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Original Studies |
Medical Research Council Human Reproductive Sciences Unit (A.P., A.S.M.) and Department of Reproductive and Developmental Sciences (H.O.D.C.), University of Edinburgh Centre for Reproductive Biology, Edinburgh EH3 9ET, United Kingdom; and Department of Reproductive Medicine, Westmead Hospital (P.J.I.), University of Sydney, Westmead, New South Wales, Australia 2145
Address correspondence and requests for reprints to: Prof. Alan S. McNeilly, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH3 9ET, Scotland, United Kingdom. E-mail: a.mcneilly{at}ed-rbu.mrc.ac.uk
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Abstract |
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We conclude that low-dose estradiol treatment given as TES suppresses ovarian activity as measured by inhibins B and A by reducing the secretion of LH and FSH during breast-feeding for several weeks. This supports the concept that suckling-induced suppression of the GnRH system is associated with an enhancement of the negative effects of estradiol on the hypothalamic GnRH system. Furthermore, because the contraceptive efficacy of breast-feeding is complicated by the unpredictable early return of ovarian activity in some women, TES could be the basis for the development of a novel contraceptive for breast-feeding women.
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Introduction |
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Even though the estradiol concentration remains low during lactational amenorrhea, there is evidence of small follicles developing in the ovaries (12) that secrete small amounts of estradiol (1, 13). We suggested that maintenance of lactational infertility, the only physiological suppressor of fertility in women, was related to a suckling-induced enhancement of the negative feedback effects of estradiol on GnRH release during breast-feeding (13). Subsequently we have shown that short-term low-dose estradiol treatment markedly decreased plasma gonadotropin concentrations and pulsatile LH secretion in breast-feeding women 12 weeks postpartum (PP) (2). This confirmed a previous study that showed an enhanced negative feedback effect of single injection high-dose estrogen treatment in breast-feeding women (14). Furthermore, in lactating cynomolgus monkeys, ovariectomy resulted in a marked rise in gonadotropin concentrations even though ovarian activity was suppressed at the time of ovariectomy (15), although continued suckling alone may maintain suppression of gonadotropin secretion for some time (1). All this supports the concept that minimal amounts of estradiol secreted by the small follicles suppress gonadotropin secretion during lactation. If LH secretion resumes, this may stimulate limited ovarian estradiol secretion from the small follicles present, but, because of the enhanced sensitivity to negative feedback, this low level of estradiol will switch off further GnRH/LH, stopping the follicles developing further. In this way, prolonged periods of ovarian suppression can be maintained during breast-feeding (1, 13).
In previous studies on breast-feeding and fertility, ovarian activity has been monitored mainly by changes in urinary steroid output and occasionally by ultrasonography and plasma estradiol concentrations. The development of specific assays for inhibins A and B has enabled us to monitor the functional activity of small follicles in the ovaries during prolonged exogenous estradiol treatment in breast-feeding women. Inhibin B is secreted by the small growing follicles, whereas inhibin A is mainly a product of the selected follicle destined to ovulate and can, therefore, be used to monitor the return of ovarian activity (16, 17).
The aim of this study was to determine whether estradiol administered in low doses transdermally could induce long-term suppression of ovarian activity in breast-feeding women.
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Subjects and Methods |
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Of the six women who discontinued the study, four were in the Barrier group, whereas the TES and PL groups were reduced by a single drop-out each. No reason for these discontinuations were required, however, some of the women had simply stopped breast-feeding. Four women in the TES group suffered from irritation of the skin due to the patch. Two of these women continued in the study after changing the brand of the patch (Evorel, 50 µg/24 h; Janssen Pharmaceuticals-Cilag, High Wycombe, UK), whereas two women had to discontinue the study after 5 weeks because of a continuous rash. The hormone concentrations and other findings from these individuals have been included in the study until they dropped out.
The study was approved by the Lothian Research Ethics Committee, and an informed written consent was obtained from all of the women.
