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Endocrinology, doi:10.1210/en.2007-0046
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Endocrinology Vol. 148, No. 9 4432-4439
Copyright © 2007 by The Endocrine Society

Rising Follicle-Stimulating Hormone Levels with Age Accelerate Female Reproductive Failure

Kirsten J. McTavish, Mark Jimenez, Kirsty A. Walters, Jennifer Spaliviero, Nigel P. Groome, Axel P. Themmen, Jenny A. Visser, David J. Handelsman and Charles M. Allan

ANZAC Research Institute (C.M.A., K.J.M., M.J., K.A.W., J.S., D.J.H.), University of Sydney, Concord Hospital, New South Wales 2139, Australia; School of Biomolecular Sciences (N.P.G.), Oxford Brookes University, Oxford OX3 0BP, United Kingdom; and Department of Internal Medicine (J.A.V., A.P.T.), Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. Charles M. Allan, ANZAC Research Institute Research Institute, University of Sydney, Concord Hospital, New South Wales, 2139 Australia. E-mail: charles{at}anzac.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rising serum FSH levels is one of the earliest signs of human female reproductive aging. Whether or not elevated FSH remains a passive reflection of a diminishing ovarian follicle pool or actively contributes to declining female fertility with age has not been established. We therefore investigated female reproduction in mice expressing progressively rising serum levels of transgenic human FSH (Tg-FSH, 2.5–10 IU/liter) independently of follicle depletion. We show that serum LH and estradiol levels and uterine size remained normal in Tg-FSH females, whereas ovarian weight and corpora lutea number were significantly increased up to 1.3- and 5-fold, respectively. Furthermore, the monotrophic FSH rise produced a striking biphasic effect on female fertility. Tg-FSH females less than 22 wk old delivered increased litter sizes, then beyond 23 wk, litter sizes decreased rapidly culminating in premature infertility despite continued ovary follicle development, and increased ovulation and uterine embryo implantation sites as well as normal serum levels of anti-Mullerian hormone, a marker of ovarian follicle reserve. We found that rising circulating Tg-FSH produced premature infertility by increasing embryo-fetal resorption and parturition failure with age. Thus, our Tg-FSH mice present a novel paradigm to investigate selective contributions of elevated FSH to age-related female infertility, which revealed that rising FSH levels, despite no exhaustion of ovarian reserve, actively accelerates female reproductive aging primarily by postimplantation reduction of embryo-fetal survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FEMALE REPRODUCTIVE FUNCTION is reliant on pituitary-derived FSH and LH. FSH controls cyclic recruitment of small growing follicles, supports follicle development to preovulatory stage, and confers sensitivity to LH-stimulated ovulation and luteinization (1). Increasing basal levels of circulating FSH throughout the menstrual cycle, especially in the early follicular stage, is one of the earliest signs of human reproductive aging (2, 3, 4, 5, 6, 7, 8), preceding any changes in serum LH or estradiol before menopause (2, 5, 6, 9). Elevated FSH is associated with shorter follicular phase (4, 5, 8) and cycle length (7, 8, 10, 11) in aging women (late 30s to mid 40s) before the menopause transition, coinciding with the marked decline in fecundity (12, 13). Paradoxically, higher FSH is also linked to familial or age-related dizygotic twinning (14, 15) and is elevated in women carriers of fragile X syndrome (16, 17, 18) who display an increased incidence of dizygotic twinning but earlier onset of ovarian failure (18, 19, 20). Rising levels of serum FSH with age is at least in part to due to declining inhibin B secretion (6, 7, 21). Inhibin B is known to suppress pituitary FSH secretion, so reduced inhibin production by a decreasing growing follicle population (as ovarian reserve is depleted with age) may elevate FSH secretion (6). Whether or not rising FSH represents a passive reflection or a direct effector of age-related changes in human follicle dynamics, ovulatory cycles, and fecundity remains conjectural.

