1 Departments of Obstetrics and Gynaecology, 2 Human Genetics, 3 Epidemiology, and 4 Chemical Endocrinology, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: diminished ovarian reserve/fragile X premutation/FSH/inhibin B/premature ovarian failure
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
With expanding knowledge and progression in diagnostics and therapeutics, counselling should be given, not only about the genetic risks, but also about preimplantation genetic diagnosis (PGD) and, therefore, IVF. For this reason premutation carriers should be counselled about the risk of fertility problems related to POF.
Presently, little is known about the endocrinology of premenopausal premutation carriers and the spontaneous pregnancy rates or pregnancy rates after IVF procedures. To determine whether young fragile X premutation carriers show endocrine signs of premature ovarian dysfunction, we investigated the endocrine profiles of young women from fragile X families who were using combined oral contraceptives, and those who did not. To our knowledge, only one pilot study (Braat et al., 1999) and one larger study (Murray et al., 1999
) have been conducted on the endocrinology of premenopausal premutation carriers not using combined oral contraceptives. The latter study determined endocrine profiles in blood samples taken on one occasion between days 1 and 10 of the menstrual cycle from premenopausal premutation carriers and women not carrying a premutation aged 1753 years. The premenopausal fragile X premutation carriers showed higher serum concentrations of FSH than the other women (i.e. full mutations and controls). The present study, however, focused on women aged <40 years, because these women are at risk of developing POF, which occurs, by definition, prior to the age of 40 years. Hence, we studied the endocrine profiles in women with menstrual cycles and in those who were using oral contraceptives.
Women who are using combined oral contraceptives will not show any overt signs of diminished ovarian function. Owing to the administration of oestrogens and the monthly withdrawal bleeding, most women who are using combined oral contraceptives will not know whether they are peri- or post-menopausal. Combined oral contraceptives suppress the hypothalamicpituitaryovarian axis (Mishell et al., 1977). Suppression of FSH release seems to be an exclusive effect of 17
-ethinyl oestradiol and inhibin B, whereas the release of LH is modified by both 17
-ethinyl oestradiol and the progestin components. Combined oral contraceptives inhibit normal ovarian function, result in anovulation and cause withdrawal bleeding. During the pill-free interval, the function of the hypothalamicpituitaryovarian axis is restored. Serum concentrations of FSH, LH and 17ß-oestradiol start to rise, with a parallel increase in inhibin B (Renier et al., 1998
). Gonadotrophin concentrations return to the normal early follicular phase concentrations (Cohen and Katz, 1979
, 1981
), although the 17ß-oestradiol concentrations are still lower than those in the early follicular phase (Van der Spuy et al., 1990
).
We analysed the serum concentrations of FSH and 17ß-oestradiol in women aged <40 years. The serum inhibin B concentration was also assessed to evaluate whether inhibin B was decreased in women who were carrying a fragile X premutation. Inhibin B is produced by (pre)antral follicles (Roberts et al., 1993). In the early follicular phase, inhibin B has an inhibitory effect on the FSH secretion. As the ovary ages, the number of follicles declines. It has been suggested that a decrease in inhibin B is the earliest marker of reproductive ageing (Welt et al., 1999
). However, the clinical value of inhibin B testing is doubted by other groups (Corson et al., 1999
), and therefore remains controversial.
Studies demonstrating that, with increasing age, FSH starts to rise did not evaluate whether FSH concentrations are, in fact, a measure of ovarian reserve. However, they documented that FSH concentrations increase at the same time that the incidence of diminished ovarian reserve increases (Sharara et al., 1998). Most data concerning the predictive value of FSH are from studies in an assisted reproductive technology setting, in which the predicting value of basal FSH is evaluated for pregnancy rates, cancellation rates, etc., during treatment (Scott et al., 1995; Sharara et al., 1998
). Recently, it was demonstrated that the predictive value of FSH in predicting ovarian response was 79%, again in assisted reproductive technology setting (Creus et al., 2000
). In such studies, women with an FSH concentration of
15 IU/l have lower spontaneous pregnancy rates as well as lower pregnancy rates after IVF procedures than women with a serum FSH concentration of <15 IU/l (Scott and Hofmann, 1995
). We evaluated whether more premutation carriers aged <40 years demonstrated a serum FSH concentration of
