Inhibin: a candidate gene for premature ovarian failure

Andrew N. Shelling1,7, Karen A. Burton1, Ashwini L. Chand1, Cynthia C. van Ee1, John T. France1, Cynthia M. Farquhar1, Stella R. Milsom2, Donald R. Love3, Ksenija Gersak5, Kristiina Aittomäki6 and Ingrid M. Winship4

1 Research Centre in Reproductive Medicine, Department of Obstetrics and Gynaecology and 2 Fertility PLUS, National Women's Hospital, Auckland, 3 School of Biological Sciences and 4 Department of Molecular Medicine, University of Auckland, Auckland, New Zealand, 5 Department of Obstetrics and Gynecology, University Medical Centre, Ljubljana, Slovenia and 6 Department of Clinical Genetics, Helsinki University Central Hospital, Helsinki, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Premature ovarian failure (POF) occurs in 1% of all women, and in 0.1% of women under the age of 30 years. The mechanisms that give rise to POF are largely unknown. Inhibin has a role in regulating the pituitary secretion of FSH, and is therefore a potential candidate gene for ovarian failure. Using single-stranded conformation polymorphism (SSCP) and DNA sequencing, DNA samples were screened from 43 women with POF for mutations in the three inhibin genes. Two variants were found: a 1032C->T transition in the INHßA gene in one patient, and a 769G->A transition in the INH{alpha} gene in three patients. The INHßA variant appears to be a polymorphism, as there was no change in the amino acid sequence of the gene product. The INH{alpha} variant resulted in a non-conservative amino acid change, with a substitution from alanine to threonine. This alanine is highly conserved across species, and has the potential to affect receptor binding. The INH{alpha} variant is significantly associated with POF (3/43 patients; 7%) compared with control samples (1/150 normal controls; 0.7%) (Fisher's exact test, P < 0.035). Further analysis of the inhibin gene in POF patients and matched controls will determine its role in the aetiology of POF.

Key words: infertility/inhibin/mutation detection/ovarian failure/premature


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Premature ovarian failure (POF) is a condition causing amenorrhoea, infertility, hypo-oestrogenism and elevated gonadotrophin concentrations in women under the age of 40 years. It is a common condition that occurs in 1% of all women, and in 0.1% of women before the age of 30 years (Coulam et al., 1986Go). Two significant consequences of POF are the loss of fertility, and the clinical effects of hypo-oestrogenism. Intermittent ovarian function has been reported in some women, and pregnancy can occur in ~5–10% of patients subsequent to diagnosis (Kalantaridou et al., 1998Go). Low concentrations of oestrogen from a young age appear to increase the risk of osteoporosis and coronary heart disease (Conway, 1997Go).

The best known causes for ovarian failure are sex chromosome abnormalities such as Turner syndrome. In most patients with POF and normal female chromosome constitution, however, no cause can be identified (for recent reviews see Conway, 1997; Anasti, 1998; Kalantaridou et al., 1998; Shelling, 2000). Iatrogenic agents, such as chemotherapy or radiotherapy, are known to reduce follicle numbers and cause POF. Although autoimmune diseases are seen in 10–20% of women with POF (Conway, 1997Go), the role of autoimmunity remains controversial in patients without other signs of autoimmune endocrine disease. Infections, such as mumps, have also been suggested to cause oophoritis resulting in ovarian failure. In a minority of cases a specific causative factor can be identified. These include galactosaemia, enzyme deficiencies and defects of gonadotrophin signalling. Approximately 5% of women with POF have a positive family history (Conway, 1997Go), suggesting an inherited predisposition to POF. Candidate genes or loci that have been suggested to cause either familial or sporadic POF include genes on the X chromosome (POF1, POF2, FMR1) and 3q22-3q23 in families with POF and blepharophimosis (recently reviewed by Shelling, 2000).

Most women with POF are found to have follicles, but they do not appear to respond to normal gonadotrophin stimulation (Conway, 1997Go). However, very few mutations have been identified in gonadotrophin hormones or their receptors. The loss of function mutation, 566C->T, identified in the FSH receptor (Aittomaki et al., 1995Go) was found to cause ovarian failure with amenorrhoea in a group of Finnish families. It appears that this FSH receptor mutation is rare elsewhere, as it has not been detected in other populations (Conway, 1997Go; Layman et al., 1998Go). Ovarian resistance has also been seen in association with a premature stop codon in the LH receptor gene (Latronico et al., 1996Go). These data demonstrate the obvious importance of the FSH axis in ovarian function, and the possibility that abnormalities in the FSH response genes may be candidates for POF.

