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Original Article |
Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington 98105; and Reproductive Endocrine Unit, Department of Medicine, Massachusetts General Hospital (P.M.S.), Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Nancy A. Klein, M.D., Division of Reproductive Endocrinology, 4225 Roosevelt Way NE, Suite 305, Seattle, Washington 98105. E-mail: nklein{at}u.washington.edu.
Abstract
This study sought to determine whether the shortened follicular phase in ovulatory older women is secondary to advanced (i.e. earlier) or accelerated (i.e. more rapid) folliculogenesis. Normal ovulatory women, aged 40–45 yr (n = 15) and 20–25 yr (n = 13), underwent daily venipuncture and transvaginal ultrasonography throughout the follicular phase of a spontaneous menstrual cycle (control cycle) and after pituitary down-regulation with a GnRH agonist (study cycle). As expected, the older subjects in the control cycles demonstrated an elevated d 3 FSH and a shortened follicular phase compared with the younger subjects. After release from hypothalamic-pituitary-ovarian axis suppression, the early follicular phase FSH peak occurred earlier (6.8 vs. 9.8 d; P < 0.01) and was of a greater magnitude (12.1 vs. 6.5 mIU/ml; P < 0.01) in the older subjects. The time from release of suppression until the subsequent LH surge was also shorter (17.5 vs. 20.8 d; P < 0.01) in the older group. However, the time from FSH peak to LH surge was similar in the older and younger groups (10.7 vs. 11.0 d; P = 0.74). Compared with younger women, older subjects had normal follicular phase levels of estradiol and inhibin A and lower levels of inhibin B in both control and study cycles. We conclude that the shortened follicular phase observed in older ovulatory women is due to earlier dominant follicle selection, independent of hormonal influences from the preceding luteal phase.
REPRODUCTIVE AGING IS a continuum that begins many years before absolute dysfunction occurs. An important manifestation of the aging process is a progressive decline in female fertility that begins in the latter part of the third decade and becomes exponential after age 35 yr (1, 2). Such fertility impairment precedes overt signs of hypothalamic-pituitary-ovarian (HPO) axis dysfunction such as menstrual irregularity, with the majority of women continuing to have regular, ovulatory menstrual cycles well into their 40s (3, 4). In the years preceding the menopausal transition, regular ovulation is maintained, while menstrual cycles progressively shorten (4, 5, 6). This phenomenon is due to a shortening of the follicular phase without any associated change in the length of the luteal phase length (5, 7). An additional hallmark of advancing reproductive age is the rise in FSH unaccompanied by a rise in LH (monotropic FSH rise) (6, 7, 8, 9). The onset of the monotropic FSH rise and earlier ovulation appear to be temporally associated with an acceleration in the rate of follicle atresia that ultimately leads to depletion of the follicular reserve (10). Thus, the monotropic rise in FSH and the shortened follicular phase serve as clinical indicators of advancing reproductive age and rapidly declining fertility (11).
During the normal menstrual cycle, FSH rises in the late luteal or early follicular phase and typically reaches a peak in the early follicular phase (12, 13). With selection and subsequent development of the dominant follicle, FSH falls to a relatively low level until the midcycle gonadotropin surge. The dominant follicle in eumenorrheic women of advanced reproductive age is relatively healthy, grows to a normal size, produces normal or elevated levels of estradiol (E2), and secretes normal levels of progesterone after luteinization (7, 8, 14). These normal characteristics of the older dominant follicle suggest that the earlier ovulation in older women is not due to an intrinsic defect in the follicle, but to the extrafollicular hormonal milieu. The endocrinological changes most consistently described in studies of reproductive aging are an increased absolute level of FSH (6, 7, 8, 9), an earlier rise in the early follicular phase FSH (7), and a decline in early follicular phase levels of inhibin B (15, 16, 17, 18).
The current study was conducted to determine whether the shortened follicular phase in ovulatory older women is secondary to advanced (i.e. earlier) or accelerated (i.e. more rapid) folliculogenesis. We hypothesized that the earlier dominant follicle development in older women is secondary to the earlier rise in follicular FSH compared with that in younger controls, and that the absolute rate of follicular growth would be similar in both ages. In other words, we hypothesized that follicle development in women of advanced reproductive age is advanced rather than accelerated. Hormonal and intraovarian effects from the preceding menstrual cycle can potentially confound studies of dominant follicle recruitment and maturation. To eliminate any such influence from the preceding cycle, we suppressed each subject’s HPO axis by standard GnRH agonist down-regulation. After HPO axis suppression was documented, we compared the recovery of hormonal, follicular, and menstrual function in older, ovulatory subjects compared with a younger control group.
