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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

January 2000
Volume IV, Number 1
ISSN: 1537-6583
Pages: 064-073

Estradiol/Progesterone Substitution In The Luteal Phase Improves Pregnancy Rates In Stimulated Cycles-But Only In Younger Women

Norbert Gleicher, M.D., Theresa Brown, Ph.D.**, Alan Dudkiewicz, Ph.D.*, Vishvanath Karande, M.D., Ramaa Rao, M.D., Martin Balin, M.D., Ph.D., David Campbell, B.S.**, Donna Pratt, M.D.

From the Division of Reproductive Endocrinology and Infertility, the Division of Laboratories* and the Section of Information Systems and Medical Statistics **, the Center for Human Reproduction and the Foundation for Reproductive Medicine, Chicago, Illinois, USA.

*SART, Society of Assisted Reproductive Technologies is a special interest group of the American Fertility Society, Birmingham, Alabama

Correspondence: N. Gleicher, M.D., The Center for Human Reproduction, 750 N. Orleans Street, Chicago, IL 60610, Tel: 312-649-9686, Fax: 312-649-9645

Capsule

An investigation whether hormone substitution with progesterone and estradiol would be more successful in improving pregnancy rates.

Abstract

Innumerable studies have attempted to demonstrate that hormonal support of the luteal phase during ovulation induction cycles improves pregnancy rates. None has, however, so far been able to confirm the validity of such treatment conclusively, possibly because most studies only utilized progesterone substitution. Since luteal phase endometrium also requires estradiol support, this study attempted to investigate whether hormone substitution with progesterone and estradiol would be more successful in improving pregnancy rates. Amongst approximately 7500 consecutive ovulation induction cycles were identified prospectively which were characterized by a precipitous drop of luteal phase serum estradiol levels by more than 50% over a 48 hour period within 10 days from hCG administration. Those cycles were prospectively randomized to oral micronized estradiol substitution (Group I) or not (Group II), while both groups received routine progesterone substitution of the luteal phase. Cycles were then evaluated in regards to the occurrence of chemical, ectopic and clinical pregnancies. One hundred sixty-three Group I cycles resulted in 34 pregnancies (20.9%), which compared favorably to 21 pregnancies in 167 Group II patients (12.6%) (x²[1] = 4.06; p < 0.04). The advantage for Group I cycles (29/95 pregnancies, 31%) vs. Group II cycles (16/105, 15%) became even more pronounced when only women up to age 35 years were evaluated. Estradiol substitution maintained a significant advantage until age 38 (x² [1] = 6.87; p < 0.009). While gravidity did not affect pregnancy success, estradiol substitution in Group I benefited nulliparous (23% pregnancy rate) over multiparous women (12% pregnancy rate) (x² [2] = 6.86; p< 0.03). This association was, however, age-dependent. A combined estradiol and progesterone substitution of the luteal phase of ovulation induction cycles increases the overall pregnancy rate. Since estradiol substitution was initiated in this study only once a precipitous drop in serum estradiol levels had already taken place, an even larger improvement in pregnancy rates could conceivably be possible if earlier estradiol substitution of the luteal phase is initiated. A further expansion of investigations of similar protocols for routine ovulation induction and in vitro fertilization (IVF) cycles may be indicated, especially in women below age 38 years and in nulliparous females.

Introduction

The endometrium is composed of glandular/ epithelial and stromal components. In the luteal phase of the menstrual cycle, these two endometrial components are mostly separately controlled by progesterone and estradiol, respectively (Mascar 1988).

Recent data have suggested that, especially in stimulated cycles, glandular and stromal components of the luteal phase endometrium are frequently out of phase (Blasco 1994). Empirically, clinicians have therefore for quite some time assumed that added hormonal support in the luteal phase can beneficially affect pregnancy rates. Such an assumption has, however, been primarily made in regards to progesterone, the hormone considered dominant in the luteal phase. This led to the wide utilization of luteal phase support with progesterone in in vitro fertilization (IVF) cycles and the hormone’s frequent use in stimulation cycles with clomiphene citrate and/or gonadotropins, even though its effectiveness in improving pregnancy rates has remained controversial (Soliman et al., 1994).

