Human Reproduction, Vol. 17, No. 2, 295-298,
February 2002
© 2002 European Society of Human Reproduction and Embryology
Sequential hormonal supplementation with vaginal estradiol and progesterone gel corrects the effect of clomiphene on the endometrium in oligo-ovulatory women
1 Center for Reproduction at Gramercy, MacGregor Medical Association, Houston, Texas, USA and 2 Department of Obstetrics and Gynecology, Nyon, Switzerland
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Abstract |
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BACKGROUND: We investigated the possibility of correcting the endometrial alterations induced with clomiphene citrate (CC) by vaginal hormonal supplementation (HS) with estradiol (E2) and progesterone gel. METHODS: Oligo-ovulatory women were prospectively randomized into four groups receiving either 50 mg (groups 1 and 2) or 100 mg (groups 3 and 4) of CC from cycle day 3–8. Groups 2 and 4 also received vaginal E2 cream 0.1 mg twice daily from day 8 until the LH surge and vaginal progesterone gel, starting 3 days after ovulation. All participants had an endometrial biopsy performed 10 ± 1 days after ovulation. RESULTS: All biopsies in the HS groups (2 and 4) showed complete predecidual changes, and were `in-phase' with findings normally made 10 days post-ovulation (± 2 days of clinical dating). However, without HS (groups 1 and 3), only 4/6 and 3/6 biopsies showed predecidual changes in women receiving 50 and 100 mg of CC. CONCLUSION: The addition of vaginal E2 and progesterone to CC ovulation induction regimens normalizes the alterations in endometrial morphology. Hormonal treatment combining vaginal E2 and progesterone may improve endometrial receptivity in CC cycles and ultimately yield higher pregnancy rates.
Key words: clomiphene citrate/endometrial morphology/oligo-ovulation/vaginal estrogen/vaginal progesterone
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Introduction |
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Clomiphene citrate (CC), a synthetic, non-steroidal estrogen receptor agonist and antagonist has been used to induce ovulation in patients suffering from chronic oligo-anovulation (Adashi, 1984
). CC therapy induces ovulation by increasing pituitary gonadotrophin output via a blockage of the normal negative feedback exerted by estrogens on hypothalamic GnRH release (Kerin et al., 1985). The resulting increase in circulating FSH and LH levels triggered by CC administration initiates follicular maturation and ultimately leads to ovulation. Treatment with CC at a dosage of 50 or 100 mg/day for 5 days succeeds in inducing ovulation in 50–75% of women suffering from chronic oligo-anovulation, but the number of pregnancies achieved is much lower than would be expected based on ovulation rates (Gysler et al., 1982).
Adverse effects of CC on the endometrium are likely to be the primary cause of the suboptimal pregnancy rates seen when ovulation is induced with CC. It has long been recognized that women who receive CC for ovulation induction demonstrate a delay in endometrial stromal development, resulting in out of phase endometrial biopsies performed in the late luteal phase. Specifically, 10–12 days after ovulation, the predecidual changes induced by progesterone that characterize this later part of the luteal phase are lacking in a large fraction of women who used CC (Wentz, 1980; Cook et al., 1984; Fritz et al., 1987; Massai et al., 1993). Moreover, studies of the endometrium after exposure to CC have shown a high incidence of histological features consistent with hypo-estrogenic effects (Bonda, 1992), suggesting that the lack of progestational effects seen in the late luteal phase may stem from anti-estrogenic effects resulting in insufficient development of estrogen receptors (ER) and progesterone receptors (PR). Supporting this latter hypothesis were findings that supplementing the hormonal environment during the late luteal phase with exogenous progesterone did not normalize late luteal endometrial biopsies (de Ziegler and Bouchard, 1993).
The purpose of this study was to examine the effects on endometrial morphology of a timed sequence of vaginal hormone supplementation (HS) with estradiol (E2) and progesterone gel following CC therapy and to determine if this regimen can correct the endometrial anomalies seen in CC cycles. We postulated that to restore normal endometrial morphology in CC cycles, it was first necessary to neutralize the anti-estrogenic effects of CC on the endometrium as soon as follicular maturation was initiated, i.e. on cycle day 8.
