1 Department of Biology and Genetics, Medical Faculty, University of Milan, Via Viotti 3/5, 20133 Milan, 2 First Department of Obstetrics and Gynaecology, University of Milan, 3 Department of Clinical and Biological Sciences, Ospedale di Circolo, Varese and 4 Department of Experimental and Environmental Medicine and Medical Biotechnology, University of MilanBicocca, Monza, Italy
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: inhibin/mutation/ovarian failure/premature ovarian failure/sterility
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
From the observation that some POF women carry chromosomal abnormalities and that several families show at least two relatives experiencing POF, the disease can be considered as a genetic disorder. Pedigree studies of affected families suggest that the idiopathic POF condition may be inherited as an autosomal dominant sex-limited transmission or X-linked with incomplete penetrance (Vegetti et al., 1998; Van Kasteren et al., 1999
).
Although the pathogenesis of POF is still unknown, several cellular mechanisms can be suggested as responsible for the disease, including a reduced primordial follicle pool, an accelerated follicular atresia, and an alteration of follicular recruitment and maturation (Christin-Maitre et al., 1998). Some of these cellular events could be correlated to high FSH levels (te Velde et al., 1998
). Thus, FSH and other genes implicated in the FSH signalling, or in the control of the FSH level, have been considered responsible for POF.
Supporting this hypothesis, the analysis of the FSH receptor gene revealed a point mutation in exon 7 in Finnish families affected by primary amenorrhoea (Aittomaki et al., 1995). However, further studies on different cohorts of POF patients did not identify the above mutation and must be unique to the Finnish population (Layman et al., 1998
; Conway et al., 1999
).
Inhibins and activins are multifunctional hormones, belonging to the transforming growth factor-ß superfamily of proteins, which inhibit or stimulate respectively, the synthesis and secretion of FSH (Ling et al., 1985; Miyamoto et al., 1985
; Rivier et al., 1985
; Robertson et al., 1985
; Ling et al., 1986
; Vale et al., 1986
). Furthermore, inhibins increase the number of ovarian follicles (O et al., 1989
), and inhibit oocyte meiosis (Hsueh et al., 1987
), whereas activins cause follicular atresia (Woodruff et al., 1990
), and induce granulosa cell proliferation in vitro (Rabinovici et al., 1990
).
Inhibins (INH) are heterodimeric glycoproteins constituted by an inhibin subunit and by one of the two related ß subunits (ßA or ßB), to produce respectively INHA and INHB (Ling et al., 1985
; Miyamoto et al., 1985
; Rivier et al., 1985
; Robertson et al., 1985
). Activins are homodimeric glycoproteins composed of inhibin ßA or ßB subunits (Ling et al., 1985
; Vale et al., 1986
).
The synthesis of the three inhibin subunits (, ßA and ßB) is restricted to the granulosa cells of the ovary from early stages of folliculogenesis (Drummond et al., 1996
). The comparison of perimenopausal with mid-reproductive-aged women by INHA, INHB and activin assays, indicates that a decrease in both INHA and INHB, or an increase in activin A (Danforth et al., 1998
; Santoro et al., 1999
), is responsible for the high level of FSH characteristic of reproductive ageing. The observed variation of inhibin:activin ratio is probably due to the deficit of the
subunit production, which leads to the preferential formation of activin homodimers (Santoro et al., 1999
). Thus, the POF condition could be a consequence of mutations in the INH
gene, which causes a decrease in the amount of bioactive inhibin, and consequently an increase in FSH concentration.
Recently, the analysis of the INH gene has revealed a missense mutation (769G
A transition) in exon 2 of the gene, occurring in three out of 43 POF women (Shelling et al., 2000
). The mutation has been hypothesized to impair the binding of INHA and INHB to their receptor, and therefore to inhibit the activation of the signal transduction pathway.
We analysed 205 women affected by ovarian failure to evaluate the possible association between the 769GC transition and hypergonadotrophic amenorrhoea, to further test the concept that INH
can be considered a gene involved in ovarian dysfunction.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The POF status was defined as the cessation of ovarian function for a period of >6 months, before or at the age of 40 years, and FSH concentration 40 IU/l detected on two different occasions. The EM condition is defined as the occurrence of menopause before the age of 45 years.
