1 Department of Reproductive Medicine, Division of Obstetrics, Neonatology and Gynecology, 2 Department of Endocrinology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, 3 Department of Internal Medicine and 4 Department of Public Health, Erasmus University Medical Center Rotterdam, The Netherlands
5 Present address: Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands
6 To whom correspondence should be addressed. e-mail: p.vanzonneveld{at}azu.nl
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Abstract |
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Key words: ageing/female fertility/follicle development/hormones/menstrual cycle
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Introduction |
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Although much is known about hormonal patterns and follicle development in the normal menstrual cycle, it is difficult to define cycle abnormalities unless there is no follicle growth at all or no ovulation. In the present study, observations in young and fertile women were used as a reference of cycle normality. The cycles of these women were considered as normal unless definite abnormalities could be seen, such as absence of follicle development or ovulation.
The aims of the present study were to: (i) obtain reference values for cycle normality from a group of relatively young fertile women who have regular menstrual cycles; and (ii) evaluate whether follicle growth, ovulation, hormonal patterns and endometrial development in older women differ from this reference group, and, if present, whether such differences may explain the age-related loss of fertility.
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Materials and methods |
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Experimental design
A relatively older group of women who previously were normally fertile [n = 26; median age 42 (range 4146) years] was compared with younger women, the reference group [n = 35; median age 31 (range 2234) years]. Investigations were started in the midluteal phase of the first study cycle. The luteal phase was assumed to have started when a temperature rise on the BBT chart had been observed. The temperature was considered to be elevated if it was higher than the temperature on any of the six preceding days (World Health Organization, 1967). From the seventh day after the temperature shift, the volunteers visited the authors research department every 2 or 3 days for ultrasound scans in which the size and number of all follicles
2 mm in diameter, were counted and measured, and endometrial thickness was determined. Blood samples were taken at each visit and stored for determination of FSH, LH and estradiol (E2). Inhibin A and B were also determined in those blood samples closest to the start of the menstrual period. After menstruation had started in the subsequent cycle (cycle 2), the volunteers returned on cycle day 2, 3 or 4 for ultrasound scanning and for E2, FSH, LH, inhibin A and B measurements. Thereafter, the same measurements (except for inhibin A and B) were performed every 23 days. When the dominant follicle had reached a mean diameter of at least 14 mm, ultrasound scans were performed and blood samples taken daily until ovulation had occurred. Ovulation was defined as a complete disappearance of the follicle or a reduction of its mean diameter by at least 5 mm (Janssen-Caspers et al., 1986
; Check et al., 1990
). After ovulation, an ultrasound scan was performed in the midluteal phase, and blood was taken at the same time for measurement of progesterone. The duration of the follicular phase was defined as the period of time (in days) from and including the day the menstrual period started, until and including the last day that the dominant follicle was present before ovulation. The duration of the luteal phase was considered to be the interval following ovulation up to and including the day before the onset of menstruation.
Hormone assays
Blood sampling and transvaginal sonography were performed on the same days. Hormone concentrations were measured in plasma (E2, FSH, LH and progesterone) and serum (inhibin A and B) specimens stored at 20°C until processed. E2 concentrations were assayed using a microparticle enzyme immunoassay (MEIA) kit (Abbott Laboratories, Abbott Park, IL, USA) and a semi-automated IMx analyser. Between-run coefficients of variation (CV) for E2 were 10.1, 7.0 and 6.9% at 533, 1354 and 4197 pmol/l respectively (n = 49, 49 and 30). Concentrations of FSH, LH and progesterone were measured using a fully automated AxSYM immunoanalyser (Abbott Laboratories) according to the manufacturers instructions. All specimens from each volunteer were analysed in the same run, with all assays based on MEIA technology. The standard of the LH assay was calibrated against the WHO First International Reference Preparation for human LH (68/40), whereas that of the FSH assay was referenced against the WHO Second International Reference Preparation for human FSH (78/549). For progesterone, the inter-assay CV was 14.1% at 3.5 nmol/l, 7.8% at 19.0 nmol/l, and 9.9% at 71.2 nmol/l (n = 51). For LH, the between-run coefficients of variation were 5.5, 7.2 and 7.9% at 4.8, 39 and 83 IU/l respectively (n = 48). For FSH, the between-run CV was 6.0, 6.6 and 8% at levels of 5.0, 25 and 75 IU/l respectively (n = 46). Inhibin A and inhibin B levels were measured using an immunoenzymometric assay (Serotec, Oxford, UK) (Groome et al., 1996). Intra- and inter-assay CVs for inhibin A and inhibin B assays were <7.7 and <8.0%, and <14.6 and <14.0% respectively.
