1 Department of Reproductive Medicine, Division of Perinatology and Gynecology, University Medical Center Utrecht, 2 Department of Public Health, Faculty of Medicine, Erasmus University, Rotterdam, 3 Department of Clinical Chemistry, Free University Medical Center, Amsterdam, Division of Reproductive Medicine, 4 Department of Obstetrics and Gynecology and 5 Department of Internal Medicine III, Erasmus University Medical Center, Rotterdam, The Netherlands
6 To whom correspondence should be addressed at: Department of Reproductive Medicine, Division of Perinatology and Gynecology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. e-mail: gabriellescheffer{at}hotmail.com
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Abstract |
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Key words: antral follicles/GnRH agonist stimulation test/inhibin B/reproductive ageing/transvaginal sonography
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Introduction |
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The number of antral follicles and the total ovarian volume as measured by transvaginal ultrasound (Lass et al., 1997; Tomas et al., 1997
; Chang et al., 1998
; Ng et al., 2000
; Bancsi et al., 2002
), basal FSH (Muasher et al., 1988
; Scott et al., 1990
; Bancsi et al., 2000
), inhibin B (Seifer et al., 1997
; Corson et al., 1999
; Hall et al., 1999
; Creus et al., 2000
), estradiol (E2) (Evers et al., 1998
; Mikkelsen et al., 2001
) and the E2 and inhibin B response to exogenous GnRH agonist (GAST) (Winslow et al., 1991
; Avrech et al., 1996
; Galtier-Dereure et al., 1996
; Ranieri et al., 1998
; Ravhon et al., 2000
) or FSH stimulation (EFORT) (Fanchin et al., 1994
; Dzik et al., 2000
; Fabregues et al., 2000
; Elting et al., 2001
) have all been mentioned in the literature to predict declining fertility related to reproductive ageing. Most of these studies were performed in infertility populations, using pregnancy rates or response to ovarian hyperstimulation in IVF as outcome measures. However, the establishment of a pregnancy is influenced by many more, partly unknown, factors. To what extent ovarian ageing per se contributes to the reproductive failure of an individual couple remains a matter of speculation.
Studies concerning physiological ovarian ageing in women without fertility problems have only been performed in a limited number of relatively young women (Schipper et al., 1998) or have compared small groups of relatively aged women with very young controls (Klein et al., 1996a
,b). It therefore seems warranted to evaluate the aforementioned sonographic and hormonal test parameters in a large group of normal women of different ages with proven natural fertility. Since such women are likely to represent the age-related decline of the reproductive potential in the normal population, the assumption that their chronological age approximates reproductive age seems justified. Chronological age, therefore, was used as the outcome variable to identify, first, those factors that show the best correlation with age, second, to obtain insight into how the various predictive parameters correlate with one another, and third, whether or not one or more variables would improve the predictive performance of the best one.
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Materials and methods |
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Transvaginal sonography measurements
Transvaginal sonography of the ovaries was carried out on cycle day 1, 2, 3 or 4. All sonography measurements were performed by the same observer (G.S.) using the 7.5 MHz transvaginal probe on a Toshiba Capasee SSA-220A (Toshiba Medical Systems Europe BV, Zoetermeer, The Netherlands). Examination of the ovary was established by scanning from the outer to the inner margin (Pache et al., 1990; van Santbrink et al., 1995
). All follicles 210 mm in size were measured and counted in each ovary. The sum of both counts was the antral follicle count. Follicle size was calculated from two or three perpendicular measurements depending on the diameter (
6 or >6 mm). The volume of each follicle was calculated by applying the equation of the volume of an ellipsoid (LxWxDx
/6). By adding all volumes of follicles up to 10 mm in size in both ovaries, the total follicular volume was obtained (Haning et al., 1982
). Mean follicular volume was calculated by dividing the total follicular volume by the number of follicles counted. The volume of the left and right ovary was assessed by measuring the diameter of the ovarian contour in three perpendicular directions and applying the equation for the volume of an ellipsoid (D1xD2xD3x
/6) to calculate ovarian volume. Total ovarian volume was then obtained by summing the volume of the left and right ovary. Intra- and inter-observer variations of the antral follicle count and ovarian volume assessment have been published elsewhere (Scheffer et al., 2002
).