The glycoprotein, inhibin, is a potential candidate for POF due to its role in the negative feedback control of FSH, which has a pivotal role in the recruitment and development of ovarian follicles during folliculogenesis. Inhibin is structurally related to the transforming growth factor (TGF)-ß superfamily, a group of multi-functional growth and differentiation factors. The mature inhibin is a 31–32 kDa heterodimeric glycoprotein consisting of an 18 kDa {alpha}-subunit linked by two disulphide bonds to one of two 14 kDa ß-subunits (Vale et al., 1988Go). There are two forms of inhibin; inhibin A ({alpha}-ßA), and inhibin B ({alpha}-ßB). The homodimer of the ß-subunit is the glycoprotein activin, which has an opposing function to inhibin. The inhibin subunits are encoded by three separate genes: INH{alpha}, INHßA and INHßB, which map to 2q33-qter, 2cen-q13 and 7p15-p14 respectively (Barton et al., 1989Go).

The main function of inhibin in women is its regulation of pituitary FSH secretion. A decline in serum inhibin concentrations has also been shown to occur when the ovarian follicular reservoir begins to subside (MacNaughton et al., 1992Go). Recent evidence suggests that changes in inhibin secretion are responsible for an increase in FSH, and may be a marker of ovarian reserve (Burger et al., 1998Go; Hofmann et al., 1998Go; Reame et al., 1998Go). It has been demonstrated that an increase in FSH secretion coincides with an increased rate of follicular depletion during the menopausal transition (Richardson et al., 1987Go).

The hormonal patterns of POF patients also implicate inhibin as being causative in the disease mechanism. A defect in inhibin secretion has been reported in women with POF (Pampfer and Thomas, 1989Go), and inhibin concentrations were lower in women with ovarian failure during both anovulatory and ovulatory rebound cycles compared with infertile women with normal ovulatory cycles (Buckler et al., 1991Go). It has also been noted that women with impending POF had lower follicular and luteal phase inhibin concentrations (Halvorson and Decherney, 1996Go).

It is proposed that a functional mutation in any of the inhibin genes would lead to a decrease in the amount of bioactive inhibin. This loss would result in an increase in the concentrations of FSH by removing the negative feedback to the pituitary, leading to premature depletion of follicles, and hence result in POF. We show that a 769G->A transition in the INH{alpha} gene occurs in ~7% of POF patients, and is associated with POF at a very early age.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient information
A total of 43 women with POF was recruited for study in the Departments of Obstetrics and Gynaecology in Auckland, New Zealand, and Ljubljana, Slovenia. For the purpose of this study, POF was defined as cessation of menses for a duration of 6 months or longer before the age of 40 years, and a FSH concentration of >40 IU/l. All patients underwent a clinical assessment that included taking a complete medical and gynaecological history, and recording age at menses, menstrual history, and age at menopause. A serum gonadotrophin assessment was performed, and all patients were analysed for cytogenetic abnormalities. Five patients from the New Zealand samples had a family history of POF, defined as having more than one affected primary relative. Normal control samples were anonymous DNA samples from the general population, and would include males and females of various ages.

DNA extraction
Genomic DNA was extracted from 10 ml samples of blood. Lymphocytes were isolated from blood samples using the NYCOMED LymphoprepTM kit (Nycomed, Oslo, Norway). Cells were incubated at 65°C for 1 h with 3.5 ml guanidine hydrochloride solution (6 mol/l), 250 µl ammonium acetate solution (7.5 mol/l), 50 µl proteinase K solution (10 mg/ml) and 250 µl sodium sarcosyl solution (20% w/v). Cells were added to 2 ml of cold chloroform and then centrifuged at 2500 g for 3 min. The top layer was collected, and 10 ml of cold absolute ethanol added to precipitate the DNA. DNA was stored in 200 µl Tris–EDTA buffer at 4°C.

