Endometrial preparation: lessons from oocyte donation

Modern trend$ Edward E. Wallach, M.D. Associate Editor FERTILITY AND STERILITY@ Copyright

©

Vol. 66, No.6, December 1996

1996 American Society for Reproductive Medicine

Printed on acid-free paper in U. S. A

Endometrial preparation: lessons from oocyte donation

Johnny S. Younis, M.D.*t Alex Simon, M.D.:j: Neri Laufer, M.D.:j: Poriya Hospital, Tiberias, and Hadassah University Hospital, Mount Scopus, Jerusalem, Israel

Objective: To gain insight into the physiology of human endometrial development after artificial preparation with estrogen (E) and P, before oocyte donation. Design: Review and analysis of relevant studies published in the last decade, identified through the literature and Medline searches. Results: Oocyte donation represents a unique in vivo experimental model in the human that permits the study of endometrial development under controlled variable conditions. Early studies have shown that adequate endometrial preparation can be achieved by sequential E and P only. The successful implementation ofthe simplified approach to oocyte donation demonstrated that satisfactory endometrial receptivity is not dependent on incremental administration of E and P and similarly can be achieved by fixed dosages of these steroids. Moreover, numerous clinical oocyte donation studies have shown that both physiologic and supraphysiologic levels of E and P have resulted in good endometrial development and pregnancy rates, underlining the relative insensitivity of the endometrium to extreme hormonal conditions. In addition, it has been clarified that the endometrium is tolerant of some manipulations during the follicular phase. Contrary to morphological studies that demonstrated preservation of endometrial preparation after luteal E depletion, preliminary evidence suggests that the functional capacity of the endometrium could be affected adversely. Conclusion: In contrast to early oocyte donation studies, which indicated a correlation between morphologic integrity and functional capacity of the endometrium, some evidence presented in this review demonstrates that adequate endometrial morphology does not always imply normal endometrial receptivity. Fertil Steril® 1996;66:873-84 Key Words: Endometrial preparation, oocyte donation, endometrial receptivity

Throughout the last decade, oocyte donation has become a well-accepted modality of assisted reproduction therapy in situations that previously were defined as incurable. Since the development of this new technology, the methodology of treatment has been simplified considerably, mainly to overcome logistic problems in dealing with the donor on the one hand and the recipient on the other. Along with these treatment modifications, new insights into the

Received December 28, 1995. * Poriya Hospital. t Reprint requests: Johnny S. Younis, M.D., Reproductive Endocrinology Clinic, Department of Obstetrics and Gynecology, Poriya Hospital, Tiberias, Israel (FAX: 972-6738306). :j: Department of Obstetrics and Gynecology, Hadassah University Hospital. Vol. 66, No.6, December 1996

fundamental physiology of endometrial preparation have been gained. Moreover, it has become apparent that oocyte donation treatment, especially in patients with ovarian failure, represents a unique in vivo experimental model in the human, which has the potential to answer unresolved questions in reproductive physiology. Unlike the natural ovarian-menstrual cycle, in oocyte donation procedures, uterine development in the recipient is dissociated from ovarian gametogenesis and steroidogenesis in the donor. The endometrial effects of estrogen (E) and P are isolated from other ovarian secretory activities and, therefore, oocyte donation treatment in ovarian failure patients allows assessment of the relative roles of E and P on endometrial proliferation and differentiation. Moreover, precise manipulation of E and P allows Younis et a1. Endometrial preparation

873

ity of the endometrium to support implantation and sustain embryonic growth. SIMPLIFIED APPROACH OF ENDOMETRIAL PREPARATION

II I

1

,I

5

~ E2 (4 mglday)

Ii

II

15

21

28

E2 + P (25mglday) ~

Figure 1 Mimicking the natural cycle for endometrial preparation.

the study of endometrial growth and the effects of these steroids on the process of implantation and early pregnancy maintenance. This review will survey the pertinent literature from the last decade, relating particularly to endometrial preparation in the human. MIMICKING THE NORMAL CYCLE

The basic assumption in the initial clinical trials of oocyte donation was that, to ensure optimal endometrial development, normal implantation, and sustained embryonic growth, the artificial preparation ofthe recipient's endometrium must imitate the hormonal milieu of the natural cycle as closely as possible (1). The first oocyte donation attempts (2, 3) used exogenous E and P to prepare the endometrium, in variable dosages that simulated the natural cycle. Estrogen was administered incrementally in the artificial follicular phase with a late follicular peak, a postovulatory fall, and a second midluteal rise. Progesterone was started on day 15-the day of donation, increased on day 17, and reduced again on day 27 ofthe recipient's cycle (Fig. 1). In those cycles in which ET took place, E and P were maintained at midluteal phase levels to avoid the late luteal phase decrease in steroid levels observed in nonconceptual cycles. The successful implementation of this oocyte donation protocol in the pioneering studies of Lutjen et a1. (2) and Navot et a1. (3) in patients with ovarian failure or in women with no ovaries showed that sequential E and P therapy alone, with no other ovarian products, was sufficient for endometrial preparation. Moreover, it became evident that sequential E and P supplementation was capable of producing physiological levels of those steroids, which in turn resulted in normal endometrial maturation, as seen by light and electron microscopy (3). Most importantly, it was shown that these morphologic changes were coupled with a functional capabil874

