Unipath Limited*, Priory Business Park (FM), Bedford, MK44 3UP and Unilever Research Colworth (LJA), Sharnbrook, Bedford MK44 1LQ, UK
1 To whom correspondence should be addressed:; Email: fernando.miro{at}unipath.com
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Abstract |
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Key words: estrogens/follicular phase/follicle-stimulating hormone/menstrual cycle/urinary hormones
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Introduction |
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The length of the menstrual cycle is not stable throughout reproductive life. One of the first manifestations of reproductive aging is a reduction in the length of the follicular phase (Sherman and Koreman 1975; Lenton et al., 1984
; Klein et al., 1996
). Because FSH levels rise in parallel to this reduction of the follicular phase, an attractive explanation for this phenomenon is that the higher FSH accelerates follicular development (Lenton et al., 1988
; Klein et al., 1996
, 2002
; Miro et al., 2004a
). On the other hand, others have suggested that the reduction in length might instead be the result of earlier onset of FSH rise during the lutealfollicular transition (van Zonneveld et al., 2003
).
During the luteal phase of the human menstrual cycle, the corpus luteum inhibits follicular development by secreting the hormones estradiol and inhibin A, known to suppress FSH production (Ross et al., 1970; Groome et al., 1996
). In this way, the rise in FSH might well be related to a drop in the production of these suppressive hormones.
The main aim of this study is to determine whether the FSH rise during the lutealfollicular phase transition occurs on a particular day. Additionally we intended to determine the possible relationship with levels of luteal hormones, and to investigate potential changes in the onset of FSH rise due to reproductive ageing. For this purpose, we use a large database consisting of daily variations in urinary hormonal markers in 102 women during several cycles. For identifying the day of FSH rise, we employed two different analytical approaches. First, we developed an algorithm that retrospectively detects the first sustained rise in FSH during the cycle. Second, we compared the variation in mean FSH levels during the last 14 days of the cycle.
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Materials and methods |
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Each woman collected daily first morning void urine samples (Lasley et al., 1994; Santoro et al., 2003a
) for several successive cycles. Samples were kept in universal specimen bottles containing sodium azide as preservative (0.1% volume). Volunteers were asked to keep the samples refrigerated until delivered to the laboratory (on a weekly basis). On arrival, the specimens were aliquoted and stored at 4°C until the hormonal analyses were performed.
Hormonal analyses
All samples were analysed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrone 3-glucuronide (E1G) and pregnanediol 3-glucuronide (PdG) by immunoassay, using AutoDelfia® (Perkin Elmer Life Sciences, Cambridge, UK) following in house established and validated protocols, as previously described (Miro et al., 2004a, b
).
Data preparation and analyses
The concentrations of FSH, LH and E1G were adjusted in order to compensate for urine volume fluctuations as previously described (Miro et al., 2004b). Briefly, the ln(PdG) profiles were smoothed using cubic spline, and the residuals were used to adjust the concentrations of the other three hormones. The advantage of the smoothing is that it has the effect of reducing local (23 days) fluctuations with no bias towards particular shapes over a long time interval. This was achieved by using the SAS/IML Splinec routine with a smoothing parameter of 10 (Littell et al., 1996
; Miro et al., 2004b
).
FSH results are expressed as international units/litre (IU/l) and E1G as ng/ml.
Parameters, algorithms and statistics
The onset of FSH rise (FSHr) was determined by an algorithm applied to smoothed-adjusted FSH values. The algorithm retrospectively detects the first day when a sustained increase in FSH concentration takes place, based on the change in FSH slope relative to the current FSH level. This change in slope is measured by the parameter surge size (S) for each day within the interval between LH peaks in successive cycles, excluding the last 10 days.
Right slope (RS) is the slope of the line joining the current day with the day 3 days later, and left slope (LS) is the slope of the line joining the current day with the day 2 days earlier.
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FSHr found by the algorithm is defined in relation to the first day of menses, which is regarded as day 1.
Comparison of relative variation in FSH and E1G during the luteal phase
To determine the proportional changes in FSH and E1G during the lutealfollicular phase transition, relative changes in FSH and E1G during the last 14 days of the cycle were analysed. To do this, concentrations of FSH and E1G during this interval were divided by the maximum value occurring on each cycle. Consecutive values were compared in order to determine the first significant rise (for FSH) and fall (for E1G).
FSH rise and reproductive ageing
To study the possible effect of reproductive ageing on FSHr, we analysed consistent variation of FSHr with two different parameters related to reproductive ageing: chronological age and initial levels of FSH (iFSH) in the cycle (mean value on days 15 of the cycle).