Understanding the contributing factors to age-related declining female fecundity has important clinical relevance considering greater proportions of women in developed countries delay childbearing until at least 30 yr of age (13, 22). Advancing our knowledge of FSH effects may have profound implications for human female fertility, including ovarian follicle recruitment and depletion with age (1, 6, 8), follicle recovery/preservation and quality during in vitro fertilization (23) and maturation (24), or after chemotherapy (25) as well as timing of onset of age-related diseases (cardiovascular disease and osteoporosis) associated with ovarian failure. Increased FSH levels may accelerate female reproductive aging by increasing recruitment of small growing follicles and thereby accelerating final depletion of the diminishing follicle reserve (26). Yet an exponential decline in female fertility begins several years before menopause (12, 13), raising the possibility that rising FSH may directly influence ovarian function or female reproductive aging before exhaustion of ovarian reserve.

A major limitation to evaluating this hypothesis has been the lack of in vivo animal models described to selectively investigate the progressive effects of rising basal FSH levels on female fertility. We previously developed a transgenic (Tg) mouse model that expressed pituitary-independent human FSH at levels providing physiological ovarian follicular and hormone responses (27, 28). We now reveal that female Tg mice exhibit progressively increasing levels of circulating FSH, which is not a direct consequence of ovarian follicle depletion, providing a unique opportunity to examine the impact of rising basal FSH upon female reproduction with increasing age. The reproductive aging profile of this Tg-FSH paradigm reveals that elevated FSH levels can have a direct role in accelerating the decline of female fertility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Tg-FSH mice expressing pituitary-independent human FSH via the rat insulin II gene promoter were created and maintained as described (27). Animals were mated to obtain heterozygous Tg-FSH and non-Tg wild-type (WT) littermate age-matched controls as determined by genotyping tail DNA (27). All studies were approved by the Sydney South West Area Health Service Animal Welfare Committee and performed in accordance to the National Health and Medical Research Council code of practice and the New South Wales Animal Research Act (1985). By terminal ketamine-xylazine anesthesia, blood was collected via cardiac puncture and serum stored at –20 C, and dissected tissues were weighed and fixed overnight in 4% paraformaldehyde at 4 C and then transferred to 70% ethanol.

Hormone assays
Serum Tg-FSH and endogenous mouse FSH and LH levels were determined using species-specific DELFIA (dissociation-enhanced lanthanide fluoroimmunoassay) kits (Perkin-Elmer, Turku, Finland) as described (27, 29), with detection limits of 0.05 IU/liter human (Tg) FSH, 0.2 ng/ml mouse FSH, and 70 pg/ml mouse LH, respectively. Serum estradiol levels were determined by DELFIA kit (Perkin-Elmer) after diethyl ether extraction, with a functional sensitivity of 12 pM (30). Serum anti-Mullerian hormone (AMH) levels were determined by a validated mouse AMH ELISA, with a detection limit of 7 pg/ml (31). Interassay coefficient of variation was less than 10% for all hormone assays, except the estradiol assay, which was less than 15%. Hormone levels were measured in the diestrous stage of the estrous cycle determined by vaginal epithelial cell smears; singly housed females exposed to male bedding to encourage cycling had epithelial cells gently collected daily in 20 µl sterile PBS, which were transferred to glass slides and stained with 0.05% Trypan Blue for microscopy. Mice were smeared for 2 wk consecutively around 12, 26, 40, and 52 wk of age.

Ovary RNA and real-time PCR
Whole-ovary RNA (4 µg) extracted using TRI reagent (Sigma Aldrich Inc., St. Louis, MO) was treated with RNase-free DNase (Invitrogen, Carlsbad, CA) and then reverse-transcribed using Superscript III (Invitrogen) according to the manufacturer’s methods. Quantitative real-time PCR analysis of cDNAs was performed on a Corbett RotorGene 2000 (Corbett Research, Sydney, Australia) using TaqMan primers (mouse AMH, 18S, Wbscr1; Applied Biosystems, Foster City, CA) with Platinum qPCR Supermix (Invitrogen) as recommended. Reaction steps were 50 C for 2 min and 90 C for 2 min and then 50 cycles of 95 C for 15 sec and 60 C for 45 sec. Standards were assigned an arbitrary value, and mean relative AMH mRNA expression standardized to housekeeping gene (18S and Wbscr1) values in each sample.

