Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice

Abstract

Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called “egg infertility.” A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.

Women of advanced maternal age (AMA) (age ≥ 35) have an elevated risk of oocyte chromosome segregation errors. This leads to oocyte and embryonic aneuploidy that causes infertility, trisomic miscarriages, and trisomic birth defects that lead to serious disabilities and neonatal death. These problems are collectively referred to as “egg infertility” (1). Egg infertility increases exponentially with age due to increased rates of oocyte chromosome segregation errors. Egg infertility is now a significant public health problem, with 1 in 5 Unites States women now attempting her first pregnancy after 35 (2). By age 42, up to 87% of embryos are aneuploid, and 40%–50% of women experience miscarriages and infertility (3–5). The root causes of egg infertility are not well understood. There is no prevention and no cure.

As ovarian reserve diminishes, serum FSH becomes elevated throughout the menstrual cycle (6–9). From the midthirties to the early forties, high FSH occurs in the context of regular cycles and the diminution of fertility that ensue before the perimenopausal onset of irregular cyclicity. Elevation of FSH is strongly associated with diminished fertility. Because FSH mediates the process by which oocytes prepare for meiotic chromosome segregation (10–13), it is plausible that FSH levels may play a role in regulating the fidelity of chromosome segregation. For many years, physicians in the field of obstetrics and gynecology have favored the notion that FSH is not a cause of AMA oocyte aneuploidy and infertility, and there is considerable controversy as to whether rising FSH is strictly an epiphenomenon or a cause of AMA aneuploidy and infertility (5). There is strong evidence that degenerative processes inherent in the oocyte, including cohesin defects, telomere shortening, mitochondrial dysfunction, reactive oxygen species, and defects in the spindle apparatus and checkpoint machinery, contribute to egg aneuploidy (5, 14–16 and references therein). It is not known whether these molecular aging processes inherent in the egg entirely explain egg infertility, or whether elevation of endogenous FSH contributes to oocyte meiotic errors in AMA women.

Ovarian follicles are receptive to FSH during the period of growth that occurs for about the last several cycles before the egg is ovulated (about 19 d in mice and up to about 84 d in women) (11, 17, 18). FSH plays key roles in orchestrating follicle and oocyte growth and maturation from the preantral stage onward (19–23). Exposure of the follicle to endogenous and exogenous stimuli early in follicle growth has an important impact on the quality of the egg at the time it is ovulated (24). Chronic exposure to high endogenous FSH during the period of oocyte growth may be a cause of the decline in egg quality in AMA women.

Chromosome misalignments and spindle aberrations visualized in fluorescence microscopy are highly predictive of impending aneuploidy (25–27). If elevation of endogenous FSH during the period of oocyte growth increases the likelihood of chromosome and spindle misalignments, then administration of FSH activity for a chronic period during the period of oocyte growth would be predicted to increase rates of chromosome misalignments and spindle aberrations in ovulated oocytes. Conversely, therapeutic lowering of FSH during the period of oocyte growth should decrease rates of chromosome misalignments and spindle aberrations. This FSH-lowering therapy before the pregnancy attempt should also increase fertility. We call this regimen hormone normalization therapy.

Activin is a dimeric member of the TFGβ superfamily that is expressed in ovary, the pituitary, and other tissues (28). Activin stimulates pituitary FSH mRNA expression and FSH hormone secretion and also has other functions. ActRIIB is a transmembrane receptor for activin. ActRIIB:Fc (alias ACE-031 and ACVR2B) is a cloned soluble chimera of ActRIIB that has the extracellular domain, lacks the transmembrane and cytoplasmic kinase domains, and is fused to an IgG Fc sequence (29, 30). ActRIIB:Fc is an “activin decoy receptor.” It sequesters activin and inactivates activin signaling.

Recently, we reported that the senescence-accelerated mouse-prone-8 (SAMP8) is a model with many human-like attributes of midlife female reproductive aging (31). Like midlife women, midlife SAMP8 females have elevated FSH levels, elevated incidence of oocyte spindle aberrations, and diminished fertility, compared with young mice (31). These characteristics make SAMP8 useful as a model to test the impact of modulating FSH levels on egg infertility, for determining whether ActRIIB:Fc administered for the duration of mouse oocyte growth will lower FSH, prevent chromosome and spindle misalignments and increase litter sizes, and whether high gonadotropin activity in the form of eCG will increase chromosome and spindle misalignments in midlife SAMP8 mice.

In addition to the association of FSH elevation with egg infertility, rising basal FSH is associated with ovulation of fewer oocytes in response to exogenous FSH stimulation (11). If endogenous FSH elevation through the period of oocyte growth that characteristically occurs in midlife plays a role in diminished oocyte yield and viability that is seen in aging subjects, then eCG given for several consecutive estrous cycles should reduce the number of viable ovulated oocytes and increase the number of nonviable oocytes. Conversely, ActRIIB:Fc should lower FSH and increase the number of viable oocytes while maintaining a lower percentage of nonviable oocytes that are ovulated.

