menstruation and normal reproductive function. On days subtle rise in LH is only observed at a later stage and to a lesser degree (Lee et al., 1988), and LH does ...
Human Reproduction vol.12 no.6 pp.1152–1155, 1997
Utility of follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol and FSH:LH ratio in predicting reproductive age in normal women
You Kon Kim1,2, Samuel K.Wasser1,3, Victor Y.Fujimoto1, Nancy A.Klein1, Donald E.Moore1 and Michael R.Soules1 1Department
of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Box 354793, University of Washington School of Medicine, Seattle, WA 98195, USA 2Present
address: Department of Obstetrics and Gynecology, Hallym University School of Medicine, Seoul, South Korea 3To
whom correspondence should be addressed
The relative efficacy of follicle stimulating hormone (FSH), luteinizing hormone (LH), FSH:LH ratio and oestradiol is evaluated as a predictor of ovarian reserve (reproductive age) in normal women. Serum levels of FSH, LH, oestradiol and FSH:LH ratios were measured during menstrual cycle days 1–4 in younger (20–25 years; n J 23) and older (40– 45 years; n J 32) reproductive age women with regular menstruation and normal reproductive function. On days 1–4, mean levels of FSH, oestradiol and FSH:LH ratios were significantly higher in older compared with younger women. FSH increased in concentration across cycle days in both age groups. A significantly lower LH value in younger versus older women was found only on day 1. Oestradiol showed no change across days in the younger group, but increased significantly from day 1 to day 4 in the older group. FSH values on days 1 or 2 were the best single predictor of age differences. However, the best prediction of age differences was obtained by using the combination of FSH and LH (as opposed to the FSH:LH ratio) on day 1 of the menstrual cycle. Key words: FSH:LH ratio/gonadotrophins/reproductive ageing
Introduction At birth the human ovary has between 0.5 and 13106 primordial follicles, with regression to near zero by the time of menopause (Richardson et al., 1987; Faddy et al., 1992; Gougeon et al., 1994). By the end of the reproductive lifespan, the ovary loses its functional capacity (ovulation efficiency and hormone secretion). An early secondary effect of this attenuation of ovarian secretory capacity is a monotropic rise in early follicular (EF) phase follicle stimulating hormone (FSH) (Sherman and Korenman, 1975; Sherman et al., 1976; Reyes et al., 1977; Lee et al., 1988; Klein et al., 1996a) associated with a shortened follicular phase (Lenton et al., 1984; Klein et al., 1996a,b; Santoro et al., 1996). The elevation of early follicular phase FSH represents a standard clinical marker of reduced ovarian reserve and diminished responsiveness of the 1152
ovary to ovulation induction (Scott et al., 1989; Toner et al., 1991). Several methods have been used to evaluate ovarian reserve, such as cycle day 3 (EF phase) serum FSH values (Scott et al., 1989; Toner et al., 1991), the clomiphene citrate challenge test (Loumaye et al., 1990; Tanbo et al., 1992; Scott et al., 1995), the gonadotrophin releasing hormone (GnRH) agonist stimulation test (Winslow et al., 1991), and EF phase oestradiol levels (DeGeyter et al., 1993; Licciardi et al., 1995). Although these tests have reasonable predictive values, normal results do not necessarily ensure optimal responses to exogenous gonadotrophin stimulation. Furthermore, the assignment of day 3 as the most sensitive day for clinical testing appears to be arbitrary. Accordingly, we examined changes in endocrine measures of ovarian reserve on days 1–4 of the menstrual cycle. The pituitary gland secretes FSH and luteinizing hormone (LH) in a pulsatile manner in response to GnRH. While the FSH rise occurs relatively early in reproductive ageing, a subtle rise in LH is only observed at a later stage and to a lesser degree (Lee et al., 1988), and LH does not demonstrate a sustained increase until after the menopause (Sherman et al., 1976; Lee et al., 1988). However, a recent study in older ovulatory women showed a decreased LH response to GnRH stimulation compared with younger women (Fujimoto et al., 1996). This finding suggests that LH synthesis and secretion may be different in older ovulatory women, despite the fact that no significant changes in basal serum LH levels were demonstrated. This being the case, both FSH and LH may contribute information relevant to ovarian reserve. In support of this concept, it has recently been reported that day 3 FSH:LH ratio is useful in predicting the outcome of an in-vitro fertilization (IVF) cycle in women with a normal day 3 FSH (Mukherjee et al., 1996). Another study has shown a subtle increase in LH with age (Ebbiary et al., 1994). In order to understand better the relationship between FSH and LH as it pertains to the ageing female, we evaluated the EF phase FSH, LH, oestradiol and FSH:LH ratio in normal ovulatory younger (control group) and older (study group) women on days 1–4 of the menstrual cycle.