Transvaginal ultrasound
All transvaginal ultrasound measurements were performed by the same observer (G.J.S.) using a 7.5 MHz transvaginal probe on a Toshiba Capasee SSA-220A (Toshiba Medical Systems Europe BV, Zoetermeer, The Netherlands). The ovary was examined by scanning from the outer to the inner margin in longitudinal cross-sections (Pache et al., 1990). All follicles were measured and counted. Mean follicle diameters were calculated from two or three perpendicular measurements depending on the diameter (
6 mm in two directions; >6 mm in three directions) by taking the mean of the measurements. In the analysis of dominant follicles, only follicles with a mean diameter
10 mm were included because follicles <10 mm may initially grow, but fade away later on (Pache et al., 1990
). Follicles of 210 mm diameter were considered as antral non-dominant follicles. By including follicles up to 10 mm diameter, the maximal antral follicle cohort is represented. Good accuracy was found in an in-vitro study of small cystic structures, as well as good intra-observer reproducibility of antral follicle numbers and sizes of dominant follicles in seven patients (Pache et al., 1990
). An inter-observer reproducibility study performed in 37 volunteer women showed an adequate reproducibility of small follicle numbers (Scheffer et al.., 2002
), while in an earlier study a good correlation was observed between the calculated volume of pre-ovulatory follicles and the volume of aspirated follicular fluid (OHerlihy et al., 1980
). Endometrial thickness was expressed as the total sonographic thickness of the two layers of the endometrium.
Methods of analysis
Sonographic and hormonal observations were started in the luteal phase (cycle 1) because events in the luteal phase are likely to influence the follicular phase of the subsequent cycle (cycle 2). In order to synchronize hormonal and ultrasound data obtained during the course of an ovarian cycle, several time-markers were used. The time after the BBT-rise was used in cycle 1 to describe the subsequent events of the luteal phase. The nadir before a temperature rise was considered as day 0. The start of the menstrual period in the subsequent cycle was used to describe the follicular phase prospectively, and the day of the LH peak (the day the highest LH level was reached during the LH surge) to investigate the follicular phase retrospectively. Finally, the progesterone determinations in this cycle were timed 7 days after ovulation. Ideally, a time-marker is a well-defined, clearly recognizable event, but any of the markers mentioned had its drawbacks. The lack of precision of the BBT-rise as a marker of ovulation is well known (Barrett and Marshall, 1969; Dunson et al., 1999
). The time point at which a menstrual period is considered to start is a subjective choice of the woman, especially when there is no abrupt start of the bleeding as often is the case. It is also uncertain whether the onset of shedding of the endometrium corresponds with the hormonal events, or also depends on endometrial factors. The LH peak value can be identified by using the highest value of daily results, as was performed in the present study. However, the duration and magnitude of the LH surgeand thereby the day of the highest valuemay vary. In the luteal phase and in the first half of the follicular phase, the volunteers were seen every 23 days, which may result in missing values on several days in graphical presentations of data. For that reason, in some parts of the cycles, data of two consecutive days (and in the beginning of the second cycle of cycle days 14) were pooled. If results of 2 days were available, only the value of one of these days was used after random selection. Data are provided as mean values ± 1.96 SEM (the 95% confidence interval of the mean) in Table I and Figure 1. Statistical analysis was carried out using SPSS (Statistical Package for Social Sciences) for Windows (release 10.0.7). For comparisons of results from different phases of the cycle between age groups, the MannWhitney U-test was applied. P-values < 0.05 (two-sided) and < 0.01 were considered statistically significant and highly significant respectively.
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Results |
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Among the 35 younger women, the results of hormonal determinations and ultrasound of cycle 2 were incomplete in one woman. This cycle must have been ovulatory because the woman became pregnant. In the younger group one cycle (2.9%) was judged as abnormal, having a cycle duration of 16 days and a luteal phase of only 4 days. In the older group, two abnormal cycles (7.7%) were observed, and both showed a persisting follicle. The proportion of abnormal cycles in both groups was not statistically different (P = 0.57). The conception cycle with missing data, and the three cycles considered abnormal, were excluded from further analysis. The remaining 33 cycles in the younger age group, and 24 cycles in the older age group were considered normal ovulatory. A dominant follicle (>10 mm in diameter) was recognized in these 57 cycles; this showed a constant growth of 1.6 mm per day and reached a mean diameter of 15.726.4 mm before ovulation. In the follicular phases of these cycles, there was a steady increase in E2 concentrations.
