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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4569-4574
Copyright © 2004 by The Endocrine Society

Evidence of Early Ovarian Aging in Fragile X Premutation Carriers

C. K. Welt, P. C. Smith and A. E. Taylor

Reproductive Endocrine Unit (C.K.W., P.C.S.), Department of Medicine, Massachusetts General Hospital, Boston Massachusetts 02114; and Pfizer Global Research and Development (A.E.T.), Groton Laboratories, Groton, Connecticut 06340

Address all correspondence and requests for reprints to: Corrine Welt, Reproductive Endocrine, BHX 511, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: cwelt{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Up to 28% of female fragile X premutation carriers develop premature ovarian failure. To test the hypothesis that fragile X premutation carriers with ovulatory menstrual cycles exhibit hormone changes characteristic of early ovarian aging, 11 regularly cycling fragile X premutation carriers, 24–41 yr old (34.5 ± 5.7 yr, mean ± SD), drew daily blood samples across one menstrual cycle. LH, FSH, estradiol, progesterone (P4), inhibin A, and inhibin B levels were compared with levels in 22 age-matched, regularly cycling women, 23–41 yr old (34.6 ± 5.8 yr), at each cycle stage.

Total cycle (26.1 ± 1.0 vs. 28.2 ± 0.4 d; P < 0.05) and follicular phase length (12.9 ± 0.8 vs. 14.5 ± 0.4 d; P < 0.05) were decreased in fragile X premutation carriers compared with age-matched controls, whereas luteal phase length was similar (13.2 ± 0.5 vs. 13.7 ± 0.3 d; P = not significant). FSH was elevated across the follicular (21.9 ± 3.5 vs. 11.2 ± 0.5 IU/liter; P < 0.001) and luteal phases (14.6 ± 3.9 vs. 7.9 ± 0.5 IU/liter; P < 0.05) in fragile X premutation carriers compared with age-matched controls. Inhibin B in the follicular phase (77 ± 11 vs. 104 ± 6 pg/ml; P < 0.05) and inhibin A (3.4 ± 0.7 vs. 5.8 ± 0.5 IU/ml; P < 0.01) and P4 [7.3 ± 1.0 vs. 10.1 ± 0.7 ng/ml (23.2 ± 3.0 vs. 32.1 ± 2.3 nmol/liter); P < 0.05] in the luteal phase were decreased in fragile X premutation carriers compared with age-matched controls, whereas there was no difference in estradiol or LH.

In summary, despite regular ovulatory cycles, FSH was increased in fragile X premutation carriers compared with age-matched controls. The increased FSH was accompanied by decreased inhibin B in the follicular phase and inhibin A and P4 in the luteal phase. These hormonal changes suggest that fragile X premutation carriers exhibit early ovarian aging despite regular menstrual cycles. Early ovarian aging in fragile X premutation carriers likely results from decreased follicle number and function, as reflected by lower inhibin B, inhibin A, and P4 levels.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PREMATURE OVARIAN FAILURE (POF), defined as amenorrhea before the age of 40 yr and in the presence of an elevated FSH level, affects 1% of reproductive-age females (1). Although the underlying cause of POF can be identified in some patients, the majority of POF cases remain unexplained (2). The diagnosis of POF is based on amenorrhea; however, the course is variable with folliculogenesis (78%), ovulation (46%), and pregnancy (14%) occurring in a significant proportion of patients (3). Importantly, the ovulation rate is better in patients with a shorter time from diagnosis (3). Thus, early identification of patients at risk for POF may be important for fertility outcome.