Hormone assays
Plasma concentrations of LH and FSH were measured by RIAs, as
described previously (3, 5), with assay sensitivities of
0.8 and 0.9 U/L, respectively, and within-assay coefficients of
variation (CV) of 4.6% and 5.0%, respectively. Plasma estradiol
concentrations were measured by RIA following diethyl ether extraction,
as described previously (18, 19). The sensitivity of the
assay was 10 ng/L, and the inter- and intra-assay CV were 15.8% and
4%, respectively. Inhibins A and B concentrations were measured using
a two-site enzyme-linked immunosorbent assay based on the use of plates
coated with specific monoclonal antibodies to inhibins ßA and ßB
subunits (16, 17). Another monoclonal antibody to inhibin
-subunit conjugated to alkaline phosphatase was used for detection.
The CV were less than 5% within plate and less than 7% between plates
for both assays. The sensitivity of the assay was 10 ng/L for inhibin B
and 2 ng/L for inhibin A.
Urinary estrone glucuronide and pregnanediol glucuronide were measured as described previously and corrected for the amount of creatinine in each sample (5). All samples were assayed in the same assay, with an intra-assay CV of less than 10%.
Ultrasonography
The ultrasonography was performed using a Siemens Sonoline SI-250 machine (Siemens plc, Bracknell, UK) with a 7.5/5-MHz vaginal probe. Several women (TES, n = 3; PL, n = 5; Barrier, n = 9) were unwilling to undergo vaginal ultrasound and had abdominal ultrasonography. For consistency of method, only the vaginal ultrasound scan data are reported. Both ovaries were scanned, and the size of the biggest follicle estimated by the average of two measurements in different planes. The endometrial thickness was measured in the standard manner, as the thickness of the double-layer of endometrium between the myometrial-endometrial junction on either side of the midline.
Statistical analysis
Statistical analyses of the data were carried out after logarithmic transformation. Results within a treatment group at different time points were compared using ANOVA with repeated measures. Comparisons between groups were performed by using two-way ANOVA. Data are presented as mean ± SEM. A P value less than 0.05 was selected to indicate significant difference.
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Results |
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The women breast-fed their babies 8.5 ± 0.3 times per day,
on average, in the beginning of the study 6 weeks PP (Fig. 1
). The frequency steadily decreased so
that at 12 weeks PP the babies were fed 6.8 ± 0.3 times per day
and at 18 weeks PP 5.8 ± 0.5 times per day. There were no
significant differences between the groups, except for the transient
decrease in feeding frequency in the PL group during weeks 15 and 16 PP
(P < 0.05).
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The data from the Barrier control group is shown in Fig. 2. There was no significant change in the
plasma concentrations of estradiol, FSH, or inhibin B from weeks 6–18
PP, the duration of the treatment in the TES and PL groups. In
contrast, plasma concentrations of LH increased significantly
(P < 0.05) from week 6 to week 10 PP and then did not
change significantly thereafter. The mean ±
SEM for the Barrier group is shown as the
shaded area in each panel in Fig. 3.
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The changes in plasma concentrations of estradiol, LH, inhibin B,
and FSH in the TES and PL groups in comparison with the changes in the
Barrier group are shown in Fig. 3
.
Plasma estradiol concentrations were in the low follicular phase range
throughout the study. Surprisingly, TES did not significantly increase
the plasma estrogen levels (Fig. 3). The apparent rise after 2 weeks of
TES did not reach statistical significance. The high estrogen levels at
14 weeks PP in the TES and PL groups are a result of a single
exceptionally high level in TES and two high levels in the PL
group.
Measurement of the endometrial thickness by ultrasonography showed that
in the TES group the endometrium was significantly (P
< 0.05) thicker after 4 weeks of treatment (6.2 ± 0.9 mm
vs. 4.5 ± 0.4 in Barrier), after which the
measurements showed parallel endometrial growth (Table 1). Within the TES group the values were
significantly (P < 0.05) greater at 10 and 14 weeks PP
compared with the initial value at 6 weeks PP.