No LH surge was observed in one cycle; this was in 42-year-old volunteer who had high levels of FSH and LH present in the follicular phase. During the luteal phase, progesterone levels rose to a normal value of 68 nmol/l, but there was no good reason to consider this cycle as abnormal. Thus, data obtained from 33 cycles in the younger women, and from 24 cycles in the older women were compared.
Values of sonographic events and hormonal levels on relevant days of cycles 1 and 2 of both age groups are presented in Table I. In general, results in the older age group were remarkably similar to those in the reference group of young women. All women in the elderly group ovulated, though the mean diameter of the dominant follicle just before ovulation was almost 2 mm less than that in the reference group women. Some of the differences found were in line with previously published observations, including a shorter duration of the follicular phase, elevated early follicular FSH, and a trend to decreased inhibin B levels in the early follicular phase of the older age group. The difference of 2 days at the first appearance of the dominant follicle is in line with the shorter follicular phase in the older women. In the early follicular phase, much fewer small antral follicles (210 mm) were observed in the older age group, but without any difference in their mean volume. The lower LH peak level in the older age group has not been described previously. In both age groups, inhibin B levels were increased after the LH peak, whereas progesterone levels in the luteal phase and endometrial thickness either did not differ at all or were perhaps slightly higher in the older age group.
The results of hormone measurements, and the diameters of small antral and dominant follicles are presented in Figure 1, in relation to the three time-markers as defined. Results of the luteal phase in the first cycle are shown in relation to the rise in BBT, with results of the follicular phase in the second cycle plotted from the start of the menstrual period as well as in retrospect from the day of the LH peak level in order to correct for the observed difference in the length in the follicular phases between the two age groups.
The dominant follicle can be observed from cycle day 8 in the older age group, and from day 10 in the younger age group. On the corresponding days of the cycle (Figure 1, 1B), the mean follicle diameter in the older group exceeds that of the younger women, and seems to be advanced in the older group by about 2 days relative to the younger group. However, when the diameters are grouped in relation to the LH peak (Figure 1, 1C), the development of the dominant follicle appears to be remarkably similar except for the last 3 days when growth in the older women slows down slightly. As a result, in the older women the dominant follicles are 2 mm smaller, on average, on the day before ovulation (Table I).
The evolution of E2 levels was in complete agreement with the growth pattern of the dominant follicle (Figure 1, 2B and 2C).
Mean FSH levels in the midluteal phase (7 days after the BBT-shift; Figure 1, 3A) showed comparable low values, rising thereafter both earlier and higher in the older age group. This earlier rise in FSH was also present on the combined cycle days 14, followed by a rise in the younger age group (Figure 1, 3B). During the following days all FSH levels declined, which concurred with the identification and subsequent growth of the leading follicle, and rising E2 levels. The gradual rise after cycle days 78 in the older, and after cycle day 12 in the younger group was caused by the FSH surge which accompanies the LH surge that occurs much earlier in the older group. In relation to the LH peak level as a time-marker (Figure 1, 3C), the late luteal and early follicular FSH rises could be seen 910 days before the day of the LH peak (day 0) in both groups.
LH levels did not differ between the two age groups, except for a lower peak level during the LH surge (P < 0.05) in the older group.
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Discussion |
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There is convincing evidence available which indicates that the fertility of women aged >40 years is seriously decreased (Bongaarts, 1982; Wood, 1989
; Holman et al., 2000
), and the second aim of the present study was to seek an explanation for this phenomenon. The results obtained do not provide obvious clues, however, as the majority of women had seemingly normal, ovulatory cycles with normal estradiol and progesterone levels. In addition, the growth curves of the dominant follicle in both older and younger women were very similar until some days before ovulation. The diameter of the dominant follicle before ovulation was slightly smaller in the older women, but whether this is an indication of diminished follicle quality is unclear.