Complete medical and gynaecological history, including age at menarche and previous menses, were undertaken for all patients. Family history was reviewed during genetic counselling, and family members were traced back three generations. All the patients included in this study were phenotypically normal and considered idiopathic because they did not show any POF-related conditions (ovarian surgery, previous chemo- or radiotherapy, autoimmune diseases or metabolic disorders such as galactosaemia). Sixty-five POF patients had a family history of premature menopause, with at least one relative experiencing POF (n = 48) or early menopause (EM) (n = 17). Conversely, the remaining 92 patients were classified as having sporadic POF. Among the patients with primary amenorrhoea, six patients (four families) showed a familial condition.
None of the patients carried structural or numerical chromosome anomalies, as evaluated by karyotype analysis based on high resolution banding technique, on at least 30 metaphases. A total of 100 peripheral blood samples was obtained from woman who experienced physiological menopause; this cohort was used as the control group. All the women included in the study gave their informed consent to review their medical history and to collect a peripheral blood sample suitable for further cytogenetic and molecular analysis.
DNA extraction and PCR
Genomic DNA was extracted from 1 ml of peripheral blood by the proteinase K method as previously described (Marozzi et al., 1999). Two regions of the INH
gene were analysed by PCR. The first region of 444 bp, which includes the coding region of exon 1 (268 bp), 119 bp of the 5'-UTR, and 57 bp of the first intron, was amplified by primers INH
ex1F (5'-AAGGGTAGAAGAGGGTGGGTGTGG) and INH
ex1R (5'-CATGCTGTGCCTTGCTTTTCTCA). The amplification of a second region of 601 bp, which includes part of exon 2, was carried out by primers INH
F and INH
R, as previously described (Shelling et al., 2000
). Moreover, an internal region of the exon 2 comprising 243 bp was amplified by employing primers INH
1F and INH
1R (Shelling et al., 2000
). PCR reactions were carried out using 100 ng of genomic DNA as a template in the presence of 50 pmol of both primers, in a final volume of 50 µl. After denaturation at 94°C for 5 min, the samples underwent 30 cycles of amplification (94°C denaturation for 45 s, 5865°C annealing for 45 s, 72°C extension for 1 min); the last cycle was followed by 10 min extension at 72°C.
Restriction fragment length polymorphism (RFLP) analysis and sequencing
The 5'-UTR and exon 1 region of the INH gene was analysed by sequencing the PCR product from patient and control DNA samples obtained using primers INH
ex1FINH
ex1R. Conversely, exon 2 PCR products obtained by primers INH
1FINH
1R were analysed for the Bst71I restriction enzyme polymorphism (RFLP), since the occurrence of the 769G
A transition abolishes the Bst71I restriction site. The digestion was performed as previously described (Shelling et al., 2000
), and the products analysed by 3% agarose gel electrophoresis. Wild-type DNA yields three fragments of 85, 25 and 134 bp, whereas a homozygous sample for the mutation gives only two fragments of 85 and 159 bp. DNA samples showing the occurrence of the RFLP were further analysed by sequencing the PCR products obtained using primers INH
F and INH
R (Shelling et al., 2000
). Sequencing was performed on PCR products purified by nucleospin extraction kit (Macherey-Nagel, Düren, Germany) following the Big-Dye terminator sequencing protocol consisting of 50 ng DNA, 2 µl Big Dye terminator mix, and 3.2 pmol primers (Applied Biosystems, Foster City, CA, USA). All fluorescent traces were analysed using the Applied Biosystem Model 3100 DNA Sequencing System.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The seven POF patients carrying the 769GA mutation had a mean age of menopause onset of 35.6 ± 3.7 years (range 3040), whereas the mean age of menopause experienced by their mothers was 44.3 ± 5.8 years (range 3650) (Table II
). All the women carrying the 769G
A mutation and belonging to the group of familial POF showed a maternal transmission of the disease (patients 65, 55 and 162), whereas the POF women with a family history of EM showed either a maternal or paternal transmission of the disease (patients 77 and 96 respectively) (Table II
).
