Serum GDF9 and BMP15 as potential markers of ovarian function in women with and without polycystic ovary syndrome.

OBJECTIVE: Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are critical paracrine regulators of female fertility and are predominantly expressed by oocytes. However, it is unknown if serum concentrations reflect changes in ovarian function and/or reproductive endocrine disorders. This study aimed to determine if serum GDF9/BMP15 are associated with ovarian, pituitary, oestrogenic, androgenic and metabolic characteristics and the ovarian pathologies, polycystic ovarian morphology (PCOM) and polycystic ovary syndrome (PCOS). DESIGN: Women aged 21-45 years (n = 381) were included from a cross-sectional study at the National University Hospital, Singapore. PATIENTS: Participants were volunteers and patients with possible PCOS. MEASUREMENTS: Anthropometric measurements, transvaginal ultrasound scans and serum sampling were performed and a questionnairecompleted. Serum GDF9 and BMP15 concentrations were matched with menstrual cycle length, ovarian protein and steroid hormone production, pituitary hormone production and metabolic assessments in women with PCOM or PCOS and those with neither (control). RESULTS: Serum GDF9 and BMP15 were detectable in 40% and 41% of women, respectively and were positively correlated with each other (r = 0.08, p = 0.003). GDF9, but not BMP15, was positively correlated with ovarian volume (p = 0.02) and antral follicle count (AFC) (p = 0.004), but not with anti-Müllerian hormone (p = 0.05). However, serum GDF9 and BMP15 concentrations were not significantly different between control, PCOM and PCOS women, nor associated with androgenic or metabolic PCOS features. However, the relationship between GDF9 and AFC differed between control, PCOM and PCOS women (p = 0.02). CONCLUSIONS: Serum GDF9 and BMP15 concentrations somewhat reflect ovarian but not androgenic or metabolic characteristics of PCOS, with increased GDF9 reflecting high AFC as seen in PCOM/PCOS.

Serum Concentrations of Oocyte-Secreted Factors BMP15 and GDF9 During IVF and in Women With Reproductive Pathologies.

Oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n = 154) and from perimenopausal and postmenopausal women (n = 28). Serum concentrations of BMP15 and GDF9 were analyzed in women relative to age, anti-Müllerian hormone, number of oocytes retrieved, and polycystic ovary syndrome (PCOS) after superovulation for in vitro fertilization. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/mL, respectively), and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64-fold and 15-fold, respectively, between women, but they did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15 concentration, was associated with oocyte number retrieved in patients without PCOS (P = 0.018). GDF9 and BMP15 associations with oocyte number differed significantly (P < 0.05) with PCOS status. GDF9 concentrations were lower in poor responders (women with fewer than four oocytes retrieved or with cancelled cycles; P = 0.020). Serum BMP15, but not GDF9, was lower in women >55 years of age, compared with women of reproductive age (P < 0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro.

The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.

Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery.

Failure of the endometrium to achieve receptivity results in infertility, and it is also a rate-limiting step in in vitro fertilization (IVF) success. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. Although a number of gene array studies have defined mRNA changes across the cycle, with infertility, and in IVF cycles, these have not generally been informative due in part to the subsequent regulation of transcription and posttranslational modifications of the proteins. State-of-the-art proteomic technologies now enable analysis of changes in the endometrium and its secretome related to cycle phase and associated with infertility. These techniques include two-dimensional differential in-gel electrophoresis, isobaric tags for relative and absolute quantitation, and multiplex analyses of selected panels of markers. Subsequent definition of cellular location, timing of production of identified proteins, and their regulation by steroid hormones and blastocyst-derived factors provide indications of their functions and their relationship to the establishment of pregnancy. Proteins discovered by proteomic analyses and fully evaluated will provide the differentiative profiles necessary to inform clinical practice and serve as an end point for optimizing stimulation cycles in IVF clinics as well as more clearly defining the molecular mechanisms underlying successful implantation.

Inhibins and activins in blood: predictors of female reproductive health?