Younis et a1. Endometrial preparation

Serhal and Craft (4) introduced a simplified regimen for endometrial preparation using a fixed dose ofE and P sequential therapy. In addition, the duration of the follicular phase was manipulated depending on the availability of donated oocytes. They maintained the recipients on a fixed E replacement regimen for 2 to 4 weeks before the expected donation, thus allowing more flexibility in the timing of the transfer (Fig. 2). This simple protocol of oocyte donation resulted in satisfactory pregnancy rates (PRs) (4-6), and subsequent histologic studies of endometrial specimens using the above protocol demonstrated adequate endometrial development (7). Comparable clinical pregnancy and implantation rates were confirmed by other investigators (8-11). Furthermore, in a retrospective analysis, there was no difference in PRs per ET between the fixed-length and the variablelength protocols (12). This simplified endometrial preparation regimen eliminated the need for meticulous embryonic-endometrial synchronization between donor and recipient on the one hand and the need for embryo cryopreservation technologies on the other (13, 14). Moreover, it has become evident that the human endometrium is not affected adversely when exposed to a fixed dose of sequential E and P, and its developmental capacity is preserved. This approach also demonstrated that the endometrium is tolerant of varying durations of the follicular phase, without adverse effects on its functional receptivity. MANIPULATING THE FOLLICULAR PHASE Modifying the Duration

The main theoretical advantage of the simplified method of embryonic-endometrial synchronization is

0.0 E.T

15 P.O. micronized Estradiol (4mg/day)

Figure 2

28 E + I.M. Progesterone (50 mglday)

The simplified approach of endometrial preparation.

Fertility and Sterility®

the ability to lengthen or shorten the follicular phase beyond its physiologic limits (1). In the natural cycle, short or prolonged follicular phases are uncommon-only 7% are <11 days and 5% are >20 days (15). In the original study by Serhal and Craft (4), the follicular phase was kept to between 14 and 28 days. To explore the limits of follicular phase length manipulations, several workers assessed the endometrium's ability to undergo adequate secretory transformation under P stimulation after various estrogen-priming durations (7, 16). Navot et al. (16) studied 12 women with ovarian failure. Six were studied throughout 13 cycles with a short follicular phase consisting of 6 days of E administration, and three other women were studied during 6 cycles with a long follicular phase consisting of21 to 35 days ofE stimulation. These two groups were compared with a control group of nine patients studied throughout 18 cycles based on 14 days of E priming. Midluteal and late luteal biopsies were morphologically comparable between both the short and long follicular groups on the one hand and the control group on the other. A phenomenon found by this group in the midluteal biopsies was a lag in glandular maturation that was caught up at the late luteal phase. These mild endometrial inadequacies do not affect functional capacity. In another study, Younis et al. (7) investigated the limits of prolonged endometrial preparation in 18 patients with ovarian failure. These women were divided prospectively and randomly into three groups. All groups were treated with a fixed dose of oral micronized E2 and estriol (at a 2:1 ratio), 4 mg/ d, for 21, 28, and 35 days, respectively, before P administration. During treatment, no patient suffered from breakthrough bleeding. Late follicular biopsies in all patients showed a normal proliferative endometrium with no signs of glandular cystic hyperplasia. Midluteal biopsies showed a secretory endometrium comparable to results obtained with the natural cycle. Several authors subsequently questioned whether the morphologic integrity seen after manipulated follicular phase durations reflects functional capacity-namely implantation rates and PRs (11, 17, 18). Younis et al. (11) retrospectively analyzed 51 recipient cycles of oocyte donation in ovarian failure patients, using the simplified method of endometrial preparation. A logistic regression analysis was performed, so as to isolate the relative impact of the various factors on PRo The success rate was found to correlate closely with the duration of E stimulation. Pregnancy rates were 7.7%, 52%, and 7.7% after short (4 to 11 days), intermediate (12 to 19 days), and long (20 to 29) follicular phases, respectively. In Vol. 66, No.6, December 1996

another study, Shapiro et al. (17) studied a group of 12 patients with ovarian failure undergoing oocyte donation using the simplified approach. Five clinical pregnancies were achieved. The duration ofE preparation in the pregnant group was 12 ± 1.6 days (mean ± SD) compared with 23 ± 3.9 days in the nonpregnant group (P < 0.05). Conversely, Navot et al. (18) prospectively studied 60 recipient cycles of oocyte donation: 27 short and 33 long follicular phase cycles. There was no difference in the implantation rates between the short (5 to 10 days) and the long (21 to 42 days) estrogen-priming durations. However, early pregnancy loss was significantly higher in the short protocol, 52.9%, compared with 18.8% in the long protocol, suggesting an adverse effect of the short cycle on endometrial functional receptivity. Taken together, these studies further explore the limits ofthe follicular phase duration. Morphological studies demonstrated that reducing the duration of the follicular phase to as short as 6 days or, conversely, increasing the duration to as long as 35 days, did not affect adversely endometrial development. On the other hand, it seemed that endometrial receptivity was best preserved when the follicular phase was kept between 12 and 19 days. This functional tolerance needs further evaluation and substantiation. Manipulating E2 Levels

