms or malignant conditions were excluded.Group 3 comprised 30 healthy, fertile women (median age 30 years; range, 25–35 years). All women were required to have regular ovulatory menstrual cycles, to be healthy with normal body mass index, and to have no past or current reproductive endocrine problems. Luteinizing hormone, FSH, and E2 concentrations were measured in the early follicular phase. Dominant follicle development and ovulation were monitored by transvaginal ultrasound. Complete medical and gynecologic history were undertaken for all patients. Appropriate routine tests were performed to exclude internal diseases.Serum concentrations of LH, FSH, and E2 were determined by specific immunoassays. Women with adrenal, thyroid, and pituitary disorders were excluded from the study. All women were required to be taking no medications or exogenous hormones. The study protocol was approved by the institutional review board, University of Mainz, Germany,and written consent was obtained from all volunteers. Blood specimens were collected at 9 am by venipuncture after overnight fasting. After centrifugation for 10 minutes, the serum was stored at −70°C until it was assayed. Serum concentrations of LH, FSH, and E2 were measured by a fully automated immunoassay system (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions.Serum concentrations of inhibin A, inhibin B, pro-αC, and activin A were measured by specific, commercial, two-site ELISAs (Serotec, Oxford, United Kingdom) according to the manufacturer's recommendations as described previously (14Groome N.P. Illingworth P.J. O'Brien M. Priddle J. Weaver K. Mc Neilly A.S. Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.J Clin Endocrinol Metab. 1995; 80: 2926-2932Crossref PubMed Google Scholar, 15Knight P.G. Muttukrishna S. Groome N.P. Development and application of a two-site enzyme immunoassay for the determination of “total”activin-A concentrations in serum and follicular fluid.J Endocrinol. 1996; 148: 267-279Crossref PubMed Scopus (259) Google Scholar, 16Groome N.P. Illingworth P.J. O'Brien M. Pai R. Rodger F.E. Mather J.P et al.Measurement of dimeric inhibin B throughout the human menstrual cycle.J Clin Endocrinol Metab. 1996; 81: 1401-1405Crossref PubMed Scopus (850) Google Scholar). The ELISAs had the following measurable ranges: concentration of inhibin A was 3.9–500 pg/mL, concentration of inhibin B was 15.6–1,000 pg/mL, concentration of pro-αC was 1.56–200 pg/mL, and concentration of activin A was 0.078–5.0 ng/mL. Intra- and interassay coefficients of variation were <10% for inhibin A, <8% for pro-αC, and <7% for inhibin B and activin A. The detection limit was <3.9 pg/mL for inhibin A, <15.6 pg/mL for inhibin B, <2.0 pg/mL for pro-αC, and <100 pg/mL for activin A. Cross-reactions for each assay with the various inhibin-related proteins were <1%.Statistical analysis of the data was performed by analysis of variance and the Wilcoxon signed rank test. P<.05 was considered statistically significant. The data were analyzed with commercial software (SPSS 10.0; SPSS, Chicago, IL).Women with POF and postmenopausal women showed low levels of inhibin A (<3.9 pg/mL) and inhibin B (<15.6 pg/mL), whereas levels of inhibin A (36.7 ± 10.5 pg/mL) and inhibin B (62.2 ± 21.4 pg/mL) were within the normal range in fertile controls. No statistically significant difference in the levels of activin A was observed in all three study groups (POF: 0.43 ± 0.19 ng/mL; postmenopausal women: 0.39 ± 0.12 ng/mL; controls: 0.38 ± 0.09 ng/mL). As illustrated in Figure 1, pro-αC levels in women with POF (76.6 ± 28.3 pg/mL) were significantly higher than in postmenopausal women (35.4 ± 26.9 pg/mL), but pro-αC levels increased significantly in fertile controls (135.2 ± 18.8 pg/mL, P<.05).This retrospective study showed that serum inhibin A and inhibin B levels are below the detection limit and thereby present as markedly low in women with premature ovarian failure and in postmenopausal women. In accordance with previous studies, it can be assumed that elevated FSH and decreased E2 levels are associated with a definitive lack of ovarian function, which is accompanied by low levels of inhibins (14Groome N.P. Illingworth P.J. O'Brien M. Priddle J. Weaver K. Mc Neilly A.S. Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.J Clin Endocrinol Metab. 