Materials and Methods

SAMP8 mouse colony

Initial breeding pairs of SAMP8 male and female mice were provided from the colony of Dr John Morley and Dr Susan Farr at St. Louis University School of Medicine. Mice were housed and bred in-house at the University of Maryland School of Medicine animal facility in Baltimore and cared for using University of Maryland School of Medicine Institutional Animal Care and Use Committee-approved animal use protocols according to National Research Council Guidelines, with extra measures taken to promote regular estrous cyclicity of the SAMP8 female mice as described (31). Midlife SAMP8 female mice in all test groups ranged from 5.8 to 8.7 months of age at the time of terminal bleeds and oocyte harvests (mean age of 6.85 mo). Young SAMP8 mice ranged from 2.3 to 3.2 months (mean age of 2.82 mo).

Blood collection, serum preparation, and quantitation of serum FSH levels

Survival bleeds and terminal bleeds, preparation of mouse sera, and quantitation of serum FSH concentrations with a Milliplex MAP Rat Pituitary magnetic bead panel (RPTGMAG-86K), were performed as described (31). Pooled sera from ovariectomized (OVX) rats were assayed alongside test samples to assess interassay variability and normalize measurements between assays. Duplicate FSH measurements were performed for each serum sample (31).

Purification of ActRIIB:Fc

ActRIIB:Fc was expressed in cultured Chinese hamster ovary cells and purified from conditioned media as described (32).

Mouse ovariectomies, and treatments with ActRIIB:Fc

SAMP8 female mice aged 7.4–7.6 months were OVX as described (33). Mice were permitted to recover on average for 2.3 weeks before commencement of injections. Eighteen OVX mice were injected with phosphate-buffered saline (PBS) vehicle control and then, 1 hour later, subjected to a survival bleed. They were then divided into 2 test groups comprised of 9 mice per group. Mice were injected ip with 4- or 10-mg/kg ActRIIB:Fc. Bleeds were performed 24 and 96 hours after ActRIIB:Fc injection. Mice were 8.9–9.1 month old at the time of blood collection.

Treatment of cycling midlife SAMP8 female mice with ActRIIB:Fc and collection of blood sera for FSH measurements

Estrous cycle phases of mice were determined by daily vaginal smearing and cytologic analyses (31). Midlife mice were injected ip with PBS (vehicle control). Survival bleeds were collected the next morning of estrus, which occurred 1–4 days after the PBS injection. Immediately after blood collection, 7-mg/kg ActRIIB:Fc in PBS was injected ip into regularly cycling SAMP8 mice. Blood was drawn 1–4 days later the next morning of estrus and processed to generate serum samples.

ActRIIB:Fc “booster” injections were continued every 3–4 days after the initial injection. The booster dose of ActRIIB:Fc was calculated to restore the serum ActRIIB:Fc concentration of 7 mg/kg, according to the equation for exponential decay:

$$\text{A}={\text{A}}_{\text{o}}{\text{e}}^{-\text{t}/\tau }$$

where t = time elapsed since the previous injection (3 or 4 d); A = final serum concentration after the elapsed time t since the previous injection; A₀ = (7 mg/kg) (initial serum concentration of ActRIIB:Fc at the time of the first injection); and τ = 15.87, the constant for exponential decay for ActRIIB:Fc assuming an 11-day half-life of ActRIIB:Fc in mice (34). The booster dose of ActRIIB:Fc was (7.0 mg/kg − A) (weight of mouse in kg), where A is the expected fraction of the dose of ActRIIB:Fc remaining after the decline in ActRIIB:Fc concentration due to the elapse of 3–4 days since the previous injection.

ActRIIB:Fc injections were continued every 3–4 days until approximately 3 weeks of treatment had been given. The day of terminal blood collection was 19–24 days after the start of treatment, the exact day determined by the day that estrus occurred. This coincided with the morning that oocytes were collected.

Oocyte collection from cycling mice

Untreated SAMP8 mice and SAMP8 mice treated with ActRIIB:Fc were naturally cycling as determined by vaginal smearing and cytology for several consecutive estrous cycles. Mice were injected ip with 5 IU human chorionic gonadotropin (hCG) the afternoon of proestrus to induce ovulation. Freshly ovulated oocytes were collected from ovarian ampullae on the morning of estrus, 14–16 hours after hCG administration.

Chronic administration of eCG to midlife SAMP8 female mice

eCG was purchased from the National Hormone and Peptide Program. The eCG preparation had a specific FSH activity of 2000 IU/mg. Mice were injected ip with 5-IU eCG. A second dose of 5-IU eCG was administered a week later, followed by a third injection a week thereafter. Forty-eight hours later, mice were injected with 5 IU of hCG (National Hormone and Peptide Program). The duration of eCG administration was thus 17 days, equivalent to the duration of approximately 3–4 consecutive estrous cycles in SAMP8 mice. A control group of mice was not treated with eCG but was given 5 IU of hCG the afternoon of proestrus. Freshly ovulated oocytes were recovered from ovarian ampullae 14–16 hours after hCG injection.

Scoring of oocyte morphology

Freshly ovulated oocytes were scored for morphology (31). Oocytes were scored as morphologically normal if they were healthy and derived from intact cumulus-oocyte complexes. They were scored as morphologically abnormal if they were denuded, postovulatory aged (POA), apoptotic (fragmented), or dead at the time of retrieval.

