Hum. Reprod. Advance Access originally published online on January 5, 2006
Human Reproduction 2006 21(5):1154-1160; doi:10.1093/humrep/dei452
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Controversial role of inhibin 
-subunit gene in the aetiology of premature ovarian failure
1 Instituto de Biología y Medicina Experimental (IBYME-CONICET), 2 Centro de Investigaciones Endocrinológicas (CEDIE), Hospital de Niños R. Gutiérrez, 3 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and 4 Centro Nacional de Genética Médica (ANLIS), Buenos Aires, Argentina
5To whom correspondence should be addressed at: Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina. E-mail: sundblad{at}dna.uba.ar
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BACKGROUND: Premature ovarian failure (POF) is characterized by hypergonadotropic amenorrhoea before the age of 40. Inhibin
-subunit (INH) gene is proposed as a candidate gene due to its role in negative feedback control of FSH. METHODS: Polymorphism –16C>T of INH gene was studied in 61 POF patients and 82 controls above 40 years old (C > 40). Substitution 769G>A was studied in 59 POF patients, 76 C > 40 and 73 controls below 40 years old (C < 40). RESULTS: No significant difference in risk of POF development for –16T allele was found when comparing idiopathic POF (I-POF) with C > 40 (Odds ratio = 1.46; 95% confidence interval = 0.63–3.19). Implication of –16C>T polymorphism in serum inhibin levels was analysed in 46 controls, and no significant differences (P > 0.05) were found between CC and CT + TT genotype groups when comparing either mid-follicular phase Pro-C and inhibin B values or mid-luteal phase Pro-C and inhibin A values. Heterozygosity for substitution 769G>A was found in 1 of 59 POF woman, 2 of 76 C > 40 and 6 of 73 C < 40. Presence of this substitution in a relevant number of control subjects is herein described for the first time. CONCLUSION: Our results indicate that –16C>T and 769G>A variants in INH gene may not be associated to POF disease.
Key words:
inhibin -subunit gene/inhibin levels/premature ovarian failure
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Premature ovarian failure (POF) is a syndrome clinically defined by ovarian failure before the age of 40 years. It is characterized by primary or secondary amenorrhoea, hypoestrogenism and elevated gonadotrophin serum levels (Moraes-Ruehsen and Jones, 1967
). This syndrome is very heterogeneous with a multicausal pathogenesis, and any of the following—chromosomal, enzymatic, iatrogenic, autoimmune or infectious aberration—may be the cause of the disease (Hoek et al., 1997). Incidence of POF among women in reproductive age is estimated in 1% (Coulam et al., 1986).
Owing to frequent inheritance in families, it was suggested that POF may be a genetic disorder (Coulam et al., 1983; Mattison et al., 1984; Conway et al., 1995). In support of this hypothesis, the FSH receptor (FSHR) has long been considered the foremost candidate gene for POF (Aittomaki et al., 1995; Gromoll et al., 1996; Beau et al., 1998; Touraine et al., 1999; Doherty et al., 2002; Allen et al., 2003; Meduri et al., 2003). However, in a previous study, and in accordance with several other authors (Whitney et al., 1995; Liu et al., 1998; Conway et al., 1999; de la Chesnaye et al., 2001; Takakura et al., 2001; Tong et al., 2001), we found no mutations in exons 1–10 of FSHR gene in 20 women with POF from among our population and postulated that FSHR isoforms might not be associated to disease risk (Sundblad et al., 2004).
The glycoprotein inhibin may be a potential candidate for POF due to its role in the negative feedback control of FSH, which has a pivotal role in ovarian follicle recruitment and development during folliculogenesis (Shelling et al., 2000). Dimeric inhibins are glycoproteins predominantly produced in the gonads (Burger, 1992), and are composed of - and 
-subunits. Heterodimerization of -subunit with either form of the -subunit, A and B, generates dimeric inhibin A and inhibin B, respectively. The homodimers composed of A- or B-subunits are the glycoproteins activins (Ying, 1988). It has been proposed that a large reservoir of inhibin -subunit (INH) in the ovary may be necessary to ensure dimeric inhibin formation, instead of dimerization of -subunits to produce activins (Findlay et al., 2001). Inhibins and activins are members of the transforming growth factor superfamily. Since the development of specific assays, profiles of serum levels of inhibins throughout the menstrual cycle have duly been described. Inhibin A is primarily secreted by the mature follicle and corpus luteum; it remains low during the early and mid-follicular phase and predominates in the luteal phase of the cycle. Conversely, inhibin B is a product of early pre-antral follicles; it presents maximal levels in the early follicular phase, falls before ovulation and remains low during the luteal phase (Groome et al., 1994, 1996; Sehested et al., 2000).