Inhibins A and B are gonadal factors which are important in fertility. Their use as predictors of female reproductive health has centred on their application to ovarian cancer, Anorexia Nervosa, Down Syndrome and preeclampsia. Inhibin B also provides an index of the endocrine feedback relationship between the ovary and pituitary particularly when the ovarian follicle reserve is low. These applications are relevant in monitoring the onset of the menopause transition, ovarian recovery following chemotherapy and disturbances in pubertal development. Currently, these applications have only found widespread use in Down Syndrome and ovarian cancer. Activins, on the other hand, appear to have a limited application.

Hemochromatosis and ovarian cancer.

Evaluation of: Gannon PO, Medelci S, Le Page C et al. Impact of hemochromatosis gene (HFE) mutations on epithelial ovarian cancer risk and prognosis. Int. J. Cancer 128(10), 2326-2334 (2011). The frequency of two mutations (C282Y and D62H) of the hemochromatosis gene were investigated in women with ovarian cancer. A single allele mutation of the C282Y but not the H63D gene product was detected in 8-9% of women with benign ovarian tumors (n = 124) and ovarian cancers (n = 360) compared with 2.5% for controls (n = 80) representing a 4.9-fold increase in risk. With high-grade serous ovarian cancers (n = 179), the survival rate of women with a single allele C282Y mutation was reduced from 39 to 19 months. These results implicate mutations of the hemochromatosis gene in the generation and severity of ovarian cancers, which may have prognostic value.

Generation of a specific activin antagonist by modification of the activin A propeptide.

Elevated activin A levels in inhibin-deficient mice promote the development of gonadal tumors and induce cachexia by reducing muscle, liver, stomach, and fat mass. Because activin A is an important regulator of tissue growth, inhibiting the actions of this TGFß family ligand may halt or reverse pathology in diseased tissues. In this study, we modified the activin A propeptide to generate a specific activin antagonist. Propeptides mediate the synthesis and secretion of all TGFß ligands and, for some family members (e.g. TGFß1), bind the mature growth factor with high enough affinity to confer latency. By linking the C-terminal region of the TGFß1 propeptide to the N-terminal region of the activin A propeptide, we generated a chimeric molecule [activin/TGFß1 propeptide (AT propeptide)] with increased affinity for activin A. The AT propeptide was 30-fold more potent than the activin A propeptide at suppressing activin-induced FSH release by LßT2 pituitary gonadotrope cells. Binding of the AT propeptide to activin A shields the type II receptor binding site, thereby reducing Smad2 phosphorylation and downstream signaling. In comparison with the commonly used activin antagonists, follistatin (IC(50) 0.42 nM), soluble activin type II receptor A-Fc (IC(50) 0.47 nM), and soluble activin type II receptor B-Fc (IC(50) 0.91 nM), the AT propeptide (IC(50) 2.6 nM) was slightly less potent. However, it was more specific, inhibiting activin A and activin B (IC(50) 10.26 nM) but not the closely related ligands, myostatin and growth differentiation factor-11. As such, the AT propeptide represents the first specific activin antagonist, and it should be an effective reagent for blocking activin actions in vivo.

Feedback regulation by inhibins A and B of the pituitary secretion of follicle-stimulating hormone.

Inhibins A and B are gonadal factors that negatively regulate FSH synthesis by the anterior pituitary. Across the menstrual cycle, women show a strong inverse correlation between circulating FSH and inhibin B, estradiol, and anti-Mullerian hormone (AMH), but not with inhibin A. Estradiol is believed to provide a tonic inhibitory effect while the inhibitory role of AMH is unknown. In human males, inhibin B is the primary testicular factor regulating FSH with limited effects by gonadal steroids. In vitro and in vivo studies in rats indicate that inhibin B is more biologically active than inhibin A but showed a lower affinity for the activin type II receptors and the co-receptor, betaglycan, suggesting an alternative mechanism. While this review reinforces the important role inhibin plays in regulating FSH, the observed differences in mode of action of inhibins A and B and their interplay with other gonadal factors are still poorly understood.

Changes in serum antimüllerian hormone levels across the ovulatory menstrual cycle in late reproductive age.