Polymerase chain reaction (PCR)
PCR primers were designed spanning the mature peptides of each inhibin gene, i.e. for INH{alpha} (nucleotides 841–1242) (Mayo et al., 1986Go), INHßA (nucleotides 1167–1528) (Mason et al., 1986Go) and INHßB (nucleotides 717–1061) (Mason et al., 1986Go) using the Primer Select module in the DNAStar computer program from Lasergene 1994 (DNASTAR Inc., Madison, WI, USA). Primers flanking the whole region were designed to give one large fragment for each gene, which was used for DNA sequencing. Smaller overlapping fragments of 200–300 bp were designed that spanned the functional region and were used for single-stranded conformation polymorphism (SSCP) analysis. The primers flanking each fragment are shown in Table IGo. The primers were dissolved in sterile water to give a final concentration of 20 mol/l.


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Table I. Primers for mutation detection and sequencing. Primers (forward and reverse) flanking each fragment in the three inhibin genes. The size of the PCR products generated by each set of primers is indicated. The location of the primers is shown with reference to corresponding nucleotides in the corresponding genes. PCR primers were designed spanning the mature peptides of each gene for INH{alpha} (nucleotides 841–1242) (Mayo et al., 1986Go), INHßA (nucleotides 1167–1528) (Mason et al., 1986Go) and INHßB (nucleotides 717–1061) (Mason et al., 1986Go) using the Primer Select module in the DNAStar computer program from Lasergene 1994 (DNASTAR Inc., Madison, WI, USA)
 
PCR conditions
PCR was carried out using Taq DNA polymerase (Qiagen GmbH, Hilden, Germany) and PCR buffer. Genomic DNA (100 ng) was amplified in a 25 µl volume reaction containing 2.5 µl of PCR buffer (1x), 25 nmol of each dNTP, 5 nmol of forward and reverse primers, and 0.125 µl Taq DNA polymerase. ß-Globin was used as positive control, and a nil DNA reaction was used as a negative control for all PCR reactions. Standard PCR conditions comprised 94°C denaturation for 1 min, 58°C annealing for 1 min, and 72°C extension for 1 min for 30 cycles. To ensure that a single band of expected size was present after amplification, electrophoresis of 5 µl of each PCR product was carried out in a 1.5% agarose gel and visualized under UV light using an ethidium bromide stain.

Single-stranded conformation polymorphism (SSCP)
An initial group of samples from 12 patients was analysed by SSCP analysis, to determine whether this was a suitable mutation detection strategy. The remaining 31 samples were analysed by DNA sequencing only. The PCR products were diluted 1/10 with sterile water. Equal volumes of diluted sample and 2x formamide loading buffer were heated to 95°C for 3 min to denature the samples, and immediately placed on ice to prevent DNA strands from re-annealing. A 3 µl aliquot of each sample was electrophoresed alongside non-denatured and denatured controls. The SSCP gels consisted of 1x Tris–borate–EDTA (TBE) buffer, 8% or 10% polyacrylamide, with or without glycerol (5%). Setting agents were 15 µl (25% w/v) ammonium persulphate and 15 µl N,N,N',N-tetramethylethylenediamine (TEMED) for every 10 ml of non-denaturing gel. Electrophoresis was performed at room temperature (20–24°C), using 0.5x TBE running buffer. Mini gels (BioRad mini-protean II cell; BioRad, Hercules, CA, USA) were electrophoresed for 2–3 h at 170 V and large gels (BioRad SequiGen Sequencing cell) were electrophoresed overnight at 200–300 V. The DNA was visualized using silver staining. Gels were fixed in equal volumes of 40% ethanol and 10% acetic acid for a least 30 min, followed by two 15 min washes in a second mixture of 10% ethanol and 5% acetic acid. Fixation was followed by a 15 min wash in a K2Cr2O7-based oxidizer. Gels were then washed in distilled water until the yellow coloration of the oxidizer was completely removed. The gels were then stained in 20 ml of silver reagent dissolved in 180 ml of distilled water for 20 min. Development of the gels was performed by washing in distilled water for 1 min, followed by three washes in 200 ml aliquots of developer, and a final 5 min wash in 5% acetic acid. The gels were washed to remove the acetic acid, transferred to filter paper (3 MM; Whatman, Clifton, NJ, USA), dried and stored.