It is now clear that female carriers of the fragile X syndrome premutation are at high risk for POF (4, 5, 6, 7, 8, 9, 10). Fragile X syndrome is one of the most common causes of mental retardation and is characterized by hyperactivity, autism-like behavior, attention deficits, and features including an elongated face with a prominent jaw and large ears. Fragile X syndrome results from expansion of an unstable CGG repeat in the 5' untranslated region of the fragile X mental retardation (FMR)1 gene (11, 12, 13). Normally, seven to approximately 54 CGG repeats are present in the 5' untranslated region of FMR1 (14). When greater than 200 repeats are present, the promoter is methylated, resulting in failure of transcription, absence of the FMR protein (FMRP), and the full fragile X syndrome (13, 15, 16). Premutations are repeat lengths between 55 and 200, which appear to increase transcription but decrease translation, resulting in decreased FMRP (17, 18). The premutations tend to expand to full mutations when passed through the mother, with a frequency related to the number of repeats (11, 19, 20, 21). Repeat lengths between approximately 46–60 are termed intermediate, and they very rarely progress to a full mutation (19).

Previous studies demonstrate that fragile X premutation carriers have a 12–28% rate of POF (4, 5, 6, 7, 8, 9, 10). Paradoxically, they also have an increased rate of twinning (4, 22, 23). These findings are reminiscent of normal reproductive aging in which dizygotic twinning is increased, presumably related to the elevated FSH levels resulting from decreased ovarian feedback (24). Recent studies suggest that FSH is elevated in fragile X premutation carriers (25, 26, 27). However, these studies were small (25) and examined levels on only 1 d in the follicular phase, potentially missing FSH changes in some cycles (26, 27). The sampling frequency was also inadequate to evaluate changes in estradiol (E2) and the inhibins (26, 27). Thus, the prevalence and severity of the reproductive hormone abnormalities have not been well characterized in fragile X premutation carriers, nor has a mechanism of POF been delineated.

To test the hypothesis that fragile X premutation carriers exhibit hormonal changes characteristic of early reproductive aging while still experiencing regular menstrual cycles, a comprehensive evaluation of reproductive hormone levels across the menstrual cycle was performed in fragile X premutation carriers and age-matched controls. The hormone changes demonstrated suggest early reproductive aging in fragile X premutation carriers and suggest that early reproductive aging is more common than previously suspected in these women.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Fragile X premutation carriers (n = 11), 24–41 yr old (34.5 ± 5.7 yr; mean ± SD), with body mass indices of 20–42 kg/m2 (24.7 ± 6.4; mean ± SD), were studied. Subjects were mothers of affected children (n = 9), a cousin of a known premutation carrier (n = 1), and an infertility patient with a high FSH level (n = 1). All subjects had ovulatory menstrual cycles, 21–35 d by report, and no history of amenorrhea except in the setting of pregnancy or breastfeeding.

Control subjects (n = 22) were selected from a previous study of normal reproductive aging (28) and were matched for age to the fragile X premutation carriers (34.6 ± 5.8 yr; mean ± SD; range, 23–41 yr). The control subjects had no personal or family history of POF or mental retardation and had a normal body weight, with body mass indices of 18–27 kg/m2. All control subjects had a history of regular 25- to 35-d menstrual cycles, with evidence of ovulation as indicated by a serum progesterone (P4) level of more than 6 ng/ml (19.1 nmol/liter).

Fragile X premutation carriers and control subjects had no history of excessive exercise, had a normal TSH and prolactin, and were on no hormonal medication for 3 months. The study was approved by the Subcommittee on Human Studies of the Massachusetts General Hospital (MGH), and all subjects gave written informed consent before participation.

Subjects had daily blood samples for one menstrual cycle at MGH or at a location close to their home. In a subset of subjects (fragile X premutation carriers, n = 4; and control subjects, n = 21), transvaginal ultrasounds (Toshiba SAL 727B, 5 MHz convex array transducer, Tokyo, Japan) were performed starting in the late follicular phase to document the number of dominant follicles. The number and maximum diameter of all follicles of more than 10 mm were recorded. Seven fragile X premutation carriers were unable to have transvaginal ultrasounds because the distance to MGH was too great.