Breeding, superovulation, and embryo culture
Breeding analysis used randomly selected 7- to 10-wk-old Tg and WT females housed with fertile males for at least 1 yr. For induced ovulation and fertilization, 12- and 26-wk-old Tg and WT females received 10 IU pregnant mare serum gonadotropin (Folligon; Intervet, Bendigo, Australia) and then 10 IU human chorionic gonadotropin (Organon, Syndey, Australia) 47 h later and were mated overnight with fertile males. Oocyte-cumulus complexes were harvested into M2 media (Sigma) 20 h after human chorionic gonadotropin, cumulus cells were removed using hyaluronidase, and single-cell embryo/oocyte health (general morphology and presence of pronuclei and polar bodies) and number were determined using Nomarski optics. Embryos were transferred into M16 media (Sigma) and cultured overnight at 37 C with 5% CO2 to assess two-cell-stage development using Nomarski optics.

Uterine embryo implantation sites and parturition
To assess embryo implantation and postimplantation embryo and fetal survival, 9- and 23-wk-old Tg and WT females were mated with fertile males until a copulatory plug was observed. At parturition or 21 d postcoitum (dpc), Tg and WT dams were culled and dissected ovaries fixed overnight in 4% paraformaldehyde at 4 C. Pups or resorbing embryos/fetuses in utero were counted in dissected uterine horns. Implantation sites were counted by dissecting microscope after staining with 10% ammonium sulfide for 5 min, followed by a water rinse and 5 min in 10% potassium hexacyanoferrate II with 0.5% HCl to visualize hemorrhagic alteration at each site (32).

Ovarian follicle and zona pellucida remnant quantification
Fixed ovaries embedded in hydroxymethylmethacrylate resin (Technovit 7100; Kulzer and Co., Friedrichsdorf, Germany) were sectioned (25 µm) according to manufacturer’s recommendations using a Polycut S microtome (Reichert Jung, Nossloch, Germany) and stained with periodic-acid-Schiff, hematoxylin, and Scott’s bluing solution. Follicles and zona pellucida remnants were counted by microscope (x40 oil objective) using stereo investigator software (MicroBrightfield, Williston, VT) and optical dissector counting in every ovary section (33). Follicles were classified as follows: small preantral, oocyte and one and a half to two layers of cuboidal granulosa cells; large preantral, oocyte surrounded by three to five layers of cuboidal granulosa cells; small antral, oocyte surrounded by over five layers of cuboidal granulosa cells and/or one or two small areas of follicular fluid; large antral, oocyte and single large antral cavity; and preovulatory, single large antrum and oocyte surrounded by cumulus cells at the end of a mural granulosa cell stalk. Only follicles containing an oocyte with a visible nucleolus were measured, to avoid repetitive counts. Total ovarian corpora lutea counts were determined by morphology consistent with luteinized follicles (33) using aligned captured microscope (x2 objective) images from every fourth ovarian section.

Statistical analysis
Data analysis was performed using SPSS (Chicago, IL) and NCSS (Statistical Analysis and Data Analysis Software, Kaysville, UT) software using one-way ANOVA or two-way ANOVA (as indicated), supplemented with Tukey-Kramer post hoc tests. Significance was set at P < 0.05. Values or figures are all presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Tg-FSH on body, ovarian, and uterine weights
Progressively increasing body weights of Tg-FSH and WT females were similar at all ages examined, except for a small but significant reduction (13%, P < 0.05) in weights of 26-wk-old Tg-FSH relative to WT females. Ovary weights of Tg-FSH and WT mice were similar at 5 wk of age, but Tg ovaries were larger than WT at 12 (24%, P < 0.05) and 40 (27%, P < 0.05) wk of age and then returned to WT ovary weight at 52 wk of age (Fig. 1AGo). There was no significant difference (two-way ANOVA, P = 0.09) in the age-related increase in uterine weights of Tg-FSH and WT females (Fig. 1BGo).


Figure 1
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  		FIG. 1. Effect of Tg-FSH on reproductive organs and hormones. A and B, Ovary weights (A), using means of both ovary weights per mouse and uterine weights (B) from female Tg-FSH (bullet) and WT ({circ}) mice 5–52 wk of age (n = 7–15 mice per group); C–F, serum levels of Tg-FSH (C), mouse LH (D), mouse FSH (E), and estradiol (F) in female Tg-FSH (bullet) and WT ({circ}) mice from 5–52 wk of age (n = 6–9 mice per group), collected during diestrous stage of estrous cycle. *, Significant difference (P < 0.05) between age-matched Tg-FSH and WT mice.