DNA sequencing
All 43 samples were analysed by DNA sequencing. The large PCR fragments, INH{alpha} (601 bp), INHßA (529 bp) and INHßB (586 bp), that spanned the entire functional region of each of the three inhibin genes (Table IGo), were used as templates for DNA sequencing. Samples were purified with Promega's Wizard PCR Preparations DNA Purification System (Promega Corp., Madison, WI, USA), and sequencing was performed using an Applied Biosystems Model 377 automated sequencer (PE Biosystems, Foster City, CA, USA) and 2 µl of template DNA.

Characterization of variants
The PCR products from ethnically matched control samples were analysed for the variant 769G->A in the INH{alpha} gene subunit. Restriction fragment length polymorphism (RFLP) analysis used Bst71I as the restriction enzyme to determine if this variant was a naturally occurring polymorphism, or a mutation that may be responsible for POF. DNA with a known sequence was used as negative control DNA, while DNA from a patient shown by sequence analysis to be a carrier of the variant 769G->A acted as a positive control for the RFLP test. The restriction enzyme digest was undertaken in 1x restriction buffer using 2.5 U of Bst71I, 0.2 µl of acetylated bovine serum albumin (BSA), 5 µl of PCR product, and sterile water to give a total reaction volume of 20 µl. The reaction mixtures were incubated at 37°C for 1–2 h, electrophoresed in a 2% agarose gel, and stained with ethidium bromide. To allow better separation of the fragments, some of the samples were also electrophoresed in an 8% polyacrylamide gel and subsequently stained with SYBR Gold (Molecular Probes Inc., Eugene, OR, USA). The undigested DNA control was also incubated in a reaction mixture containing all the above reagents except the Bst71I enzyme, and electrophoresed alongside the digested PCR products. The wild-type INH{alpha}1 PCR product yields three fragments of 85 bp, 25 bp and 134 bp when digested with Bst71I. In the presence of the 769G->A variant, the enzyme recognition site CGTCG(n)12 is abolished, and hence yields only two fragments of 85 bp and 159 bp. A heterozygous sample will display all four fragments.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients (n = 43) with POF were screened for mutations in the three inhibin genes. Two variants were detected in the INHßA1 and the INH{alpha}1 fragments using SSCP analysis in the initial group of 12 unrelated New Zealand POF patients (data not shown). The fragments INH{alpha}2, INH{alpha}3, INHßA2, INHßA1, INHßB1, INHßB2 and INHßB3 did not reveal any migration variants in any of the patient samples when compared against the wild-type DNA.

The migrational shift detected in the INHßA1 fragment was caused by a silent substitution at nucleotide 1032C->T (Figure 1aGo). This variant did not change the amino acid sequence of the inhibin beta A subunit as it occurred in the third position of the codon, causing a GGC (glycine) to GGT (glycine) alteration.



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Figure 1. (a) Electropherogram displaying the sequence of the INHßA1 variant compared with the wild-type sequence. Arrows indicate a C to T sequence change in the variant, and the corresponding nucleotide in the wild-type sequence. (b) Electropherogram displaying the sequence of the INH{alpha}1 variant compared with the wild-type sequence. Arrows indicate a G to A sequence change in the variant, and the corresponding nucleotide in the wild-type sequence.

 
Direct DNA sequencing of the PCR product confirmed the INH{alpha}1 variant detected using SSCP. The variant was the result of a G->A missense substitution at nucleotide 769 (Figure 1bGo) that alters codon 257 from GCT to ACT, resulting in an alanine to threonine amino acid substitution in the INH{alpha} gene subunit. To confirm that the INH{alpha} 769G->A variant was not a sequencing error, the INH{alpha}1 fragment was amplified from both the original DNA sample, DNA extracted from a second blood sample, and each was sequenced in both directions. Again, the same sequence variation was identified. Finally, sequencing was performed on the INH{alpha} (601 bp), INHßA (529 bp) and INHßB (586 bp) amplification products from all 12 patients, and no additional variants were found.

In order to extend the above study, DNA was collected from a further six affected women with POF from New Zealand, and 25 from Slovenia. DNA was also collected from seven women with primary amenorrhoea (from Slovenia). The fragments INH{alpha} (601 bp), INHßA (529 bp) and INHßB (586 bp) were amplified from these 31 POF patients and seven primary amenorrhoea patients, and each was sequenced. Another two patients were found to carry the INH{alpha} 769G->A variant. No other sequence variants were identified in any of the POF patients, nor from DNA obtained from the seven women with primary amenorrhoea.