In contrast, the substitution of the luteal phase with estradiol has attracted only limited attention and has not become common practice amongst fertility centers (Smitz et al., 1993; Lewin et al., 1994). Infertility treatments, utilizing artificial cycles, have demonstrated conclusively that estradiol (and progesterone) support in the luteal phase will result in the development of normal endometrial architecture (Navot et al., 1986; Li et al., 1994). This observation alone suggests that some patients, and possibly primarily those with out of phase endometrium due to inappropriate estradiol stimulation of the glandular component, can be expected to benefit from luteal phase substitution with estradiol. In order to investigate this hypothesis, a prospective study was designed to investigate the premise that luteal phase estradiol substitution in addition to progesterone substitutions in women with apparent luteal phase estradiol deficiency may improve pregnancy rates.

This study reports on the prospective investigation of women under various ovarian stimulation protocols, who were randomly substituted in their luteal phase may, in fact, improve pregnancy rates in ovarian stimulation cycles if combined with standard progesterone support.

Material and Methods

In prospective fashion over a one year period, 330 consecutive ovarian stimulation cycles were identified (amongst approximately 7500 cycles), which demonstrated, between days 1 and 10 post-human menopausal gonadotropin (hCG) administration (day 0), a precipitous decrease in serum estradiol levels of at least 50% over a 48 hour period. Serum estradiol levels were routinely obtained every other day from day +4 on.

Women who qualified for this study had a mean age of 33.96 ! 4.27 years (range 23.8 to 43.9). Their gravity ranged from 0 to 8, with 159 (48%) being nulligravidas. Parity ranged from 0 to 3 with 257 (78%) being nulliparous. They were randomly assigned to oral micronized estradiol substitution (Group I) or no substitution (Group II), after informed consent had been obtained. Group I contained 163 and Group II 167 cycles, after 7 cycles had to be disqualified during statistical evaluation because of protocol violations (Group I, 5 and Group II, 2).

Amongst 330 ovarian stimulation cycles, 188 (55%) were cycles stimulated with gonadotropins, which were in over 95% accompanied by intrauterine inseminations. One hundred thirty seven (42%) were IVF cycles, universally representing ovarian stimulation by gonadotropins after suppression of ovarian function with a gonadotropin releasing agonist, and 10 (3%) involved stimulation with clomiphene citrate.

Groups I and II were identical in age (t[328] = 0.09), gravity (x²[7] = 6.42) and parity (x²[3] = 0.73) and demonstrated identical distribution amongst the three cycle treatment options.

Patients in Groups I and II were routinely substituted with progesterone in oil during the luteal phase according to the protocol of Meldrum et al. (1987), if an IVF cycle. Patients in clomiphene citrate and gonadotropin cycles were substituted with 25 to 100 mg of progesterone in oil only if progesterone levels in the luteal phase on days +4 or +6 were below 30 pgms/ml.

Patients in Group * were substituted with either 4 or 6 mg of oral micronized estradiol daily (Estrace, Mead Johnson Laboratories, Princeton, N.J.) if serum estradiol levels, prior to the 50% decline, were below or above 2000 pgms/ml, respectively. Whenever estradiol substitution was started and the patient had not been on appropriate progesterone substitution, the patient concomitantly was placed on 50 mg progesterone in oil.

Serum hCG levels were obtained from day +8 on in two day intervals. Pregnancies were defined as "chemical" if at least two consecutive hCG rises were noted; as "clinical," if a pregnancy was confirmed by ultrasound and as "ectopic" if such a diagnosis was confirmed either by laparoscopy or hysterosalpingography (Gleicher et al. 1992).

Statistical analyses utilized included Student’s t-test, chi-square and discriminant analysis.

Results

Amongst 163 estradiol treated cycles (Group I), a total of 34 pregnancies occurred, for a total pregnancy rate of 20.9% per cycle. Amongst 167 control cycles (Group II), only 20 (12/6%) conceptions occurred. This difference was statistically significant (x²[1] = 4.06; p < 0.04). A breakdown in pregnancies is shown in Table 1.

Patients with "advanced maternal age", defined as 35 years or above, benefited less from estradiol substitution than women below age 35. Amongst 95 cycles, treated with estradiol in the younger subgroup, 29 pregnancies occurred, for a conception rate of 31% per cycle. In contrast, amongst 105 women below age 35, who were not treated with estradiol, only 16 (15%) conceived. This difference was statistically highly significant (x²[1] = 6.87; p < 0.009). Women above age 35 continued to benefit statistically from estradiol treatment until age 38. If women were classified by age according to SART* criteria (age 40 as cutoff), estradiol treatment was ineffective, both above and below age 40 (Table 2).