Two indicators were used to determine whether eggs were mature (in MII), (31). One was the presence of the polar body (PB)1. Sometimes the PB1 is degenerated, although the egg is in fact mature. Polarity is a distinctive characteristic of the cortical architecture of the mature egg that can be seen in the microscope (35) (and references therein). It is still visible even if the PB1 has degenerated. Scoring of polarity thus permitted us to accurately access the maturation status of each oocyte.

Scoring of oocyte chromosome misalignments and spindle aberrations

Oocytes were denuded of cumulus granulosa cells (GCs) and underwent removal of the zona pellucida, fixation, immunofluorescent staining of chromosomes and spindle microtubules, and scoring of chromosome and spindle organization at ×400 power in immunofluorescence microscopy, according to the methods described (31). Oocytes were scored as having very misaligned chromosomes or spindle aberrations according to criteria described (31).

Analyses of SAMP8 litter sizes after treatment with ActRIIB:Fc

Intact female SAMP8 mice aged 5.5–6 months were injected ip with 7-mg/kg ActRIIB:Fc. This was followed by booster injections of ActRIIB:Fc administered every 3–4 days to maintain the initial administered dose of ActRIIB:Fc for 21–24 days. On the day that the last injection was administered, the female mice were paired for 11 days with intact proven male SAMP8 mice aged 4.75 months. By these means, regularly cycling females had the opportunity to attempt pregnancy on 2 consecutive estrous cycles. Males were then removed from the female cages. Daily pup checks were initiated 10 days later and continued for an additional 14 days.

Females were approximately 6.2–6.7 months old at the initiation of pairing with the male, and were 7.0–7.7 months old at parturition. Pups were counted daily each morning by 10 am. Litter sizes were compared with those of untreated SAMP8 female mice where the untreated females, and the males were of the same age ranges as the experimental mice.

Statistical analyses

Differences were considered significant if P < .05 (95% confidence level). P values comparing yields of oocytes from midlife SAMP8 mice treated or untreated with eCG (total yields of oocytes, viable oocytes, or nonviable oocytes compared between each of the 2 test groups) were calculated with the Mann-Whitney test. Comparisons of litter sizes between test groups of dams treated with ActRIIB:Fc vs untreated dams were also subjected to the Mann-Whitney test. Mann-Whitney tests were chosen due to nonnormal distributions of egg yields and litter sizes in the test groups of treated and untreated mice.

P values comparing oocyte yields from untreated midlife SAMP8 mice, midlife SAMP8 mice treated with ActRIIB:Fc, and untreated young SAMP8 mice, were calculated using Kruskal-Wallis tests (nonparametric ANOVA). This test was chosen due to the multiple comparisons, and because the data do not conform to parametric assumptions (those assumptions being equal standard deviations and underlying normality). P values comparing mean FSH concentrations for blood sera from ActRIIB:Fc-treated and untreated mice at different ActRIIB:Fc treatment time points in OVX or cycling mice were also calculated with multiple comparison tests (one-way parametric ANOVA or Kruskal-Wallis), depending on whether the dataset met parametric assumptions.

P values comparing the fractions of oocytes with chromosome misalignments and comparing the fractions of oocytes with spindle aberrations between untreated midlife SAMP8 vs midlife SAMP8 treated with eCG were calculated using Fisher exact tests. The Fisher exact test was also used to compare the fractions of oocytes with chromosome misalignments and comparing the fractions of oocytes with spindle aberrations between untreated midlife SAMP8 mice vs midlife SAMP8 mice treated with ActRIIB:Fc.

Results

Seventeen-day treatment with eCG significantly reduces the yield of viable ovulated oocytes and increases the yield of nonviable oocytes in midlife SAMP8 mice

Midlife SAMP8 mice were administered consecutive weekly treatments with eCG for 17 days to maintain high FSH activity, a duration equivalent to 3–4 estrous cycles, the approximate duration of FSH-sensitive growth in mice (16, 19, 36–39). Ovulation induction with hCG was then performed the afternoon of proestrus in this test group, and in a control group of mice that did not receive eCG. Freshly ovulated oocytes were collected. There was no significant change in total egg yield (Figure 1, A and C). Oocytes were scored as morphologically normal or abnormal.

Figure 1.

eCG does not alter total yield of oocytes in midlife SAMP8 mice but markedly decreases the fraction of viable oocytes and increases the fraction of nonviable oocytes. A, Number of oocytes/mouse with or without eCG treatment. −, no eCG, gray bars; +, eCG treated, black bars. Total number of eggs/mouse were scored, as well as morphologically normal eggs/mouse and abnormal eggs/mouse. B, Percent of normal or abnormal eggs/mouse with or without eCG treatment. P values were calculated using 2-sided Mann-Whitney tests, each comparing individual test groups with the untreated control group. For this and all subsequent figures, error bars are SEM, and P values showing significant differences (<.05) are shown in bold.

The number of normal oocytes in the eCG-treated mice decreased by 5.62 oocytes per mouse, a 57% decrease (P < .0001) (Figure 1, A and B). The number of abnormal oocytes greatly increased, by 8.26 oocytes per mouse, a 1007% increase (P < .0001) (Figure 1). Nearly all abnormal oocytes were apoptotic or dead (207/226, 92.5%), the remainder attributable to denuded oocytes. No oocytes were POA. The terms “normal” and “abnormal” are thus used interchangeably with the terms “viable” and “nonviable,” respectively.