15 IU/l than women not carrying a premutation.
As FSH concentrations increase with age in women with regular menstrual cycles (Lee et al., 1988), we evaluated whether premutation carriers with normal cycles had higher FSH concentration than women not carrying the premutation with normal cycles, after correcting for age.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We selected women from our ongoing study who either menstruated at least once every 3 months or were using combined oral contraceptives. All participants gave informed consent for the study, which was approved by the institutional review board. As in a previous paper (Murray et al., 1999), the participants were further stratified into premutation carriers and the other women. The latter group comprises women who were not carrying a premutation, i.e. the women with either a normal or a fully-mutated FMR1 gene (as from here designated as: the other women). As serum FSH concentrations are higher in women who smoke cigarettes (El-Nemr et al., 1998
) we stratified the women into two subcategories, i.e. women who were currently smoking and those who were not (ex- and non-smokers).
Study protocol
In this study, normal menstrual cycles were defined as regular cycles with no more than 5 days intercycle time variation and a length of 2432 days. Cycles that deviated from these criteria were considered to be abnormal. Venous blood samples were taken on the third day of the menstrual cycle for women with menstrual cycles. For the women who were using oral contraceptives, blood samples were taken on the last day of the 7 day pill-free interval. The rationale for these sampling days was day 3 for comparison with previous studies and day 7 as the point at which the basal FSH concentration had returned to that of pretreatment early follicular phase concentrations (Cohen and Katz, 1981). Serum FSH and LH concentrations were measured using the random access analyser AxSym (Abbott Laboratories, Chicago, IL, USA), which is a fully automated analyser system based on microparticle enzyme immunoassay (MEIA) technology. Serum 17ß-oestradiol and progesterone concentrations were measured with radioimmunoassays developed in the endocrinology laboratory of this hospital and included preanalytical diethylether extraction of the serum specimens followed by Sephadex-LH20 chromatography and subsequent radioimmunoassay (Thomas et al., 1977
). Serum inhibin B assays were performed using the Inhibin B Dimer assay kit (Serotec, Oxford, UK). With this assay, the lowest detection limit of inhibin B is 4 ng/l. In order to perform statistical analyses we considered inhibin B concentrations of <4 ng/l to be 2 ng/l.
Statistical analysis
Means and SD were compared using independent sample t-tests. We also used multiple linear regression analyses to compare the endocrine profiles of the premutation carriers with those of the other women, while adjusting for the effects of age and smoking. To stabilize the variance, we first transformed our data by taking the natural logarithm (ln) of the serum concentrations. Sometimes more than one carrier from the same family was participating in this study. To avoid familial influences on FSH concentrations, we reanalysed the data while including only one randomly selected premutation carrier per family. In case of more than one woman not carrying the premutation (the other women), the analysis was conducted in an identical manner, thus only including one randomly selected other woman. A 2 test was used to compare the significance of differences in the number of premutation carriers with a serum FSH of
15 IU/l to the number of the other women with a serum FSH concentration of
15 IU/l. All analyses were performed with the Statistical Package for Social Sciences software (SPSS), version 9.0. P < 0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Women with menstrual cycles (n = 45)
A total of 17 women were carriers of the fragile X premutation, while 28 were not (other women). The latter group comprised 16 women with a normal FMR1 gene and 12 who had a full mutation. One premutation carrier had a serum progesterone concentration of 16 nmol/l on day 3 of the menstrual cycle. This concentration probably reflected the luteal phase concentration of the previous cycle, so this woman was excluded from further analysis. The mean age of the premutation carriers and the other women was 36 ± 2.2 and 34 ±4.7 years respectively (Table I). Six (37.5%) premutation carriers and six (21.4%) of the other women were currently smoking. The serum FSH in the premutation carrier group was in the range 5.439.1IU/l, while that in the other women was 1.812.9 IU/l (Figure 1
).
|
|
Since our analyses sometimes included more than one premutation carrier or other women from the same family, we reanalysed our data as described in above. It was demonstrated that after correction for possible familial influences on FSH, the premutation carriers had significantly higher FSH concentrations than the other women (P = 0.002).
In addition, we assessed serum inhibin B concentrations. In the premutation carrier group, they were in the range 2.0148 ng/l, while in the other women they were 2.0 169 ng/l. The mean serum inhibin B concentration was 52.8 ± 42.6 ng/l in the premutation carriers, and 70.7 ± 37.8 ng/l in the other women (Table I), which was not significantly different. The concentrations of 17ß-oestradiol were evenly distributed over the two groups.