The monomeric inhibin form Pro-C is found in the circulation at a higher concentration than that of inhibins A and B (Groome et al., 1995; Bergada et al., 1999). It has not yet been established whether this peptide may have a physiological role since it lacks biological action related to the regulation of FSH synthesis. On the other hand, inhibins A and B may act as endocrine factors, suppressing hypophyseal FSH biosynthesis and secretion (de Jong, 1988; de Kretser and McFarlane, 1996; Burger and Robertson, 1997), as well as intraovarian paracrine factors (Ying, 1988; Mather et al., 1992; Weiss et al., 1993). They contribute to the regulation of FSH secretion in the normal menstrual cycle, a process that allows the timely ovulation of a single mature follicle. Conversely, activins appear capable of direct pituitary stimulation of FSH secretion (Ying, 1988).
Several pieces of evidence suggest that the decrease in dimeric inhibin levels observed during perimenopause, and the concomitant increase in activin A, may be responsible for the high level of FSH characteristic of reproductive ageing (Klein et al., 1996; Danforth et al., 1998; Reame et al., 1998; Santoro et al., 1999). The reduction of inhibin : activin ratio observed during this period is likely due to an -subunit production deficit, which leads to the preferential formation of activin homodimers (Santoro et al., 1999). Thus, it has been suggested that POF could be a consequence of mutations in the INH gene, which may cause a decrease in the amount of bioactive inhibin, thereafter increasing the FSH concentration (Shelling et al., 2000; Marozzi et al., 2002).
INH gene maps to 2q33-qter and is composed of two exons. Two polymorphic sites were identified in this gene: –16C>T in the 5'UTR and 675C>T in exon 2, the latter in complete linkage disequilibrium with the former (Montgomery et al., 2000). Marozzi et al. (2002) studied –16C>T polymorphism and found that the derived frequency of T allele was significantly lower in POF population than in the control group. On the other hand, Shelling et al. (2000) identified a missense mutation (769G>A) in exon 2 of this gene, in 3 of 43 women with POF. This substitution was subsequently described in 7 of 157 Italian women with POF (Marozzi et al., 2002) suggesting an association between the 769G>A variant and the development of POF.
The purpose of the present work was to study INH gene as a putative candidate gene for POF in a cohort of patients from Argentina.
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Subjects
The population studied comprised 61 patients with POF, all with normal 46,XX karyotype. Among these patients, 18 presented POF in association with autoimmune diseases (AAD–POF patients). Fifteen of the AAD–POF patients presented autoimmune thyroiditis, two of them associated to vitiligo, two patients presented systemic lupus erythematosus and one presented myasthenia gravis. The remaining 43 POF patients were considered idiopathic (I-POF patients) because they did not show any other POF-related condition (i.e. ovarian surgery, previous chemo- or radiotherapy or metabolic disorders such as galactosemia). Patients presenting antibodies directed to FSHR, determined by radioreceptor assay (Chiauzzi et al., 2004
), were excluded from the present work. Sixteen patients had a family history of premature menopause, whereas the remaining 45 were classified as having sporadic POF. Patients had been characterized as POF due to amenorrhoea for over a year starting before the age of 40 (range, 15–39 years old) and serum FSH level above 40 mIU/ml (normal follicular phase levels, 2–9 mIU/ml) in two consecutive determinations. Nine patients presented primary amenorrhoea and 52 secondary amenorrhoea. Plasma 17-estradiol levels were below 15 pg/ml (normal follicular phase levels, 20–120 pg/ml).