Although only 2/294 (0.7%) of oocytes from untreated midlife mice lacked a PB, 26/106 (24.5%) of oocytes from the eCG-treated mice lacked a PB (P < .0001, 2-sided Fisher exact test). The presence of polarity in an egg is seen when an egg matures to the MII stage. 292/294 (99.3%) of oocytes from the untreated group displayed polarity and 105/106 (99.1%) of oocytes from the eCG-treated group displayed polarity (P = 1.000). These data suggest that the oocytes in both test groups matured to the MII stage and that eCG may have induced degeneration of the first PB1.

Chronic treatment of midlife SAMP8 female mice with eCG increases rates of chromosome misalignments and spindle aberrations

Midlife mice were treated to maintain high FSH activity by repeated administration of eCG for 17 days, the approximate duration of oocyte growth. eCG significantly increased the rate of chromosome misalignments in midlife mice by 2.27-fold (P < .0001) (Figure 2, A and B). eCG significantly increased the rate of spindle aberrations by 2.1-fold (P = .0299) (Figure 2, A and C).

Figure 2.

Seventeen days of eCG treatment increases the frequency of chromosome misalignments and spindle aberrations in ovulated oocytes of midlife SAMP8 mice. First column, Entire oocyte is shown. Second, third, and fourth columns, Meiotic spindle is enlarged to show detailed structures. Images of chromosomes and spindles were captured from channels at the wavelengths of 4′6′-diamidino-2-phenylindole (DAPI) and fluorescein, respectively. Third column is a merged image of chromosomes and spindles. Background was subtracted using ImageJ software. Row A, Organized chromosomes and spindles in an oocyte from midlife mice. Row B, Misaligned chromosomes and spindles in an oocyte from midlife mice. Rows C and D, Misaligned chromosomes and spindles in oocytes from midlife mice treated with eCG. Arrows show chromosomes diverging from the metaphase plate and microtubules diverging from the spindle. Note the long thin spindle in D from eCG-treated mice. B, eCG treatment for 17 days increases the rate of chromosome misalignments in midlife SAMP8 mice. P values were calculated with a Fisher exact test. Fold increase in chromosome misalignment rate was calculated as the ratio of the percentage of oocytes from eCG-treated mice with misalignment chromosomes to the percentage of oocytes from untreated mice with misaligned chromosomes. C, Treatment with eCG for 17 days increases the rate of spindle aberrations in midlife SAMP8 mice.

ActRIIB:Fc suppresses FSH in OVX SAMP8 mice

SAMP8 mice were given ActRIIB:Fc to assess its ability to lower serum FSH and identify an effective dosage range. Responses of OVX mice were tested in initial studies to eliminate variability inherent in estrous cyclicity of baseline FSH levels. Mice were given a preinjection survival bleed and then injected with 4- or 10-mg/kg ActRIIB:Fc. Bleeds were performed 24 and 96 hours later. Mean FSH in mice given 4-mg/kg ActRIIB:Fc displayed 65.5% suppression after 24 hours vs untreated control (P < .001), and 43.2% suppression after 96 hours (P < .01) (Figure 3, A and B). Mice given 10-mg/kg ActRIIB:Fc displayed 80.9% FSH suppression after 24 hours of 10-mg/kg ActRIIB:Fc treatment (P < .001), and 81.1% FSH suppression after 96 hours of treatment (P < .001). No significant differences were found between mean FSH levels in test groups treated for 24 vs 96 hours with ActRIIB:Fc at either 4 or 10 mg/kg (P > .05), indicating persistent FSH suppression by ActRIIB:Fc across the 4-day timeline of ActRIIB:Fc treatment.

Figure 3.

ActRIIB:Fc suppresses serum FSH levels in OVX SAMP8 mice. P values were computed using a Kruskal-Wallis test. Percent coefficient of variation (% CV) ranged from 0% to 16.9% for FSH measurements in all test groups. A and B show graphical and table data representations, respectively. P values were calculated with Kruskal-Wallis tests.

ActRIIB:Fc suppresses FSH in cycling midlife female SAMP8 mice

Based on the data from OVX mice, we predicted that an ActRIIB:Fc dose between 4 and 10 mg/kg would provide sustained suppression of FSH for several weeks in cycling mice while not suppressing FSH to a degree that would abolish estrous cyclicity during the period of treatment. Cycling mice were given 7-mg/kg ActRIIB:Fc. FSH levels were suppressed by 73.6% on the successive day of estrus, 1–4 days after the initial injection (P < .001) (Figure 4).

Figure 4.

ActRIIB:Fc suppresses FSH in cycling midlife SAMP8 mice. Survival bleeds were drawn the morning of estrus. Mice at 0 time were injected with PBS vehicle control, and blood was collected 1 hour later. n, number of mice tested per group. P values were calculated with a one-way ANOVA test. % CV ranged from 0% to 14.72% for FSH measurements in all test groups.