 
Serum Tg-FSH, mouse gonadotropin, and estradiol levels
Serum levels of pituitary-independent Tg-FSH were positively correlated (r = 0.78; P < 0.001) with increasing age, exhibiting a 4-fold rise from 5 (2.7 ± 1.4 IU/liter) to 52 (9.9 ± 1.3 IU/liter) wk of age (Fig. 1CGo). Expression of Tg-FSH was shown to significantly decrease (61–91%) serum levels of endogenous mouse FSH during diestrus at all ages examined (Fig. 1EGo). Serum levels of LH (Fig. 1DGo) and estradiol (Fig. 1FGo) during diestrus were equivalent in Tg-FSH and WT females at all ages.

Age-related changes to fertility and estrous cycles of Tg-FSH females
Tg-FSH had a marked biphasic effect upon female fertility. Female mating determined by the presence of copulatory plugs was not inhibited in older Tg-FSH mice. Young Tg-FSH females (≤22 wk old) had significantly larger litters (10.4 ± 0.9 vs. 7.6 ± 0.3 pups per litter, P < 0.001) with no significant difference in time between litters (P = 0.18) relative to WT controls (Fig. 2AGo). However, older Tg-FSH females (23–44 wk old) produced decreased litter sizes (4.5 ± 1.3 vs. 8.0 ± 0.4 pups per litter, P < 0.001) and exhibited premature infertility with last litters at 34.1 ± 4.3 wk compared with the WT females remaining fertile at 52 wk of age, with a rapid decline in cumulative live-birth rate by Tg females from 23 wk of age compared with WT females (Fig. 2BGo).


Figure 2
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  		FIG. 2. Breeding performance of Tg-FSH females. Fertility data comparing Tg-FSH (bullet) and WT (bullet) females up to 52 wk of age. A, Total pups born per litter plotted with age of dam at day of birth, with fitted curves for the Tg-FSH (solid line) and WT (dashed line) data; B, cumulative live-birth rate from Tg-FSH (n = 8) and WT (n = 12) females with age.

 
Estrous cycle length was equivalent in 12-wk-old Tg-FSH and WT females (5.2 ± 0.5 vs. 5.2 ± 0.3 d, P = 0.9). There was a significant reduction in estrous cycle length at 26 compared with 12 wk of age in both Tg (21% decrease, P < 0.05) and WT mice (12%, P < 0.05). However, cycle length was significantly shorter in 26-wk-old Tg compared with 26-wk-old WT mice (4.1 ± 0.1 vs. 4.6 ± 0.1 d, P < 0.005). In older Tg females, cycle length was similar (4.1 ± 0.2 vs. 5.0 ± 0.4 d, P = 0.099) or equivalent to normal (4.5 ± 0.2 vs. 4.6 ± 0.3 d, P = 0.76) at 40 or 52 wk old, respectively, compared with age-matched WT females.

Effect of Tg-FSH on ovarian follicle growth and corpora lutea number
Ovarian histology showed all stages of follicle development including the presence of primordial follicles and corpora lutea in 12- to 52-wk-old Tg-FSH and WT mice (Fig. 3AGo). Total corpora lutea counts were significantly increased in Tg compared with WT mice at 12 (4.8-fold, P < 0.001) and 26 (1.5-fold, P < 0.05) wk of age, demonstrating increased ovulation despite markedly reduced fertility in 26-wk-old Tg females (Fig. 3BGo). Corpora lutea counts were equivalent in 52-wk-old Tg and WT mice (Fig. 3BGo). Quantitation of growing follicle populations at diestrus in 26-wk-old females, corresponding to the age of rapidly declining fertility, showed a small but significant reduction (17%, P < 0.01) in small preantral follicle numbers in Tg-FSH relative to WT mice; however, there was no significant difference in the number of larger preantral to antral or preovulatory follicles in Tg-FSH compared with WT mice (Fig. 3CGo). The total numbers of zona pellucida remnants, indicative of overall follicle atresia, were not significantly different in ovaries from 26-wk-old Tg and WT mice (Fig. 3CGo).