Eighty-three women above the age of 40 (C > 40) with normal menstrual history and without premature menopause were also included in this study as controls. All but one were of proven fertility, and only four were below 45 years of age. For the study of the implication of –16C>T polymorphism in serum inhibin levels, 46 controls were selected according to the following criteria: (i) age below 40; (ii) regular menses (duration, 25–35 days); (iii) no family history of premature or early menopause; (iv) no family history of autoimmune disorders; and (v) no consumption of oral contraceptives or other hormonal medications at the time of inclusion in the study. Weight and height of each subject was registered, and body mass index was calculated according to the formula: weight/(height)2. Twenty-nine per cent of these women were of proven fertility. Two blood samples were taken from each volunteer, the first between days 7 and 10 (mid-follicular phase) and the second between days 21 and 24 (mid-luteal phase) of the menstrual cycle. The exact day of blood extraction at mid-follicular and mid-luteal phases was determined for each subject—among the referred days—according to the duration of her own menstrual cycle. From each blood sample, DNA was isolated as described below and serum was used for hormone determinations. FSH, estradiol, inhibin B and Pro-C values were measured on day 7–10 serum samples (mid-follicular phase), whereas progesterone, inhibin A and Pro-C levels were determined on day 21–24 serum samples (mid-luteal phase).
In addition, 73 normally menstruating women below the age of 40 (C < 40), without family history of premature or early menopause and with no history of autoimmune disorders, were also included for 769G>A study. Thirty-three of them were of proven fertility.
The protocol was approved by the Institutional Review Board of the Instituto de Biología y Medicina Experimental. Informed consent was obtained from all patients and controls.
DNA analysis
DNA was isolated from peripheral blood leukocytes by standard methods. Two regions of INH-subunit gene (NM_002191
[GenBank]
) were amplified from genomic DNA by PCR with specific oligonucleotide primers. The first region of 240 bp (fragment A), which includes 140 bp of 5'UTR and 100 bp of exon 1, was amplified by primers AF (5'-GACTGGGGAAGACTGGATGA-3') and AR (5'-TCACCTTGGCCAGAACAAGT-3'). The second region of 396 bp (fragment B), which comprises part of exon 2, was amplified by primers BF (5'-AGCAGCCTCCAATAGCTCTG-3') and BR (5'-AGCTCCTGGAAGGAGATGTTC-3'). Genomic DNA (200ng) was amplified in a 50 ml volume reaction containing 1 x PCR buffer (Invitrogen, Carlsbad, CA, USA), 2mM MgCl2 (Invitrogen), 2.5% DMSO (Merck KgaA, Darmstadt, Germany), 0.2 mM of each dNTP (Promega, Madison, WI, USA), 0.5 mM of each specific primer and 1.5U of Taq DNA polimerase (Invitrogen). After an initial denaturation at 90ºC for 1 min, the samples underwent 35 cycles of amplification (94ºC denaturation for 40 sec, 57ºC annealing for 30 sec, 72ºC extension for 1 min); the last cycle was followed by 7 min extension at 72ºC.
Polymorphism –16C>T in 5'UTR was screened in the samples by restriction enzyme analysis using SpeI (New England Biolabs, Ipswich, MA, USA). Briefly, fragment A was amplified by PCR and 3 µl of purified PCR product were digested overnight at 37°C with 5 U of SpeI, electrophoresed on 8% polyacrylamide gels, stained with ethidium bromide and photographed. Presence of the 240 bp fragment indicated a homozygous variant for C, whereas presence of two fragments of 120 bp corresponded to homozygous variant T.
Substitution 769G>A of exon 2 was analysed by digestion of fragment B with two different restriction enzymes. Two microlitres of purified PCR product were digested overnight at 37°C with 5 U of BsrFI and analysed as described above. The restriction site that renders two fragments of 340 and 56 bp is abolished in the mutated allele. In addition, 3 µl of purified PCR product were digested overnight at 37°C with 3 U of Fnu4HI, electrophoresed on 15% polyacrylamide gels, silver stained and dried. The 396 bp fragment renders four fragments of 153, 107, 51 and 25 bp—among others of lower molecular weight—in the wild-type allele, whereas the allele with substitution 769G>A renders four fragments of 153, 107, 76 and 51 bp, among others of lower molecular weight. In addition, all heterozygous samples—together with DNA from two control individuals—were further analysed by direct sequencing. New PCR products were purified by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bio-Sciences, Cardiff, Wales, UK), following the Big Dye terminator sequencing protocol (Applied Biosystems, Foster City, CA, USA) and analysed using ABI prism DNA analyser.
Hormone measurements in serum
Follicular phase serum FSH was measured by enzymeimmunoassay (EIA) using a FSH EIA kit (Immunometrics, London, UK). Intra- and inter-assay coefficients of variance (CV) were 5.3 and 19.4, 3.9 and 11.1, and 3.6 and 12.6%, for low, medium and high pools, respectively. Assay sensitivity was 0.5 mIU/ml (second IRP 78/549).
