Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo

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1 FERTILITY AND STERILITY VOL. 76, NO. 5, NOVEMBER 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo Diana Valbuena, M.D., a Julio Martin, Ph.D., a,b Jose Luis de Pablo, Ph.D., b José Remohí, M.D., a,b Antonio Pellicer, M.D., a,b,c and Carlos Simón, M.D. a,b Instituto Valenciano de Infertilidad Foundation and Valencia University, Valencia, Spain Received January 29, 2001; revised and accepted April 23, Supported by grant FISss 00/0643 from the Spanish Government, Madrid, and Instituto Valenciano de Infertilidad Foundation. Presented in part at the 47th Annual Meeting of the Society for Gynecological Investigation, March 22 25, Reprint requests: Carlos Simón, M.D., Instituto Valenciano de Infertilidad, Guardia Civil, 23, Valencia, Spain (FAX: ; csimon@interbook.net) a Instituto Valenciano de Infertilidad Foundation (FIVIER). b Department of Pediatrics, Obstetrics and Gynecology, Valencia University. c Department of Gynecology and Obstetrics, Hospital Peset, Valencia, Spain /01/$20.00 PII S (01) Objective: To investigate whether the deleterious effect of E 2 on embryonic implantation is due to a direct effect on the endometrium, on the embryo, or both. Design: Prospective, controlled in vitro study. Setting: Tertiary infertility center. Patient(s): Fertile patients in the luteal phase with histologically normal endometrium who were attending the infertility clinic as oocyte donors (n 14). Intervention(s): E 2 dose-response (0, 10 8,10 7,10 6,10 5, and 10 4 M) and time course (day 2 vs. day 5) experiments were performed in an in vitro embryo adhesion assay composed of human polarized endometrial epithelial cells obtained from fertile patients and mouse embryos. Main Outcome Measure(s): Blastocyst formation rate and embryo adhesion rate. Results: Monolayers of polarized endometrial epithelial cells expressed ER at the mrna level. The E 2 dose response of blastocysts with polarized endometrial epithelial cells (n 235) demonstrated a progressive reduction in embryonic adhesion that was statistically significant at 10 6 M. When polarized endometrial epithelial cells were treated alone with increasing doses of E 2 for 3 days and E 2 was then removed and blastocysts added (n 410), embryonic adhesion was not significantly reduced, except at 10 4 M. When 2-day mouse embryos (n 609) were treated with increasing E 2 concentrations until day 5, the rate of blastocyst formation significantly decreased at a concentration 10 6 M, and embryonic adhesion decreased when blastocysts (n 400) were obtained at a concentration 10 7 M. Time course experiments of embryos cultured for 2 days with polarized endometrial epithelial cells (n 426) showed that the adhesion rate was higher at E 2 levels of 10 7,10 6 and 10 5 M compared with embryos cultured for 5 days (n 495). Conclusion(s): High E 2 levels are deleterious to embryo adhesion in vitro, mainly because they have a direct toxic effect on the embryo that may occur at the cleavage stage. (Fertil Steril 2001;76: by American Society for Reproductive Medicine.) Key Words: Adhesion, receptivity, embryo, estradiol. The influence of high levels of E 2 during embryonic implantation is still controversial. We and others have reported that controlled ovarian hyperstimulation (COH) used for IVF inhibits embryo implantation in humans (1 7) and mice (6, 8). However, other investigators found that high E 2 levels are not detrimental to IVF outcome (9 11) or the two events do not correlate (12 14). It has been proposed that high E 2 levels after COH impair endometrial receptivity (1 7) because oocyte quality, fertilization rate, and embryo cleavage (until day 2) were normal in patients with a high response (3) and the quality of embryos and the implantation rate seemed normal in subsequent frozen-thawed embryo transfer (7). Indeed, low implantation rates in high responders can be improved by using a step-down regimen in a subsequent cycle to decrease E 2 levels (5). These clinical observations are reinforced by the morphologic changes on standard histological techniques (15) and scanning electron microscopy observed in the secretory endometrium of patients undergoing COH (16). This controversy is important not only to determine adequate management of high responders (5), but also to justify minimal ovar- 962