To the best of our knowledge, the effect ofE 21evels throughout the follicular phase on endometrial development and function has not been examined directly in oocyte donation programs. There have been several studies that evaluated endometrial maturation using morphologic and ultrasonographic measures of the endometrium, however, the results were not consistent (17, 19). Sauer et al. (19) histologically assessed the endometrium on day 26 of the cycle in 19 agonadal patients who had received different E and P regimens. A high dose incremelltal E regimen (6 mg/d oral micronized E 2) was show'n to affect adversely the secretory endometrium, in contrast to a lower E incremental regimen (1 to 3 mg/d oral micronized E 2). Serum E 21evels during the follicular phase were not reported, however, serum E2 and P levels at the time of endometrial biopsy were 193.1 ± 28.8 pg/mL (mean ± SD) and 25.6 ± 7.4 ng/mL in the first regimen compared with 115.9 ± 24.8 pg/mL and 29.4 ± 4.8 ng/mL (conversion factors to SI units are as follows: E 2, 3.671; P, 3.18) in the second. Biopsies of patients enrolled in the first regimen all demonstrated glandular abnormalities consistent with excessive E stimulation, including stratified glandular epithelium, intraluminal papillary excrescences, stromal edema, and lack of pseudodecidualized stroma. Younis et aI. Endometrial preparation

875

j,

Table 1 The Effect of E2 Levels on Clinical Pregnancy Rate Timing of

E, No. of cycles

Natural Navot, et al. 1986 (3) Lutjen, et al. 1986 (20) Asch, et al. 1988 (22) Simplified Ben-Nun, et al. 1989 (9) Kogosowski, et al. 1990 (10) Navot, et al. 1991 (18) Younis, et al. 1992 (11) Shapiro, et al. 1993 (17)

* Conversion factor

8 8 8 12 26 60 51 12

levels*

Clinical pregnancy rate

pg/mL

%

Peak Peak Peak

920 298 513

25 50 75

Last day 7 days after Peak Mean Last day

657 543 243 748 877

30 61 55 29 41

measurements

E,

to SI unit, 3.671.

Conversely, Shapiro et al. (17) enrolled 11 agonadal women into a prospective clinical trial to evaluate two different E regimens. Patients received either 4 or 8 mg/d of micronized E 2. Late follicular E2 levels were 901 ± 140 pg/mL and 1,789 ± 601 pg/mL in the 4- and 8-mg groups, respectively. A favorable endometrium (defined by ultrasound as an endometrium of >6 mm thick with a triple-line pattern) was achieved more quickly with the high dose of E stimulation. In the early studies of oocyte donation treatment in agonadal patients, endometrial preparation imitated the natural cycle as closely as possible. As a result, E2 levels throughout the follicular phase, including the E2 surge, were within the physiologic range, usually <600 pg/mL (20, 21). Endometrial function was clearly satisfactory, as evidenced by acceptable rates of implantation and pregnancy. Later studies, especially in programs using the simplified approach of endometrial preparation, reached supraphysiologic follicular E2 levels (>600 pg/mL), without any apparent adverse effect on the clinical outcome (9-11, 17, 18). Table 1 summarizes several studies dealing with oocyte donation therapy. Only studies that included sufficient data on follicular E2 levels have been chosen. Some used the "natural" approach (2, 20, 22) and others used the simplified approach (9-11, 17, 18) of endometrial preparation. It is obviously impossible to evaluate simply the differences between these studies; in addition to E2 levels, many other factors could have affected the clinical PR (11). Moreover, random, mean, or peak E2 levels, especially when the duration of the follicular phase is not fixed, do not assess actually the total target organ exposure to E throughout the priming phase. Despite these reservations, the clinical PRs achieved in all the studies summarized in Table 1 are favorable, all were >26% per cycle, suggesting that endometrial function was not altered markedly by the different 876

Younis et al. Endometrial preparation

E 21evels. Furthermore, Younis et al. (7), in a retrospective study of 61 oocyte donation cycles, evaluated the effect on clinical outcome of mean follicular E2 levels. Estradiol levels in that group ranged between 186 and 1,964 pg/mL but were shown not to affect the PR. Similarly, Shapiro et al. (17) showed that E2 levels did not differ between the pregnant and the nonpregnant groups in a study of 12 agonadal patients undergoing oocyte donation. In summary, although targeted studies have not been performed to evaluate directly the effect of E2 levels on the morphological and functional capacities of the endometrium, indirect evidence supports the notion that the endometrium is tolerant to different E2 exposures and that incremental or fixed E2 levels, even in the supraphysiologic range, do not seem to have a significant affect on endometrial preparation. Prospective direct studies are needed to confirm this impression. It is suggested that future studies evaluate the area under the curve so as to examine the total target organ exposure to E 2, for each endometrial stimulation protocol (18). MANIPULATING THE LUTEAL PHASE Modifying the Quantity of P