Mid-follicular phase serum estradiol was measured by radioimmunoassay using a COAT-A-COUNT Estradiol kit (Diagnostic Products Corporation, Los Angeles, CA, USA). Intra- and inter-assay CV were 7.0 and 8.1, 4.3 and 6.8, and 4.0 and 4.2% for low, medium and high pools, respectively. Assay sensitivity was 8 pg/ml.
Mid-luteal phase serum progesterone was measured by radioimmunoassay using a COAT-A-COUNT Progesterone kit (Diagnostic Products Corporation). Intra- and inter-assay CV were 8.8 and 9.7, 4.0 and 5.7, and 2.7 and 3.9% for low, medium and high pools, respectively. Assay sensitivity was 0.02 ng/ml.
Serum inhibin A, B and Pro-C were measured using a two-site enzyme-linked immunosorbent assay (Oxford Bio-Innovation, Oxon, England) specific for each peptide as previously described (Groome et al., 1994, 1995, 1996). Recombinant inhibins A and B (Genentech, San Francisco, CA, USA) and a partially purified (>75% purity) Pro-C preparation were used as standards. Assay sensitivity was 7 pg/ml for inhibin A, 15 pg/ml for inhibin B and 2 pg/ml for Pro-C. Intra- and inter-assay CV were <10% for the three assays.
All samples from an individual woman were measured in duplicate in the same assay run.
Statistical analysis
Odds ratio (OR) and 95% confidence interval have been calculated to estimate the relative risk of T allele and the different genotype combinations. Unpaired t-test was used to compare hormonal values in different genotype control groups. Mann–Whitney U-test was used when appropriate.
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A mutation study of the INH
gene was carried out in POF patients and control subjects.
Our analysis revealed the presence of polymorphism –16C>T already reported (Montgomery et al., 2000). The PCR product (fragment A) comprising nucleotide –16 was digested with SpeI (Figure 1). Allelic frequencies and genotypes found in the 61 POF patients and in 82 control individuals above 40 years of age (C > 40) are summarized in Table I. The T variant was considered the putative genetic risk factor for OR calculation. We were unable to demonstrate an association between this variant and the risk of being affected with I-POF (Table I).
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In addition, the implication of the single nucleotide polymorphism in serum inhibin levels was analysed in subjects not affected by the disease. We evaluated inhibin A, inhibin B and Pro-C peptide levels and correlated these data to the genotype of –16C>T polymorphism in 46 cycling women below 40 years of age. Given that T allele was considered the putative genetic risk variant, samples were divided into two groups: (i) CC genotype (n = 34) and (ii) CT + TT genotypes (n = 12). Clinical characteristics and hormonal profile of control subjects from both groups are summarized in Table II. Figure 2 shows serum inhibin levels (mean ± SEM in pg/ml) determined in the different genotype groups. No significant differences (P > 0.05) were found between both groups in mid-follicular phase Pro-C or inhibin B levels (CC: 146.30 ± 18.80 versus CT + TT: 210.55 ± 44.63 and CC: 134.90 ± 12.63 versus CT + TT: 130.44 ± 20.85, respectively) and mid-luteal phase Pro-C and inhibin A levels (CC: 671.01 ± 68.61 versus CT + TT: 768.06 ± 86.44 and CC: 38.09 ± 3.92 versus CT + TT: 42.73 ± 7.86, respectively).
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Substitution 769G>A of exon 2 was analysed by restriction enzyme digestions in 59 of 61 patients and in 76 of 83 control women above 40 years. PCR product comprising nucleotide 769, fragment B, was digested with BsrFI (Figure 3A) and/or with Fnu4HI (Figure 3B). Two of 76 controls and 1 of 59 POF patients, an AAD–POF woman with associated autoimmune thyroiditis, were heterozygous for this mutation (Table III). In addition, 73 cycling women below 40 years of age were also evaluated for the presence of this mutation. We found six of these women heterozygous for 769G>A substitution (Table III). The presence of this variant was further confirmed in all heterozygous samples by direct sequencing (Figure 4). Characteristics of individuals carrying the INH 769G>A variant are summarized in Table IV. Serum FSH values of four of six heterozygous 769G>A women below the age of 40 were measured, and all of them were in the normal range (2–9 mIU/ml).