|
Almost all the patients recruited in this study were also screened for DNA variations in the 5'-UTR and exon 1 of the INH gene. The analysed region spans 444 bp; the forward primer was located 25 nucleotides downstream of the transcription start, and the reverse primer located in the first intron, 57 nucleotides downstream of the end of exon 1. The analysis, carried out by direct DNA sequencing, showed a 129C
T transition in the 5'-UTR. This transition was found in 28 out of 142 POF patients (19.7%), eight out of 34 EM patients (23.5%), and in three out of 11 primary amenorrhoea patients (27%). Due to the unavailability of DNA, the same analysis was carried out on 69 DNA samples from the control group; this analysis revealed the T allele in 23 women (33%). The result indicates that the prevalence of the C variant in POF patients (80.3%) is higher than expected (66.7%) (Fisher's exact test, P = 0.014).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Women showing ovarian failure were subclassified into three groups of patients: affected by POF (n = 157), by EM (n = 36), and by primary amenorrhoea (n = 12 women). The molecular analysis indicated that seven POF and three primary amenorrhoea patients were heterozygous for the transition, whereas both EM patients (n = 36) and controls (n = 100) did not have the INH 769G
A variant. Since the women belonging to the control group experienced physiological menopause, the obtained results strengthen the concept of the INH
gene as a candidate for ovarian failure. Furthermore, the absence of the 769G
A transition in patients affected by the EM condition, defined as the cessation of ovarian function between 40 and 45 years of age, allows us to suggest that the mutation has an effect on the timing of ovarian failure manifestation.
The incidence of the mutation within both the POF (sporadic and familial) (seven out of 157) and primary amenorrhoea (three out of 12) patients is statistically significant, being respectively 4.5% (Fisher's exact test, P = 0.030) and 25% (Fisher's exact test, P < 0.0001). Previous work suggested that POF and EM conditions represent a variable expression of the same genetic disease (Torgerson et al., 1997; Tibiletti et al., 1999
). In this regard, the inclusion of the POF patients with familial EM into the group of familial POF gives a statistically significant likelihood of finding the mutation in the familial condition (5/65; 7.7%) (Fisher's exact test, P < 0.001), compared with the control group (0/100). The prevalence of the mutation in familial versus sporadic POF could be explained by the inheritance of a particular genetic background, which, in association with the 769G
A variant, could lead to POF.
The observed median age of POF onset in patients carrying the mutation was 35.6 ± 3.7 years. This figure is higher than that previously reported (Shelling et al., 2000) for three POF patients carrying the same mutation (20 ± 4). The small number of analysed patients may represent a possible explanation for the discrepancies observed on the median age of POF onset. Furthermore, we identified a high percentage of primary amenorrhoea patients with the mutation (three out of 12). One possibility to explain how the 769G
A variant might be involved in causing infertility, is to hypothesize that in primary amenorrhoea patients the mutation is highly penetrant.
A further contribution to the concept of POF as a heterogeneous disease can be derived from the analysis of families showing the inheritance of both POF and INH gene mutation. In one family, we found the co-segregation of the 769G
A mutation with POF (Figure 1C
), but in another family the proband inherited the mutation from the father, whereas the mother affected by POF did not carry the mutation (Figure 1D
). In this case, the POF manifestation in the mother is not correlated to the INH
gene mutation, and thus she represents a phenocopy of her daughter, probably due to the genetic heterogeneity of the disease. Conversely, the previous analysis of another POF family showed the occurrence of the mutation in a woman experiencing physiological menopause (Shelling et al., 2000
). The occurrence of the mutation without POF manifestation could be explained through incomplete penetrance.
To investigate whether other sequence variations within the INH gene might be linked to the development of POF, we also analysed the 5'-UTR and exon 1. We only detected a single nucleotide polymorphism in the 5'-UTR: a 129C
T transition. The derived frequency of the C allele (80.3%) was significantly higher (Fisher's exact test, P = 0.014) in the POF population than in the control group (66.7%). Interestingly, none of the analysed patients simultaneously carried the 769G
A variant and the T allele. Moreover, the frequency of the C allele in the POF group is comparable with that observed in a large population of women from 326 pedigrees showing an elevated frequency of dizygotic (DZ) twin pregnancy (Montgomery et al., 2000
). Mothers of DZ twins have a higher incidence of spontaneous multiple ovulation and elevated FSH concentrations (Martin et al., 1991
). FSH release is controlled by a negative feedback mediated by the INH
peptide produced by the ovary; thus, a reduced concentration of the INH
peptide is thought to result in elevated levels of FSH and multiple ovulation. Although the IHN
5'-UTR variant was not found in linkage with DZ twinning, it is possible to speculate that in the POF population this variant may contribute, in concomitance with variations in other genes, to the increased rate of follicular depletion (Richardson et al., 1987
). The implication of the single nucleotide polymorphism in follicle depletion could be derived by the evaluation of INH
peptide levels (McConnell et al., 1998
) in carriers of the C allele, but not affected by the disease.