Blood samples were obtained from all fragile X premutation carriers for determination of CGG repeat length in the FMR1 gene. A multiplex PCR was performed in which the 5' end of the forward primer was fluorescently tagged, and the products resulting from the PCR were compared with an internal standard for sizing (29, 30). Samples that revealed a single band for FMR1 were subjected to a Southern blot protocol after restriction digest (29).

Assays

LH and FSH were analyzed by RIA (31, 32) or by a two-site monoclonal, non-isotopic, continuous access immunoassay system (AxSYM, Abbott Laboratories, Abbott Park, IL) calibrated with the same reference preparation used in the RIA (33). LH and FSH levels were not significantly different using the two assays across the dynamic range of the assays and across the menstrual cycle within individual subjects as described previously (34). LH and FSH levels are expressed in international units per liter, as equivalents of the Second International Reference Preparation 71/223 of human menopausal gonadotropins. For the AxSYM LH, the interassay coefficients of variation (CVs) were 5.3, 5.5, and 7.4% for quality control sera containing 5.6, 26.2, and 69.0 IU/liter, respectively. For the LH RIA, the interassay CVs were 23.2, 6.0, and 11.4% for quality control sera containing 2.9, 10.3 and 24.6 IU/liter, respectively. For the AxSYM FSH, the interassay CVs were 6.9, 7.1, and 6.3% for quality control sera containing 4.3, 35.4, and 79.5 IU/liter, respectively. For the FSH RIA, the interassay CVs were 12.2, 8.4, and 11.8% for quality control sera containing 2.9, 7.7, and 21.9 IU/liter, respectively. E2 was measured using a microparticle enzyme immunoassay on a continuous access immunoanalyzer (AxSYM, Abbott Laboratories). P4 was measured using a chemiluminescence immunoassay system (Immulite Diagnostic Products Corporation, Los Angeles, CA). For the E2 assay, the interassay CVs were 10.2, 6.5, and 8.2% for quality control sera containing 81, 284, and 683 pg/ml (297, 1042, and 2507 pmol/liter), respectively. For the P4 assay, the interassay CVs were 14.4, 10.6, and 10.8% for quality control sera containing 1.5, 3.2, and 14.3 ng/ml (4.8, 10.2 and 45.5 nmol/liter), respectively.

Inhibin A was measured in duplicate by ELISA (Serotec, Oxford, England) as previously described (35). The assay uses a lyophilized human follicular fluid calibrator standardized as equivalents of the World Health Organization recombinant human inhibin A preparation 91/624, and values are reported as international units per milliliter. The limit of detection of the assay was 0.6 IU/ml. The interassay CV values for the dimeric inhibin A assay were 11.7 and 10.3% for quality control sera containing 5.01 and 10.01 IU/ml, respectively.

Inhibin B was measured as single samples by ELISA (Serotec) as previously described (36). The limit of detection of the inhibin B assay (mean ± 2 SD of multiple zero standard measurements) was 15.6 pg/ml. The intraassay CV was 4–6% and the interassay CV was 15–18% for quality control sera containing 121, 250, and 723 pg/ml, respectively. All samples with levels in excess of 500 pg/ml were appropriately diluted. For all assays, all samples for a given individual were run in the same assay.

Data analysis and statistics

Data were centered to ovulation for comparison of hormonal dynamics across the follicular and luteal phases of the menstrual cycle using three of four of the following criteria: 1) day of LH peak; 2) day of the midcycle FSH peak; 3) day of or after the midcycle E2 peak; and 4) day the P4 doubled from baseline or reached 0.6 ng/ml (37). Menstrual cycles were standardized to a 28-d cycle length, with the day of ovulation centered to d 0 and mean hormone levels determined in the early (d –13 to –9), mid (d –8 to –5), and late (d –4 to –1) follicular phase and early (d 1–4), mid (d 5–9), and late (d 10–14) luteal phase, as previously described (38). The mean value approximates the area under the curve for hormonal values at each cycle stage based on the trapezoidal rule. Hormone data from each cycle phase were log normalized and compared between fragile X premutation carriers and controls using two-way repeated measures ANOVA with Tukey post hoc testing. Relationships between hormone levels, age, and number of CGG repeats were evaluated using Pearson or Spearman correlations, as appropriate.