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During menopausal transition, the increase in FSH secretion coincides with an increment in follicular depletion rate (Richardson et al., 1987
). A decline in serum inhibin concentration has also been shown to occur when the ovarian follicular reservoir begins to subside (MacNaughton et al., 1992), being almost undetectable in post-menopausal women (Buckler et al., 1991). Indeed, it was suggested that this decrease in inhibin A and B tones may be responsible for the high level of FSH, characteristic of reproductive ageing (Klein et al., 1996; Reame et al., 1998; Santoro et al., 1999). Studies on hormonal patterns of POF patients have implicated inhibin as being involved in the disease mechanism. A defect in inhibin secretion has been reported to be involved in POF and natural menopause (Pampfer and Thomas, 1989). In addition, Petraglia et al. (1998) found that serum inhibin A and B levels in POF women were similar to those observed in post-menopausal women.
Considering that reduction of the inhibin : activin ratio observed during menopause could probably be due to a deficit of the INH production (Santoro et al., 1999), POF condition could be thought of as a consequence of mutations in the INH gene, which may cause a decrease in the amount of bioactive inhibin, and consequently, an increase in FSH concentration. Two previous studies have suggested a promising involvement of the INH gene in the aetiology of POF (Shelling et al., 2000; Marozzi et al., 2002).
Two polymorphic sites were identified in this gene: –16C>T in 5'UTR and 675C>T in exon 2, the latter in complete linkage disequilibrium with the former (Montgomery et al., 2000). Marozzi et al., (2002) studied –16C>T polymorphism and found that the derived frequency of the T allele was significantly lower in POF population than in the control group. In the present work, we were unable to demonstrate any association between the T variant and the risk of being affected with POF. On the other hand, we measured serum monomeric and dimeric levels of inhibins in control cycling women. Inhibin levels were found to be similar in women with different polymorphism genotypes, all of them in the normal range (Groome et al., 1995; Sehested et al., 2000), suggesting that ovarian production of inhibins may not be affected by –16C>T allelic variant adding support to the concept that the 5'UTR allelic variant might not be involved in abnormal ovarian function.
Presence of the 769G>A substitution in exon 2 of INH gene was described for the first time by Shelling et al. (2000) in 3 of 43 women with POF compared to 1 of 150 controls. Later on, Marozzi et al. (2002) reported that 769G>A transition was significantly more frequent in POF patients (7 of 157) than in control women (0 of 100). The substitution has been hypothesized to impair the binding of inhibin to its putative receptor (Shelling et al., 2000). Recently, this mutation has also been found in 9 of 80 POF patients and 0 of 100 control subjects from India (Dixit et al., 2004). However, it was found neither in 84 POF patients nor in 100 control subjects from Korea (Jeong et al., 2004). Moreover, the functional significance of the amino acid variant at codon 257 is still unknown, given that no functional studies have been performed to date. In the present study, we investigated the presence of substitution 769G>A in exon 2 of INH gene in order to determine if it could contribute to explain the pathophysiology of POF patients from among our population. In accordance with a study in Korean POF patients (Jeong et al., 2004), we were unable to demonstrate a significant association between this mutation and ovarian failure. We observed only one patient, an AAD–POF woman, and 2 of 76 control women above 40 years old with the mutation described by Shelling et al. (2000).
It is noteworthy that some isolated cases of 769G>A substitutions were previously described in normal women. Studying 150 DNA samples from the general population—males and females of various ages—as controls, Shelling et al. (2000) found a normally menstruating 26-year-old female heterozygous for this substitution. In addition, the authors also reported that a patient’s mother was found to be a carrier of the variant and had undergone normal menopause at 55 years. Another young 26-year-old woman, with regular menses but a carrier of 769G>A substitution, was described by Marozzi et al. (2002). Even though occurrence of mutation without POF manifestation was explained by these authors through incomplete penetrance, these findings could imply that this substitution is also present in control subjects, becoming a putative polymorphism with no clinical consequences. In view of these considerations, we therefore included a group of 73 normal cycling women below 40 years of age in the analysis, and found this substitution in six of them. Presence of this variant in a relevant number of control subjects is herein for the first time described. Even though the possibility of POF development cannot be ruled out in these control subjects, the following should be considered: (i) 769G>A substitution frequency (8.2%) observed in this group is definitively above POF incidence, estimated in 1% of women of reproductive age; and (ii) if these heterozygous 769G>A control women were to develop POF, a similar frequency might be expected to be found in POF patients. It is interesting to note that Shelling et al. (2000) and Dixit et al. (2004) found that all POF patients with 769G>A substitution developed POF before the age of 25. The authors therefore suggested that this substitution could be associated to a relatively severe early onset of POF. If so, the possibility of POF development in our control heterozygous women between the ages of 27 and 34 (Table IV) might be even lower. Nevertheless, considering that Marozzi et al. (2002) found that the mean age of menopause onset of the seven patients carrying 769G>A substitution was 35.6 ± 3.7 years, an appropriate follow-up of our heterozygous control women below 40 should be carried out.