1995; 80: 2926-2932Crossref PubMed Google Scholar, 17Burger H.G. Dudley E.C. Hopper J.L. Groome N. Guthrie J.R. Green A et al.Prospectively measured levels of serum follicle-stimulating hormone, estradiol, and the dimeric inhibins during the menopausal transition in a population-based cohort of women.J Clin Endocrinol Metab. 1999; 84: 4025-4030Crossref PubMed Scopus (351) Google Scholar). Serum concentrations of activin A were not significantly different in all study groups, a finding that supports the speculation that this proteohormone could not be used to differentiate between women with premature ovarian failure and postmenopausal women. Inhibin A is primarily secreted in the luteal phase; inhibin B is the physiologically relevant form of inhibin during the follicular phase (5Petraglia F. Zanin E. Faletti A. Reis F.M. Inhibins: paracrine and endocrine effects in female reproductive function.Curr Opin Obstet Gynecol. 1999; 11: 241-247Crossref PubMed Scopus (27) Google Scholar). There is a regulation of these proteohormones through FSH and insulin-like growth factor I, whereas FSH stimulates inhibin secretion in cultivated granulosa cells (18Seifer D.B. Gardiner A.C. Lambert-Messerlian G. Schneyer A.L. Differential secretion of dimeric inhibin in cultured luteinized granulosa cells as a function of ovarian reserve.J Clin Endocrinol Metab. 1996; 81: 736-739Crossref PubMed Scopus (64) Google Scholar, 19Welt C.K. Schneyer A.L. Differential regulation of inhibin B and inhibin A by follicle-stimulating hormone and local growth factors in human granulosa cells from small antral follicles.J Clin Endocrinol Metab. 2001; 86: 330-336Crossref PubMed Scopus (95) Google Scholar).The data presented support the hypothesis that such para- and autocrine effects in women with premature ovarian failure are not present. It is noteworthy that in all three study groups measurable serum levels of pro-αC could be established, and in women with premature ovarian failure these levels were significantly higher than in postmenopausal women. Whereas the finding in women with premature ovarian failure is the first reported in the literature, the data obtained for postmenopausal women confirm the findings of a study showing measurable serum levels of pro-αC in postmenopausal women (10Cobellis L. Luisi S. Pezzani I. Reis F.M. De Leo V. Petraglia F. Serum inhibin A, inhibin B, and pro-alphaC levels are altered after surgically or pharmacologically induced menopause.Fertil Steril. 2002; 77: 745-749Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar).To date, little is known about the role of the α subunit precursor pro-αC. The primary location for synthesis of pro-αC is the ovary. Pro-αC reaches its highest serum level during the luteal phase of the cycle. Several reports have suggested that pro-αC plays a role in ovarian tumorigenesis. Serum levels of pro-αC were increased in 41% of women with epithelial ovarian cancer and were 2-fold to 10-fold higher than in healthy postmenopausal women. In the same context, increased levels of pro-αC were found in the peritoneal fluid of both malignant and benign ovarian neoplasms (20Menon U. Riley S.C. Thomas J. Bose C. Dawnay A. Evans L.W et al.Serum inhibin, activin, and follistatin in postmenopausal women with epithelial ovarian carcinoma.Br J Obstet Gynaecol. 2000; 107: 1069-1074Crossref Scopus (42) Google Scholar, 21Ala-Fossi S.L. Mänpää J. Bläuer M. Aine R. Tuohimaa P. Punnonen R. Inhibin A, B and pro-αC in serum and peritoneal fluid in postmenopausal patients with ovarian tumors.Eur J Endocrinol. 2000; 142: 334-339Crossref PubMed Scopus (12) Google Scholar). A recent study has demonstrated that serum levels of inhibin A and pro-αC were significantly higher in women with amenorrhea induced by a GnRH-analogue for treatment of endometriosis and in women with amenorrhea induced by antineoplastic chemotherapy than those observed in menopause (10Cobellis L. Luisi S. Pezzani I. Reis F.M. De Leo V. Petraglia F. Serum inhibin A, inhibin B, and pro-alphaC levels are altered after surgically or pharmacologically induced menopause.Fertil Steril. 