In addition we investigated the relationship between increasing levels of iFSH and follicular phase length, and compared with the relationship between increasing iFSH and FSHr. To do this, we distributed the cycles into three categories according to iFSH as: 5;>5 to
10 and >10 U/l. In a previous study, we found a progressive reduction in follicular phase length with increasing FSH based on these three categories, thus, we regard them an appropriate reference for this study (Miro et al., 2004a
). Follicular phase length was considered as the interval from day of FSH rise to the day of the LH peak.
Statistical comparisons
To determine the effect of chronological and reproductive age on FSHr, regression analysis with ANOVA was used. All other comparisons were based on one way ANOVA.
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Results |
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Cycles were divided into three categories according to iFSH (5;>5 to
10 and >10 U/l). Total follicular phase length (from FSHr to day of LH peak) was compared between the three categories. A consistent reduction in follicular phase length with increasing iFSH category was found: 18.1, 17.2 and 16.6 days respectively, P< 0.0001. In contrast, there was no significant difference in FSHr between the same three iFSH categories: 4, 4 and 3.99 days, P=0.9947 Figure 4).
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Discussion |
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An early study comparing variation in circulating levels of FSH during the lutealfollicular transition in conception and non-conception cycles, shows a distinct change to positive slope in the levels of circulating FSH about 1112 days after the LH peak in non-conception cycles (Lenton et al., 1982). A similar, more recent, study found that in non-conception cycles, FSH profiles, both in serum and urine, show consistently a shift to positive slope around day 11 after the LH peak, or 4 days before menses (Qiu et al., 1997
). Others reported a rise in FSH around 3 days before menses in non-conception cycles (Welt et al.,1997
). Finally, a more recent study based on 35 young healthy women locates the FSH rise about 1112 days after the shift in body basal temperature (van Zonneveld et al., 2003
).
Clear changes in the activity of the gonadotrophin-releasing hormone pulse generator occur during the lutealfollicular transition, with an abrupt increase on day 11 after the LH peak (Hall et al., 1992; Welt et al., 2003
), and the corpus luteum produces at least two hormones (inhibin A and estradiol) known to suppress FSH secretion. The relative effect of these FSH inhibitory hormones has been investigated thoroughly.
In non-human primates injection of inhibin A during the early follicular phase suppresses FSH (Molskness et al., 1996), however, the effects were weak, and the concentrations used considerably high. Other studies using more physiological concentrations of inhibin A failed to exert any significant suppressive effect on FSH during the lutealfollicular transition (Fraser and Tsonis, 1994
). Studies in humans suggest an absent or trivial role for inhibin A on FSH secretion during the luteal phase of the cycle (Le Nestour et al., 1993
; Lahlou et al., 1999
; Welt et al., 2003
) and that estradiol is the only significant regulator of FSH during the lutealfollicular transition (Hall et al., 1992
; Le Nestour et al., 1993
; Lahlou et al., 1999
; Welt et al., 2003
). Interestingly, we found that the first significant rise in FSH and drop in E1G occur on the same day, suggesting a close association between both episodes.
A reduction in the length of the follicular phase is one of the early known effects of reproductive ageing. Since this reduction occurs in parallel to an elevation in the initial levels of FSH, it has been hypothesized that an acceleration in follicular development occurs due to the higher FSH levels (Lenton et al., 1988; Klein et al., 2002
; Miro et al., 2004a
). Further support to the connection between reduction in follicular phase length, accelerated follicular development and higher FSH has been provided by studies showing comparatively higher mitotic index in granulosa cells from hyper-stimulated women, as well as those over 40 years of age (Gougeon and Testart, 1990
; Gougeon, 1998
).
An alternative interpretation to the accelerated follicular development hypothesis has been proposed, whereby the reduction in length is the result of earlier onset of FSH rise (van Zonneveld et al., 2003). In support of this view is a study reporting that follicular growth starts earlier in older women (Santoro et al., 2003b
). We found no effect of chronological age, or initial FSH levels on the onset of FSH rise, moreover, there was an inverse relationship between initial levels of FSH and total follicular phase length (from day of FSH rise to LH peak). An explanation for these discrepancies might be that the above study compared statistical changes in the magnitude of daily FSH levels, and thus, the shift is found first in the group with the higher FSH (i.e. the older one). Our approach, though, is based on changes in the slope, and therefore, not subjected to this effect.
Since the age-related acceleration in follicular growth appears to affect events involved in the early follicular phase (Klein et al., 2002; Miro et al., 2004a
), including follicle recruitment and selection, these results are clinically relevant. For instance, further research is needed to determine whether FSH levels prior to menses provide a better indicator of ovarian reserve than the conventional day 3 post-menses approach, or whether hyperstimulation treatment for infertility might benefit from starting 4 days before the onset of menses. Finally, it seems worth investigating the contraceptive potential of manipulating this stage of the cycle.
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Notes |
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References |
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Submitted on July 28, 2004; accepted on September 10, 2004.