It has been hypothesized that this mutation may be sufficient to impair binding affinity of inhibin to its putative receptor, leading to a subsequent inability to regulate the FSH level by negative feedback (Shelling et al., 2000). Consequently, it might be plausible to find relative higher levels of FSH in control women found to be heterozygous, as compared with subjects without such substitution. However, in the present study, heterozygous 769G>A women below 40 showed normal FSH values. On the other hand, the only patient identified to date in the literature to be homozygous for 769G>A substitution did not present a particular severe phenotype, with FSH levels similar to those presented by other POF patients with a wild-type genotype (Dixit et al., 2004). These two observations, together with the fact that in our study 769G>A frequency in POF patients was similar to—or even lower than—that found in control women, may suggest that this substitution in INH gene might not be involved in the aetiology of POF. Nevertheless, in order to better define the role of 769G>A in POF development, the number of patients and controls included in the analysis should be increased.
It has long been recognized that POF could be associated with nearly all organ-specific autoimmune diseases as well as with some nonorgan-specific disorders (de Moraes et al., 1972; Coulam, 1983; La Barbera et al., 1988; Betterle et al., 1993; Hoek et al., 1997; Forges et al., 2004). The frequent relationship between POF, Hashimoto’s thyroiditis, Addison’s disease and other autoimmune diseases suggests that all of them are different manifestations of a general immunologic disorder, and might imply an autoimmune mechanism acting as the aetiologic agent in these POF patients (Coulam, 1983; La Barbera et al., 1988). Indeed, patients with a record of autoimmune diseases are usually excluded when I-POF is the target of study. Nevertheless, considering that the exact role of autoimmunity in the pathophysiology of this disease still remains controversial (Hoek et al., 1997; Forges et al., 2004), in this work patients with autoimmune-associated diseases were analysed in a separate group. Surprisingly, the only POF patient found to be heterozygous for 769G>A substitution belongs to this AAD–POF group. This outcome might question the role of INH gene in POF aetiology, reinforcing our findings in controls. Alternatively, it could add further support to the concept of the multifactorial nature of the disorder, if a role of INH gene was ultimately confirmed.
It has been proposed that changes in inhibin secretion due to alterations in INH gene could be responsible for the increase in FSH levels found in POF patients (Shelling et al., 2000; Marozzi et al., 2002), thus becoming the cause of the disease mechanism. Alternatively, the reduction in inhibin tones could be a natural consequence of the reduction in the number of follicles in these patients’ failing ovaries, like in physiological menopause. Considering our results, low inhibin tones might be thought of as a consequence of premature follicular depletion, or alternatively, the decreased inhibin levels could indeed be responsible for high FSH levels characteristic of POF patients—becoming the cause of the disease mechanism—deriving from some alteration(s) in the regulation mechanism(s) of inhibin secretion, instead of from defects in the INH gene itself.
In conclusion, in contrast to previous studies (Shelling et al., 2000; Marozzi et al., 2002; Dixit et al., 2004) and in accordance to Jeong et al. (2004), our results might indicate that –16C>T and 769G>A variants in INH gene may not be associated with POF disease. Other studies on the general population, as well as more detailed functional studies, are required to elucidate if substitution 769G>A is a ‘normal’ variant of INH gene. In addition, further investigations into a larger number of POF patients from different populations should be conducted, in order to define the role of INH gene in the aetiology of POF.
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The study was supported by a grant from Alberto J. Roemmers Foundation and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. We thank Dr S. Belli and the División de Endocrinología, Hospital Durand and Dr L. Alba, Departamento de Genética Clínica, Centro Nacional de Genética Médica, Buenos Aires, Argentina, for help in patient recruitment and diagnosis.
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Submitted on June 2, 2005; resubmitted on November 15, 2005; accepted on November 21, 2005.
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