To the best of our knowledge, targeted studies to evaluate endometrial development and function with various dosages of P have not been performed in oocyte donation programs. Sauer et al. (19) morphologically compared two different endometrial preparation protocols in ovarian failure patients. The two protocols were identical with respect to follicular and luteal E therapy, but varied in the P replacement. One hundred fifty milligrams per day of 1M P in oil was used in the first group and 160 mg/day of P using vaginal P-containing pessaries was used in the second. Mean luteal serum P levels of 48.8 ± 10.4 and 29.4 ± 4.8 ng/mL, respectively,; were achieved. Better histologic results were achieved with the first protocol, suggesting an advantage for excessive luteal P levels during endometrial preparation. However, indirect evidence from other clinical studies using the in vivo model of oocyte donation does not seem to agree with these results. In several studies of oocyte donation, P replacement therapy during the luteal phase was tailored to mimic the natural cycle: 1M P in oil (3, 22) or intravaginal P pessaries (2) were used, in dosages ranging from 26 to 100 mg/d. Physiological levels of P in the serum usually were achieved, ranging from 18 to 27 ng/mL in one study (3) and from 6 to 19 ng/mL in the other (22). Furthermore, when using the simplified approach of oocyte donation therapy with a fixed dose Fertility and Sterility®

Table 2 The Effect of P Levels on Clinical Pregnancy Rate No. of cycles Natural Navot, et al. 1986 (3) Lutjen, et al. 1986 (20) Asch, et al. 1988 (22) Simplified Serhal and Craft, 1987 (4) Leeton, et al. 1989 (8) Ben-Nun, et al. 1989 (9) Kogosowski, et al. 1990 (10) Navot, et al. 1991 (18) Younis, et al. 1992 (11) Shapiro, et al. 1993 (17)

* Values

8 8 8 17 14 12 26 60 51 12

P dosage and administration route

25 to 50 1M 50 to 100 vaginally 25 to 50 1M 100 1M, 300 by mouth 50 1M, 100 to 200 vaginally 100 1M 100 1M 50 to 100 1M 50 to 100 1M 100 1M

Serum P levels*

Clinical pregnancy rate

ng/mL

%

18 ± 27 6 ± 19 18 ± 3

25 50 75

41 56 45 39 16

41 36 30 61 55 29 41

± ± ± ± ±

22 14 15 19 0

are means ± SD. Conversion factor to SI unit, 3.18.

of P for endometrial preparation during the luteal phase, a higher dosage ofP usually was used, reaching supraphysiologic levels of P in the serum (4, 911, 17, 18). Both regimens-the "natural" and the simplified-achieved favorable clinical PRs, indicating that the quantity of luteal P administered was of little importance (Table 2). Furthermore, Younis et al. (11), in a retrospective analysis, did not find luteal P levels to be a significant factor affecting pregnancy or implantation rates. Hence, although some preliminary histologic evidence supported the use of a higher quantity of P to achieve a better morphological outcome, indirect evidence from clinical studies using oocyte donation does not support those earlier findings. It seems that P levels of approximately 10 ng/mL are sufficient to sustain morphological and functional endometrial development. The quantity of P during the luteal phase needed to achieve effective implantation and reproduction is of particular interest in women> 40 years of age, in whom low pregnancy and implantation rates could be attributed to either oocyte senescence or an age-related decline in endometrial receptivity. In recent years, considerable efforts have been invested in investigating this issue, mainly using the oocyte donation model. Most authorities today believe that a decline in oocyte quality plays a major role in the reproductive performance of older women (6, 2326); whether uterine receptivity is a contributing factor in the reduced fertility of the older women remains controversial. Studies in the subhuman mammalian species have shown that the endometrium undergoes marked age-related morphologic and physiologic changes that are thought to be responsible, at least in part, for the decline in reproductive capacity (27 35). Morphologically, as age advances, there is an increase in collagen content, a reduced number of Vol. 66, No.6, December 1996

stromal cells, reduced tissue deoxyribonucleic acid content, and fewer estrogen receptors on the surface of endometrial cells (27-32). Furthermore, when embryos from young animals are transferred to older recipients, there is a markedly decreased implantation rate, implying reduced endometrial receptivity with aging (33-35). In the human, on the other hand, the situation is much less clear cut, with different studies yielding contradictory results. Sterzik et al. (36) found more out-of-phase endometria in women older than 35 years during unsuccessful IVF cycles than in younger women. Moreover, ultrasound assessment ofthe late follicular phase has shown a thinner endometrium during ovarian stimulation in older women (37). On the other hand, Sauer at al. (38) designed a prospective study in ovarian failure patients in three separate age groups: 25 to 39, 40 to 49, and 50 to 60 years old. Similar E and P replacement regimens yielded equivalent results in terms of endometrial histology, ultrasonographic appearance, and steroid receptor markers in all groups, suggesting that the endometrium maintains its a,bility to respond adequately to hormone replacement therapy through the sixth decade of life. Clinical studies using the oocyte donation model to evaluate the contribution of the endometrium to age-related reproductive failure in women are divided between pros (26, 39-44) and cons (23-25, 45-48). However, the vast majority of those studies have significant methodological shortcomings. Some are simply observational, others are retrospective, and the remainder were not controlled adequately. Navot et al. (47) undertook a well-designed prospective study in which the same-cohort oocytes obtained from one young donor during a specific cycle were evenly distributed among "younger" and "older" oocyte recipients. Clinical pregnancy and delivery rates were similar in both groups, indicating that Younis et al. Endometrial preparation