Materials and methods Subjects Sixty-six normally cycling women age 20–25 [group Y (younger), n 5 29] and 40–45 years [group O (older), n 5 37] were recruited for a series of studies on reproductive ageing (Sherman and Korenman, 1975; Reyes et al., 1977). All subjects were healthy and were of normal weight for height (body mass index of 18–24 kg/m2). All subjects had evidence of regular ovulation based on (i) regular menstrual cycles every 25–35 days, (ii) a prestudy biphasic basal © European Society for Human Reproduction and Embryology
Age-related changes in early follicular phase
body temperature graph, and (iii) serum progesterone of .10 nmol/ l. Evidence of dominant follicle development in the study cycle was confirmed by serial ultrasound examinations of follicle growth and daily measurement of serum oestradiol. Exclusion criteria included any medications (including no exogenous hormones within 6 weeks of the study), any past or current reproductive endocrine problems (e.g. galactorrhoea, hirsutism) or infertility, and participation in .5 h/week of aerobic exercise. All subjects had normal serum prolactin (,20 µg/l) and testosterone (,3.5 nmol/l) levels in the cycle immediately preceding the study cycle. Informed consent was obtained from each participant. The study protocol was approved by the University of Washington Human Subjects Review Committee. Hormone assay In the study cycle, daily blood samples were obtained between 0700 and 1000 h by venipuncture beginning with the first day of menstrual bleeding (day 1) and continuing daily until ovulation. For practical reasons, an arbitrary cut-off of 2200 h was used for day 1. Subjects who did not have blood drawn on day 1 were excluded from the analysis (six younger and five older subjects). Serum was isolated and frozen in aliquots at –4°C for subsequent analysis. All samples from an individual were analysed in a single assay. Samples were analysed in duplicate for LH (Second International Standard) and FSH by an immunoradiometric method (MAIA clone; Serono Laboratories, Geneva, Switzerland). The inter-assay coefficients of variation were 12 and 14%; intra-assay coefficients of variation for LH and FSH were 5 and 9% respectively. The double-antibody radioimmunoassay for oestradiol was performed in duplicate using reagents supplied by ICN Biomedical, Inc. (Costa Mesa, CA, USA). The antibody cross-reacts 20% with oestrone, 1.5% with oestriol, and ,1% with all other steroids. The inter- and intra-assay coefficients of variation were 16 and 7% respectively. Serum progesterone concentrations were determined in duplicate by solid phase radioimmunoassay using reagents supplied by Diagnostic Systems Laboratories, Inc. (Webster, TX, USA). The antibody crossreactivity is ,5% with all other steroids. The inter-assay and intraassay coefficients of variation were 13 and 11% respectively. Statistical analysis Separate repeated measures analyses of variance were used to examine the relation between each of the dependent variables: FSH, LH, the FSH:LH ratio, and oestradiol concentrations against the main effects of age (i.e. younger versus older women) and cycle day (i.e. across the first 4 days of the menstrual cycle). The interaction between age and cycle day also was examined in each analysis. Simple regression analyses were then used to generate standardized scores that allowed direct comparison between each of these measures, on each day of the menstrual cycle, to determine which single measure and day best discriminated between these two age groups. These standardized scores reflect the amount of variance explained using simple regression analyses to predict younger versus older subjects based on FSH, LH, the FSH:LH ratio, and oestradiol values, on each day of the menstrual cycle. A hierarchical multiple regression analysis was then used to predict age differences based on FSH and LH concentrations, in that order, on each cycle day.