A rapid RFLP screen was developed to identify the presence of the INH{alpha} 769G->A variant. This variant abolished a Bst71I restriction enzyme site. An RFLP analysis of the 244 bp INH{alpha} amplification products from DNA samples of 150 ethnically matched normal individuals (100 from New Zealand and 50 from Slovenia) showed only 134 bp and 85 bp fragments, except in a single sample which showed heterozygosity. The patients with the 769G->A variant demonstrated heterozygosity with fragment lengths of 159 bp, 134 bp and 85 bp (Figure 2Go). According to Fisher's exact test, the likelihood of finding a variant in the POF patient samples (3/43; 7%) was significantly different from finding a variant in a group of matched controls (1/150; 0.7%) (P < 0.035).



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Figure 2. RFLP analysis of the INH{alpha}1 fragment using Bst71I. Undigested DNA gives a single band at 244 bp. Wild-type (WT) yields three bands of 134, 85 and 25 bp. Homozygosity for the variant yields two fragments of 150 and 85 bp. A heterozygote carrier will have all four fragments (150, 134, 85 and 25 bp). The variant is heterozygous as it yields both the wild-type and variant fragments when digested. The 25 bp fragment is undetectable on this 8% polyacrylamide gel. Lane 1, marker lane; lane 2, undigested DNA; lanes 3–5, POF patients identified by DNA sequencing to be heterozygous for the variant; lane 6, normal control sample; lanes 7–9, POF patients shown by DNA sequencing to be homozygous normal for the variant.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
POF is a condition affecting 1% of women under the age of 40 years (Coulam et al., 1986Go), with most of the cases being idiopathic (Conway, 1997Go). POF can result from a defect in either the development or function of the ovaries. We suggest that mutations in one of the inhibin genes allow a period of normal ovarian function before the onset of early POF.

The 1032C->T transition in the INHßA gene (Figure 1aGo) identified in one of 12 New Zealand patients was not found to change the amino acid sequence of the polypeptide product, and is most likely a synonymous substitution creating a silent polymorphism. However, this particular nucleotide is conserved in porcine, bovine and human INHßA genes (Mason et al., 1986Go), and this patient had menopause at age 25 years.

The 769G->A variant was initially identified in the INH{alpha} gene (Figure 1bGo) in a single New Zealand patient by SSCP. This results in the substitution of alanine for threonine at codon 257. A further two probands were subsequently identified by DNA sequencing to be heterozygous for this variant. RFLP analysis suggests that the INH{alpha} 769G->A variant is rare in the normal population, as only one of 150 control samples from New Zealand and Slovenia was positive for this variant. This single positive control sample came from a 26-year-old female who could be a normal carrier or, alternatively, may be predisposed to the development of POF.

In a separate study to identify inhibin gene polymorphisms, the sequence analysis of the INH{alpha} gene in 20 control samples did not identify a DNA sequence change at this position (M.Kudo and A.J.Hsueh, Stanford University Medical Center, Stanford, CA, USA, personal communication). The alanine to threonine change is a non-conservative substitution, and results in the addition of an aliphatic hydroxyl group in the side chain of the functional group. The human INH{alpha} gene demonstrates 80% homology with equine, bovine, porcine, ovine, rat and mouse sequences (Yamanouchi et al., 1995Go). When the amino acid sequences of the INH{alpha} protein of these species were compared, the alanine at codon 257—the site of the mutation (Figure 3Go)—was conserved in horse, porcine, ovine, mouse, bovine, possum and chicken species; however, the rat sequence contained serine. The amino acid sequence surrounding codon 257 is also highly conserved. These data suggest that alanine at codon 257 has an important role in the function of the protein. It is interesting to note that in this study of 43 patient samples, there was only one other DNA variant identified, implying that the inhibin genes are highly conserved, and nucleotide changes as part of natural variation are not well tolerated.