Endocrine testing
Blood sampling was performed on the same day as the sonography examination. Hormone concentrations were measured in plasma (E2 and FSH) and serum (inhibin B). Specimens were stored at 20°C until processing. A random subgroup of 40 women underwent a GnRH agonist stimulation test in the cycle subsequent to the one in which the basal endocrine and ultrasound characteristics were measured. A single s.c. injection of 100 µg of triptorelin (Decapeptyl; Ferring, Hoofddorp, The Netherlands) was administered at day 3 of the cycle (Ranieri et al., 1998). Blood samples were taken immediately before and 24 h after GnRH agonist administration. The administered dose is assumed to provide maximal stimulation of the pituitary with mean peak levels of 52 IU/l for LH and 25 IU/l for FSH at 4 h after the injection and LH levels of 12.0 IU/l and FSH levels of 10.3 IU/l after 24 h (Broekmans et al., 1993
). E2 concentrations were assayed with a microparticle enzyme immunoassay (MEIA) purchased from Abbott Laboratories (Abbott Park, IL, USA) and using 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 were measured with the use of the MEIA technology on a fully automated AxSYM immunoanalyser (Abbott Laboratories) according to the manufacturers instructions. All specimens of each volunteer were analysed in the same run. The standard of the FSH assay was referenced against the World Health Organization Second International Reference Preparation for human FSH (78/549). The between-run CV of the FSH assay was 6.0, 6.6 and 8% at levels of 5.0, 25 and 75 IU/l (n = 46). Inhibin B levels were measured using an immuno-enzymometric assay (Serotec, Oxford, UK) (Groome et al., 1996
). Intra- and inter-assay CV for the inhibin B assay were <14.6 and <14.0% respectively.
Statistical methods
Statistical analysis was performed by using SPSS (Statistical Package for Social Sciences) for Windows (release 6.1.3) and GLIM (Generalized Linear Interactive Modelling; NAG, Oxford, UK). For the study group as a whole, three age groups were distinguished: a young group of women aged <35 years, an old group of women aged >40 years and a middle group of women with ages in between. Responses of inhibin B and E2 in the GAST group were defined as the serum or plasma level 24 h after GnRH agonist (cycle day 4 value) minus the baseline level before GnRH agonist was injected (cycle day 3 value). Comparison between subgroup variables was performed by the MannWhitney U-test or the Wilcoxon signed rank test, when appropriate. The correlations between age, the various endocrine and the sonographic parameters in the total group as well as in the GAST group were presented as a correlation matrix. In addition, in the total group an agglomerative cluster analysis on the absolute values of the correlations was performed, in order to visualize the strength of correlations between two variables, and to obtain better insight into the clustering of variables.
Using linear regression, univariate correlations of all variables with chronological agethe outcome variable in this studywere calculated for the total group. Subsequently, partial correlations were determined in a stepwise fashion after controlling for the effect of variables having the strongest correlation with chronological age in previous steps. The square of the correlation of a variable with chronological age was calculated as a reflection of the fraction of variation of chronological age explained by this variable and was considered a direct measure of its predictive performance. The additional predictive effect of the subsequent variables, after the best one had been selected, was calculated by multiplying the squares of the partial correlations with the remaining unexplained fraction.
For all statistical analyses, P < 0.05 was considered as significant.
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Results |
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Discussion |
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It is generally accepted that reproductive ageing is directly related to the remains of the stock of primordial follicles, which is established during fetal life. This pool progressively empties as a woman grows older and is (almost) completely exhausted when menopause is reached (Faddy et al., 1992). In a previous study we showed that the pattern of age-dependent loss of antral follicle numbers is strikingly similar to that of the primordial follicle pool (Scheffer et al., 1999
). It seems plausible, therefore, that the number of antral follicles, as assessed by sonography, reflects what is left of the primordial follicle pool and, thus, the reproductive age of an individual woman.
The number of antral follicles correlated much better with the age of the women evaluated in this study, than other presumed basal markers for reproductive age, including FSH, inhibin B, E2 and ovarian volume. As expected, total follicular volume appeared to be a simple derivative from number of follicles, evidenced by a strong and positive correlation with numbers of follicles (r = 0.69) and a negative correlation with reproductive age (r = 0.34). We were surprised, however, to find that the mean follicular volume considerably increased with age and had almost doubled in the old age group in comparison with the young ones. The correlation of mean follicular volume, a derivative of the total follicular volume and the number of follicles, with age, therefore, is positive (r = 0.37). E2 levels are also positively correlated with age. Apparently, the few antral follicles which are still present in the early follicular phase in older aged women, are not only larger but also produce more E2 than the many, but smaller, antral follicles present at a younger age. Several studies have shown that the follicular phase of the menstrual cycle in older aged women becomes considerably shorter (Lenton et al., 1984; Klein et al., 1996a
; van Zonneveld et al., 2003
). This phenomenon has been postulated as suggesting accelerated growth of antral follicles in older women (Klein et al., 1996a
). Since the diameter on the day of ovulation and the mean follicular growth per day were almost the same in older and younger women (van Zonneveld et al., 2003
), we suggest that follicular development in older women is not accelerated but advanced. Dominant follicle growth in older women is likely to have started already in the luteal phase of the previous cycle before menstruation starts. This earlier start of development is in accordance with data from the literature, indicating that the intercycle FSH rise is not only higher, but also starts earlier in older women (Klein et al., 1996a
; van Zonneveld et al., 2003
). Advanced development of follicular growth fully explains why at the onset of the menstrual cycle, antral follicles are larger and, though lower in number, produce higher E2 levels in older compared with younger women.