Subjects and Methods
Experimental subjects
As part of a series of studies of normal reproductive aging, we recruited healthy ovulatory women, aged 40–45 yr (n = 16) and 20–25 yr (n = 15), for participation. All subjects were required to have regular menstrual cycles (cycle intervals of 21–35 d), normal body mass index (18–24 kg/m2), and absence of medical or reproductive disorders (including any history of infertility) and to demonstrate midluteal serum levels of PRL below 20 ng/ml, progesterone greater than 10 nmol/liter, and testosterone less than 3 nmol/liter in a prestudy cycle. For the duration of the study, all subjects were either sexually abstinent or used nonhormonal methods of contraception (e.g. barrier method or intrauterine device). Written consent was obtained from each participant, and monetary compensation was provided for all volunteers. The protocol was reviewed and approved by the University of Washington human subjects review board.
Materials and methods
Study protocol. As a control cycle, all subjects underwent daily venipuncture and serial transvaginal ultrasonography to assess dominant follicle development in the follicular phase of a spontaneous, natural cycle. For the study cycle, nafarelin acetate (400 µg, intranasally, daily) was initiated 7 d after urinary LH kits detected the midcycle gonadotropin surge in the preceding cycle. When suppression of the HPO axis was confirmed by a serum E2 level below the detection limit of the assay (73 pmol/liter), nafarelin was continued for an additional 5 d to ensure a uniform down-regulation of the HPO axis. Beginning the day after discontinuation of nafarelin, daily venipuncture for E2 analysis was performed. Once the serum E2 concentration reached or exceeded 367 pmol/liter, daily transvaginal ultrasound examinations were performed until dominant follicle collapse was observed. The study cycle was initiated within 6 months of the control cycle.
Assays.
All serum samples from a particular subject were analyzed in duplicate in the same assay to minimize the effects of intraassay variability. Serum FSH levels were determined using a solid phase two-site monoclonal ELISA (DELFIA, Wallac Inc., Gaithersburg, MD). The inter- and intraassay coefficients of variation (CV) were 4.6% and 2.3%, respectively. The RIA for serum E2 was performed using reagents supplied by ICN Biomedicals, Inc. (Costa Mesa, CA). The inter- and intraassay CV were 18% and 9%, respectively. A solid phase sandwich ELISA (Serotec, Oxford, UK) was used to measure inhibin B, based on the use of plates coated with a monoclonal antibody specific for the inhibin ß subunit. Inhibin B is detected using a second monoclonal antibody specific for the inhibin 
-subunit, and the assay procedure, according to the manufacturer, involves specimen pretreatment with reagents provided in the kits (sodium dodecyl sulfate and peroxide). The analytical sensitivity (e.g. minimum for the ßA-subunit of inhibin) was determined with a horseradish peroxidase-labeled monoclonal antibody specific for the -subunit used for detection. The assay standard provided by the manufacturer was calibrated using WHO’s First International Standard for Inhibin (recombinant human inhibin; lot 91/624), and results are reported as international units per milliliter of this reference material. The assay was controlled in duplicate using aliquots of specimens containing 2.00 or 9.56 IU/ml, respectively. Based upon these quality controls the between assay CV were 8.7% and 3.3%, respectively, over the 57 assays used to obtain the data reported herein.
Statistical analysis.
For outcomes with a single data value from each individual (e.g. follicular phase length), means from the two cohorts were compared using a two-sided t test. An = 0.05 was selected to indicate a significant difference. For outcomes with several data values from each individual (e.g. serial E2 levels), the sample means were compared by ANOVA with repeated measures. Based on the results of previous studies, we expected the variability in time to onset of the LH surge to be approximately 20%. Therefore, assuming a coefficient of variation of 20%, we estimated that we would have an 80% chance of finding a difference as small as 20% with 15 subjects in each group.