Results are expressed as mean ± SEM unless otherwise indicated. A P value < 0.05 was considered significant, except for analyses of correlations, in which a P value < 0.01 was considered significant to account for multiple variables.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
All fragile X premutation carriers had an ovulatory menstrual cycle during the study. Menstrual cycle length was significantly decreased in fragile X premutation carriers compared with age-matched controls. A decreased follicular phase length accounted for the shortening of the menstrual cycle (Table 1Go). There was no difference in luteal phase length between the two groups. In the control subjects and the subset of fragile X premutation carriers who underwent an ultrasound (n = 4), only one dominant follicle was present.


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  		TABLE 1. Cycle characteristics in fragile X premutation carriers and regularly cycling women1

 
FSH was increased across the entire cycle in the fragile X premutation carriers compared with age-matched controls (Fig. 1Go and Tables 2Go and 3Go). Inhibin B was decreased in the early and midfollicular phases; and in the early luteal phase, inhibin A was decreased in the early and midluteal phases, and P4 was decreased across the luteal phase in fragile X premutation carriers compared with age-matched controls (Fig. 1Go and Tables 2Go and 3Go). There was no difference in LH or E2 between the two groups.



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  		FIG. 1. Mean ± SE daily inhibin B (Inh B), FSH, E2, inhibin A (Inh A), LH, and P4 levels in fragile X premutation carriers (n = 11; •) compared with age-matched control subjects (n = 22; shading, mean ± 1 SD). Conversion factors to Systeme International units: E2, 3.671; P4, 3.18 (*, P < 0.05)

 

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  		TABLE 2. Mean follicular phase LH, FSH, inhibin B, inhibin A, and E2 levels1

 

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  		TABLE 3. Mean luteal phase FSH, inhibin B, inhibin A, E2, and P4 levels1

 
Although all fragile X premutation carriers had a history of ovulatory cycles and had an ovulatory menstrual cycle during the study, one subject had 4 months of amenorrhea after enrollment in the study, despite a history of consistently regular 23- to 28-d cycles, and one subject with a history of infertility and an elevated FSH level had irregular cycles upon closer analysis, varying from 14–45 d. When these subjects were removed to include only subjects with regular, ovulatory cycles in the analysis (n = 9), FSH remained significantly increased across the cycle (early follicular phase, 21.6 ± 3.2 vs. 12.0 ± 0.6 IU/liter; midfollicular phase, 17.0 ± 3.2 vs. 11.6 ± 0.8 IU/liter; late follicular phase, 13.1 ± 1.2 vs. 8.8 ± 0.6 IU/liter; early luteal phase, 15.3 ± 1.7 vs. 11.1 ± 1.1 IU/liter; midluteal phase, 7.9 ± 0.7 vs. 6.1 ± 0.6 IU/liter; late luteal phase, 8.1 ± 0.8 vs. 6.7 ± 0.4 IU/liter; P < 0.01); and P4 was lower across the luteal phase [early luteal phase, 5.9 ± 0.9 vs. 8.5 ± 0.8 ng/ml (18.9 ± 2.9 vs. 27.0 ± 2.7 nmol/liter); midluteal phase, 12.6 ± 1.5 vs. 17.0 ± 1.6 ng/ml (39.9 ± 4.8 vs. 54.1 ± 4.9 nmol/liter); late luteal phase, 4.8 ± 0.7 vs. 7.1 ± 0.7 ng/ml (15.3 ± 2.3 vs. 22.6 ± 2.2 nmol/liter); P < 0.05] compared with age-matched controls.