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). Plasma FSH decreased
significantly (P < 0.01) from 6.1 ± 0.8 U/L to
3.3 ± 0.6 U/L after 2 weeks of treatment and was significantly
(P < 0.01) lower in the TES group compared with the PL
group throughout the remaining period of treatment. Plasma LH
concentrations showed no initial difference between the treatment
groups. Estrogen treatment decreased plasma LH concentrations
significantly (P < 0.01) within the TES group at all
time points, and the concentrations were significantly (at least
P < 0.05) lower than both the PL and Barrier groups
after 4, 6, 8, and 10 weeks of estrogen treatment.
Plasma inhibin B concentrations were initially similar in all groups
and did not change significantly during the study in the PL group.
However, estrogen treatment rapidly and significantly
(P < 0.01) decreased plasma inhibin B from 48.9
± 7.3 to 15.5 ± 5.8 ng/L within 2 weeks of the start of
treatment in the TES group. Plasma inhibin B was significantly lower in
the TES group compared with the PL and Barrier groups at all time
points from 8 weeks onward during the treatment. Plasma concentrations
of inhibin A showed marked deviations within the groups and remained
relatively low at all times (Table 2).
The plasma concentrations were significantly lower in the TES group
compared with the PL group at 8, 12, 14, and 18 weeks PP. During TES,
inhibin A levels did not exceed 7 ng/L, whereas 21% of samples in the
PL and Barrier groups exceeded 7 ng/L (range, 3–45).
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).
However, at 14 weeks PP, the largest follicle in the TES group was
significantly (P < 0.01) smaller than that of the
Barrier group. Furthermore, a significant (P < 0.05)
increase in follicle size was detected from 6 weeks PP to 10 and 14
weeks PP in the Barrier group.
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Changes in individual subjects
The changes in breast-feeding pattern, urinary steroids, and
plasma concentrations of estradiol, inhibins A and B, and LH and FSH in
two representative individual women using either a PL patch or TES are
shown in Fig. 4. In these women the
breast-feeding pattern and rates of introduction of either formula or
solid food are very similar. However, in the PL-treated woman, urinary
estrogen fluctuates with a number of samples exceeding 10 µg/L,
whereas in the TES woman no levels exceeded this value. Over the first
3 weeks of TES treatment plasma estradiol levels were increased and
then declined to be equivalent to those in the woman on the PL patch.
TES was associated with a dramatic decline in LH, FSH, and inhibins B
and A, with a rapid increase in all these parameters at the end of TES
treatment at 18 weeks. In contrast, plasma inhibin B levels were
elevated to concentrations equivalent to those in the early follicular
phase of the menstrual cycle in the woman with the PL patch.
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Discussion |
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). In our previous study
using 100 µg/24 h patches, the estradiol concentrations were measured
daily. The concentrations were elevated during the first 2 days and
then returned near the control level (2). A similar
initial increase at the beginning of the treatment in the present study
is possible. However, if such an increase exists, it is unlikely to
last for more than 2 days and would be of a lower magnitude than we
observed previously because we are using half the dose of estradiol
used in our previous study. Furthermore, the failure to demonstrate
higher plasma levels of estradiol in the TES women compared with
controls probably reflects a reduction in endogenous estradiol
secretion from the small ovarian follicles due to the suppression of
both FSH and LH. We did not measure estradiol in the breast milk, but significant increases would seem unlikely as the elevation in plasma concentration remained small and transient, and thereafter, there was no difference in plasma concentrations of estradiol between the groups. This is an important issue because women who chose to breast-feed usually do not want to expose their babies to extra steroids. There is previous data suggesting that estrogen treatment inhibits lactation (20, 21), but the amounts of estrogen used were substantial compared with those in the present study (22), and, because the plasma estrogen levels were not significantly elevated, this concern is unsubstantiated. Estradiol has been used to treat women with severe postnatal depression (23). This treatment was effective using a 200 µg/24 h dose, but these women were not breast-feeding. Blood estradiol concentrations were measured in few of the women and found to be substantially higher than those at LH peak, but still within the physiological range. Thus, although doses of estradiol higher than 50 µg/24 h may be needed to treat some symptoms of the hypoestrogenic state of lactation, the results of the present study confirm that low doses of estradiol are sufficient to block the ovarian function and would be expected to alleviate many of the symptoms of the hypoestrogenic state associated with lactation because the dose of TES used is standard for hormone replacement therapy. However, there may be different thresholds for the effects of estradiol in breast-feeding women, and this will require further study.