Results
Two younger and one older women were dropped from the study due to failure to achieve HPO axis suppression. Baseline characteristics of the control cycle for the remaining subjects are shown in Table 1
. As expected, the older subjects demonstrated a shortened follicular phase as well as an elevated FSH and decreased inhibin B on cycle d 3 of the control cycle compared with the younger subjects. In both control and study cycles, all subjects in each age group developed a dominant follicle with ultrasound evidence of subsequent follicle collapse after the midcycle LH surge, demonstrating apparently normal dominant follicle development and ovulation in both natural cycles and after discontinuation of GnRH suppression. The duration of Synarel (Searle, Skokie, IL) required to achieve suppression as defined by the study protocol was somewhat longer for younger controls (range, 9–20 d; mean, 12.8 ± 0.8 d; total dose, 5.1 ± 0.3 mg) compared with older subjects (range, 9–13 d; mean, 11.0 ± 0.3 d; total dose, 4.4 ± 0.1 mg; P = 0.05 for both duration and dose).
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For the purposes of this study, we divided the follicular phase into two intervals: early follicular phase (defined as the time from the onset of menses or discontinuation of GnRH agonist suppression until the early follicular phase FSH peak) and late (defined as the time from the early FSH peak until the midcycle gonadotropin surge). Follicular phase intervals (total, early, and late) are shown in Fig. 1. After release from HPO axis suppression, the older cohort reached an early follicular FSH peak sooner (6.8 ± 0.7 vs. 9.8 ± 0.6 d, respectively; P < 0.01), thus yielding a shorter early follicular phase. Additionally, the older cohort had a shorter time from release of suppression to the subsequent LH surge (17.5 ± 0.9 vs. 20.8 ± 0.7 d, respectively; P < 0.01), for an overall shorter total follicular phase. However, the time from the early follicular phase FSH peak to the midcycle LH surge (late follicular phase) was similar in the older and younger groups (10.7 ± 0.7 vs. 11.0 ± 0.8 d, respectively; P = 0.74). In both older and younger subjects, the time from discontinuation of nafarelin to subsequent LH surge was longer than the follicular phase of the control cycle (older, 17.5 vs. 12.9 d; younger, 20.8 vs. 14.8 d).
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FSH profiles surrounding the early follicular phase FSH peak for both the control and study cycles are depicted in Fig. 2. The peak early follicular phase FSH level was higher in older subjects in both the control (12.0 ± 1.4 vs. 6.8 ± 0.3 mIU/ml, respectively; P < 0.01) and study (12.1 ± 1.9 vs. 6.5 ± 0.4 mIU/ml, respectively; P < 0.01) cycles. No differences in the magnitude of the FSH peak were observed between the control and study cycles within each age group (Fig. 2). Therefore, the magnitude of the FSH peak appears to be independent of the influence of the preceding luteal phase in both age groups.
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The predominant secretory products of the dominant follicle are E2 and inhibin A. Figure 3 depicts follicular phase concentrations of E2, normalized to the LH surge, for both age groups in control and study cycles. The pattern and amount of E2 secreted by the dominant follicle are similar between study and control cycles in both age groups. Although the slope of the E2 rise and size of the dominant follicle were not different between groups, the midcycle peak E2 was greater in older women in both control (1175 ± 73 vs. 973 ± 76 pmol/liter, respectively; P = 0.05) and study cycles (1351 ± 91 vs. 1061 ± 94 pmol/liter, respectively; P < 0.05). In both cycles, older subjects also demonstrated normal inhibin A concentrations in the follicular phase, with no differences observed between control and study cycles (Fig. 4). Older subjects had higher midcycle concentrations of inhibin A in both control (3.44 ± 0.45 vs. 2.50 ± 0.25 IU/ml, respectively; P = 0.08) and study (3.32 ± 0.37 vs. 2.54 ± 0.26 IU/ml, respectively; P = 0.10) cycles, but this increase did not reach statistical significance. Thus, the dominant follicle of an older ovulatory woman secretes normal or elevated concentrations of both E2 and inhibin A. The secretion of these follicular hormones is unaffected by previous HPO axis suppression.
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). Note that not all subjects had sufficient aliquots of serum to perform inhibin A and/or B assays; therefore, the number of subjects analyzed is noted in the each figure legend.
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Prior studies have demonstrated a progressive shortening of the follicular phase with advancing age despite apparently normal dominant follicle size, secretory capacity, and ovulation (5, 7, 14). Possible explanations include either earlier (advanced) selection or more rapid (accelerated) development of the dominant follicle. In either case, factors that determine the length of the follicular phase potentially include alterations in luteal phase physiology (affecting paracrine and/or endocrine signaling mechanisms) or changes in early follicular phase HPO interactions. The current study was designed to eliminate the effects of the preceding luteal phase by suppressing the HPO axis to a similar degree. In this way the follicular phase could be examined from a similar starting point.