877

2

Various experimental protocols have been used, including ovariectomy, antiestrogen therapy, and treatment with antibodies against E. Meyer et al. (54) found that high doses of P were effective in maintaining pregnancy in rhesus monkeys that had been ovariectomized 2 to 6 days after ovulation. Hodgen (Hodgen GD, abstract) was able to allow implantation and sustain pregnancies after transfer of donated embryos in 4 of 11 P-treated rhesus monkeys who were ovariectomized after ovulation. Good and Moyer (55) systematically studied the effects ofvarious doses of E and P on the developmental capacity of the secretory endometrium in ovariectomized rhesus monkeys. When physiological doses of P were tested against reduced concentrations ofE2, stromal development was enhanced but the glands were small and poorly developed. These findings suggested that a small amount ofE may be necessary for the development of the progestational endometrium. Furthermore, Bosu and Johansson (56) observed that, of six ovariectomized monkeys, P replacement maintained pregnancy in only one animal. In addition, several antiestrogenic compounds, given immeThe Need for E2 diately after mating, have been shown to block pregnancy in the rhesus and bonnet monkeys (57, 58). Although the role of follicular E2 and luteal P in Ravindranath and Moudgal (58) showed that tamoxsuccessful implantation and maintenance of early ifen administration during the post ovulatory period pregnancy in the human has been well established, offemale mated bonnet monkeys inhibited the estabthe specific role ofluteal E2 still is undetermined. It lishment of pregnancy in most of the animals tested. has been well documented that E2 in the follicular In a subsequent study, the same group was able phase is responsible for the proliferation of surface to prevent pregnancy using polyclonal antibodies epithelium, glands, stroma, and blood vessels in the against E in four of five treated bonnet monkeys zona functionalis of the endometrium. In addition, compared with only one of five controls (59). All told, follicular E2 is known to induce the synthesis of spethe animal data in primates, specifically the moncific proteins in the endometrium, including estrokey, regarding the need for E2 during the luteal gen and Preceptors (49, 50). It also has been estabphase, is controversial. lished that luteal phase P is the hormone principally To investigate this question in the human, de responsible for the secretory endometrial transforZiegler et al. (60) and Younis et al. (61) recently mation that is necessary to allow implantation and examined the effect ofE 2 depletion during the luteal maintain pregnancy in most species (49-51). Howphase on endometrial development and maturation ' ever, the exact role of E2 in the luteal phase in the (Fig. 3). Both groups independently showed that luhuman is undetermined. It is unclear whether E2 teal E2 depletion in the human did not seem to have secretion by the corpus luteum is obligatory for noran adverse effect on the morphological development mal morphologic development of the endometrium of the endometrium. Stopping the E treatment in and whether it is crucial for successful implantation the luteal phase in agonadal patients and reducing and pregnancy maintenance. the E2 concentrations to hypogonadic levels did not The literature on this issue suggests that the E cause any distinguishable changes in the morphorequirement in the luteal phase appears to differ logic features of the midluteal or late luteal endofrom species to species. In rodents, E seems to be metrial biopsies compared with controls (61). These essential for implantation: rat and mouse blastoresults also were corroborated by transmission cysts can implant only after the rise ofluteal E secreelectron microscopy, which showed typical charaction, which occurs around day 4 of gestation (52). teristics of early and late secretory endometrium, Without this nidatory estrogen, eggs will be maindespite mean luteal E levels of 21 ± 5 pg/mL (61). tained in a state of metabolic quiescence as long as Furthermore, endometrial maturation, assessed by enough P is available (53). In primates, most of the E and P receptors identified by immunocytochemisdata on luteal E requirements have been obtained try, showed a typical distribution seen on day 24 of in monkeys and seem to be controversial (54-59).

the capacity to conceive appears to be independent of endometrial aging. On the other hand, Meldrum (26), recently has reported results of oocyte donation in recipients under and over age 40 years. There was a marked decrease in ongoing-delivered pregnancies in the women> age 40 years. However, when the luteal P dosage was increased from 50 to 100 mg/d, recipients older than age 40 years had a marked and significant increase in successful PRs, comparable to those in recipients younger than age 40 years. Those results were consistent with data from another study (44) suggesting a correctable cause of age-related decline in endometrial receptivity. Overall, it seems that waning oocyte quality is the principal factor responsible for the age-related decline in female fertility potential. The contribution of endometrial receptivity to this age-related decline is still controversial. In practice, oocyte donation using oocytes from young donors, together with an increased dosage of P to the aging recipient, reverses both of those defects.