Mice received booster shots of ActRIIB:Fc every 3–4 days to maintain ActRIIB:Fc levels between approximately 5.8 and 7 mg/kg for a period of 19–24 days, and then terminal bleeds were performed the morning of estrus (“3-wk treatment group”). Mice continued to cycle during ActRIIB:Fc treatment. FSH levels the morning of estrus in the 3-week treatment group were 47.3% lower than in vehicle-treated mice (P < .05) (Figure 4). LH levels were not significantly different between vehicle-treated mice and mice treated with ActRIIB:Fc (our unpublished data). Mice appeared healthy and showed no signs of pathology in necropsies immediately after terminal bleeds were performed. Mice have been treated with 10 mg/kg of ActRIIB:Fc for over a month without toxicity (S.-J. Lee, unpublished data).

ActRIIB:Fc restores the yields of oocytes in midlife SAMP8 mice, approximating the yields in young SAMP8 mice

Freshly ovulated oocytes were recovered from ActRIIB:Fc-treated midlife mice. Their morphologies were compared with those from untreated control mice. Nearly all oocytes in both test groups were mature eggs with a PB. No oocytes exhibited POA. Treatment with ActRIIB:Fc for 19–24 days significantly increased the total yield of oocytes from 10.64 oocytes/mouse to 14.33/mouse (P < .01) (Figure 5, A and B). The yield of viable oocytes increased commensurately, from 9.86/mouse to 12.73/mouse (P < .01). The yield of nonviable oocytes/mouse was not significantly affected (0.7727/mouse vs 1.6/mouse; P > .05).

Figure 5.

Treatment of midlife SAMP8 female mice with ActRIIB:Fc enhances oocyte yield and approximates the yields from young SAMP8 mice. A, Oocyte yields were compared between untreated midlife mice (n = 22), midlife mice treated with 7-mg/kg ActRIIB:Fc for approximately 3 weeks (n = 15; 19–24 d), and untreated young mice (n = 20). B, Test groups are comprised of midlife mice; midlife mice treated with ActRIIB:Fc (“Midlife + Act”); and young mice. Gray bar graphs, Viable oocytes. Black bar graphs, nonviable oocytes. P values were calculated with a Kruskal-Wallis test.

A total of 191/215 (88.8%) of oocytes from midlife mice treated with ActRIIB:Fc were viable. This is not significantly different than the 217/234 (92.7%) of oocytes that were viable in untreated mice (Fisher exact test, P = .1023). This indicates that the increase in oocyte yield caused by ActRIIB:Fc is primarily caused by an increase in the number of viable oocytes per mouse.

In previous studies, we reported a significant decline with age in the total yield of oocytes in the untreated mice with age, from 12.05/young mouse to 10.64/midlife mouse (P = .0155) (31). The number of viable oocytes correspondingly declined from 11.25/young mouse to 9.86/midlife mouse (P = .0119). The fraction of viable oocytes in ActRIIB:Fc-treated midlife mice (191/215; 88.8%) did not differ significantly from that of young untreated mice (225/241; 92.9%, P = .0986). ActRIIB:Fc-treated midlife mice had a total oocyte yield of 14.22 per mouse vs 12.05 per young untreated mice (P > .05). The yield of viable oocytes from ActRIIB:Fc-treated midlife mice was 12.73 per mouse, vs 11.25 per untreated young mouse (P > .05). These results show that ActRIIB:Fc restores total oocyte yield and yield of viable oocytes to the yields of the young mice.

Treatment of midlife SAMP8 mice with ActRIIB:Fc prevents oocyte chromosome misalignments and spindle aberrations

Treatment with ActRIIB:Fc suppressed the rates of chromosome misalignments 2.33-fold (P = .0060) (Figure 6). The fraction of oocytes from ActRIIB:Fc-treated mice with misaligned chromosomes was also 1.79-fold lower than that which was observed in young mice (P = .0200). A 14.5-fold higher rate of spindle aberrations was observed in midlife mice compared with young mice (P = .0003). ActRIIB:Fc treatment of midlife mice suppressed the rate of spindle aberrations by 2.8-fold (P = .0200) (Figure 6). The rate of spindle aberrations mice after ActRIIB:Fc treatment was statistically indistinguishable from that of young mice (P = .1179, not significant). ActRIIB:Fc therefore suppresses the rate of chromosome misalignments even lower than it is for young mice, and it restores the rate of spindle aberrations in midlife mice to that of young mice.

Figure 6.

Treatment of midlife SAMP8 mice with ActRIIB:Fc for 19–24 days restores organization of chromosomes and spindles in ovulated oocytes. Cycling mice were untreated (n = 22), or treated with ActRIIB:Fc (n = 15) for 19–24 days. Mice continued to cycle for the duration of ActRIIB:Fc treatment. Mice were injected with hCG on the afternoon of proestrus. Freshly ovulated oocytes were recovered from ovarian ampullae. Oocytes were fixed and stained for examination of chromosomes and spindles. A, Fluorescence microscopy of oocytes from midlife mice. Rows A and B, Oocytes from untreated mice. Rows C and D, Oocytes from mice treated with ActRIIB:Fc for 19–24 days. Note the spindle in row B from the untreated midlife test group has an oblate spheroidal configuration (flattened poles and short z-axis relative to the x- and y-axes at the metaphase plate), compared with the barrel shape of the other spindles (long z-axis). B. Reduced incidence of chromosome misalignments in oocytes from ActRIIB:Fc-treated mice. C, Reduced incidence of spindle aberrations in oocytes from ActRIIB:Fc-treated mice. P values were computed with a Fisher exact test.