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Materials and methods |
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Study patients
Patients presenting to the Center for Reproduction at Gramercy (Houston, TX, USA) for ovulation induction with CC and intrauterine insemination (IUI) were offered the option of participating in this study. Thirty-seven women (aged 21–38 years) with oligo-amenorrhoea were enrolled as candidates in the study. To be enrolled, patients needed to have to have oligo-amenorrhoea (menstrual cycles >40 days apart), body mass index (BMI) <36 kg/m2, a fasting glucose (mg%)/insulin (µIU/ml) ratio of
4.5, documented patent Fallopian tubes, a normal uterus, and not have previously failed to ovulate in response to CC. Candidates were then screened on cycle day 2–4 with an endocrine profile [HCG, LH, FSH, prolactin, dehydroepiandrosterone sulphate (DHEA-S), testosterone and thyroid stimulating hormone (TSH)]. None had received hormonal treatment (except progesterone to induce menses) within 6 weeks of screening. The protocol followed ethical guidelines established by the Declaration of Helsinki, revised 1983, and was approved by the Western Institutional Review Board. Each patient gave written informed consent.
Treatment design
All patients who met screening criteria underwent a transvaginal sonogram on day 2 or 3 of the menstrual cycle to rule out ovarian cysts, defined as any sonoluscent structure measuring >15 mm in mean diameter. Patients with a satisfactory ultrasound and negative serum pregnancy test were assigned, using a computer-generated random numbers table, to one of four treatment groups (Table I). Groups 1 and 2 received 50 mg oral CC (Serophene®; Serono Laboratories, Randolph, MA, USA) and groups 3 and 4 received 100 mg oral CC for 5 days starting 3 days after spontaneous or induced menses. Groups 2 and 4 also received HS with vaginal E2 0.1 mg twice daily (Estrace Cream®; Bristol Myers Squibb Company, Princeton, NJ, USA) and 90 mg/day of progesterone (1.125 g of vaginal progesterone gel, Crinone® 8%; Serono Laboratories).
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Starting on cycle day 8, groups 2 and 4 began vaginal E2 therapy. All patients started to perform urinary ovulation detection testing starting on cycle day 10 using a commercially available urine LH detection kit (Assure®; Conception Technologies, San Diego, CA, USA) until an LH surge was detected. An ultrasound was performed on day 12–14 to assess follicular growth and endometrial thickness in all patients. A blood sample was also obtained by venipuncture in all patients on cycle day 12–14 and the specimen analysed for E2 and progesterone concentrations. Patients using vaginal E2 discontinued estrogen therapy with detection of a LH surge. A blood sample was obtained in all patients 3 days after the detected LH surge for determination of progesterone concentrations and confirmation of ovulation. All patients with documented ovulation (progesterone level >4 ng/ml) 3 days after the LH surge pursued the study. Women in groups 2 and 4 started Crinone® therapy 3 days after the LH surge. All women who failed to ovulate 22 days after initiating CC therapy (or in whom ovulation was not confirmed after LH surge detection) discontinued the study. Ten (±1) days after the LH surge, all participating patients returned to the clinic for an endometrial biopsy. Pathologists who were blinded to study treatment evaluated the endometrium and dated their findings according to the normal sequence of changes previously described in the luteal phase (Noyes et al., 1950
).
Hormonal–endometrial analyses
To analyse serum concentrations of E2 and progesterone, a competitive chemiluminescent immunoassay was used (ACS-estradiol-6 and progesterone; Chiron Diagnostics, Medfield, MA, USA). The detection level for E2 was 10 pg/ml with inter-assay precision of 4.2%; the detection level for progesterone was 0.1 ng/ml, with inter-assay precision of 4.7%. Endometrial biopsies were interpreted and dated according to Noyes criteria (Noyes et al., 1950). Ten (± 1) days after ovulation predecidual changes of the endometrial stroma were expected. A delay >2 days in endometrial transformation was considered as abnormal and qualified as out of phase by reference to prior studies on luteal phase defect (Jones et al., 1970; Kennan et al., 1989).
Statistical analyses
Fishers's exact test was used to analyse differences in the endometrial biopsy data between groups. For comparisons of differences in ages, BMI, hormone measurements, day of LH peak, follicular recruitment and endometrial thickness between the four CC treatment groups, data were analysed using one-way analysis of variance (ANOVA) followed by Duncan's multiple comparison tests in those cases where overall treatment differences were significant (SuperANOVA; Abacus Concepts, Berkeley, CA, USA). A P value of < 0.05 was regarded as statistically significant.
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Results |
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Of 37 women initially enrolled in the study, 13 (35%) withdrew before study inclusion due to failure to ovulate; 24 completed the study cycle (six in each treatment arm; Table I
). As per inclusion criteria, all study patients (100%) ovulated in response to CC treatment.
The age and BMI were similar for all CC treatment groups with mean age (± SEM) of 30.8 ± 0.6 years (range 26–38) and BMI of 28.3 ± 1.2 kg/m2 (range 19–36) (F test, not significant). As shown in Table II, baseline endocrine parameters (LH, FSH, prolactin, DHEA-S, testosterone and TSH) were not different among the four groups.