To evaluate whether the premutation carriers with normal cycles had endocrine signs of diminished ovarian function, we first excluded the data from the women with abnormal menstrual cycles (n = 7). We subsequently analysed the data from the women with normal cycles (n = 37). The mean serum FSH concentration was 11.0 ± 7.89 IU/l in the regularly menstruating premutation carriers, and 6.92 ± 2.71 IU/l in the other women (see Table I). Multiple linear regression analyses demonstrated that these premutation carriers had significantly higher serum FSH concentrations than the other women with a normal cycle (P = 0.029). A serum FSH of
15 IU/l was observed in one out of 12 premutation carriers, but in none of the 25 other women (P > 0.05). There were no significant differences in serum inhibin B and 17ß-oestradiol.
Women on oral contraceptives (n = 34)
All these women were using low-dose (<50 µg 17-ethinyl oestradiol) combined oral contraceptives. A total of 13 women were premutation carriers, while the 21 other women were not. In the latter group, 11 women had a normal FMR1 gene and 10 had a full mutation.
The mean age of the premutation carriers and other women was 34 ± 5.3 years and 31 ± 4.5 years respectively (Table I). Six (46%) premutation carriers and five (24%) of the other women were currently smoking.
Endocrine profiles were assessed in the blood samples obtained on the last day of the 7-day pill-free interval. Serum FSH ranged from 6.0 to 54 IU/l in the premutation carrier group and from 3.5 to 16 IU/l in the other women (Figure 2). The mean serum FSH concentration was 20.4 ± 13.5 IU/l in the premutation carriers and 8.35 ± 3.5 IU/l in the other women (Table I
). After adjusting for age and smoking behaviour, linear regression analysis demonstrated that the premutation carriers still had significantly higher serum FSH concentrations (P = 0.001). We observed a serum FSH of
15 IU/l in significantly more of the premutation carriers than in the other women (
2 test; P = 0.013). Overall, this was the case in seven (54%) out of the 13 premutation carriers, and in two (9.5%) out of the 21 other women (Figure 2
). We observed one 34 year old premutation carrier who had an FSH concentration of 54 IU/l. After correcting for possible familial influences in FSH, it was shown that the carriers had significantly higher FSH concentrations than the other women (P = 0.001).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A serum FSH concentration of 15 IU/l was more frequently observed in premutation carriers (both oral contraceptive users, and non-users) aged <40 years than in other women, which suggests that these premutation carriers have a poorer prognosis for future pregnancy. However, most data concerning the predictive value of FSH are from studies in an assisted reproductive technology setting. It remains questionable whether these findings also apply to women who are not being treated for infertility problems.
An item of concern was that sometimes more than one female per family participated in our study. We investigated whether familial bias factors influencing FSH concentrations were present in our data. It was found that after correction for multiple familial participation premutation carriers had higher FSH concentrations than the other women. This was the case in both carriers who were using oral contraceptives and in those who were not.
Although inhibin B was assumed to be the first marker of ovarian ageing (Welt et al., 1999), clinical relevance is still elusive. Furthermore, to our knowledge, there are no reports on the interpretation of serum inhibin B concentrations in the pill-free interval. Nevertheless, we measured inhibin B and found no statistically lower concentrations in premutation carriers than in the other women; neither in the contraceptive users, nor in the women with menstrual cycle. This is in line with a recent study (among a comparable study population) that did not find a significant association either, although the authors consider their inhibin data to be inconclusive (Murray et al., 2000
). Therefore, it remains questionable whether the assessment of inhibin B has any clinical relevance.
Young premutation carriers who are using combined oral contraceptives will not be able to recognize peri-menopausal signs, due to the administration of exogenous oestrogens. In the present study, we demonstrated that unrecognized ovarian dysfunction, or even worse, POF (as was the case in the premutation carrier with a serum FSH of 54 IU/l) was present in female carriers of the fragile X premutation using combined oral contraceptives. To our knowledge, this is the first report on unrecognized diminished ovarian function in women who are using combined oral contraceptives.