The association between an estradiol benefit and young age even holds if neither chemical nor ectopic pregnancies are considered as positive pregnancy outcomes. Specifically, amongst 95 women below age 35, 10 (11%) achieved a clinical pregnancy if treated with valerate, while amongst 106 cycles not treated with estradiol in this age group, only 6 (6%) achieved a clinical gestation (x²[2] = 6.90; p < 0.03).

Gravidity did not affect the success of estradiol therapy. However, parity was statistically significantly correlated to estradiol treatment success. Nulliparous patients had higher pregnancy rates if treated with estradiol (n = 28; 23%) than those who did not receive such treatment (n = 16; 12%) (x²[2] = 6.86; p < 0.03).

Since nulliparous women are usually younger than mulitparous females, a discriminate analysis was performed, which confirmed parity as a dependent factor of age. Uniparity versus greater than uniparity, did not demonstrate an estradiol benefit, which thus was exclusively associated with nulliparity.

Neither gonadotropin cycles, IVF cycles nor clomiphene citrate cycles alone demonstrated a significant benefit of estradiol treatment. This was, however, with great likelihood due to small sample sizes, which did not allow adequate statistical power. In fact, in gonadotropin stimulation as well as in IVF cycles, involving women below age 35, estradiol treatment almost achieved statistical significance (x²[1] = 3.80; p > 0.10 and x²[1] = 3.06; p < 0.10, respectively). Only 10 clomiphene citrate cycles had qualified for this study, too small a number to allow statistical power.

Discussion

This is, to our knowledge, the first study in the literature which conclusively demonstrates that the combined hormone substitution with estradiol and progesterone in the luteal phase of ovulation induction cycles improves pregnancy rates. Hormone substitution with progesterone alone represents routine care in a majority of IVF cycles worldwide and is also widely utilized in ovulation induction cycles. Its efficacy as a single agent has, however, remained controversial (Soliman et al 1994).

Considering the known physiology of luteal phase support by the female hormones estradiol and progesterone, ¹ this should not surprise. Progesterone, while considered the dominant hormone of the luteal phase, affects largely the stromal components of the endometrium. In contrast, estradiol primarily supports the glandular endometrial structures (Mascar 1988). A benefit from progesterone substitution can be expected if the stromal components are insufficiently supported, and therefore out of phase. The selection support of luteal phase endometrium by progesterone can on occasion, however, further enhance the endometrial discrepancies between glands and stroma, frequently observed in ovulation induction cycles (Blasco 1994, Benadiva and Metzger 1994).

In animal models at least some data suggest that progesterone administration alone maintains pregnancy in the luteal phase only rarely. Bosu and Johansson (1975) maintained pregnancy in only one out of six ovariectomized monkeys after progesterone substitution. Further evidence for the need of estradiol comes from several experiments which utilized antiestrogenic compounds in the luteal phase. Two such studies suggest that antiestrogens will significantly inhabit the occurrence of pregnancy if administered in the luteal phase (Ravindranath and Moudgal 1987, Morris et al. 1967). Younis, et al. (1994), recently suggested that similar data in humans was still lacking, however.

A reasonable argument can therefore be made that any hormonal support in the luteal phase of stimulated cycles has to include both progesterone and estradiol substitution. This was the basis for the here utilized protocol. Progesterone substitution was given in routine fashion, either universally to all IVF patients or selectively during ovulation induction cycles. The difference in this study to earlier ones therefore lies in the concomitant administration of estradiol substitution with natural progesterone on a per need basis.

Need for estradiol substitution was defined as a precipitous drop in luteal phase serum estradiol levels, probably as much as result of deficient corpus luteum function as is low luteal progesterone. Substitution was then given like in artificial cycles of embryo recipients (Navot et al. 1986, Li et al. 1994). In such recipient IVF cycles, it has been well established that a combination of estradiol and progesterone is needed to maintain a proper endometrial milieu for implantation and early pregnancy maintenance. Those cycles have also established the safety of such a treatment approach (Navot et al. 1986, Navot et al. 1991).

