1 Obstetrics and Gynecology Epidemiology Center and 2 Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Boston, MA 02115, USA
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Abstract |
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Key words: body mass index/estradiol/gonadotrophins/menstrual cycle/ovulation
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Introduction |
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That measurement of reproductive hormones at a single point during the cycle might be informative was suggested by data indicating that basal hormone concentrations measured during the early follicular (i.e. menstrual) phase of the cycle were correlated with success after IVF. A high basal FSH (and, curiously, a high basal estradiol) predicted fewer oocytes recovered and lower chance of pregnancy (Muasher et al., 1988; Scott et al., 1989
; Pearlstone et al., 1992
; Licciardi et al., 1995
; Smotrich et al., 1995
; Witt, et al., 1995
; Buyalos et al., 1997
). Thus, measurement of basal FSH and estradiol, as potential markers for ovarian reserve, offers an approach to the study of the ovarian life cycle that is easier to use in large populations and yields simpler descriptive statistics. However, because recent studies (Burger, 1994
; Santoro et al., 1996
) suggest that there may be considerable variation in hormone concentrations from cycle to cycle, it is important to monitor hormones over more than one cycle. In this study, basal hormone concentrations were measured at entry, 6 and 12 months later in women aged 3645 years. Description of reproductive hormones in this group may contribute to a greater understanding of the ovarian life cycle during a critical age period when fertility declines markedly.
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Materials and methods |
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From those who completed the initial survey, cohorts of women with and without current or past depression were invited to participate in a prospective study that included structured clinical psychiatric interviews, baseline and semi-annual medical questionnaires, and menstrually timed collection of blood specimens. The cohort of non-depressed women, who were the focus in this study, were those who scored <16 on the CES-D and did not meet criteria for a lifetime history of major depression based upon a Structured Clinical Interview for the Diagnostic and Statistical Manual (SCID-IV). Approximately 30% of the initially screened non-depressed women were eventually enrolled in the Harvard Study of Moods and Cycles. Among the women not enrolled, about half were unwilling to make the sizeable commitment required for participation in the Study of Moods and Cycles which entailed bi-annual menstrually timed blood donations and questionnaires. The remaining were ineligible for enrolment due to pregnancy, menopausal status, or loss to follow-up after the initial screening assessment.
The depressed and non-depressed cohorts were followed every 6 months over a 36 month follow-up period and were asked to provide early follicular phase blood specimens and to participate in medical and psychiatric follow-up interviews. The medical questionnaires assessed menstrual history including age at menarche, average cycle length currently and during the first 5 years after menarche, pregnancy and breastfeeding history, and oral contraceptive (OC) or hormone use. A variable called estimated ovulatory cycles was calculated, which has previously been found to be a predictor of risk for menopause (Cramer and Xu, 1996). This variable was calculated (for a premenopausal woman) by subtracting the woman's age at menarche from her current age which estimates her maximum `ovulatory years.' This was reduced by potential `anovulatory years' approximated by years of OC use, number of liveborns (roughly equating a term pregnancy with a year of anovulation), and total years of breastfeeding. This `adjusted ovulatory years' is multiplied by 365 and divided by the women's average cycle length (during the first 5 years after menarche) to yield the estimated number of ovulatory cycles. The cycle length that women recalled having during their first five years of menstrual life was used rather than the current cycle length, which on average would have been around age 40 years. It was felt that cycle length in early rather than late reproductive life was more relevant to the number of ovulations experienced during the majority of reproductive life.
For the prospective assessment of basal hormone concentrations, blood was drawn on any one day of the first 5 days of the menstrual cycle with day 1 defined as the first day of menstrual bleeding. The specimen was drawn during the first period after study enrolment and was scheduled every 6 months over the 3 year period of the study. Reminder calls were placed to women around the estimated time of their 6 monthly anniversary periods at which time the regularity of the menstrual cycle and the occurrence of events such as pregnancy, breastfeeding, and use of oral contraceptives or other hormones was reassessed. Because the blood draw had to accommodate holidays and interviewer's and subject's schedules, it was impossible to do the blood test on precisely the same menstrual day for each woman.