In women who have just started using oral contraceptives, the suppressed FSH concentrations return to the normal baseline concentration (of the control cycle) during the pill-free period (Cohen and Katz, 1979; Cohen and Katz, 1981
). In peri- or post-menopausal women who have just started using oral contraceptives, FSH is not restored to the previously established control baseline concentration during the short pill-free interval (Creinin, 1996
). All these reports compared the baseline concentration of FSH with the hormone concentration at the end of the pill-free interval. Thus, it seems reasonable to conclude that endocrine profiles assessed on the last day of the pill-free interval do not necessarily reflect the (non-suppressed) baseline concentrations. As the present study lacked data on baseline FSH, it was not possible to determine whether the serum FSH values returned to baseline. It is possible that women with high serum FSH concentrations may have even higher baseline concentrations of FSH. In the present study, the premutation carriers had higher serum FSH concentrations and so we expect that especially the premutation carriers will have even higher baseline FSH concentrations.
Four women were using combined oral contraceptives to regulate their irregular cycles: one premutation carrier (36 years, FSH 34.9 IU/l) and three other women (aged 25, 28 and 28 years, with serum FSH concentrations of 9.2, 6.7 and 7.1 IU/l respectively). It is possible that, at that time, these irregular cycles were caused by diminished ovarian function. In order to minimize the risk of potential selection bias (by including women who were taking combined oral contraceptives for diminished ovarian function), we also performed a multiple regression analysis which excluded these four women from the analysis. The FSH concentrations remained significantly higher in the premutation carriers (P = 0.006).
In order to analyse the endocrinology of normally menstruating women, we had to exclude seven women from this analysis because they reported having abnormal menstrual cycles (four premutation carriers and two other women). In the women with abnormal cycles, the premutation carriers only differed from the other women in having significantly higher serum FSH concentrations (t-test; P = 0.043). In the premutation carriers the mean serum FSH was 27 IU/l, while the mean serum inhibin B was 11.1 ng/l. In the other women, these concentrations were 4.40 IU/l and 79.7 ng/l respectively (Table I). In our view, it seems that especially the premutation carriers with abnormal cycles demonstrated signs of ovarian ageing, which may have caused these cycle abnormalities. However, the numbers were small; further research may help to elucidate this issue.
Figures 1 and 2 depict a few outlying observations, which may represent a subset of women who had ovarian dysfunction. All these outlying observations are accounted for by premutation carriers. However, not all premutation carriers had endocrine signs of ovarian reserve diminution, and therefore it is possible that the group of premutation carriers could have been divided in at least two subgroups with different phenotypes and, possibly, genotypes. Recently, we found that women with a paternally-inherited premutation (PIP) have a high risk for developing POF, whereas women with a maternally-inherited premutation (MIP) do not (Hundscheid et al., 2000
). We evaluated whether a parent-of-origin effect could be detected in the endocrine profiles. Therefore, we analysed the data on all the premutation carriers in whom the parental origin of the premutation could be determined. We found no associations between ovarian dysfunction and the parental origin of the premutation. The numbers, however, were too small to draw any firm conclusion. Further research is warranted to determine whether women with a PIP have a different endocrine profile from other women.
In conclusion, premutation carriers demonstrated increased serum FSH concentrations in comparison with the other women. This applies to premutation carriers with regular cycles as well as to those who were using oral contraceptives. Moreover, serum FSH concentrations of 15 IU/l were more frequently observed in premutation carriers, irrespective of the use of oral contraceptives. Both findings suggest that premutation carriers show endocrine signs of diminished ovarian function, which is in line with the observation that premutation carriers have a high risk of developing POF. Women with an FSH of
15 IU/l have lower spontaneous pregnancy rates as well as lower pregnancy rates after IVF than women with a serum FSH of <15 IU/l (Scott and Hofmann, 1995
). Hence, premutation carriers need to be informed about this risk, especially if they are considering PGD, which implies IVF. In the case of mild FSH elevations, it might be possible to achieve pregnancy with assisted reproductive technology. At present, we can only advise young carriers of the fragile X premutation not to postpone starting a family for too long.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Braat, D.D.M., Smits, A.P.T. and Thomas, C.M.G. (1999) Menstrual disorders and endocrine profiles in fragile X carriers prior to 40 years of age. Am. J. Med. Genet., 83, 327328.[ISI][Medline]
Cohen, B.L. and Katz, M. (1979) Pituitary and ovarian function in women receiving hormonal contraception. Contraception, 20, 475487.[ISI][Medline]
Cohen, B.L. and Katz, M. (1981) Further studies on pituitary and ovarian function in women receiving hormonal contraception. Contraception, 24, 159172.[ISI][Medline]
Corson, S.L., Gutmann, J., Batzer, F.R. et al. (1999) Inhibin-B as a test of ovarian reserve for infertile women. Hum. Reprod., 14, 28182821.