When examined individually, all 11 fragile X premutation carriers had an elevated FSH more than 1 SD above the mean for age-matched controls (28), and 10 of 11 (91%) had a FSH level more than 2 SD above the mean on at least 1 d in the follicular phase. Similarly, 8 of 11 (73%) had a FSH level more than 1 SD and 5 of 11 (45%) had a FSH level more than 2 SD above the mean in the luteal phase. Importantly, 5 of 11 (45%) had a history of infertility as defined by 1 yr of unprotected intercourse without a pregnancy.

The number of CGG repeats ranged from 56–135. There was no correlation between FSH and the number of CGG repeats in fragile X premutation carriers, whether d-3 FSH or the average FSH at any cycle phase was evaluated. There was no relationship between CGG repeat number and inhibin A, inhibin B, E2, LH, or P4 at any cycle phase. Finally, there was no significant relationship between FSH in the follicular phase or on d 3 and age in fragile X premutation carriers.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Recent studies demonstrate that FSH is higher in fragile X premutation carriers than in regularly cycling controls and is higher than the upper limit of normal in 8–14% of premutation carriers with regular menstrual cycles (25, 26, 27). Our intensive examination of reproductive hormone levels across the menstrual cycle extends these findings, providing evidence that the FSH abnormalities in fragile X premutation carriers are more prevalent than previously suspected. FSH was elevated over 2 SD above the mean for regularly cycling women on at least 1 d in 91% of the fragile X premutation carriers we examined. Furthermore, inhibin B, inhibin A, and P4 were decreased, suggesting decreased follicle number and impaired follicular or luteal function (39). Thus, ovarian dysfunction characteristic of early reproductive aging may be more common than previously suspected in fragile X premutation carriers.

The hormone changes in reproductive aged fragile X premutation carriers with ovulatory cycles are more marked than those demonstrated during normal reproductive aging (28). Premutation carriers with regular menstrual cycles exhibit a greater than 1 SD FSH increase and inhibin B decrease compared with age-matched subjects in the follicular and luteal phases. In contrast, E2 levels are not higher than levels in age-matched controls in the late follicular phase. The relatively normal E2 levels in the setting of increased FSH levels are similar to the pattern in regularly cycling women after age 44 yr, when FSH continues to rise and E2 decreases from elevated to normal (40). Inhibin A and P4 levels are clearly decreased, whereas E2 is preserved in the luteal phase in fragile X premutation carriers, similar to the more subtle changes seen in reproductive-age women studied longitudinally (28). The more marked decrease in inhibin B and inhibin A coincident with the greater increase in FSH in the presence of preserved E2 in fragile X premutation carriers compared with age-matched controls provides further support for inhibin A and inhibin B as important negative feedback regulators of FSH, similar to previous studies in reproductive aging (28, 41, 42, 43, 44).

The lower inhibin B levels, in conjunction with the higher FSH levels in the early follicular phase of fragile X premutation carriers, suggest that ovarian dysfunction is related to a decreased number of follicles. Increased FSH levels in reproductive aging (28) have a clear relationship to decreased follicle number, as demonstrated by anatomical (45, 46, 47) and ultrasound (48, 49) data. The decreased follicle number results in decreased ovarian negative feedback on FSH (28). The FSH negative feedback is mediated by serum inhibin B levels, which reflect the aggregate output from the granulosa cells of the growing cohort of small antral follicles, as demonstrated by in vivo (34, 50) and in vitro studies (51). Taken together, the changes in FSH and inhibin B suggest decreased follicle number in fragile X premutation carriers compared with controls, and suggest that a decreased initial follicle number or increased atresia may account for POF in these women.

The decreased inhibin A and P4 in fragile X premutation carriers suggest granulosa cell dysfunction. Inhibin A levels reflect both follicle/granulosa cell number and gonadotropin stimulation from each granulosa cell (34, 50, 51). Because corpus luteum granulosa cell number is fixed, the decreased inhibin A and P4 levels in the luteal phase of fragile X premutation carriers reflect defective luteinized granulosa cell function and/or decreased cell number in the corpus luteum compared with controls. Thus, there is evidence for both deficient steroid and protein production from the corpus luteum of fragile X premutation carriers. In contrast, E2 is preserved, suggesting that aromatase function is normal or that lower aromatase levels are compensated by increased FSH stimulation.