If ActRIIB:Fc improves oocyte quality and prevents aneuploidy by lowering FSH, then overriding the FSH-lowering effect of ActRIIB:Fc by coadministration of eCG should decrease oocyte quality. Midlife mice cotreated with ActRIIB:Fc and eCG displayed a significant reduction in the number of viable oocytes from 12.7 oocytes/mouse in ActRIIB:Fc-treated mice (n = 15 mice; 191/215 [88.8%]) oocytes viable), to 5.3/mouse in eCG/ActRIIB:Fc-cotreated mice (P = .0007, Mann-Whitney test; n = 11 mice; 57/137 [41.6%] oocytes viable). The yield of viable oocytes declined below that observed in midlife mice treated with a single dose of eCG alone (7.6/mouse, P = .0123, n = 18 mice). The number of nonviable oocytes concomitantly increased, from 1.6/mouse in the ActRIIB:Fc-treated test group, to 7.2/mouse in the ActRIIB:Fc + eCG-cotreated test group (n = 11 mice; P < .0001).

The midlife mice cotreated with eCG and ActRIIB:Fc exhibited a tendency to increased chromosome misalignments, from 11/159 (6.9%) in mice treated with ActRIIB:Fc alone, to 5/42 in eCG-ActRIIB:Fc-cotreated mice (11.9%; 1.72-fold increase). They also exhibited a tendency to increased rates of spindle aberrations, from 4/152 (2.6%) in ActRIIB:Fc-treated mice to 3/38 in the eCG-ActRIIB:Fc-cotreated mice (7.9%; 3.0-fold increase). These differences were not statistically significant (P > .05). Although the number of oocytes per mouse permitted sufficient statistical power to compare oocyte yields between the test groups, the number of oocytes recovered in the eCG-ActRIIB:Fc-cotreated group was too small to permit conclusions comparing rates of chromosome and spindle misalignments (unpaired 2-tailed t tests, 80% power). These data are consistent with the notion that ActRIIB:Fc improves oocyte viability due at least in part to its FSH lowering effect. Future studies will employ a large number of animals cotreated with ActRIIB:Fc and eCG to obtain sufficient numbers of oocytes to compare the rates of chromosome and spindle misalignments with to that of the ActRIIB:Fc-treated group.

ActRIIB:Fc improves fertility in midlife SAMP8 female mice

Because ActRIIB:Fc increases the yield of ovulated oocytes and prevents oocyte chromosome misalignments and spindle aberrations, we predicted that treatment of the midlife mice with ActRIIB:Fc would restore some of the fertility that is lost with age. Litter sizes increased from a mean of 5.06 pups/litter to 6.22 pups/litter and from a median of 5 pups/litter to a median of 7 pups/litter (P = .0384) (Table 1). These data demonstrate that ActRIIB:Fc increases fertility in midlife SAMP8 mice.

Table 1.

ActRIIB:Fc Increases Litter Sizes in Midlife SAMP8 Mice

Parameter	Midlife Untreated		Midlife + ActRIIB:Fc
Number of litters	33		18
Number of pups	167		112
Mean pups/litter	5.06		6.22
Median pups/litter	5.000		7.000
P value		.0384

Dams were 6–8 months old. Only dams with litters were counted, because mating plugs are often not visible in SAMP8 mice. P values were computed with a Mann-Whitney test.

Discussion

ActRIIB:Fc, gonadotropins, and egg infertility

High basal FSH levels correlate with oocyte and fetal aneuploidy, live-born trisomies, reduced implantation rates, infertility, and miscarriages (8, 40–54). FSH concentrations are higher within follicles harboring aneuploid oocytes than within follicles harboring euploid oocytes (55). Several other studies have found no association between egg infertility and basal FSH (49, 56–58). eCG has high FSH activity (59). Treatment of mice with eCG or FSH causes oocyte degeneration, chromosome and spindle defects, increased aneuploidy, lower implantation rates, impeded embryonic development, fetal malformation, and fetal demise (60–68). Adverse impacts of FSH were seen in 8 different mouse strains. Ewes superovulated with FSH display higher rates of oocyte morphologic abnormalities than naturally cycling ewes (69). High-dose FSH is associated with low birth rates (70, 71). Increased aneuploidy rates in human oocytes, embryos, and spontaneous abortuses are seen after high-dose FSH in vivo and in vitro (72–75).

Midlife SAMP8 mice have higher levels of FSH throughout their cycles than young SAMP8 mice, and they have elevated rates of oocyte spindle misalignments and diminished litter sizes (31). Here, we show that chronic treatment of midlife SAMP8 mice with ActRIIB:Fc lowers FSH, increases the yield of viable oocytes, prevents oocyte chromosome and spindle misalignments, and increases fertility. Conversely, 2.5 weeks of treatment with eCG was performed in order to subject the midlife mice to chronic elevation of FSH activity for a duration that is analogous to that which occurs in AMA women. This treatment lowers total yield of oocytes, shifts them to a population comprised predominantly of dead and dying oocytes, and increases their rates of chromosome and spindle misalignments. Although this regimen likely raises FSH activity to supraphysiologic levels, the approach of pushing a system to an amplified degree is often employed in characterization of biological phenomena. Future studies will provide smaller increases in FSH activity designed to emulate the more moderate extent to which FSH is increased in AMA women with diminished ovarian reserve.