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As shown in Table III
, end follicular phase E2 levels were higher in patients receiving 100 mg CC plus HS (group 4) as compared with 50 mg CC alone (group 1). Progesterone levels were similar for the four groups (Table III).
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There was a slight difference in the day of occurrence of the LH surge between the groups with and without HS. The mean day of LH peak was 13.8 in women receiving HS compared with 14.8 in patients without HS (Table IV
). There was no difference in the number of follicles >10 mm on cycle day 12–14 between the groups, but there was a trend for more follicles to be recruited with HS (Table IV). No differences in endometrial thickness were observed at mid-cycle among any of the treatment groups.
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As seen in Table V
, all biopsies in the HS groups (2 and 4) showed complete predecidualization of the endometrial stroma and were therefore read as 100% in-phase (6/6 and 6/6 respectively). In contrast, in women not receiving HS (groups 1 and 3), only 4/6 and 3/6 women respectively had an in-phase biopsy. The difference in number of in-phase biopsies between women who received HS and those who did not reached statistical significance (P < 0.01, Fisher's exact test).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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While CC is recognized as the primary therapy for infertility linked to anovulation (Gysler et al., 1982
), a striking discrepancy has long been recognized between ovulation and conception rates in CC-treated women. The anti-estrogenic effects of CC on estrogen-dependent tissues such as the endometrium (Wentz, 1980; Cook et al., 1984; Fritz et al., 1987; Yagel et al., 1992; Massai et al., 1993; de Ziegler and Bouchard, 1993) is the primary explanation for this discrepancy. The present study confirmed the prior observation that biopsies from CC-treated women have a high percentage of delayed endometrial maturation.
Given its properties, it is reasonable to postulate that endometrial maturation is suboptimal secondary to the anti-estrogen effect of CC. Following the same principles, prior attempts have been made to co-administer an estrogen preparation in order to restore proper endometrial priming prior to exposure to endogenous and exogenous progesterone during the luteal phase (Yagel et al., 1992). Gerli observed that supplementing CC with exogenous estrogen increased endometrial thickness and decreased the risk of spontaneous abortion in oligomenorrhoeic women who used CC for ovarian stimulation (Gerli et al., 2000). Adding support to this hypothesis, Hurd et al. demonstrated in a different trial that when CC was used for ovarian stimulation for IVF–embryo transfer, luteal support with both E2 and progesterone significantly increased conception rates compared with no luteal support (Hurd et al., 1996). Recognizing the need for proper E2 priming, we designed a study using hormonal supplementation with E2 and progesterone in order to correct the endometrial disorders that progesterone alone fails to achieve. Both hormones were administered vaginally in order to optimize endometrial concentrations, since evidence supports that vaginally administered E2 and progesterone result in higher endometrial concentrations of these hormones than when administered otherwise (Miles et al., 1994; Bulletti et al., 1997; Tourgeman et al., 1999). Our results clearly showed that exogenous vaginal E2 and progesterone improved endometrial morphology of CC-treated patients.
Discrepancies exist, however, between various published reports on endometrial morphology in CC cycles, as some investigators have failed to observe any morphological abnormalities in the mid-luteal (Jones et al., 1970) or even in the late luteal phase (Lamb et al., 1972; Hecht et al., 1990). A likely explanation for this finding is that the markedly varying E2 levels which occur in ovarian stimulation cycles using CC may on occasion overcome the inherent anti-estrogenic properties of clomiphene on the endometrium. It is conceivable, however, that in some cycles where CC is used, suboptimal levels of E2 may not suffice in negating the effects of CC on the endometrium.
This study demonstrated that hormonal supplementation with vaginal E2 and progesterone resulted in in-phase endometrial development in 100% of women following CC-induced ovulation. Hence, our findings suggest that adding vaginal estrogen followed by progesterone supplementation to CC in ovulation induction regimens for oligo-ovulatory women is a valuable option for maximizing endometrial morphology without greatly increasing costs or monitoring for the patient.
Large scale studies using simplified derivatives of our sequential vaginal E2 and progesterone supplementation regimen for CC cycles should assess the practical value (in terms of pregnancy rates) of normalizing endometrial morphology with exogenous hormones.
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Acknowledgements |
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This study was supported by an unrestricted educational grant from Columbia Laboratories. It was presented in part at the 16th Annual Meeting of the European Society of Human Reproduction and Endocrinology, Bologna, Italy, June 25–28, 2000.
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Notes |
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3 To whom correspondence should be addressed at: Woman's Center for Fertility and Advanced Reproductive Medicine,9000 Airline Highway-Suite 670, Baton Rouge, LA 70810, USA. E-mail: res-keh{at}womans.com
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accepted on October 11, 2001.
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