Figure 3
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  		FIG. 3. Effect of Tg-FSH on ovarian follicle growth and corpora lutea number. A, Ovary tissue sections (20 µm) periodic-acid-Schiff and hematoxylin stained from 12-, 26-, or 52-wk-old Tg-FSH and WT female mice shown at same magnification. Clearly identified corpora lutea are indicated by asterisks. Primordial follicles were present in 52-wk-old WT (i) and Tg-FSH (ii) ovaries. B, Total ovarian corpora lutea counts from diestrous stage-matched 12-, 26-, and 52-wk-old Tg-FSH (black bars) and WT (white bars) mice (n = 3 mice per group). *, Significant difference (P < 0.05) between age-matched Tg-FSH and WT mice. C, Ovarian follicle quantitation in 26-wk-old Tg-FSH or WT females. Total numbers of small preantral (SPA), large preantral (LPA), small antral (SA), large antral (LA), and preovulatory (PO) follicles and zona pellucida remnants (ZPR), indicative of follicle atresia, were compared in subfertile Tg-FSH (black bars) and WT (white bars) mice. *, Significant differences (P < 0.05) between follicles or ZPR; n = 3 mice per group.

 
Effect of Tg-FSH on fertilization, superovulation, and early embryo development
Effects of rising Tg-FSH on oocyte recovery were examined in superovulated 12- or 26-wk-old females (Table 1Go). Significantly higher numbers (two-way ANOVA, P < 0.05) of oocytes were collected from superovulated Tg-FSH compared with WT females, and total oocyte numbers decreased with age (two-way ANOVA, P < 0.001). Total numbers of oocytes recovered from Tg-FSH females were equivalent (P = 0.395) at 12 wk of age, during the period of increased litter size, and then were higher (50%, P < 0.05) at 26 wk of age vs. WT values (Table 1Go). Examination of single-cell embryos or oocytes collected from superovulated females mated with fertile males revealed that the percentage of healthy fertilized embryos from 12- and 26-wk-old Tg-FSH females were significantly reduced (two-way ANOVA, P < 0.02) compared with the percentage from 12- and 26-wk-old WT females. However, the percentage of the healthy fertilized embryos from 12- and 26-wk-old Tg-FSH vs. WT females that developed to the two-cell stage were not significantly different (two-way ANOVA, P = 0.3).


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  		TABLE 1. Effect of Tg-FSH expression on oocyte recovery, fertilization, and early embryo development

 
Ovarian AMH mRNA and serum AMH levels in Tg-FSH mice
AMH was recently shown be to a marker of the ovarian early follicle pool (31, 34, 35). Total ovary AMH mRNA levels by real-time PCR compared with two housekeeping genes (18S or WBscr1) generated equivalent results. Ovarian AMH mRNA expression significantly declined in aging Tg-FSH and WT females (two-way ANOVA, P < 0.001), and expression levels were lower (two-way ANOVA, P < 0.05) in aging Tg relative to WT ovaries (Fig. 4AGo). Serum AMH levels were also significantly reduced with increasing age in Tg (r = –0.63) and WT (r = –0.67) females (two-way ANOVA, P < 0.001). Serum AMH levels remained similar in 5- to 40-wk-old Tg-FSH relative to WT females, but were significantly reduced (62%, P < 0.05) in 52-wk-old Tg-FSH compared with WT mice (Fig. 4BGo).


Figure 4
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  		FIG. 4. Expression of AMH in Tg-FSH females. Ovary AMH mRNA expression (n = 3–7 mice per group) (A) and serum AMH levels (n = 4–9 mice per group) (B) in 5- to 26-wk-old Tg-FSH (black bars) and WT (white bars) mice. Whole-ovary mRNA AMH expression was measured relative to housekeeping Wbscr1 gene, using TaqMan primers by real-time PCR. *, Significant difference (P < 0.05) between age-matched Tg-FSH and WT mice.