Further investigations are required to confirm our preliminary observation on the preferential occurrence of the 769GA mutation in familial POF. In the case of validation of this observation, the INH
mutation analysis can represent a useful diagnostic marker and allow prevention of the early onset of ovarian failure by replacement of the inhibin hormone. The mutation analysis could also be important to allow family members to anticipate the decision of conception.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conway, G.S., Conway, E., Walker, C., Hoppner, W., Gromoll, J. and Simoni, M. (1999) Mutation screening and isoform prevalence of the follicle stimulating hormone receptor gene in women with premature ovarian failure, resistant ovary syndrome and polycystic ovary syndrome. Clin. Endocrinol., 51, 9799.[ISI][Medline]
Coulam, C.B., Adamson, S.C. and Annegers, J.F. (1986) Incidence of premature ovarian failure. Obstet. Gynecol., 67, 604606.[Abstract]
Christin-Maitre, S., Vasseur, C., Portnoi, M.F. and Bouchard, P. (1998) Genes and premature ovarian failure. Mol. Cell. Endocrinol., 145, 7580.[ISI][Medline]
Danforth, D.R., Arbogast, L.K., Mroueh, J., Kim, M.H., Kennard, E.A., Seifer, D.B. and Friedman, C.I. (1998) Dimeric inhibin: a direct marker of ovarian aging. Fertil. Steril., 70, 119123.[ISI][Medline]
Drummond, A.E., Dyson, M., Mercer, J.E. and Findlay, J.K. (1996) Differential responses of post-natal rat ovarian cells to FSH and activin. Mol. Cell. Endocrinol., 122, 2132.[ISI][Medline]
Hsueh, A.J., Dahl, K.D., Vaughan, J., Tucker, E., Rivier, J., Bardin, C.W. and Vale, W. (1987) Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Proc. Natl Acad. Sci. USA, 84, 50825086.[Abstract]
Layman, L.C., Amde, S., Cohen, D.P., Jin, M. and Xie, J. (1998) The Finnish follicle-stimulating hormone receptor gene mutation is rare in North American women with, 46,XX ovarian failure. Fertil. Steril., 69, 300302.[ISI][Medline]
Ling, N., Ying, S.Y., Ueno, N., Esch, F., Denoroy, L. and Guillemin, R. (1985) Isolation and partial characterization of a Mr, 32,000 protein with inhibin activity from porcine follicular fluid. Proc. Natl Acad. Sci. USA, 82, 72177221.[Abstract]
Ling, N., Ying, S.Y., Ueno, N., Shimasaki, S., Esch, F., Hotta, M. and Guillemin, R. (1986) A homodimer of the beta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone. Biochem. Biophys. Res. Commun., 138, 11291137.[ISI][Medline]
Marozzi, A., Dalprà, L., Ginelli, E., Tibiletti, M.G. and Crosignai, P.G. (1999) FRAXA premutations are not a cause of familial premature ovarian failure. Hum. Reprod., 14, 573575.
Martin, N.G., Robertson, D.M., Chenevix-Trench, G., de Kretser, D.M., Osborne, J. and Burger, H.G. (1991) Elevation of follicular phase inhibin and luteinizing hormone levels in mothers of dizygotic twins suggests nonovarian control of human multiple ovulation. Fertil. Steril., 56, 469474.[ISI][Medline]
McConnell, D.S., Wang, Q., Sluss, P.M., Bolf, N., Khoury, R.H., Schneyer, A.L., Midgley, A.R., Reame, N.E., Crowley, W.F. and Padmanabhan, V. (1998) A two-site chemiluminescent assay for activin-free follistatin reveals that most follistatin circulating in men and normal cycling women is in an activin-bound state. J. Clin. Endocrinol. Metab., 83, 851858.