The eCG preparation we used has negligible contamination with equine LH (A. F. Parlow, personal communication). Nevertheless, in addition to its FSH activity, eCG has some LH activity inherent within the eCG molecule itself (76), so it is possible that LH activity of eCG contributes to its effects. The LH activity of hCG in rodents is much higher than that of eCG (77). SAMP8 mice given a single eCG shot followed by hCG to induce ovulation had significantly fewer viable oocytes than those given hCG to trigger ovulation without a previous superovulatory eCG stimulus (mean of 6.96/mouse cotreated with eCG+hCG vs 9.86/mouse treated with hCG alone; P = .0009). Mice treated with eCG+hCG also had significantly higher chromosome and spindle misalignment rates than mice treated with hCG alone (49/138 vs 33/193 oocytes with misaligned chromosomes, P = .0001; 20/122 vs 14/190 oocytes with spindle aberrations; P = .0112). These data suggest that the LH activity of eCG may not entirely explain its detrimental effects.

Another group of mice was given 5-IU eCG injections once per week for 17 days interspersed with 3 weekly injections of 5-IU hCG 2 days after each eCG injection, thereby maintaining a long period of elevated LH activity before oocyte retrievals. If long periods of high LH activity are detrimental to oocyte quality, then the mice treated with the 3 hCG injections interspersed with the 3 eCG injections might be expected to have fewer viable oocytes and higher rates of oocyte chromosome and spindle misalignments than mice that received 3 weekly eCG shots with only a single hCG injection 14–16 hours before oocyte recovery. However, the yield of viable oocytes/mouse given the long hCG treatment was not significantly different than the yield with only a single hCG shot (3.75/mouse vs 4.24 oocytes/mouse, P = .8978, 2-tailed Mann-Whitney test, NS). There was also no difference in rates of chromosome or spindle misalignments between mice given interspersed hCG and those who were not (chromosomes, 12/50 [24%] vs 30/101 [29.7%], 2-tailed Fisher exact test; P = .2961; spindles, 9/43 [20.9%] vs 12/91 [13.2%]; P = .3095). In other studies, it was found that endogenous mean LH levels are lower in midlife SAMP8 than they are in young SAMP8 (120.7 pg/mL in midlife mice vs 158.1 pg/mL in young mice on the day of estrus; P = .0460, and 182.6 vs 297.8 pg/mL on the day of diestrus; P = .0002). As indicated in Results, ActRIIB:Fc treatment does not significantly change LH levels.

Taken together, these data indicate that LH is not correlated with oocyte quality or fertility in SAMP8 mice. They suggest that LH activity of eCG does not play a predominant role in its detrimental effects on SAMP8 egg quality. The data are consistent with the notions that the high FSH activity of eCG and the FSH-lowering effect of ActRIIB:Fc reciprocally contribute to their effects on egg quality, and that rising FSH in midlife SAMP8 is a cause of declining egg quality and infertility. Future studies beyond the scope of this investigation will determine rates of oocyte viability, aneuploidy, and fertility in midlife SAMP8 mice given highly purified FSH for the several weeks long period of oocyte growth.

Interplay of age and long-term FSH elevation

Various literature data indicate that older subjects, both animals and humans, are more susceptible to detrimental effects of FSH on the egg and on fertility than younger subjects are Young women undergoing controlled ovarian hyperstimulation/intrauterine insemination with FSH have higher pregnancy and live-birth rates than young women undergoing natural cycles, whereas older women undergoing COHS/IUI with FSH have lower pregnancy and live-birth rates than those who underwent natural cycles (78, 79). Implantation rates are not adversely affected by high-dose FSH in young subjects, but are lowered by high-dose FSH in older subjects (71). Female mice have a biphasic response to overexpression of the FSH gene, with increased fertility in their youth, followed by decreased fertility and more pregnancy resorptions later in life (80, 81). Young but not aged rats have increased yield of ovulated oocytes induced by eCG, and the fraction of meiotically abnormal ovulated oocytes in eCG-treated aged rats is higher than in young rats (82).

In addition, we hypothesize that damage to the egg by chronic FSH elevation occurs not just in the ovulatory cycle but for perhaps the entire duration of the period of FSH-responsive oocyte growth. Mouse oocytes exhibit increasingly higher rates of spindle misalignments the longer that mice are treated with eCG before ovulation (65). They also display elevated rates of spindle misalignments many cycles after treatments were terminated. In human fertility patients, the effects of FSH stimulation often spill into the next cycle. Boue and Boue found increased rates of trisomy in abortuses conceived the cycle after fertility drugs were administered as well as the cycle they were administered (74). Several weeks of eCG treatment of young SAMP8 had no effect on the number of viable ovulated eggs/mouse, but in midlife SAMP8, it markedly reduced the yield of viable ovulated eggs/mouse. Moreover, although 17 days of eCG treatment were required to cause chromosome and spindle misalignments in young SAMP8, only 3 days of eCG treatment were required in midlife SAMP8 (L.R.B., A.C.L.M., C.L.C., I.M., unpublished data). Taken together, these data suggest that oocytes may be more severely affected by long-term FSH elevation (as in midlife women with diminished ovarian reserve) than by short term FSH elevation (such as during a 10-d infertility treatment), and that oocytes from midlife subjects may be more vulnerable to elevated FSH than those of young subjects. These data point to a complex interplay of cause and effect that may help explain why, to date, the role of FSH in egg infertility has remained confusing and controversial. Future studies will tell us whether ActRIIB:Fc treatment for the entirety of the period of FSH-sensitive egg growth is required to maximize the yield of healthy oocytes, reduce aneuploidy and increase fertility, or whether shorter treatments will also be effective.