OBJECTIVE: The aim of this study was to assess the pattern of serum antimüllerian hormone (AMH) across the normal ovulatory menstrual cycle in women in late reproductive age when ovarian follicle reserve and, hence, serum AMH levels are reduced. METHODS: Serum AMH levels were determined by enzyme-linked immunosorbent assay across the ovulatory menstrual cycle from women in mid (n = 18) and late (n = 43) reproductive life, including the menopausal transition. RESULT: : No intracycle variation in AMH level was observed in women in mid reproductive life nor in 33% (n = 14) of women with normal ovulatory cycles in late reproductive age. In the remaining cycles, a significant 2-fold decrease (P < 0.01) in AMH in 11 cycles and a significant 4.2-fold increase (P < 0.01) in 10 cycles were observed between the follicular and luteal phases. In a further eight ovulatory cycles, AMH was below the level of assay detection. As ovarian follicle reserve decreases with age and AMH levels are reduced, separate patterns of AMH are detected in the follicular and luteal phases of ovulatory menstrual cycles, presumably reflecting the intermittent pattern of the emergence of follicles close to menopause. CONCLUSIONS: It is concluded that when AMH levels are substantially reduced, as in late reproductive age, they become less reliable as markers of ovarian reserve because of the changing patterns observed in some cycles.

Gonadotropins regulate rat testicular tight junctions in vivo.

Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG +/- FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs.

Two distinct regions of latency-associated peptide coordinate stability of the latent transforming growth factor-beta1 complex.

Transforming growth factor-beta1 (TGF-beta1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-beta1 propeptide (latency-associated peptide (LAP)), and latent TGF-beta-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-beta1 complex. Initially, hydrophobic LAP residues (Ile(53), Leu(54), Leu(57), and Leu(59)), which form a contiguous epitope on one surface of an amphipathic alpha-helix, interact with mature TGF-beta1 to form the small latent complex. TGF-beta1 binding is predicted to alter LAP conformation, exposing ionic residues (Arg(45), Arg(50), Lys(56), and Arg(58)) on the other side of the alpha-helix, which form the binding site for latent TGF-beta-binding proteins. The stability of the resultant large latent complex is dependent upon covalent dimerization of LAP, which is facilitated by key residues (Phe(198), Asp(199), Val(200), Leu(208), Phe(217), and Leu(219)) at the dimer interface. Significantly, genetic mutations in LAP (e.g. R218H) that cause the rare bone disorder Camurati-Engelmann disease disrupted dimerization and reduced the stability of the latent TGF-beta1 complex.

Post-translational modifications and protein-specific isoforms in endometriosis revealed by 2D DIGE.

Endometriosis is a chronic disorder affecting approximately 10% of women in whom endometrial tissue forms painful lesions outside the uterus. It has a major impact on their physical, mental and social well-being but has no known cure, and there is no nonsurgical means of diagnosis. We have used a proteomic approach to identify proteins with altered abundance in the eutopic endometrium of endometriosis patients in the midsecretory phase of the menstrual cycle. 2D-differential in gel electrophoresis (DIGE) and mass spectrometry identified 20 proteins that were present at different levels in endometriosis patients (p < 0.05), many of which have not previously been associated with endometriosis. Protein abundance changes did not correlate well with published gene array data, emphasizing the extensive post-translational modification that occurs in this tissue. Abundance or localization changes in endometrial tissue were validated by immunohistochemistry and Western blotting for three proteins, vimentin (VIM), peroxiredoxin 6 (PRDX6), and ribonuclease/angiogenin inhibitor 1 (RNH1), while observed changes could not be confirmed for coronin 1A (CORO1A) or transgelin (TAGLN2). In addition, multiple charge and size isoforms were observed for PDRX6 and vimentin (VIM), and an additional PDRX6 isoform was observed in endometriosis patients that was below the level of detection in healthy women. Biological pathway analysis identified that cytoskeletal remodeling via keratin intermediate filaments, processing of the cystic fibrosis transmembrane receptor (CFTR), the glucocorticoid receptor subunit alpha (GCR), and heat shock factor 1 (HSF1) were all significantly over-represented features in endometriosis patients. This study highlights the highly dynamic nature of endometrial tissue and suggests that considerable post-translational modification of proteins is a key factor in the pathology of endometriosis.