 
Uterine embryo implantation and embryo development
Expression of Tg-FSH significantly increased (two-way ANOVA, P < 0.001) the number of uterine embryo implantation sites 2-fold at parturition or 21 dpc in 12- to 40-wk-old Tg compared with WT females (Fig. 5AGo). However, Tg-FSH females exhibited a significant (two-way ANOVA, P < 0.05) increase in embryo-fetal resorption compared with WT females, with 26-wk-old Tg females displaying a 7-fold rise (P < 0.05) in embryonic failure (Fig. 5BGo), which included resorbing small embryos (3–5 mm) and larger fetuses. Defective embryo development was detected as early as 10–12 dpc in 26-wk-old Tg-FSH females (data not shown). The number of pups that reached parturition or 21 dpc (i.e. delivered or remaining in utero) was nonsignificantly increased (two-way ANOVA, P < 0.07) in Tg-FSH relative to WT females (Fig. 5CGo). However, older Tg females exhibited substantial parturition failure. In particular, all closely examined pregnant 40-wk-old Tg females (n = 11) failed to deliver pups, despite the presence of viable pups in utero at 21 dpc (n = 6 dams); further analysis at 24 dpc (n = 5 dams) found that all pups died in utero and were reabsorbing.


Figure 5
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  		FIG. 5. Embryo uterine implantation and development in Tg-FSH females. Total embryo uterine implantation sites (A), embryo-fetal resorptions (B), and full-term pups (C) found at parturition or 21 dpc in 12-, 26-, and 40-wk-old Tg-FSH (black bars) and WT (white bars) mice (n = 5–10 dams per group). *, Significant difference (P < 0.05) between age-matched Tg-FSH and WT mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our Tg-FSH model has shown that increased basal FSH activity can advance the age-related onset of female infertility. Progressively rising serum Tg-FSH with age is driven by the rat insulin gene promoter (27) in parallel with normally increasing pancreatic ß-islet cell mass and insulin content in aging mice (36). Therefore, the rising Tg-FSH is pituitary independent and is not caused by declining ovarian follicle reserve, allowing a unique analysis of the direct consequences of increased FSH activity upon female reproductive ageing. Tg-FSH expression had no detectable effect on circulating LH and estradiol levels or on estrogen-sensitive uterine size. Thus, our Tg-FSH model exhibits a monotrophic FSH rise that resembles the elevated FSH with unchanged LH reported in aging women (2, 5, 9). Rising basal FSH activity in Tg female mice predominately derives from Tg-FSH, because endogenous mouse FSH levels were reduced, likely due to increased ovarian feedback (e.g. inhibin B secretion) responding to pituitary-independent Tg-FSH. We previously showed that Tg-FSH expressed in gonadotropin-deficient hypogonadal mice stimulated dose-dependent gonadal development and ovarian inhibin B secretion within normal physiological ranges (27, 28, 37, 38). As such, our Tg model contrasts with another model with supraphysiological Tg-FSH (e.g. 362,000 IU/liter) associated with elevated estradiol, enlarged uteri, cystic ovaries, female sterility, and extragonadal complications (39). Tg-FSH activity in our model did not induce such confounding effects, allowing selective investigation of a monotrophic and modest rise of basal FSH levels.

Rising Tg-FSH had a striking biphasic age-dependent effect upon female fertility. Tg breeders less than 22 wk old produced increased litter sizes and corpora lutea numbers indicative of elevated FSH activity driving increased ovulation. This pituitary-independent Tg-FSH activity was comparable to increased ovulation due to transient elevation of pituitary-derived Tg mouse FSH (40). Elevated ovulation and litter size stimulated by Tg-FSH was also consistent with proposals that higher FSH levels increase dominant follicle development and contribute to age-related or familial dizygotic twinning in women (14, 15, 41) and that increased ovarian FSH sensitivity drives above-normal ovulation rates and litter sizes in sheep with heterozygous mutations affecting the bone morphogenetic protein pathway (42). However, the increased litter sizes rapidly declined, leading to premature infertility of Tg-FSH females over 22 wk of age. The increased and then premature demise of fertility induced by rising Tg-FSH bears a resemblance to the paradox of multiple ovulation (twinning) but earlier onset of menopause associated with elevated FSH in women, such as carriers of fragile X syndrome (18, 19, 20). Women fragile X carriers also exhibit reduced inhibin levels, suggesting fewer ovarian follicle numbers and premature ovarian ageing (17).