Safety is an important issue when estradiol substitution is to be considered in the luteal phase. The disaster following uncontrolled administration of diethylstilbestrol (DES) in the 50’s, is still on everybody’s mind (Herbst et al. 1971). High levels of estradiol in the luteal phase have also been reported to be embryotoxic and to prevent implantation (Clark et al. 1985). It is therefore important to understand that the here utilized dosages are not in an embryotoxic range and are identical to those used in artificial cycles (Smitz et al. 1993, Navot et al. 1986, Li et al. 1994). Moreover, the here utilized micronized estradiol is a natural product.

Utilizing this protocol, we were able to demonstrate a significant improvement in pregnancy rates following the administration of estradiol. Since estradiol substitution was only initiated once a patient already had demonstrated a precipitous drop in estradiol levels, exceeding 50% over 48 hours, potentially even better results can be anticipated from estradiol substitution of treatment were to be started earlier in the cycle. Smit, et al. (1993), were unable to demonstrate such an effect. Their study was, however, not age stratified, which, as is shown in this study, can obscure valid statistical findings.

The beneficial effects of estradiol substitution were restricted to young women below 38 years of age. This is a very interesting observation since it correlates well with the observation of Meldrum et al. that older women benefit from higher levels of progesterone support in the luteal phase (Meldrum et al. 1992). From Meldrum’s report and our here reported data, a luteal support model can be drawn, which is graphically demonstrated in Figure 1.

As women age, a relative shift from principal need for estradiol support to a need for progesterone support takes place. It is unclear whether this reflects a change in endometrial hormone receptor activity or a changing ovarian performance in regards to hermone production. IVF data from donor oocyte cycles suggest that the latter explanation may apply, since older women demonstrate excellent uterine receptivity if oocytes from younger women are utilized (Sauer et al. 1990). Meldrum, et al. (1992), suggested, however, that older recipients do in fact suffer from decreased endometrial receptivity, which can be improved by high dose progesterone support in the luteal phase. As Benadiva and Metzger suggested (1994), in quoting the work of Good and Moyer (1968), the relative relationship of levels of estradiol and progesterone, rather than their absolute levels, may be the factors that determine the developmental potential of the endometrium. As women progress in age, the needed ratio between the tow hormones may change.

The presented data possibly also suggest that hormonal support insufficiency in the luteal phase may be a cause of primary infertility. At least in stimulated cycles young, nulliparous women appear to benefit most from hormonal support with estradiol. This observation should not automatically be extrapolated to natural cycles. It is, however, intriguing to speculate that luteal estradiol deficiency may represent a so far unknown cause of primary infertility.

We conclude that the combined administration of estradiol and progesterone in the luteal phase of ovarian stimulation cycle improves pregnancy rates over selective progesterone substitution. This effect is restricted to primiparous and/or young women below age 38. In such women the selective substitution of the luteal phase with estradiol and progesterone may therefore be indicated.

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Table 1

Number of pregnancies in estradiol treated patients (Group I) and controls (Group II) by pregnancy type and cycle stimulation protocol.

Group I demonstrated a significantly higher total pregnancy rate (n=34; 20.9%) than Group II patients (n=21; 12.6%) (x^2[1] = 4.06; p<0.04).

Individual treatment options (*, Clomiphene citrate; ^#, Gonadotropic stimulation; ⁺, In vitro fertilization) demonstrated, however, identical outcome distribution in study and control groups.

Table 2

Newer (percentage) of pregnancies in estradiol treated patients (Group I) and controls (Group II) by maternal age.

* The conception rate in Group I (29/95; 31%) was significantly higher than in Group II (16/105; 15%) (x^2[1]=6.87; p<0.009) for women of age 35 years or lower. Benefit of estradiol treatment remained statistically valid up to and inclusive of age 38. The benefit of estradiol valerate below age 35 received statistically significant even if only clinical pregnancies were considered. For details see text.

Figure 1

Changing needs in estradiol and progesterone substitution with advancing female age.

According to this study, estradiol substitution need is statistically associated with young age (38 and below) and with parity. Meldrum et al.,¹⁸ reported a need for higher progesterone substitution in women of advanced age. A change in estradiol/progesterone ration seems therefore to become necessary with increasing maternal age.