Creinin, M.D. (1996) Laboratory criteria for menopause in women using oral contraceptives. Fertil. Steril., 66, 101104.[ISI][Medline]
Creus, M., Penarrubia, J., Fabregues, F. et al. (2000) Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum. Reprod., 15, 23412346.
El-Nemr, A., Al-Shawaf, T., Sabatini, L. et al. (1998) Effect of smoking on ovarian reserve and ovarian stimulation in in-vitro fertilization and embryo transfer. Hum. Reprod., 13, 21922198.[Abstract]
Feng, Y., Lakkis, L., Devys, D. et al. (1995) Quantitative comparison of FMR1 gene expression in normal and premutation alleles. Am. J. Hum. Genet., 56, 106113.[ISI][Medline]
Hundscheid, R.D.L., Sistermans, E.A., Thomas, C.M.G. et al. (2000) Imprinting effect in premature ovarian failure confined to paternally inherited fragile X premutations. Am. J. Hum. Genet., 66, 413418.[ISI][Medline]
Imbert, G., Feng, Y., Nelson, D.L. et al. (1998) FMR1 and mutations in fragile X syndrome: molecular biology, biochemistry, and genetics. In Wells, R.D. and Warren, S.T. (eds), Genetic Instabilities And Hereditary Neurological Disorders. Academic Press, New York, USA, pp. 2753.
Lee, S.J., Lenton, E.A., Sexton, L. et al. (1988) The effect of age on the cyclical patterns of plasma LH, FSH, oestradiol and progesterone in women with regular menstrual cycles. Hum. Reprod., 3, 851855.[Abstract]
Marozzi, A., Vegetti, W., Manfredini, E. et al. (2000) Association between idiopathic premature ovarian failure and fragile X premutation. Hum. Reprod., 15, 197202.
Mishell, D.R. Jr., Kletzky, O.A., Brenner, P.F. et al. (1977) The effect of contraceptive steroids on hypothalamic-pituitary function. Am. J. Obstet. Gynecol., 128, 6074.[ISI][Medline]
Murray, A., Webb, J., MacSwiney, F. et al. (1999) Serum concentration of follicle stimulating hormone may predict premature ovarian failure in FRAXA premutation women. Hum. Reprod., 14, 12171218.
Murray, A., Ennis, S., MacSwiney, F. et al. (2000) Reproductive and menstrual history of females with fragile X expansions. Eur. J. Hum. Genet., 8, 247252.[ISI][Medline]
Renier, M.A., Vereecken, A., van Herck, E. et al. (1998) Dimeric inhibin serum values as markers of ovarian activity in pill-free intervals. Contraception, 57, 4548.[ISI][Medline]
Roberts, V.J., Barth, S., El-Roeiy, A. et al. (1993) Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle. J. Clin. Endocrinol. Metab., 77, 14021410.[Abstract]
Scott, R.T. Jr and Hofmann, G.E. (1995) Prognostic assessment of ovarian reserve. Fertil. Steril., 63, 111.[ISI][Medline]
Sharara, F.I., Scott, R.T. Jr. and Seifer, D.B. (1998) The detection of diminished ovarian reserve in infertile women. Am. J. Obstet. Gynecol., 179, 804812.[ISI][Medline]
Thomas, C.M.G., Corbey, R.S. and Rolland, R. (1977) Assessment of unconjugated oestradiol and progesterone serum concentrations throughout pregnancy in normal women and in women with hyperprolactinaemia, who conceived after bromocriptine treatment. Acta Endocrinol. (Copenh.), 86, 405414.[Medline]
Uzielli, M.L., Guarducci, S., Lapi, E. et al. (1999) Premature ovarian failure (POF) and fragile X premutation females: from POF to fragile X carrier identification, from fragile X carrier diagnosis to POF association data. Am. J. Med. Genet., 84, 300303.[ISI][Medline]
Van der Spuy, Z.M., Sohnius, U., Pienaar, C.A. et al. (1990) Gonadotropin and estradiol secretion during the week of placebo therapy in oral contraceptive pill users. Contraception, 42, 597609.[ISI][Medline]
Verkerk, A.J.M.H., Pieretti, M., Sutcliffe, J.S. et al. (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell, 65, 905914.[ISI][Medline]
Welt, C.K, McNicholl, D.J., Taylor, A.E. et al. (1999) Female reproductive ageing is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab., 84, 105111.
Submitted on July 20, 2000; accepted on November 21, 2000.