Expression of nodal signalling components in cycling human endometrium and in endometrial cancer.

BACKGROUND: The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development. METHODS: In this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas. RESULTS: Nodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues. CONCLUSION: The expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

Proteomic characterization of midproliferative and midsecretory human endometrium.

This study aimed to identify proteins differentially expressed in the human endometrium between the proliferative and secretory phases of normal menstrual cycles by 2D differential in-gel electrophoresis (DIGE). A total of 196 out of 1017 spots were differentially expressed (p < 0.05). Mass spectrometry identified 76 proteins representing 41 different gene products. Immunohistochemistry confirmed the observed changes in 3 representative proteins (Rho-GDIalpha, CLIC1, PGRMC1). Biological pathway analysis identified the Jnk and EGF signaling pathways as key regulators of protein expression in the midsecretory phase of endometrial proteome.

Ovarian status in healthy postmenopausal women: follow-up 12 months after transvaginal ultrasound.

OBJECTIVE: We have previously reported on the point prevalence of ovarian lesions detected by transvaginal ultrasound (TVU) in 515 asymptomatic women at least 5 years postmenopause. The aims of this study were to report, in the same women, on the repeatability of visualization of the ovaries (TVU) and the natural history of ovarian lesions seen at baseline but not treated surgically and to assess whether any women developed new ovarian abnormalities 12 months later. METHODS: The study involved a cohort of 515 postmenopausal women recruited from the community, at least 5 years past their last period. They were assessed at baseline and again after 12 months with TVU and serum levels of inhibin and CA-125. RESULTS: The right and left ovaries were seen on both occasions in 80% and 68% of women, respectively. Of the 49 women who had an ovarian lesion at baseline, did not undergo surgery at that time, and had a follow-up TVU, the lesion was unchanged 12 months later in 30 women. Four women developed a new ovarian lesion within the 12 months. None of the 14 women who underwent surgery on the basis of the ovarian appearance at baseline, or the 2 who had surgery on the basis of the ovarian appearance at follow-up, had an ovarian malignancy. CONCLUSIONS: The use of TVU in women at least 5 years after menopause is problematic because the ovaries cannot be visualized in all women and because TVU has the potential to identify many benign lesions that would otherwise remain undetected. These are important considerations in weighing up the risks and benefits of using TVU as a screening tool.

Atypical estradiol secretion and ovulation patterns caused by luteal out-of-phase (LOOP) events underlying irregular ovulatory menstrual cycles in the menopausal transition.

OBJECTIVE: The menopausal transition is characterized by irregular menstrual cycles and unpredictable hormone levels, including dramatic swings in estradiol (E2). An increasing number of studies have found variable high E2 and low luteal phase progesterone occur with progression of Stages of Reproductive Aging Workshop (STRAW)stage, but the cause remains unclear. To explore the causes of the erratic changes in E2, individual within-cycle secretion patterns of E2, progesterone, follicle-stimulating hormone, luteinizing hormone, inhibin A, and inhibin B were explored in detail. DESIGN: Blood samples taken three times per week over 1 1/3 menstrual cycles from 77 women aged 21 to 55 classified as mid-reproductive age (STRAW stages 5 and 4; n = 21), late-reproductive age (STRAW stages 4 and 3; n = 16), early menopausal transition (STRAW stage 2; n = 17), and late menopausal transition (STRAW stage 1; n = 23) were analyzed. RESULTS: Eleven of the 29 (37%) early and late menstrual transition ovulatory cycles exhibited a specific pattern of E2 secretion that was characterized by a second increase in E2 during the mid- and late luteal phases and that continued to a peak during the subsequent menstrual phase. This second rise and fall in E2 was typical in appearance of a normal follicular phase, except that it was superimposed on an existing ovulatory cycle(specifically during the luteal and menstrual phases). The pattern was therefore referred to as a luteal out-of-phase(LOOP) follicular event. In four of these LOOP cycles, a luteinizing hormone peak and ovulatory episode followed the second E2 peak early in the subsequent cycle. Compared with the typical ovulatory cycles, the cycles with LOOP events exhibited lower luteal phase progesterone, higher early cycle follicle-stimulating hormone, and lower early cycle inhibin B. They were also associated with abnormally short (<21 d) or long (>40 d) cycle length. CONCLUSIONS: Many of the marked increases in ovulatory cycle E2 and cycle irregularities during the menopausal transition may be due to LOOP events and appear to be triggered by prolonged high follicular phase follicle-stimulating hormone levels.