Possible alternative pathways for ActRIIB:Fc effects

Activin has been found to have a number of other functions in addition to up-regulating pituitary FSH secretion (28, 83, 84 and references therein). In the ovary, activin mediates primordial follicle formation, survival of germ cells, antral follicle growth, follicle selection, regulation of GC proliferation, up-regulation of GC FSH and ER receptor expression, oocyte maturation and developmental competence, and luteinization (85 and references therein). Activin also mediates endometrial implantation and embryogenesis, and it promotes apoptosis of fetal female germ cells, and cultured GCs (86, 87). The latter would invoke a mechanism that does not involve activin modulation of pituitary FSH secretion because these were in vitro studies. Paradoxically in other studies, treatment with activin-A decreases the incidence of atresia of cultured primary and preantral follicles, so the events that occur in response to activin modulation remain to be resolved (88). Other ligands can also bind activin receptors, including myostatin, growth/differentiation factor-11, and nodal, each with their own signaling pathways and biological functions (89). Improved oocyte yield and quality and improved fertility in midlife SAMP8 caused by ActRIIB:Fc may thus occur by mechanisms in addition to or instead of its FSH-lowering effect (Figure 7). Future studies will elucidate these pathways. Regardless of which pathways are used, modulation of the activin receptor binding site shows promise for translational drug development of novel fertility treatments.

Figure 7.

Reciprocal effects of eCG and ActRIIB:Fc on oocyte viability, chromosome and spindle misalignments configurations, and fertility may occur via FSH-dependent and/or FSH-independent pathways. A, eCG may decrease yield of viable oocytes, increase yields of nonviable oocytes, and increase aneuploidy by raising FSH activity. B, ActRIIB:Fc may increase yields of viable oocytes, decrease aneuploidy, and increase fertility by lowering FSH. ActRIIB:Fc may function via other activin-mediated pathways (dotted lines) in addition to or instead of via FSH.

Alternative treatments

Other treatments have been attempted to improve egg quality. These include oocyte supplementation with mitochondria from putative oogonial stem cells, transfer of ooplasm or mitochondria from oocytes of young subjects to oocytes of infertile subjects, and transfer of the germinal vesicle of an infertile subject to the ooplasm of a fertile subject (90–93). These treatments do not prevent aneuploidy or increase fertility, or the effects of their effects remain to be determined. Caloric restriction and antioxidant molecules vitamins C and E, N-acetyl cysteine, resveratrol, and coenzyme Q10 prevent oocyte chromosome and spindle misalignments, mitigate declining oocyte yield, and increase fertility, but only when administered over multiple months, timeframes equivalent to years in humans (94–99). Treatments of 3 weeks to 2 months in duration have not improved aneuploidy or pregnancy rates in women (100). Treatment timeframes of many months or years may work, but may be too long to be therapeutically practicable. However, 3 weeks of ActRIIB:Fc treatment is a timeframe equivalent to several human menstrual cycles, arguably more feasible in a clinical context.

With the exception of ActRIIB:Fc, the mouse studies cited above employed aged mice that had been superovulated with eCG, with no control that had not been eCG-treated. Because eCG itself decreases the yield of viable oocytes and increases rates of chromosome and spindle misalignments in aged animals more than in young animals (82), it is not actually known whether the endogenous yields of healthy oocytes and rates of oocyte aneuploidy these studies actually declined due to age. Instead, diminished yield of viable oocytes and increased aneuploidy may be artifacts of greater susceptibility of aged mice to damage induced by eCG. In contrast, ActRIIB:Fc restores declining oocyte yield and prevents oocyte chromosome and spindle misalignments that occur endogenously with age, because the ActRIIB:Fc-treated test group and the control group were not treated with eCG.

Future studies will determine whether ActRIIB:Fc works in part by improving the fidelity of meiotic chromosome segregation by increasing localization of cohesin proteins between sister chromatids or by preventing abrogation of the spindle assembly checkpoint, double-stranded DNA repair defects, mitochondrial defects, or telomere shortening, all of which exhibit declining function with age (90, 101, 102). Irrespective of the mechanism, although no therapeutic approaches have been found to prevent AMA aneuploidy and improve fertility by preventing defects in spindle assembly checkpoint, mitochondrial, cohesin, or telomere length, treatments with ActRIIB:Fc may provide a unique prospect of therapeutic benefit to prevent aneuploidy and improve fertility in AMA women. Future studies will also determine whether egg yield can be therapeutically improved with ActRIIB:Fc in women who have diminished ovarian reserve and/or are poor responders to controlled ovarian hyperstimulation.