In the older women, the follicular phase of the menstrual cycle was shorter, and in this group the dominant follicle could be identified earlier. Whether the dominant follicle has a faster growth rate in older women (accelerated growth), or starts earlier (advanced growth) (Thatcher and Naftolin, 1991; Klein et al., 1996a
) is a matter of debate in the literature. The present data clearly support the latter point of view because the growth pattern of the dominant follicle, as well as the pattern of the rise in estradiol in the follicular phase (see Figure 1, 1B/1C and 2B/2C), barely differ between older and younger women. In case of an accelerated follicle growth, a steeper slope of both the graph of dominant follicle growth and E2 rise would have been expected in the older group. The shift in time of follicle development resulting in a shorter follicular phase in the older age group might be explained by the earlier FSH rise in the luteal phase of the preceding cycle (Figure 1, 3A/3B). As a result, a dominant follicle is selected from the cohort of small recruitable follicles, and stimulated to further growth several days earlier than in the younger group. This concept is shown in Figure 2.
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Inhibin B levels showed a sharp increase on the day after the LH peak level compared with the previous day (Table I) in the younger (P = 0.006) and in the older (P = 0.036) groups. Such a rise has been previously described (Groome et al., 1996), and it has been suggested by others (Speroff et al., 1999
) that this is a result of release from the ruptured follicle. However, ovulation is not a prerequisite, as a clear rise has also been demonstrated to occur within 24 h after the induction of an artificial LH and FSH surge by a single injection of a GnRH-agonist during the early follicular phase (Scheffer et al., 2000
). This finding is supportive of the small antral follicles serving as a source for this inhibin B.
An advanced start of a cohort of early antral follicles in older women occurs in the presence of a still-functioning corpus luteum (Figure 2). It is possible that the environment for the development of pre-antral follicles during this phase of the cycle is suboptimal. A second mechanism which may explain the diminished follicle quality in older women is the limited oocyte pool model (Warburton, 1989). This hypothesis assumes that the dominant follicle, selected from the cohort of pre-antral follicles, is the one which benefits longest and most from the favourable effect of FSH during the FSH window. As the number of pre-antral follicles decreases (in the present study from a mean number of 13.6 in women aged <34 years to 4.9 in women aged 4146 years), it becomes less likely that a follicle will be present at the beginning of the FSH window. Consequently, the follicles of older women more often are not quite ready or almost too late when selected for further growth in comparison with follicles of younger women (Figure 2). Oocytes of older women, therefore, may be more likely to undergo non-disjunction at the first meiotic division. Moreover, assuming that follicles of different quality appear in the FSH window, it is more likely that a high-quality follicle is selected from a cohort of 13 (younger women) than from four (older women) follicles.
It is concluded that in spite of a dramatically decreased number of recruitable antral follicles, follicle development, hormonal events and endometrial growth are remarkably undisturbed in older women. Apparently, a grossly decreased number of antral follicles does not result in cycle disturbances until after the age of 4546 years. Because an equal endometrial thickness is present in both age groups, the involvement of a uterine factor seems unlikely. In addition, in oocyte donation the pregnancy rates depend mainly on the age of the donor, not of the recipient (Sauer et al., 1993). However, some decrease in uterine receptivity with age may be present. In recipients aged >40 years, implantation rates using oocytes from young donors could be enhanced by applying supraphysiological progesterone replacement (Meldrum, 1993
). The clear reduction in fertility of aged women is most likely explained mainly by a decrease in oocyte quality, as is reflected by an increase of chromosomal aberrations in the oocytes (Brook et al., 1984
; Gaulden, 1992
; Eichenlaub-Ritter, 1996
) and embryos (Warburton et al., 1986
; Munné et al., 1994
; Holman et al., 2000
) of older women. However, as stated above, it cannot be excluded that a decrease in oocyte quality may be the result of a decreased quality of the follicle.
There is some evidence that an advanced start of follicle development, as shown in older women, can be manipulated. One group (Le Nestour et al., 1993) showed in healthy volunteer women that the inter-cycle FSH rise can be delayed by administering physiological amounts of estrogens. In this way (hypothetically), in older women the recruitment of a new cohort of follicles, and the selection of a dominant follicle, can be pushed forward from the late luteal phase of the preceding cycle into the early follicular phase. This would result in a situation resembling that in younger women. However, whether such an approach would enhance the quality of the cohort of follicles and of the dominant follicle emerging from this cohort, resulting in a more fertile cycle, remains to be confirmed.
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Acknowledgements |
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Submitted on December 13, 2001; accepted on November 28, 2002.