2002; 77: 745-749Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar).In accordance with these results, our data support the hypothesis of residual ovarian function despite granulosa cell suppression, but to date its exact mechanism can only be speculated. At the molecular level, studies for mutation analysis of the inhibin subunits (INHα, INHβA, INHβB) indicated that in 7% of a group of 43 women with premature ovarian failure, a functional mutation of the INHα gene was evident. These results were recently confirmed in an Italian cohort study. Thus, detection of premature ovarian failure could provide an opportunity for intervention, such as timely replacement therapy of inhibins in women to delay the onset of ovarian failure (22Shelling A.N. Burton K.A. Chand A.L. van Ee C.C. France J.T. Farquar C.M et al.Inhibin: a candidate gene for premature ovarian failure.Hum Reprod. 2000; 15: 2644-2649Crossref PubMed Scopus (147) Google Scholar, 23Marozzi A. Porta C. Vegetti W. Crosignani P.G. Tibiletti M.G. Dalpra L et al.Mutation analysis of the inhibin alpha gene in a cohort of Italian women affected by ovarian failure.Hum Reprod. 2002; 17: 1741-1745Crossref PubMed Scopus (93) Google Scholar).Premature ovarian failure is an idiopathic disorder with unclear etiology. In our study population, women with premature ovarian failure had significantly higher serum levels of pro-αC than in postmenopausal women. However, the secretion of pro-αC in women with premature ovarian failure is regulated by mechanisms that are not currently identified and understood. More studies are needed to clarify these mechanisms. Premature ovarian failure (POF) is defined as a syndrome causing amenorrhea, anovulation, hypoestrogenism, and increased gonadotropin levels in women aged <40 years (1Aiman J. Smentek C. Premature ovarian failure.Obstet Gynecol. 1985; 66: 9-14PubMed Google Scholar, 2Rebar R.W. Connolly H.V. Clinical features of young women with hypergonadotropic amenorrhea.Fertil Steril. 1990; 53: 804-810Abstract Full Text PDF PubMed Google Scholar). Inhibin A, inhibin B, and activin A are glycoproteins produced by granulosa cells that have been determined to play a role in the nonsteroidal regulation of pituitary FSH secretion. These compounds are members of the transforming growth factor β superfamily (3Woodruff T.K. Besecke L.M. Groome N. Draper L.B. Schwartz N.B. Weiss J. Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner.Endocrinology. 1996; 137: 5463-5467Crossref PubMed Google Scholar, 4Pangas S.A. Woodruff T.K. Activin signal transduction pathways.Trends Endocrinol Metab. 2000; 11: 309-314Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Inhibins are heterodimeric glycoproteins consisting of an α subunit linked by two disulphide bonds to one of the two related β subunits (βA, βB), resulting in inhibin A (αβA) and inhibin B (αβB). A homodimer of the βA subunit is the glycoprotein activin A, which has a function opposite to that of inhibin. In the circulation, inhibin A, inhibin B, and activin A are detectable, as well as, named a precursor of the α chain pro-αC. Pro-αC is a monomeric pro-α subunit with no biologic activity. Inhibins control FSH secretion throughout the menstrual cycle. In particular, inhibin A is synthesized by the dominant follicle and the corpus luteum, whereas inhibin B is secreted by granulosa cells of developing antral follicles (5Petraglia F. Zanin E. Faletti A. Reis F.M. Inhibins: paracrine and endocrine effects in female reproductive function.Curr Opin Obstet Gynecol. 1999; 11: 241-247Crossref PubMed Scopus (27) Google Scholar). There are age-related changes concerning inhibin release. Low levels of both inhibin A and inhibin B are typical in older fertile and postmenopausal women. Indeed, women with POF have very low levels of inhibins. Activin A stimulates FSH synthesis and secretion and some changes in its level have been shown during the menstrual cycle in fertile women, whereas no changes are seen in postmenopausal women (6Burger H.G. Dudley E.C. Hopper J.L. Shelly J.M. Green A. Smith A et al.The endocrinology of the menopausal transition: a cross-sectional study of a population-based sample.J Clin Endocrinol Metab. 1995; 80: 3537-3545Crossref PubMed Google Scholar, 7Muttukrishna S. Child T. Lockwood G.M. Groome N.P. Barlow D.H. Ledger W.L. Serum concentrations of dimeric inhibins, activin A, gonadotrophins and ovarian steroids during the menstrual cycle in older women.Hum Reprod. 2000; 15: 549-556Crossref PubMed Scopus (75) Google Scholar, 8Petraglia F. Hartmann B. Luisi S. Florio P. Kirchengast S. Santuz M et al.