Interrelationships between ovarian and pituitary hormones in ovulatory menstrual cycles across reproductive age.

CONTEXT: Ovarian hormones regulate pituitary gonadotropin secretion across the menstrual cycle via negative and positive feedback mechanisms. The contribution of individual hormones is complex and is a continuing area of research. OBJECTIVE: The aim of the study was to identify relationships between LH/FSH and estradiol, progesterone, inhibin A, inhibin B, and anti-Mullerian hormone (AMH) in ovulatory menstrual cycles across reproductive age. DESIGN: Serum ovarian and pituitary hormones were studied in a group of young (<35 yr; n = 21) and older (>45 yr; n = 55) women. The slopes of the regression lines relating the ovarian and pituitary hormones were determined by multiple linear regression analysis and expressed with 95% confidence intervals for each ovarian hormone, with FSH and LH as independent variables. Both simultaneous and delayed (time lagged) relationships were examined. RESULTS: Clear associations were evident for the lagged prediction of FSH, with significant negative associations being evident with inhibin B and AMH in the follicular phase and with estradiol, inhibin B, progesterone, and AMH in the luteal phase. For the lagged prediction of LH, significant positive and negative associations were observed with estradiol and inhibin B, respectively, in the follicular phase and a negative association with progesterone and inhibin B in the luteal phase. CONCLUSIONS: It is concluded that in the follicular phase, inhibin B is a major feedback regulator of FSH and may also be a negative feedback regulator of LH. AMH may be indirectly involved in FSH regulation.

Anti-Müllerian hormone as a marker of ovarian reserve: an update.

In recent years there has been an increasing interest in the role of anti-Müllerian hormone or Müllerian-inhibiting substance as a marker of ovarian reserve during assisted reproduction treatment and other reproductive processes. It is concluded that anti-Müllerian hormone is superior to other markers in predicting oocyte yield in IVF and appears useful in monitoring ovarian response in a range of reproductive states and disorders. This article reviews the literature published during 2006 and 2007.

A proposed classification system for menstrual cycles in the menopause transition based on changes in serum hormone profiles.

OBJECTIVE: To characterize menstrual cycles in women in late reproductive age and the menopause transition, based on changes in serum hormone levels. DESIGN: Serum levels of estradiol, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone, inhibin A, inhibin B, and antimüllerian hormone, as previously reported as mean data grouped according to the Stages of Reproductive Aging Workshop proposals, were analyzed in 55 women aged 45 to 55 and compared with those in 21 women aged 21 to 35. RESULTS: The ovulatory cycles in the older women were divided into three types. Type 1 cycles (n = 14, 33%) were those with hormone concentrations similar to the women aged 21 to 35 except for 20-fold lower antimüllerian hormone levels. Type 2 cycles (n = 24; 53%) had increased FSH, decreased inhibin B, and increased FSH-to-inhibin B ratios but normal estradiol and progesterone levels. Type 3 cycles had the same characteristics as type 2 cycles (n = 5; 12%) in addition to lower luteal phase progesterone and increased luteinizing hormone. CONCLUSIONS: The changes in hormone levels indicated in cycle types 1 to 3 closely reflect the changes in ovarian-pituitary activity as menopause approaches and are likely to be directly attributable to a decrease in ovarian follicle reserve. The findings suggest that FSH-to-inhibin B ratios and antimüllerian hormone are distinct early indicators of the menopause transition and are likely to be useful biomarkers of impending menopause. Furthermore, this classification may provide an improved basis for the study of reproductive endocrine disorders associated with the menopause transition.