Results The mean ages of groups Y and O were 23.3 6 1.5 (n 5 23) and 42.3 6 1.7 years (n 5 32) respectively. The repeated measures analyses of variance used to compare FSH, LH, the FSH:LH ratio and oestradiol values in younger versus older
women across the first 4 days of the menstrual cycle are shown in Figure 1A–D respectively. For FSH, significant main effects were found for age (F 5 14.56; P , 0.004) and cycle day (F 5 7.78; P , 0.0001). FSH was higher in older versus younger women across all cycle days, and increased in concentration across days for younger and older women (Figure 1A). For LH, a significant main effect of cycle day (F 5 3.79; P , 0.02) was found along with a significant interaction between age and cycle day (F 5 8.74; P , 0.0001). This pattern resulted largely from a substantially lower LH value in younger versus older women on day 1 only (Figure 1B). For the FSH:LH ratio, the only significant main effect was age (F 5 4.99; P , 0.03), with the ratio being consistently higher in older versus younger women (Figure 1C). For oestradiol, there were significant main effects of age (F 5 6.63; P , 0.02) and cycle day (F 5 9.87; P , 0.0001), as well as a significant interaction between age and cycle day (F 5 8.40; P , 0.0001). Oestradiol was consistently higher in older versus younger women, and showed an increased concentration across days of the menstrual cycle in older women only (Figure 1D). The simple regression-generated standardized scores reflecting the amount of age-related variance in FSH, LH, the FSH:LH ratio and oestradiol values, on each day of the menstrual cycle, are shown in parentheses in Figure 1A–D. FSH consistently explained more of the variance in younger versus older women than did any of the other three measures on any given day of the menstrual cycle (Figure 1). Moreover, FSH values on days 1 and 2 were better predictors of these age differences than were FSH values on days 3 or 4 (Figure 1A). A significant age-related variance was found in LH values, on day 1 only (Figure 1B). However, because this difference was in the same direction as the age difference in FSH values on day 1 (Figure 1A), this age difference was not reflected by the FSH:LH ratio on day 1 (Figure 1C). We therefore used a hierarchical multiple regression analysis, adding the LH value into the regression following FSH, to determine whether the addition of LH explained more of the variance in age than did FSH alone. The addition of LH values into the regression explained an additional 9% of the variance (P 5 0.01), from 0.23 (for FSH alone) to 0.32 (for FSH and LH combined) on day 1 only. Thus, the best predictor of age differences in this study was obtained by using the combination of FSH and LH (as opposed to the FSH:LH ratio) on day 1 of the menstrual cycle. Discussion This study showed that FSH was the best single predictor of younger versus older normal ovulatory women on any of the first 4 days of the menstrual cycle. Moreover, FSH values on days 1 and 2 were better predictors of this age difference than were FSH values on days 3 or 4. This finding is not surprising, considering that older women have an earlier onset of the inter-cycle FSH rise, associated with an earlier onset of the acute follicular phase oestradiol rise and dominant follicle development, leading to higher early 1153
Y.Kon Kim et al.
Figure 1. Differences between younger (20–25 years) and older (40–45 years) normally cycling women during days 1–4 of the menstrual cycle in (A) follicle stimulating hormone (FSH), (B) luteinizing hormone (LH), (C) FSH:LH ratio and (D) oestradiol, generated by repeated measures analyses of variance. Solid bars, younger women; hatched bars, older women. Sample sizes: younger, 23 women; older, 32 women. Values in parentheses represent amount of age-related variance explained (r2) in the measure shown in that graph, on each cycle day, by simple regression analyses. *P , 0.05; **P , 0.001.
follicular phase oestradiol levels (Klein et al., 1996a,b). This early rise in oestradiol may in turn result in early suppression of pituitary FSH secretion. Basal EF phase FSH is the most commonly utilized predictor of ovarian reserve. A subtle day 3 rise in FSH suggests declining ovarian reserve in spite of regular menses, and portends decreased fertility rates in both infertile and normal women (Scott et al., 1989; Toner et al., 1991). While basal day 3 FSH has become a standard to evaluate ovarian reserve, our results suggest that it may be more useful to evaluate FSH on day 1 or 2 instead of day 3 and theoretically to begin ovulation induction earlier in the menstrual cycles of older infertile women. The serum LH level was significantly elevated in the older group on cycle day 1. This finding may be related to the fact that there is an earlier onset of the inter-cycle FSH rise (Klein et al., 1996a) associated with earlier dominant follicle development and ovulation in older ovulatory women (Lenton et al., 1984; Klein et al., 1996a,b). (While older ovulatory women have more advanced follicular development on any given follicular phase cycle day, this finding may be due to either earlier onset or an accelerated rate of follicle development.) Examining LH on cycle day 1, in addition to FSH, may improve predictive value even further, but not when examined as the FSH:LH ratio. Because the LH difference on cycle day 1 was in the same direction as the age difference in FSH values on day 1, this age 1154
difference was not reflected by the FSH:LH ratio on day 1. This finding contradicts a recent report in the medical literature that prognosis was generally poor in patients undergoing IVF cycles if the day 3 FSH:LH ratio was elevated to ù3.6, even though the day 3 FSH value was normal (Mukherjee et al., 1996). However, their cut-off value was more than 4.0 standard deviations above the ratio for our younger age group (1.40 6 0.54 versus 3.6), which was also greater than the highest FSH:LH ratio (3.42) in our older age group. These study differences may have resulted from their testing infertility patients rather than normal healthy subjects as in our study. We have demonstrated that an EF phase serum FSH level and a cycle day 1 LH level have additive predictive values for chronological age in two groups of normal women. Thus, these changes in gonadotrophin secretion patterns can be interpreted as signs of reproductive ageing. Whether such changes are indicative of premature ovarian ageing and predictive of relative subfertility and gonadotrophin resistance remains to be determined in a clinical setting. Acknowledgements We wish to thank Gretchen Davis for assistance in all phases of this study and Cydney Foote for preparation of the manuscript. This study was supported in part by grants from the National Institutes of Health (R01-HD18967 and P50-HD-12629) and by the Hallym University Medical Center.
Age-related changes in early follicular phase
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