Miyamoto, K., Hasegawa, Y., Fukuda, M., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1985) Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem. Biophys. Res. Commun.129, 396403.[ISI][Medline]
Montgomery, G.W., Duffy, D.L., Hall, J., Haddon, B.R., Kudo, M., McGee, E.A., Palmer, J.S., Hsueh, A.J., Boomsma, D.I. and Martin, N.G. (2000) Dizygotic twinning is not linked to variation at the alpha-inhibin locus on human chromosome 2. J. Clin. Endocrinol. Metab., 85, 33913395.
O, W.S., Robertson, D.M. and de Kretser, D.M. (1989) Inhibin as an oocyte meiotic inhibitor. Mol. Cell. Endocrinol. 62, 307311.[ISI][Medline]
Rabinovici, J., Spencer, S.J. and Jaffe, R.B. (1990) Recombinant human activin-A promotes proliferation of human luteinized preovulatory granulosa cells in vitro. J. Clin. Endocrinol. Metab., 71, 13961398.[Abstract]
Richardson, S.J., Senikas, V. and Nelson, J.F. (1987) Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J. Clin. Endocrinol. Metab., 65, 12311237.[Abstract]
Rivier, J., Spiess, J., McClintock, R., Vaughan, J. and Vale, W. (1985) Purification and partial characterization of inhibin from porcine follicular fluid. Biochem. Biophys. Res. Commun., 133, 120127.[ISI][Medline]
Robertson, D.M., Foulds, L.M., Leversha, L. Morgan, F.J., Hearn, M.T., Burger, H.G., Wettenhall, R.E. and de Kretser, D.M. (1985) Isolation of inhibin from bovine follicular fluid. Biochem. Biophys. Res. Commun., 126, 220226.[ISI][Medline]
Santoro, N., Adel, T. and Skurnick, J.H. (1999) Decreased inhibin tone and increased activin A secretion characterize reproductive aging in women. Fertil. Steril., 71, 658662.[ISI][Medline]
Shelling, A.N., Burton, K.A., Chand, A.L., van Ee, C.C., France, J.T., Farquhar, C.M., Milsom, S.R., Love, D.R., Gersak, K., Aittomaki, K. et al. (2000) Inhibin: a candidate gene for premature ovarian failure. Hum. Reprod., 15, 26442649.
Tibiletti, M.G., Testa, G., Vegetti, W., Alagna, F., Taborelli, M., Dalpra, L., Bolis, P.F. and Crosignani, P.G. (1999) The idiopathic forms of premature menopause and early menopause show the same genetic pattern. Hum. Reprod., 14, 27312734.
te Velde, E.R., Scheffer, G.J., Dorland, M., Broekmans, F.J. and Fauser, B.C. (1998) Developmental and endocrine aspects of normal ovarian aging. Mol. Cell. Endocrinol., 145, 6773.[ISI][Medline]
Torgerson, D.J., Thomas, R.E. and Reid, D.M. (1997) Mothers' and daughters' menopausal ages: is there a link? Eur. J. Obstet. Gynecol. Reprod. Biol., 74, 6366.[ISI][Medline]
Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Karr, D. and Spiess, J. (1986) Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature, 321, 776779.[ISI][Medline]
van Kasteren, Y.M., Hundscheid, R.D., Smits, A.P., Cremers, F.P., van Zonneveld, P. and Braat, D.D. (1999) Familial idiopathic premature ovarian failure: an overrated and underestimated genetic disease? Hum. Reprod., 14, 24552459.
Vegetti, W., Tibiletti, M.G., Testa, G., de Lauretis, Y., Alagna, F., Castoldi, E., Taborelli, M., Motta, T., Bolis, P.F., Dalprà, L. et al. (1998) Inheritance in idiopathic premature ovarian failure: analysis of 71 cases. Hum. Reprod., 13, 17961800.[Abstract]
Woodruff, T.K. and Mayo, K.E. (1990) Regulation of inhibin synthesis in the rat ovary. Annu. Rev. Physiol., 52, 807821.[ISI][Medline]
Submitted on January 11, 2002; accepted on March 14, 2002.