The mechanism behind the potential decreased follicle number and granulosa cell dysfunction in fragile X premutation carriers is unclear. FMRP is expressed in primordial germ cells in the fetus and in granulosa cells of developing follicles in adult females (52, 53). In males, the full fragile X mutation results in absence of FMRP, whereas females with full mutations in whom mRNA is transcribed from the normal but not the mutated and methylated allele have decreased FMRP. Interestingly, POF has not been demonstrated in women with full fragile X mutations (7, 9, 10), suggesting that decreased FMRP levels are not causative. Recent data demonstrate that premutation carriers have increased FMR1 mRNA levels but decreased FMRP (18, 54). Taken together, these findings suggest that the increased mRNA expression or the repeat tract itself may affect ovarian function, perhaps by binding and sequestering mRNA binding proteins (54) or mislocalizing premutation transcripts (55). Although these mechanisms allude to a potential relationship between premutation length and ovarian dysfunction, there was no correlation between premutation length and any hormone level in the current small series, and there was no relationship between premutation length and age at menopause in another recent study (56). Importantly, there was no inverse relationship between age and FSH levels in the fragile X premutation carriers in this small study, as there is in women during normal reproductive aging (28), suggesting that ovarian dysfunction related to the premutation may override this normal relationship.

The age-matched control subjects in this study were taken from a normative database of regularly cycling women, and their FMR1 CGG repeat lengths are unknown. The women did not have a history of POF or family history of mental retardation. Furthermore, the prevalence of premutation carriers is one in 259 (57). Therefore, it is unlikely that premutation carriers were included in the control group. Nevertheless, the presence of a fragile X premutation carrier in the control group would likely result in an underestimate of the FSH increase because a majority of the fragile X premutation carriers in the current study have a FSH level more than 2 SD above the mean. Taken together, it is unlikely that a large number of control subjects with normal hormone levels carried fragile X premutations, but the absence of this information remains a limitation of the current study.

The data in the current study have important clinical implications for fragile X premutation carriers. The reproductive hormone abnormalities, and thus ovarian dysfunction, in fragile X premutation carriers appear to be more prevalent than previously suggested by measurement of FSH levels on 1 d (25, 27), although the study is limited by the small subject number and the possibility that subjects with abnormalities were self-selected to participate despite the fact that they were asymptomatic. Interestingly, the abnormalities did not appear to be age related, because our two youngest subjects, 24 and 25 yr old, had the highest d-3 FSH levels. Our data suggest that further studies examining FSH, inhibin A, inhibin B, and P4 levels across the cycle in fragile X premutation carriers are warranted. They also suggest that fragile X premutation carriers should have both genetic and fertility counseling before attempting to conceive. Finally, the large number of fragile X premutation carriers with elevated FSH levels and a history of infertility suggest that the prevalence of fragile X premutations should be assessed in infertility patients with high FSH levels, particularly given the potential outcome of pregnancies achieved.


   Acknowledgments
 
We thank Dr. Stephanie Sherman at Emory University for analysis of fragile X premutation length in our subjects. We thank Dr. Patrick Sluss for his assay expertise and assistance. We also thank Katie Clapp and the FRAXA Research Foundation for advertising and helpful advice. Finally, we are deeply grateful to the fragile X premutation carriers who took the time to participate in this study despite their very demanding schedules.


   Footnotes
 
This work was supported by NIH Grant U54 HD12303–22.

Abbreviations: CV, Coefficient of variation; E2, estradiol; FMR, fragile X mental retardation; FMRP, FMR protein; MGH, Massachusetts General Hospital; P4, progesterone; POF, premature ovarian failure.

Received February 20, 2004.

Accepted June 8, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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