878

Younis et al. Endometrial preparation

Fertility and Sterility®

biopsy

biopsy

Study

Control 7 E2 4 mg/day

14

21

26

E2+ P

Figure 3 Estradiol depletion during the luteal phase in endometrial preparation.

the menstrual cycle (60). Overall, morphologic studies in the human suggest that luteal E2 does not seem to affect the developmental capacity of the endometrium. In a recent preliminary prospective study, Younis and Laufer (Younis JS, Laufer N, unpublished data) designed an oocyte donation treatment protocol to elucidate the importance of luteal E2 for embryo implantation and maintenance of early pregnancy. Sixteen amenorrheic women with premature ovarian failure, candidates for oocyte donation, were divided into two groups, eight women in each. Both groups underwent identical endometrial priming with a fixed dose of 4 mg/d oral micronized E 2, using the simplified approach. On the day of oocyte donation, P replacement therapy, 100 mg/d 1M, was administered to the two groups. Estrogen treatment was sustained in the luteal phase of the first group, but totally discontinued in the second. The two groups were comparable with regard to donors' and recipients' ages, number of donated oocytes, and number of transferred embryos. Follicular phase mean E2 levels as well as luteal phase mean P levels were similar in both groups; however, luteal E 2 levels differed significantly between the groups, being 21 : :': : 11 and 555 : :': : 108 pg/mL in the study and control groups, respectively. Two clinical pregnancies were achieved in the control group, whereas only two chemical pregnancies occurred in the study group, with maximal hCG levels of23 and 89 mIU/mL (conversion factor to SI unit, 1.00). Hence, contrary to the initial morphological results suggesting that endometrial developmental capacity was preserved after E depletion throughout the luteal phase, this new preliminary evidence suggests that the functional capacity of the endometrium in fact may be affected adversely under those conditions. Implantation has been defined as the period from the hatching of the blastocyst through the zona pelVol. 66, No.6, December 1996

lucid a to the invasion of the decidua by trophoblast cells. Implantation, in fact, is not a single process, but rather a series of inter-related developments and events that result in an intimate relationship between the embryonic tissues and the endometrium (62). In vivo, the zona pellucid a is probably digested by degradation enzymes secreted by the mature endometrium, so as to expose the pre-embryo. The ultimate, intimate relationship is achieved when the embryo is engulfed by the endometrium during invasion, during which the trophoblast penetrates into the underlying connective tissue of the decidualized endometrium (62). One may speculate that the process of hatching of the embryo through the zona pellucida, in addition to its apposition and attachment to the luminal epithelium, are not affected by luteal phase E depletion. It seems that invasion of the decidua by trophoblast cells and, most importantly, maintenance of early pregnancy may be affected adversely by the luteal phase E depletion, leading to very early abortions. MODEL FOR STUDYING ABERRATIONS OF THE NORMAL CYCLE: PREMATURE LUTEINIZATION

Premature luteinization has been presumed to be the result of increased preovulatory levels of LH and is manifested by high premature P secretion during the follicular phase (usually defined as >0.9 ng/mL); it is suspected to be associated with low implantation rate and PRs. Whereas many factors are involved in embryo implantation, they may be grouped into two general categories: those related to embryo quality and those related to endometrial receptivity. A decrease in PRs may be secondary either to an unfavorable follicular milieu, resulting in poor oocyte quality and, hence, embryo quality, or to an adverse effect on endometrial receptivity. The relative contribution of each of these factors in cases of premature luteinization remains uncertain and is a matter of some controversy. Premature luteinization occurs relatively frequently after ovulation induction and controlled ovarian hyperstimulation (COH) for assisted reproduction. Endometrial asynchrony has been reported in women treated with clomiphene citrate (CC) alone (63-65), in those treated with CC in combination with menotropins (66), with menotropins alone (36, 67), and with menotropins in conjunction with GnRH-agonist (GnRH-a) suppression (68, 69). Overall, in all those studies (36, 63-69), ovarian stimulation was associated with a mild but significant increase in P secretion during the follicular phase, which was implicated as the main cause of the adverse effect on endometrial maturation. HowYounis et al. Endometrial preparation

879

!