The present study confirms previous reports that plasma concentrations of FSH increase early in the PP period and remain within the normal menstrual range (2, 3, 5, 18). In contrast, LH levels remain suppressed throughout lactational amenorrhea (3, 5, 18). This is, at least partly, due to the erratic pattern of GnRH secretion manifest in an erratic pattern of pulsatile LH secretion. Replacement of a normal pulsatile pattern of GnRH induces follicular development and ovulation during lactational amenorrhea (3, 4). Follicular development can also be induced by LH (24). Therefore, it seems that LH rather than FSH is the limiting factor for maintaining development of functional ovarian follicles (1). In keeping with previous reports, the plasma concentrations of both LH and FSH in the PL and Barrier groups were comparable with early follicular phase values in normal menstrual cycles. Our results show that both gonadotropins were significantly suppressed during the estrogen treatment. FSH was suppressed within 2 weeks of treatment, whereas the decrease in LH concentrations took 4 weeks. Furthermore, the suppression of LH was not significant during the last week of treatment. This is an interesting finding and confirms our previous observations with short-term estradiol treatment where FSH was suppressed to a higher degree than LH (2) that there are differences in the effect of estradiol on FSH and LH secretion during breast-feeding. In the present study, we only measured basal and not pulsatile LH secretion. Previously, we showed that estradiol suppressed pulsatile secretion (2), and because LH is already suppressed to a high degree during breast-feeding, a further dramatic suppression of LH by estrogen may not be possible to detect using the infrequent sampling regime that we adopted in this study. Nevertheless, there was a clear suppression of basal LH levels indicating a prolonged period of suppression of GnRH output from the hypothalamus. The increase in LH toward the end of the treatment period may relate to alterations in suckling pattern and related reduction in the level of increase in sensitivity to estradiol-negative feedback.
The effects on FSH secretion are interesting from two aspects. The fall
in FSH is rapid presumably associated with the rapid suppression of
GnRH output and a direct pituitary effect of estradiol on FSH
production. This suppression in FSH occurs despite very low levels of
both inhibins B and A, suggesting that estradiol-negative feedback and
GnRH stimulation are more important factors in regulating FSH output in
women than inhibin at least during breast-feeding. This accords with a
previous report in which it was shown that the early PP rise in FSH is
related more closely to the falling estradiol levels than the rapidly
decreasing immunoreactive inhibin levels (25). The
mechanisms by which a plasma level of estradiol similar to controls can
suppress GnRH/LH/FSH levels during TES treatment is unclear. Certainly
suckling alone is sufficient to maintain suppression of LH secretion in
lactating monkeys for up to 30 days in the absence of the ovaries, but
increase thereafter (1). Thus, it is probable that part,
if not all, of the increase in the sensitivity to estradiol is directly
related to the suckling-induced reduction in activity of the
hypothalamic GnRH system. We have shown previously that an increase in
opioid tone within the hypothalamo-pituitary axis does not appear to be
involved in the short term, at least (2). Monitoring of
estradiol levels PP by daily measurement of urinary estrone glucuronide
secretion indicates that estradiol output fluctuates over time, even
when ovarian activity is suppressed and ovarian estradiol output is low
(26). This observation has been confirmed in the present
study and is clearly seen in the results from individual women (Fig. 4). The fact that suppression of LH and FSH occurs only in the TES
group implies that, within the same range of plasma estradiol
concentrations, a continuous rather than fluctuating plasma
concentration of estradiol can inhibit GnRH output from the
hypothalamus. During early follicular phase of the normal menstrual
cycle, treatment with ethinyl estradiol, which leads to
high levels of plasma estradiol, resulted in an increase in LH
secretion (27). Furthermore, in the normal follicular
phase levels of estradiol that are higher than those in the present
study are associated with maintained LH secretion (28).