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.Fertil Steril. 1998; 70: 907-912Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Although the pathogenesis of POF is still unknown, cellular mechanisms can be suggested to be responsible for the degree of residual ovarian function, including a reduced primordial follicle pool and an alteration of follicular recruitment and maturation (9Christin-Maitre S. Vasseur C. Portnoi M.F. Bouchard P. Genes and premature ovarian failure.Mol Cell Endocrinol. 1998; 145: 75-80Crossref PubMed Scopus (64) Google Scholar). Recently, a longitudinal study has demonstrated that women with amenorrhea induced by GnRH-analogue treatment or by chemotherapy still produce inhibin A and pro-αC, thus indicating residual ovarian activity despite granulosa cell suppression (10Cobellis L. Luisi S. Pezzani I. Reis F.M. De Leo V. Petraglia F. Serum inhibin A, inhibin B, and pro-alphaC levels are altered after surgically or pharmacologically induced menopause.Fertil Steril. 2002; 77: 745-749Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The current study was carried out to evaluate the serum levels of inhibin A, inhibin B, activin A, and pro-αC in women with POF and postmenopausal women and to compare them with serum levels in healthy fertile women. The study design included three groups of women. Group 1 comprised 30 women with POF (median age 32 years; range, 28–39 years). The POF status was defined as follows. Age <40 years, age of menarche between 10 and 12 years, secondary amenorrhea between 6 and 38 months (14.4 ± 8.2 months), E2 concentration 40 mIU/mL detected on two different occasions. Screening tests for autoimmune disease and genetic factors were carried out by clinical examination, leucocyte karyotype, and serum tests for antinuclear and antiovarian antibodies (11Wheatcroft N.J. Salt C. Ward-Milford A. Cooke I.D. Weetman A.P. Identification of ovarian antibodies by immunofluorescence, enzyme-linked immunosorbent assay or immunoblotting in premature ovarian failure.Hum Reprod. 1997; 12: 2617-2622Crossref PubMed Scopus (65) Google Scholar, 12Hoek A. Schoemaker J. Drexhage H.A. Premature ovarian failure and ovarian autoimmunity.Endocr Rev. 1997; 18: 107-134Crossref PubMed Scopus (390) Google Scholar, 13Anasti J.N. Kalantaridou S.N. Kimzey L.M. Defensor R.A. Nelson L.M. Bone loss in young women with karyotypically normal spontaneous premature ovarian failure.Obstet Gynecol. 1998; 91: 12-15Crossref PubMed Scopus (120) Google Scholar). None of the women with POF screened positive for any autoimmune disease. Group 2 comprised 30 postmenopausal women (median age 55 years; range, 50–61 years) who had experienced a natural menopause. The time between decreased menstruation and final menstruation was 2–11 years. All women had serum E2 concentrations of 40 mIU/mL. Women with climacteric symptoms or malignant conditions were excluded. Group 3 comprised 30 healthy, fertile women (median age 30 years; range, 25–35 years). All women were required to have regular ovulatory menstrual cycles, to be healthy with normal body mass index, and to have no past or current reproductive endocrine problems. Luteinizing hormone, FSH, and E2 concentrations were measured in the early follicular phase. Dominant follicle development and ovulation were monitored by transvaginal ultrasound. Complete medical and gynecologic history were undertaken for all patients. Appropriate routine tests were performed to exclude internal diseases. Serum concentrations of LH, FSH, and E2 were determined by specific immunoassays. Women with adrenal, thyroid, and pituitary disorders were excluded from the study. All women were required to be taking no medications or exogenous hormones. The study protocol was approved by the institutional review board, University of Mainz, Germany,and written consent was obtained from all volunteers. Blood specimens were collected at 9 am by venipuncture after overnight fasting. After centrifugation for 10 minutes, the serum was stored at −70°C until it was assayed. Serum concentrations of LH, FSH, and E2 were measured by a fully automated immunoassay system (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. Serum concentrations of inhibin A, inhibin B, pro-αC, and activin A were measured by specific, commercial, two-site ELISAs (Serotec, Oxford, United Kingdom) according to the manufacturer's recommendations as described previously (14Groome N.P. Illingworth P.J. O'Brien M. Priddle J. Weaver K. Mc Neilly A.S. Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.J Clin Endocrinol Metab. 1995; 80: 2926-2932Crossref PubMed Google Scholar, 15Knight P.G. Muttukrishna S. Groome N.P. Development and application of a two-site enzyme immunoassay for the determination of “total”activin-A concentrations in serum and follicular fluid.J Endocrinol. 1996; 148: 267-279Crossref PubMed Scopus (259) Google Scholar, 16Groome N.P. Illingworth P.J. O'Brien M. Pai R. Rodger F.E. Mather J.P et al.Measurement of dimeric inhibin B throughout the human menstrual cycle.J Clin Endocrinol Metab. 1996; 81: 1401-1405Crossref PubMed Scopus (850) Google Scholar). The ELISAs had the following measurable ranges: concentration of inhibin A was 3.9–500 pg/mL, concentration of inhibin B was 15.6–1,000 pg/mL, concentration of pro-αC was 1.56–200 pg/mL, and concentration of activin A was 0.078–5.0 ng/mL. Intra- and interassay coefficients of variation were <10% for inhibin A, <8% for pro-αC, and <7% for inhibin B and activin A. The detection limit was <3.9 pg/mL for inhibin A, <15.6 pg/mL for inhibin B, <2.0 pg/mL for pro-αC, and <100 pg/mL for activin A. Cross-reactions for each assay with the various inhibin-related proteins were <1%. Statistical analysis of the data was performed by analysis of variance and the Wilcoxon signed rank test. P<.05 was considered statistically significant. The data were analyzed with commercial software (SPSS 10.0; SPSS, Chicago, IL). Women with POF and postmenopausal women showed low levels of inhibin A (<3.9 pg/mL) and inhibin B (<15.6 pg/mL), whereas levels of inhibin A (36.7 ± 10.5 pg/mL) and inhibin B (62.2 ± 21.4 pg/mL) were within the normal range in fertile controls. No statistically significant difference in the levels of activin A was observed in all three study groups (POF: 0.43 ± 0.19 ng/mL; postmenopausal women: 0.39 ± 0.12 ng/mL; controls: 0.38 ± 0.09 ng/mL). As illustrated in Figure 1, pro-αC levels in women with POF (76.6 ± 28.3 pg/mL) were significantly higher than in postmenopausal women (35.4 ± 26.9 pg/mL), but pro-αC levels increased significantly in fertile controls (135.2 ± 18.8 pg/mL, P<.05). This retrospective study showed that serum inhibin A and inhibin B levels are below the detection limit and thereby present as markedly low in women with premature ovarian failure and in postmenopausal women. In accordance with previous studies, it can be assumed that elevated FSH and decreased E2 levels are associated with a definitive lack of ovarian function, which is accompanied by low levels of inhibins (14Groome N.P. Illingworth P.J. O'Brien M. Priddle J. Weaver K. Mc Neilly A.S. Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.J Clin Endocrinol Metab. 1995; 80: 2926-2932Crossref PubMed Google Scholar, 17Burger H.G. Dudley E.C. Hopper J.L. Groome N. Guthrie J.R. Green A et al.Prospectively measured levels of serum follicle-stimulating hormone, estradiol, and the dimeric inhibins during the menopausal transition in a population-based cohort of women.J Clin Endocrinol Metab. 1999; 84: 4025-4030Crossref PubMed Scopus (351) Google Scholar). Serum concentrations of activin A were not significantly different in all study groups, a finding that supports the speculation that this proteohormone could not be used to differentiate between women with premature ovarian failure and postmenopausal women. Inhibin A is primarily secreted in the luteal phase; inhibin B is the physiologically relevant form of inhibin during the follicular phase (5Petraglia F. Zanin E. Faletti A. Reis F.M. Inhibins: paracrine and endocrine effects in female reproductive function.Curr Opin Obstet Gynecol. 1999; 11: 241-247Crossref PubMed Scopus (27) Google Scholar). There is a regulation of these proteohormones through FSH and insulin-like growth factor I, whereas FSH stimulates inhibin secretion in cultivated granulosa cells (18Seifer D.B. Gardiner A.C. Lambert-Messerlian G. Schneyer A.L. Differential secretion of dimeric inhibin in cultured luteinized granulosa cells as a function of ovarian reserve.J Clin Endocrinol Metab. 