Follicular phase studies in normal women have demonstrated that FSH begins to rise in the late luteal phase, reaches a peak in the early follicular phase, and subsequently declines with the emergence of a dominant follicle (21, 22). The sonographic appearance of the dominant follicle is associated with a rise in serum E2, which, in turn, is correlated with a decline in FSH levels (23). We therefore focused on the early follicular phase FSH peak as an indicator of dominant follicle selection. In both control cycles and cycles following HPO axis suppression, the follicular phase in older women is shortened by approximately 2–3 d; specifically, the early follicular phase FSH peak occurs sooner. The results of the current study suggest that this shortened follicular phase in older women is due to an advanced recruitment (i.e. earlier selection of the dominant follicle) and not accelerated (i.e. more rapid) growth of the dominant follicle. If older women had accelerated growth of the dominant follicle, we would have expected to find a difference between the time from the early follicular FSH peak to the LH surge between groups.
A putative mechanism for the shortened follicular phase in older women is the earlier and higher FSH rise in these subjects compared with younger subjects. This phenomenon occurred in the absence of influence from the preceding luteal phase, suggesting that the onset and magnitude of the early follicular FSH elevation may be due to subtle differences in early follicular phase ovarian hormones associated with declining ovarian reserve. On the other hand, we did note that the follicular phase was longer in both age groups after nafarelin suppression compared with that in the control cycle. Although this finding is probably due in part to the time required for the pituitary to recover GnRH responsiveness (24), it also supports the concept that dominant follicle recruitment is initiated in the luteal phase of the preceding menstrual cycle (25).
A candidate hormone likely to be responsible for the observed differences in early follicular phase FSH secretion is inhibin B, a 32-kDa heterodimeric glycoprotein secreted by ovarian follicles that selectively inhibits FSH secretion. Inhibin B levels are correlated with the cohort size of developing early antral follicles (26). The diminution in inhibin B levels in older ovulatory women may result from progressive follicular atresia and the related decline in antral follicle number (10, 27). Older women with FSH elevations have lower early follicular phase levels of inhibin B, presumably a reflection of the diminishing pool of ovarian follicles (19, 20, 28). This hormonal pattern persisted after pituitary down-regulation, suggesting that the low levels of inhibin B in older women are independent of luteal phase inhibin or steroid secretion.
Normal dominant follicle size and normal follicular phase secretion of E2 and inhibin A indicate that once recruited, the dominant follicle in older women is healthy and responds to FSH stimulation. However, it appears that higher levels of FSH are necessary to recruit and maintain normal function of the dominant follicle. The higher midcycle levels of E2 reported in older ovulatory women (14, 29) may be due to more robust secretion by the dominant follicle and/or may be the result of contributions from secondary follicles. Our previous studies of follicular fluid steroid content suggest that the dominant follicle is primarily responsible for the higher circulating E2 levels observed in older premenopausal women (14). The results of the current study support a relationship between the monotropic FSH rise and early development and ovulation of the dominant follicle. Further studies are necessary to determine whether there is also a relationship among increased FSH, higher preovulatory E2 levels, and/or the accelerated rate of follicle atresia observed near the end of the fourth decade.
In conclusion, this study provides evidence that the shortened follicular phase observed in older, ovulatory women is due to shortening of the early portion of the follicular phase. The late follicular phase remains unchanged in length and hormone profile. This suggests that the growth of the dominant follicle is not accelerated, but that its selection is advanced. This study also demonstrates that the shortened follicular phase of the older, ovulatory woman is not dependent on hormonal influences of the preceding luteal phase.
Acknowledgments
We acknowledge Ms. Gretchen Davis and Ms. Laurie Guidry for their assistance with subject recruitment and project management, Mr. Patrick Clarke for his assistance with the illustrations, and Ms. Dorothy McGuinness, Mr. Arlen Sarkissian, Mr. Joseph Moy, and Ms. Sheila Mallette for their expert technical assistance with the assays.
Footnotes
This work was supported by NIA Grant RO1-AG-14579 and NICHHD Grant U54-HD29164.
Abbreviations: CV, Coefficient(s) of variation; E2, estradiol; HPO, hypothalamic-pituitary-ovarian.
Received April 22, 2002.
Accepted August 29, 2002.
References
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