While numbers of follicles already have clearly decreased in the middle-aged, hormone levels of FSH, E2 and inhibin B only become notably changed in women aged >40 years. Apparently, age-dependent hormonal changes are a relatively late phenomenon and only occur when follicle numbers are greatly reduced (te Velde and Pearson, 2002). This conclusion is in line with the results of several studies in normal volunteers. Neither maximum FSH and inhibin B concentrations in the follicular phase nor cycle day 3 FSH levels were correlated with age in normally cycling, female volunteers aged 2035 years (Schipper et al., 1998
). A study in young controls (aged 2025 years) and reproductively aged women (aged 4045 years) showed that inhibin B serum levels were only significantly lower in the reproductively aged women at the day of maximal FSH (Klein et al., 1996a
). Finally, lower early follicular inhibin B serum levels were only found in older cycling women compared with young controls (Welt et al., 1999
). All this explains why the correlations of basal FSH, E2 and inhibin B with chronological age are only weak to moderate.
Although the number of antral follicles in both ovaries appeared to have the best correlation with chronological age, basal E2 and total follicular volume slightly improved the prediction already obtained with the antral follicle count. Apparently, the predictive information provided by FSH, inhibin B and total ovarian volume is already covered by the number of follicles. Nevertheless, 90% of the explained variance of age is already obtained with the number of follicles alone, while the additional contributions of total follicular volume and E2 are 6 and 4% respectively. Such an improvement in predictive performance seems almost negligible. Therefore, the use of antral follicle counts as a single test to predict the response to controlled ovarian stimulation and the probability of pregnancy in assisted reproduction seems rational. Several studies have analysed the usefulness of antral follicle counts in this respect. In a study of IVF patients (Tomas et al., 1997) it was shown that the ovarian responsiveness is dependent on the number of small antral follicles (25 mm). In another study, patients with an antral follicle (28 mm) count of less than four appeared to have a significantly higher rate of cancellation due to poor response and no pregnancies occurred in this group of patients (Chang et al., 1998
). In several studies, logistic regression analysis has shown that the number of antral follicles is a significant predictor for the occurrence of poor ovarian response in IVF with an adequate balance between test sensitivity and specificity (Frattarelli et al., 2000
; Pohl et al., 2000
; Dumesic et al., 2001
; Hsieh et al., 2001
; Huang et al., 2001
; Bancsi et al., 2002
). Most studies, however, also revealed that pregnancy prediction from antral follicle counts, even in combination with other ovarian reserve factors, remains difficult.
It was shown that after the administration of a high dose of GnRH agonist, antral follicles greatly increase their production and release of E2 and inhibin B from the granulosa cells within 24 h. This finding is not new for E2 (Winslow et al., 1991; Ranieri et al., 1998
) and confirms results from other studies (Avrech et al., 1996
; Galtier-Dereure et al., 1996
; Ravhon et al., 2000
). As E2 and inhibin B are produced from small antral follicles present in the early follicular phase of the cycle, basal levels would reflect the size of the FSH sensitive cohort of follicles. Although this may be true for inhibin B, E2 release is much more dependent on other sources such as the remnants of the corpus luteum or an advanced growing follicle. Once stimulated by an endogenous FSH (and LH) rise in the GAST, the relation between inhibin B and the cohort size becomes magnified as evidenced by the improved correlation with the antral follicle count. For E2 the change from a negative to a clear positive correlation with antral follicle number shows that the cohort as a whole contributes to the peripheral E2 levels. These findings also confirm studies in which the instant E2 and inhibin B response to a single FSH dose administration appeared clearly related to the number of stimulated follicles in ovarian hyperstimulation for IVF (Fanchin et al., 1994
; Dzik et al., 2000
; Eldar-Geva et al., 2000
; Elting et al., 2001
). Moreover, the responses of E2 and inhibin B in the GAST are clearly better related to the chronological age of a woman when compared with baseline levels. This suggests that stimulated E2 and inhibin B may well reflect the quantitative process of reproductive ageing. As the antral follicle count is the best reflection of reproductive age when basal tests are considered, and at the same time is highly correlated with the E2 and inhibin B response in the GAST, the question arises whether the GAST may be a superior test in the prediction of outcome in assisted reproduction treatment. Back-to-back comparison with antral follicle count has not been published in the literature so far. As yet it cannot be expected that the accuracy of the test in the prediction of outcome in IVF will reach a level of supremacy that justifies the increased burden put on the patient by this test.
In summary, in the prediction of chronological age in normal women, the number of antral follicles appeared to be superior to other presumed measures of reproductive ageing. Additional, though modest, predictive information may be obtained from other endocrine or ultrasound variables. Stimulated E2 and inhibin B correlated strongly with the number of antral follicles and therefore may provide the same body of information on reproductive age. Whether these findings will help us to assess the reproductive capacity in individual subfertile women remains to be further elucidated.
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Submitted on April 27, 2001; resubmitted on September 26, 2002; accepted on November 29, 2002.