1996; 81: 736-739Crossref PubMed Scopus (64) Google Scholar, 19Welt C.K. Schneyer A.L. Differential regulation of inhibin B and inhibin A by follicle-stimulating hormone and local growth factors in human granulosa cells from small antral follicles.J Clin Endocrinol Metab. 2001; 86: 330-336Crossref PubMed Scopus (95) Google Scholar). The data presented support the hypothesis that such para- and autocrine effects in women with premature ovarian failure are not present. It is noteworthy that in all three study groups measurable serum levels of pro-αC could be established, and in women with premature ovarian failure these levels were significantly higher than in postmenopausal women. Whereas the finding in women with premature ovarian failure is the first reported in the literature, the data obtained for postmenopausal women confirm the findings of a study showing measurable serum levels of pro-αC in postmenopausal women (10Cobellis L. Luisi S. Pezzani I. Reis F.M. De Leo V. Petraglia F. Serum inhibin A, inhibin B, and pro-alphaC levels are altered after surgically or pharmacologically induced menopause.Fertil Steril. 2002; 77: 745-749Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). To date, little is known about the role of the α subunit precursor pro-αC. The primary location for synthesis of pro-αC is the ovary. Pro-αC reaches its highest serum level during the luteal phase of the cycle. Several reports have suggested that pro-αC plays a role in ovarian tumorigenesis. Serum levels of pro-αC were increased in 41% of women with epithelial ovarian cancer and were 2-fold to 10-fold higher than in healthy postmenopausal women. In the same context, increased levels of pro-αC were found in the peritoneal fluid of both malignant and benign ovarian neoplasms (20Menon U. Riley S.C. Thomas J. Bose C. Dawnay A. Evans L.W et al.Serum inhibin, activin, and follistatin in postmenopausal women with epithelial ovarian carcinoma.Br J Obstet Gynaecol. 2000; 107: 1069-1074Crossref Scopus (42) Google Scholar, 21Ala-Fossi S.L. Mänpää J. Bläuer M. Aine R. Tuohimaa P. Punnonen R. Inhibin A, B and pro-αC in serum and peritoneal fluid in postmenopausal patients with ovarian tumors.Eur J Endocrinol. 2000; 142: 334-339Crossref PubMed Scopus (12) Google Scholar). A recent study has demonstrated that serum levels of inhibin A and pro-αC were significantly higher in women with amenorrhea induced by a GnRH-analogue for treatment of endometriosis and in women with amenorrhea induced by antineoplastic chemotherapy than those observed in menopause (10Cobellis L. Luisi S. Pezzani I. Reis F.M. De Leo V. Petraglia F. Serum inhibin A, inhibin B, and pro-alphaC levels are altered after surgically or pharmacologically induced menopause.Fertil Steril. 2002; 77: 745-749Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). In accordance with these results, our data support the hypothesis of residual ovarian function despite granulosa cell suppression, but to date its exact mechanism can only be speculated. At the molecular level, studies for mutation analysis of the inhibin subunits (INHα, INHβA, INHβB) indicated that in 7% of a group of 43 women with premature ovarian failure, a functional mutation of the INHα gene was evident. These results were recently confirmed in an Italian cohort study. Thus, detection of premature ovarian failure could provide an opportunity for intervention, such as timely replacement therapy of inhibins in women to delay the onset of ovarian failure (22Shelling A.N. Burton K.A. Chand A.L. van Ee C.C. France J.T. Farquar C.M et al.Inhibin: a candidate gene for premature ovarian failure.Hum Reprod. 2000; 15: 2644-2649Crossref PubMed Scopus (147) Google Scholar, 23Marozzi A. Porta C. Vegetti W. Crosignani P.G. Tibiletti M.G. Dalpra L et al.Mutation analysis of the inhibin alpha gene in a cohort of Italian women affected by ovarian failure.Hum Reprod. 2002; 17: 1741-1745Crossref PubMed Scopus (93) Google Scholar). Premature ovarian failure is an idiopathic disorder with unclear etiology. In our study population, women with premature ovarian failure had significantly higher serum levels of pro-αC than in postmenopausal women. However, the secretion of pro-αC in women with premature ovarian failure is regulated by mechanisms that are not currently identified and understood. More studies are needed to clarify these mechanisms.