Age-related effects of Tg-FSH on female fertility may reflect increased ovulation driven in part by higher FSH accelerating growing follicle selection, which is predicted to ultimately advance depletion of follicle reserve (26), thus leading to premature infertility. Declining estrous cycle length from 12 to 26 wk of age in Tg-FSH and WT mice was consistent with the decline reported in 12- to 20-wk-old normal mice (43). However, shorter cycles in 26- to 40-wk-old Tg relative to WT females suggest that rising Tg-FSH accelerated ovarian cycling and therefore the rate of follicle recruitment. Transient Tg-FSH-induced shortening of the estrous cycle supports a direct role for elevated FSH driving the observed shortening of cycle lengths in aging women (late 30s to mid 40s) in the years preceding perimenopause (2, 7, 8, 10, 11), which also coincides with declining fecundity (12, 13). Reduced small preantral follicle numbers in 26-wk-old Tg females, in the period of rapidly declining fertility, is also consistent with accelerated follicle development. Yet ovulation (corpora lutea numbers) remained elevated in subfertile 26-wk-old Tg-FSH females, whereas numbers of large preantral, antral, or preovulatory follicles in diestrus remained normal. In the rat estrous cycle, follicles for ovulation are selected by diestrous stage (44). Therefore, our data suggest that Tg-FSH activity increased large follicle survival beyond diestrous stage up to ovulation, which is in agreement with the normal numbers of large follicles but increased ovulation observed in mice after elevated pituitary-derived Tg mouse FSH expression (40). Little is known about the survival factors of large preovulatory follicles (45), but these findings suggest that FSH actions can enhance follicle survival before ovulation.

Normal numbers of zona pellucida remnants, representing follicle atresia after development of the zona pellucida (33), indicated that 26-wk-old Tg-FSH ovaries had no marked change in overall follicle atresia. This does not exclude our proposal that higher FSH activity enhanced the survival of large follicles selected for ovulation, as changes to small numbers of large follicles on the higher background of total ovarian follicle atresia would be difficult to detect. A reduced percentage of healthy fertilized oocytes from superovulated mated Tg-FSH females relative to controls may indicate that higher FSH rescues ovarian follicles that would otherwise be excluded from selection, at the sacrifice of oocyte quality. This reduction may support the ovarian origin for age-related infertility that postulates a loss in oocyte quality and quantity (13, 22). Despite this decrease, an equivalent percentage of the healthy embryos from Tg-FSH or control females were able to progress to the two-cell stage, demonstrating that early embryo development was not markedly affected by Tg-FSH. Furthermore, our results show that ovarian follicle development or responsiveness to superovulation was not disrupted by rising Tg-FSH expression. Despite the premature infertility in aging Tg females, estrous cycling and ovulation continued, which has resemblance to declining fecundity together with higher serum FSH that occurs years before the cessation of cycling and ovulation in aging women (13, 22).

AMH is predominantly expressed in granulosa cells of small growing follicles and was recently shown to be an indicator of the ovarian pool of primordial and growing follicles in mice and a proposed measure of fertility (31, 34, 35). Declining AMH mRNA expression in Tg-FSH and WT ovaries with increasing age follows an expected decline in primordial and growing follicle numbers with age. Ovarian AMH mRNA expression was lower in Tg-FSH females, although total ovary analysis may be affected by altered follicle expression and/or percentage (e.g. altered ratio of follicles expressing to those not expressing AMH) in Tg ovaries, including more tissue (e.g. corpora lutea) not expressing AMH in Tg ovaries. Serum AMH levels declined with increasing age in both Tg and WT females, the latter supporting recent findings (31), but levels remained equal in age-matched Tg-FSH and WT females during the period (26–40 wk) of premature infertility in Tg females. Although reduced serum AMH levels in 52-wk-old Tg females relative to normal values indicate that elevated FSH activity accelerated the depletion of ovarian reserve to a small degree, this occurred well after (~26 wk) the decline of fertility. Preliminary analysis shows that there is no reduction in ovarian primordial and primary follicle numbers in 26-wk-old Tg-FSH females (McTavish, K. J., K. A. Walters, and C. M. Allan, unpublished data). Therefore, limited and delayed changes to serum AMH suggests that the premature infertility in Tg females was not due to marked changes in ovarian follicle reserve, further supported by the presence of primordial and growing follicles in ovaries of older infertile Tg females. Importantly, normal serum AMH levels in infertile Tg females show that AMH levels alone are not always a suitable indicator of female fertility, at least in the mouse.