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Figure 3. Alignment of the INH{alpha} gene subunit amino acid sequences from human, horse, porcine, ovine, mouse, bovine, possum, chicken and rat species. DNA sequences were obtained from Genbank. The arrow indicates the amino acid altered by the G->A mutation

 
The functional significance of the amino acid variant at codon 257 is unknown. Analysis of the protein structure suggests that it could interfere with receptor binding. The inhibin molecule is part of the TGF-ß superfamily, a group of multi-functional growth and differentiation factors. Overall, this family consists of ~30 members that show a conserved carboxyl terminal 7-cysteine domain with between 20 and 92% sequence identity (Griffith et al., 1996Go). The three-dimensional crystal structure of INH{alpha} is unknown; however, the structure of some other members of the TGF-ß superfamily has been determined. These are TGF-ß2 and OP-1 (oestrogenic protein-1), with an amino acid sequence homology to INH{alpha} of 24 and 26% respectively (Griffith et al., 1996Go). INH{alpha}, like the other members of the TGF-ß superfamily, has the seven conserved cysteines. However, the amino-terminal region upstream of the first cysteine is divergent from other members of the TGF-ß superfamily, and is thought to be involved in receptor binding (Griffith et al., 1996Go). The alanine to threonine change occurs within this putative receptor binding region of the final mature peptide. We suggest that this change would be sufficient to impair the binding of inhibin to its receptor resulting in an inability to activate the subsequent signal transduction pathway. Alternative hypotheses for a functional effect of this transition mutation include preventing dimer formation, cleavage of the mature peptide, or altering glycosylation.

The parents of one of the affected individuals carrying the 769G->A variant were able to provide DNA samples to determine whether the INH{alpha} variant was inherited or was a de-novo mutation. The father showed a normal DNA sequence, while the mother was found to be a carrier for the variant. It is interesting to note that she had a normal menopause at age 55 years. It might be expected in reproductive disorders that highly penetrant mutations causing infertility would not be common, and would be rapidly lost from the gene pool. Therefore, it would be expected to see incomplete penetrance in familial POF, and evidence was recently provided for the dominant transmission of POF with 79.1% penetrance in a study of 71 women (Vegetti et al., 1998Go).

The only way to be certain that a mutation causes a functional effect is to perform functional testing. Most functional studies involve the expression of receptors in a recombinant expression system accompanied by an assay with the protein of interest. However, the inhibin receptor has yet to be characterized. The activity of inhibin is currently investigated by an in-vitro bioassay based on the effect of graded doses of inhibin on FSH cell content of rat pituitary cells in culture (Robertson and de Kretser, 1989Go).

The identification of this INH{alpha} variant highlights the importance of the inhibin glycoproteins in the normal function of the female reproductive system. Women with the INH{alpha} variant had relatively severe symptoms of POF with secondary amenorrhoea at the age of 16, 20 and 24 years (Figure 4Go). It is interesting that none of the seven patients with primary amenorrhoea was found to be a carrier of the variant, which would suggest that mutations in inhibin are not a common cause of primary amenorrhoea. The three women with the INH{alpha} variant represented 7% of the study group of 43. If this were an accurate indication of the number of cases of POF caused by this mutation, then it would affect a substantial portion of young POF patients (25% of patients under the age of 25 years). Functional and further population studies of POF patients, with ethnically matched controls, are required to determine the exact contribution of this variant to POF. A genetic test, based on the Bst71I restriction analysis, could prove to be an important diagnostic tool. Detection of the mutation prior to the development of ovarian failure enables carriers to make informed decisions regarding reproductive options. Early detection would provide better opportunity for early intervention such as replacement of the inhibin hormone in patients to delay the onset of ovarian failure.



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Figure 4. Age at menopause. The age at menopause was obtained from each patient, and the distribution was plotted according to this age. The three women carrying the 769G->A variant are indicated by the solid circles.

 


    Acknowledgments
 
We would like to thank the POF patients for their involvement in this study. We would also like to thank Guy Gudex and Marian Carter at Fertility PLUS, Mary Birdsall at Fertility Associates and other clinicians for providing patients for this study. Funding was provided by Allied Foods, the University of Auckland Research Committee, the Health Research Council of New Zealand, and the Auckland Medical Research Foundation.


    Notes
 
7 To whom correspondence should be addressed at: Research Centre in Reproductive Medicine, Department of Obstetrics and Gynaecology, National Women's Hospital, Auckland, New Zealand. E-mail: a.shelling{at}auckland.ac.nz Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 8, 2000; accepted on August 24, 2000.