ever, the effect of premature P on the endometrium is confounded by the fact that ovulation induction drugs may themselves have a direct adverse effect on endometrial development (63-66). In addition, controlled ovarian stimulation could have an overall deleterious affect on endometrial receptivity (70). U sing the oocyte donation model, Hofmann et al. (71) retrospectively evaluated 68 women undergoing COH (long GnRH-a and hMG) as ovum donors, and 68 matched women with ovarian failure as ovum recipients. Twenty-one of the donors (31%) demonstrated premature luteinization. Donors with and without premature luteinization as well as recipients of the two parallel groups were comparable. Surprisingly similar implantation rates and deliveries per transfer were observed in women receiving oocytes from donors with and without premature luteinization. These findings suggest that any negative impact of premature luteinization on implantation rate in COH cycles, seems to be due not to an adverse effect on oocyte quality, but rather on endometrial receptivity. On the other hand, in IVF cycles after COH, it has been demonstrated that a subtle rise of P levels on the day of hCG administration is associated with poorer fertilization rates (68, 69, 72). Those studies seemed to indicate that even a brief exposure to higher P levels may have an adverse effect on oocyte fertilizability or, alternatively, may be a marker of low oocyte-embryo quality. To simulate premature luteinization, Ezra et al. (73) recently evaluated the effect of premature P administration on artificially prepared endometria in 16 patients with ovarian failure. The patients were randomly divided into two groups, with eight patients in each. The first group was treated with episodic P administration on days 2 and 7 (12.5 mg of P in oil) and the second group was treated with P (6.25 mg) on days 3, 4, and 5 of the artificial follicular phase (Fig. 4). Follicular E2 levels and luteal P levels were comparable between the two study groups and a matched control group; however, follicular P levels were significantly higher in the study groups than the control group (1.9 ± 4.0 and 0.2 ± 0.1 ng/mL, respectively). Endometrial biopsies demonstrated early secretory changes in the late follicular phase in 50% of the patients and stromal-glandular discrepancy in the late follicular phase in 58% of all patients in the study. Hence, isolating the effect of premature secretion during the follicular phase clearly substantiated the fact that it can result in impaired endometrial development that is uncorrectable by P supplementation during the luteal phase. The use of this unique model has provided us with evidence for the potential detrimental effect of premature P secretion in the follicular phase on endo880

Younis et al. Endometrial preparation

Control

ICI7777777777J'77T->77'7.7777~

Study

7 Follicular phase E2 14 days

14

21 Luteal phase E+P 12 days

26

Figure 4 Progesterone administration during the proliferative phase-a model of premature luteinization.

metrial receptivity. To evaluate the possible effect of premature luteinization on oocyte quality, prospective use ofthe oocyte donation model is required. ROUTE OF ADMINISTRATION

Since the introduction of oocyte donation therapy, various replacement protocols using different routes of delivery for E and P steroids have been used successfully, without consensus as to the most effective regimen (Table 3). Lutjen et al. (2) used oral E2 valerate and vaginal P suppositories for steroid replacement. Navot et al. (3) added oral micronized E2 to the armamentarium of endometrial stimulatory regimens and further modified the Lutjen et al. (2) protocol by using only 1M P for luteal phase replacement. It seems that the oral route ofE administration is the most popular for endometrial preparation during oocyte donation. Estradiol valerate has been used widely in many programs (2-4, 10, 14,20,22, 7478); alternatively, micronized E2 (11, 17, 45), micronized E2 and estrone (E l ) in a 2:1 ratio (3), or conjugated estrogens (9) have been used without any obvious advantage of one drug over another. Alternatively, the transdermal route has been used (14, 18, 79, 80), mainly sustained release E2 patches. Transdermal E2 administration has the theoretical advantage of maintaining a more physiological E2:El ratio by bypassing the intestinal and biliary tracts, thereby preventing the conjugation of E2 to glucuronide and sulfated forms, which are unavailable to the endometrium. Moreover, the transdermal route escapes the liver first pass effect, preventing the induction of hepatic enzymes that can stimulate production of antithrombin III, renin substrate, and sex hormone-binding globulin. Furthermore, this Fertility and Sterility®

Table 3

Medications and Their Route of Administration

Estrogen

Progesterone

• P in oil (Gestone) (Proluton)

• E2 valerate (Progynova)

• Vaginal rings

• • • • •

• Micronized E2

Micronized E2 (Estrace) Micronized E2 + El (Estrofem) Conjugated E (Premarin) MicroniL.ed P (Utrogestan) Dydrogesterone (Biphaston)

route of administration has not been associated with any increase in serum lipids (81). Droesch et al. (80) successfully have used transdermal E treatment in ovarian failure patients and achieved adequate endometrial development and function, as shown by histology and PRs. The only adverse effect reported was a rash at the site of the patch, requiring discontinuation of the medication in some cases (81). An alternative method for E replacement, used by the Norfolk group in the early trials of oocyte donation, was polysiloxane-impregnated vaginal rings (51). However, that technique was abandoned quickly because each new ring insertion resulted in a burst in the release of estrogen, without a steady state being achieved. Moreover, occasionally the rings caused vaginal discomfort or infections, or both, which was unacceptable to many patients. Progesterone for endometrial preparation in oocyte donation cycles traditionally has been administered either orally or parenterally. The oral route of P delivery, although convenient, and used in some oocyte donation programs (4, 76), has proven less than optimal due to rapid hepatic metabolism and poor bioavailability (82). Moreover, metabolites of orally administered P may induce hypnotic effects (83). Oral micronized P (100 mg three times) was investigated in ovarian failure patients in 12 cycles after adequate E2 priming (84). The serum P levels achieved were subphysiologic and incapable of inducing a normal secretory response in the endometrium. The most popular approaches today for endometrial preparation in oocyte donation cycles remain the vaginal or 1M routes. The 1M route remains the most reliable method of achieving predictable serum levels ofP, however, local pain at the injection site is troublesome, especially in prolonged oocyte donation treatment. On the other hand, P suppositories are associated with a discharge that is unacceptable to many patients. The use of intravaginal polysiloxaneimpregnated P cylinders was discontinued for the reasons alluded to with the E rings (51). There is controversy regarding the most effective method of P delivery. Sauer et al. (19) obtained better morphologic results on day 26 of artificially preVol. 66, No.6, December 1996