Declining fertility in Tg-FSH females was not due to a major loss of preimplantation embryos, because young fertile and older infertile Tg females exhibited significantly more uterine embryo implantation sites than normal. Consistent with increased embryo implantations in young Tg-FSH females, women less than 41 yr of age with higher FSH (>15 IU/liter) able to produce oocytes for in vitro fertilization had 3-fold higher implantation rates relative to older women with normal FSH (46). Therefore, higher FSH levels alone do not appear to directly inhibit embryo implantation. In contrast, high doses of exogenous gonadotropins in mice reduced preimplantation, implantation, and postimplantation development (47, 48, 49); however, these exogenous hormone studies did not directly examine persistent FSH elevation alone and did not distinguish between excessive FSH- or LH-dependent in vivo actions.

Rising Tg-FSH levels significantly increased postimplantation embryo and fetal resorption with age, accounting for over half of the uterine embryo implantation sites in older Tg females. Pups born to Tg-FSH females were successfully reared to weaning age; therefore, elevated FSH did not affect lactation and nurturing behavior. Therefore, progressive Tg-FSH-mediated changes to both ovarian and/or uterine function ultimately affect postimplantation embryo development or survival, leading to premature age-related female infertility. We predict that Tg-FSH activity invoked age-related changes via the ovarian FSH receptor, because evidence for direct FSH uterine action is lacking in the literature, and despite few reports of FSH receptor expression in human endometrium (50, 51, 52) and myometrium (53), we did not detect FSH receptor mRNA in mouse uterus by RT-PCR (McTavish, K. J., and C. M. Allan, unpublished data). Uterine failure due to overuse as a result of increased litter size seems not likely, because 26-wk-old virgin (mated once) or continually breeding Tg-FSH females exhibit similar fertility failure. We propose that uterine dysfunction contributing to progressive reproductive failure will be due to indirect Tg-FSH-driven ovarian effects.

Tg-FSH expression also markedly increased parturition failure with age. Older Tg-FSH females (40 wk) were unable to deliver most pups at term, leading to subsequent death of pups in utero, which accounted for about half of the detected uterine embryo implantation sites. In older women, increased pregnancy and delivery rates for assisted reproduction using oocytes from younger relative to older donors show an ovarian basis for reduced fecundity (54, 55), which is further supported by a higher incidence of chromosomal abnormalities (e.g. aneuploidy) in increasing spontaneous abortions with age (13, 22, 56). However, there is also evidence that increased numbers of spontaneous abortions occur in aging women with embryos that appear chromosomally normal (57), and analysis of embryo donor-matched IVF recipients suggest that recipient uterine factors may be important determinants for successful outcome (58). Although more modest than ovarian effects, a downward trend in birth rates for older recipients (particularly >40 yr) of assisted reproduction indicates a significant uterine effect on outcome (59). In addition, the reported rising incidence of placenta previa (60, 61), dysfunctional labor (60, 61), and uterine pathology (62, 63) may significantly contribute to declining fecundity or increasing obstetrical complications with age. Therefore, the age-related elevation in both embryo-fetal resorption and parturition failure with no maternal mortality in our Tg-FSH mice, indicative of significant uterine contribution, provides a unique in vivo paradigm to study the direct and progressive consequence of rising FSH upon regulation of both ovarian and uterine function.

In summary, Tg-FSH female mice exhibit characteristics familiar to human female aging, in which we reveal monotrophic rising FSH-induced premature female infertility despite continual ovulation and cycling and before exhaustion of ovarian follicle reserve. We propose that rising FSH reaches a threshold level to induce aberrant changes to ovarian function that impact upon both embryo quality and uterine function, leading to postimplantation failure of embryo-fetal development. Tg-FSH mice constitute a relevant model to explore effects of rising FSH upon ovary and uterus function during declining female fertility and may reveal contributing mechanisms to reduced fecundity in regularly ovulating, aging women before the menopause transition.


   Acknowledgments
 
We thank Prof Peter J. Illingworth for expert advice.


   Footnotes
 
This work was funded in part by a National Health and Medical Research Council (Australia) grant.

Disclosure Statement: The authors have nothing to declare.

First Published Online May 31, 2007

Abbreviations: AMH, Anti-Mullerian hormone; dpc, days postcoitum; Tg, transgenic; WT, wild type.

Received January 12, 2007.

Accepted for publication May 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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