Although 643 non-depressed women were enrolled in the parent study, 221 (34.4%) who did not complete three consecutive draws, 11 (1.7%) who went on hormones during the year of observation and five who had unpredictable periods throughout their reproductive life were excluded. This left 406 women who had basal hormones measured during natural cycles at study entry, at 6 and 12 months after entry. Cycle lengths at study entry for these women were between 16 and 38 days and did not vary by more than 10 days from cycle to cycle in 384 (94.6%) subjects. No subject reported 3 months between periodsthe usual definition for oligo-amenorrhoea.
Measurements of serum gonadotrophins were performed using the Coat-A-Count immunoradiometric assays (Diagnostic Products Corp., Los Angeles, CA, USA). The World Health Organization (WHO) First International Reference Preparation (68/40) of human pituitary LH was used as the standard for LH and the WHO Second International Reference Preparation (78/549) of human pituitary FSH was used as the standard for FSH. Estradiol was measured in duplicate and averaged using the Coat-A-Count radioimmunoassay. Intra-assay coefficients of variation (CV) were <4% for the gonadotrophins and <7% for estradiol. Inter-assay CV were <10% for the gonadotrophins and <15% for estradiol.
Means and 95% confidence limits on the mean for the gonadotrophins and estradiol were calculated based upon log transformed values to accommodate skewness of the hormonal distributions. Hormone concentrations were distinguished by whether they were measured at 0, 6 or 12 months and by whether they were the maximum or minimum of the three values regardless of study period. Paired t-tests were used to determine whether subjects' 6 or 12 month values differed from their baseline values or whether maximum differed from minimum values. Generalized linear modelling was used to determine whether (log transformed) entry or maximum hormone concentrations differed by categories of demographic, reproductive, or menstrual history variables after adjusting for age at study entry or cycle day of study. Significant associations were distinguished by those with a two-sided P-value of 5%. All analyses were performed using the SAS system (Statistical Analyses System Institute, SAS Campus, Cary, NC, USA).
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Results |
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Discussion |
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The results confirm that FSH concentrations increase as a woman ages (Sherman et al., 1976; Reyes et al., 1977
; Metcalf and Livesy, 1985
; Lenton et al., 1988
; Cramer et al., 1994
). Even between their baseline and 6 month period of observation, women had a small but significant increase in basal FSH and LH. The physiological basis for the increase in FSH, which begins well before the menopausal transition, is likely to be due to a declining germ cell pool with a consequent decrease in concentrations of the ovarian hormone, inhibin B, which regulates FSH secretion (Buckler et al., 1991
; Welt et al., 1999
). Unfortunately, due to the high cost and lack of availability of the inhibin assay when this study began, inhibin concentrations were not measured in this study.
The calculated variable `estimated ovulatory cycles' was also strongly related to FSH. Components of this variable include: years at risk for ovulation determined by current age and age at menarche; events associated with anovulation including pregnancies, breastfeeding, and OC use; and frequency of ovulation, as determined by cycle length. Cycle length in early reproductive life rather than late was chosen for this variable, although the two were correlated in this study (r = 0.24, P = 0.0001). Interestingly, cycle length in early reproductive life, by itself, was a strong predictor of basal FSH. It can be speculated that short cycle length in early reproductive life may signal a smaller germ cell endowment to begin with since it is known that cycle length decreases as women age and ovarian reserve decreases (Treloar et al., 1967; Lenton et al., 1984
; Munster et al., 1992
). Follicle counts performed at autopsy by Block ranged between 85 000 and 684 000 in women aged <20 years, suggesting that varying germ cell endowments are likely to occur (Block, 1952
).
An additional variable, which might also mediate its effect on FSH through germ cell depletion, is smoking. This study confirmed prior observations that current and former smokers have higher FSH concentrations (Velasco et al., 1990; Cramer et al., 1994
; Cooper et al., 1995
). Smokers undergoing IVF have poorer oocyte recovery and lower fertilization rates (Van Voorhis et al., 1992
). A logical extension of the search for factors which increase basal FSH concentrations would be that these same variables hasten age at menopause. Indeed, it is well established that smoking decreases the age at menopause (Cramer et al., 1995
) and it has also reported that the `estimated ovulatory cycles' variable predicted an earlier than normal menopause (Cramer and Xu, 1996
).