Vaginally

By mouth

1M

Transdermal • E2 patches (Estraderm)

• Vaginal rings • Micronized P (Utrogestan) • Combined estrogen + P

pared cycles using the 1M route of P administration (50 to 100 mg/d) compared with the vaginal route (100 to 200 mg/d). Conversely, Bourgain et al. (84) showed that micronized P administration by the vaginal route (300 or 600 mg/d) in ovarian failure patients, after similar E priming, resulted in better endometrial morphology on day 21 compared with the 1M route (100 mg/d). However, Miles et al. (85), recently have shown, in 20 functionally agonadal women who were candidates for oocyte donation, that achieving better P serum levels by the 1M route did not necessarily mean that there was better target organ exposure. After 6 days of P replacement therapy in these patients, serum P levels using the 1M route (100 mg/d) were 69.8 ± 5.9 compared with 11.9 ± 1.2 nglmL by the vaginal route (800 mg/d). However, endometrial concentrations of P were greater after vaginal P compared with 1M P, being 11.5+2.6 and 1.4 ± 0.4 ng P/mg protein, respectively. Ultrasonographic, histologic, and E and Preceptor analyses on day 21 were comparable in both groups. The majority of oocyte donation programs in the last decade have used the 1M route of P administration for endometrial preparation (3,4,9-11,17,18, 22, 45, 75-77), whereas others have used the vaginal route (2, 14, 20, 74, 75, 78, 79). Both methods have succeeded in obtaining favorable PRs, however, to the best of our knowledge, the superiority of one route over the other has not been tested directly in a prospective study of oocyte donation. In view of the new preliminary data that suggest the superiority of the vaginal over the 1M route (84, 85), a prospective targeted study is warranted. Recently, Shushan et al. (86) published a preliminary report examining the feasibility of a new mode of E and P treatment using combined vaginal tablets. Nine functionally agonadal patients were prepared identically by E and P. Endometrial preparation was carried out with vaginal tablets of 6 mg micronized E2 two times per day for 14 days, followed by 14 days of the combined vaginal tablets, containing 6 mg of micronized E2 and 50 mg of micronized P, two times a day. Appropriate E2 and P serum levels and adequate endometrial maturation Y ounis et al. Endometrial preparation

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were obtained following this regimen, confirming its efficacy in inducing adequate endometrial development. However, this proposed regimen has to be tested yet in actual oocyte donation treatment. CONCLUSIONS

During the last decade it has become evident that oocyte donation therapy can be used as a unique in vivo human model to explore the physiology of the endometrium. Pioneer studies have shown that only sequential E and P are essential for normal endometrial maturation and adequate embryo implantation. The simplified approach of endometrial preparation taught us that it is not necessary to expose the endometrium to physiologic incremental doses of the ovarian steroids, because a fixed dose of sequential E and P does not adversely affect endometrial receptivity. Moreover, it was shown that the endometrium is tolerant of some degree of manipulation of the duration of the follicular phase. In contrast to endometrial morphologic studies, which indicated that the endometrium was tolerant of extreme manipulations of the length of the follicular phase-from as short as 6 to as long as 35 dayssubsequent studies suggested that the receptivity of the endometrium was best preserved when the follicular phase was kept between 12 and 19 days: short follicular phases «11 days) were shown to have an adverse effect on the clinical outcome; on the other hand, the effect of a prolonged follicular phase on endometrial receptivity is unclear. Although targeted studies have not been performed to evaluate the effect offollicular E2 or luteal P levels on endometrial receptivity, clinical oocyte donation studies have shown that both physiologic and supraphysiologic levels of these steroids usually result in acceptable clinical PRs. Unlike the morphologic studies demonstrating that endometrial maturation was preserved after E2 depletion during the luteal phase, preliminary evidence suggests that, in fact, the functional capacity of the endometrium is affected adversely under those conditions. Although the pioneering work in oocyte donation indicated that there was a correlation between morphologic integrity and functional capacity of the endometrium, evidence presented in this review demonstrates that adequate endometrial morphology does not always imply normal endometrial receptivity. It is therefore mandatory that more sensitive ways of assessing endometrial functional capacity be sought. Preliminary reports of the use ofultrasonography (87) (pattern and flow), scanning electron microscopy to assess endometrial pinopodes (88), as well as endometrial proteins, such as the integrins (89), recently have been proposed for this purpose. 882

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