The mechanism remains to be determined.
In most breast-feeding women, ovarian follicles remain very small during the time frame studied, even in the absence of estrogen treatment (12). In this study, we confirmed the presence of only small follicles during lactational amenorrhea and observed that transdermal estradiol treatment causes only a slight further reduction in peak follicle diameter. We have assessed the activity of these follicles by measuring plasma inhibin B, which is an indicator of the level of activation of the small follicles present, and inhibin A to determine whether there is any progression of these small follicles toward selection as a dominant follicle. Inhibin B is normally only transiently secreted by small developing follicles in response to stimulation by FSH (17). In contrast, inhibin A is secreted mainly by the dominant selected follicle with plasma concentrations increasing to a peak just before the onset of the preovulatory LH surge, and remains elevated during the luteal phase as a result of secretion by the corpus luteum (16).
We found only low mean circulating concentrations of inhibin B, equivalent to the luteal phase of the normal menstrual cycle, during lactational amenorrhea in the PL and Barrier groups, reflecting the low level of activation of the small follicles present. However, on occasions in the PL and Barrier groups, plasma inhibin B did become substantially elevated even when follicles remained below 10 mm in diameter, indicating follicle activation. In contrast, treatment with transdermal estradiol resulted in a marked further suppression of the inhibin B concentration, thus suggesting a profound diminution in follicular activation in this group. It, thus, seems that while treatment with transdermal estradiol during lactational amenorrhea results in only a limited reduction in follicle diameter, there is a significant diminution in the level of follicular activation. However, from the present results, it is not possible to determine whether the reduced inhibin B levels are due to a decrease in numbers or growth of small follicles that cannot be detected by ultrasound, or a decrease in the inhibin biosynthetic activity of individual follicles. Because there is no follicle development beyond 10 mm, the plasma concentration of inhibin A remains low in both groups. These data also add further support to the concept that secretion of inhibin B from the ovary is closely regulated by FSH.
There was no significant difference in the length of lactational amenorrhea between the groups in the present study. Because resumption of menses occurred from 20 weeks onward in the majority of women, the suppression of ovarian activity by estrogen would seem to be short-lived once the treatment is stopped. Whether the length of lactational amenorrhea can be prolonged by estrogen remains to be determined in a future longer-lasting experiment in which treatment is prolonged beyond the normal termination of lactational amenorrhea, an unpredictable event.
In conclusion, the present study shows for the first time that ovarian activity can be suppressed for several weeks during breast-feeding by a small dose of estrogen. The duration of this hypothalamic-pituitary hypersensitivity to estrogen and the causes are not known. However, we showed previously that opioids did not seem to be involved (2). Because the main cause of lactational amenorrhea is considered to be suckling (1), the hypersensitivity to estradiol is probably lost some time during or after weaning, and will vary depending on the pattern of suckling behavior already known to influence the duration of lactational amenorrhea (1, 29). The amount of estradiol required to inhibit the gonadotropin axis during lactation seems to be less than would be required for treatment of symptoms that may be caused by the hypoestrogenic state of lactation. It would seem logical, however, that, to alleviate any such symptoms like PP depression, higher doses of estrogen may need to be used, and this would also act as a contraceptive by suppressing gonadotropin secretion.
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Acknowledgments |
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Footnotes |
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Received January 4, 2000.
Revised April 12, 2000.
Revised May 24, 2000.
Accepted July 26, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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; Barrier), a PL patch (
) or TES
(•). There were no significant differences between the groups.
) or with TES
(•), plotted in relation to the range (± SEM;
shaded area) of changes in women not receiving treatment
(Barrier, Fig. 2