Only age and BMI significantly affected the maximum LH concentration. Similar to FSH, LH increases with age; but the effect of BMI on LH may not be a simple linear one. It appears that a lower LH may occur at both extremes of weight (Table I). This association between BMI has been previously described (Bohlke et al., 1998
) and may be modulated through other hormones including insulin or leptin. Fasting insulin concentrations are positively correlated with BMI or body fat and negatively correlated with LH (Dale et al., 1992
) which could account for depressed concentrations of LH in obese women. In thin women, the association might involve the adipose hormone, leptin, which is known to be strongly and positively correlated with BMI and which also appears to have a number of hypothalamic and ovarian effects (Mantzoros et al., 1998
).
One variable which was found to significantly influence the maximum estradiol concentration was ovulatory cycles. Although the effect could be an indirect one mediated by the effect of past ovulations on FSH concentrations, it is reasonable also to consider a direct ovarian effect. A greater number of ovulatory cycles would imply that more follicles with developed granulosathecal compartments have been formed. This could impact on the number of residual cells, which have previously been luteinized and may be capable of steroid production, that remain in ovaries which have undergone a greater number of ovulations. Though highly speculative, this theory would have considerable relevance to the postulated effects of a longer reproductive life and greater number of ovulations on breast and ovarian cancer risk. Clearly, estradiol concentrations eventually decrease in all women regardless of their past reproductive history, so it will be important to describe precisely how the transition from fluctuating to low estradiol concentrations occurs.
We believe that an important observation of the current study is that there is considerable short-term variation in reproductive hormone concentrations. Instances in which wide fluctuations in reproductive hormones occurred in the same woman have been described, but mostly in women in their late forties (Burger, 1994; Santoro et al., 1996
), while the current study suggests the fluctuations begin earlier in reproductive life. The `oscillation' of basal hormones pointed out in Table IV
raises the question of what would be the physiological basis for `menopausal' concentrations of FSH and estradiol to be measured in one sampling period and low FSH and high estradiol in another. Based on the observations from Table III
, it is unlikely that this shift occurs simply at different time points during the early follicular phase of the same cycle. More likely, extremes in one cycle set up the dynamics for an opposite extreme to be observed in the next cycle. This could explain the observation from IVF clinics that both an elevated basal FSH and elevated basal estradiol are correlated with poor outcomes (Licciardi et al., 1995
; Smotrich et al., 1995
; Witt et al., 1995
). However, rather than being a distinct physiological event, an elevated basal estradiol may simply reflect the response to the high FSH from the previous cycle. Studies that monitor basal hormones in two or more consecutive cycles should be informative and clarify whether patterns tend to be repeated by the same ovary (since it is postulated that ovulation alternates between ovaries).
Limitations of the current study include the facts that the sample consisted mostly of Caucasians, excluded women with major depression, and involved a selected group of women willing to participate in a longitudinal study. These factors may limit the generalizability of the results. In addition, the key exposures which were assessed were based on recall and may be subject to error, although such biases generally obscure rather than create associations. Focusing on a single point during the cycle rather than multiple time points precluded investigation into whether all of the cycles studied were ovulatory and cycle dynamics. Logistical issues prevented collection of specimens on precisely the same day in all subjects. Finally, adrenal hormones such as dehydroepiandrosterone were not studied, which may also contribute to estrogen concentrations.
Despite these limitations, we believe this study has yielded some important conclusions. Basal FSH increases during late reproductive life and wide fluctuations in FSH and estradiol may occur, suggesting that basal hormone concentrations need to be characterized over more than one cycle. Higher basal FSH concentrations are seen in smokers and those with a shorter cycle length in early reproductive life. Estimated number of ovulatory cycles was also strongly related to FSH, providing epidemiological support for the physiological relationship between germ cell mass, inhibin, and FSH concentrations. Past ovulatory cycles also influenced maximum estradiol concentrations observed, inviting the speculation that the number of prior ovulations may also affect the steroid-producing capability of the ovary in later life. BMI may also influence hormone concentrations, especially LH, and mediate some of the known effects of BMI on fertility. Further studies involving a broader spectrum of reproductive hormones followed in consecutive cycles would advance understanding of the ovarian life cycle.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 2, 2001; accepted on September 18, 2001.