2 ian hyperstimulation (17) and late embryo transfer (18) rather than conventional agressive COH and early transfer in IVF. We investigated the effect of increasing concentrations of E 2 on embryonic adhesion by using an established embryo adhesion assay (19 21). Dose-response and time-course experiments were done to establish whether this effect is directed to the embryo, the endometrium, or both and to test whether late embryo transfer may minimize the unwanted effects of high E 2 levels. MATERIALS AND METHODS Patients This project was approved by the Instituto Valenciano de Infertilidad Review Board on the use of human subjects in research. Fourteen fertile patients 23 to 39 years of age who were attending the infertility clinic as oocyte donors consented to donate an endometrial sample for this study. Samples of endometrial tissue were obtained in the luteal phase (LH 2) of a natural cycle. Endometrial biopsy was obtained by using a Pipelle (Gynetics, Amsterdam, The Netherlands) after written consent was obtained. Patients were screened and were negative for infection with HIV, hepatitis C and B, VDRL, and mycoplasma. A portion of each specimen was stained with hematoxylin eosin for dating according to the method of Noyes et al. (22). Endometrial Cell Culture Endometrial samples were minced into small pieces less than 1 mm in size and underwent mild collagenase digestion. Endometrial stromal cells and endometrial epithelial cells were isolated as described elsewhere (18, 23, 24). Epithelial and stromal cells were cultured independently. For the epithelial cell culture, inserts (Millipore Corporation, Bedford, MA) were treated with extracellular matrix (Sigma, St. Louis, MO) in a 1:4 ratio and were sterilized overnight under ultraviolet rays the day before. Endometrial epithelial cells were grown polarized to confluence in steroid-depleted medium (75% DMEM and 25% MCDB-105 [Sigma, St. Louis, MO]) containing antibiotics that was supplemented with 10% charcoal-dextran treated fetal bovine serum and insulin (5 g/ml) (Sigma). The homogeneity of cultures was determined by morphologic characteristics and verified by immunocytochemical localization of cytokeratin, vimentin, and CD68 antigen, as described elsewhere (24). Stromal cell confluence was reached in 24 hours. For some experiments, polarized endometrial epithelial cells inserts were transported above endometrial stromal cells cultures. Culture media was changed every 48 hours until the epithelial cells reached confluence (5 6 days). Recovery of Mouse Blastocysts Ten- to 12-week-old B6C3F-1 female mice were obtained from Elevage Janvier (Le Genest, St-Isle, France) and were maintained at C on a 12-hour light/12-hour dark cycle in a conventional holding facility of the University of Valencia School of Medicine, Valencia, Spain. The animals were injected with 10 IU of intraperitoneal pregnant mare serum gonadotropin (Sigma-Aldrich, Madrid, Spain) and with 10 IU of intraperitoneal hcg (Sigma-Aldrich) 48 hours later. They were then impregnated overnight by 12-week-old males of the same strain. Mating was ascertained by the appearance of a vaginal plug on the following morning (day 1 of pregnancy). Female mice were killed at day 2 of pregnancy, and 2,589 two- to four-cell embryos were flushed from the oviducts in 14 different experiments. Depending on the objective of each experiment, some embryos were grown to the blastocyst stage in untreated or E 2 -treated embryo co-culture media (Scandinavian IVF, Göteborg, Sweden) and others were transferred to polarized endometrial epithelial monolayers that were untreated or treated with E 2. All experiments were maintained at 37 C in a 5% CO 2, 95% air-humidified incubator. Dose-Response Experiments To discern a possible endometrial or embryonic effect of increasing concentrations of E 2 on embryonic adhesion, three different experiments were designed. First, after confluence, polarized endometrial epithelial cells were cultured with co-culture medium (Scandinavian IVF), and mouse blastocysts (n 235) were co-cultured (5 per well) for 48 hours in increasing E 2 concentrations (0, 10 8,10 7,10 6, 10 5, and 10 4 M). This experiment was done to investigate an embryo and endometrial effect. Second, polarized endometrial epithelial cells were cultured in growth medium at the same E 2 concentrations as above in the absence of blastocysts for 3 days. Media and E 2 were then replaced by co-culture medium (Scandinavian IVF), and 410 mouse blastocysts (5 per well) were co-cultured in the polarized endometrial epithelial cells for 48 hours. The experiment was meant to discern an endometrial effect. To improve this in vitro model for the endometrial testing, endometrial stromal cells were added beneath polarized endometrial epithelial cells, and mouse blastocysts (n 414) were cultured under the conditions described above. Finally, 2-day mouse embryos (n 609) were cultured in increasing E 2 concentrations, and the resulting blastocysts were transferred to untreated polarized endometrial epithelial cells monolayers for 48 hours. This was done to assess an embryonic effect. Time Course Experiments To reproduce in vitro early versus late embryo transfer experiments, [1] embryos and polarized endometrial epithelial cells were treated together from day 2 to day 7 of embryonic development (n 495), or [2] embryos were cultured without E 2 until blastocyst stage while polarized endometrial epithelial cells were treated with increasing E 2, and blastocysts were transferred to the endometrium and FERTILITY & STERILITY 963

3 FIGURE 1 Representative result of reverse transcription polymerase chain reaction for ER and -actin ( -act) in polarized endometrial epithelial cells treated with increasing concentrations of E 2.C negative control; C control. culture was continued with different E 2 concentrations from day5today7(n 426). RNA Extraction and RT-PCR To analyze the expression of estrogen receptor (ER) in polarized endometrial epithelial cells, reverse transcription polymerase chain reaction (RT-PCR) oligonucleotides were designed by using Primer Designer software (Scientific and Educational Software, Stateline, PA). Total RNA was prepared from polarized endometrial epithelial cells that were cultured in increasing E 2 concentrations (0, 10 8 M, 10 7 M, 10 6 M, 10 5 M, and 10 4 M) for 48 hours. The RNA from the cells was extracted directly on the dishes by using 200 L of TRIZOL reagent (Gibco BRL, Barcelona, Spain) according to the Chomczynski and Sacchi method (25). To obtain cdna, ethanol-precipitated products were used for reverse transcription reactions in Advantage RT-for-PCR kits (CLONTECH, Palo Alto, CA). Semiquantitative PCR was developed to obtain the specific ER product. The PCR cycle, repeated 35 times, consisted of denaturation at 94 C for 1 minute, annealing at 60 C for 30 seconds, and extension at 72 C for 2 minutes. The PCR products were analyzed on 1.5% agarose gels (Pronadisa, Barcelona, Spain). For -actin, the same PCR conditions were used, except that the PCR cycle was repeated 24 times. Quantitative estimation of bands was performed by densitometric analysis with image software (1D software, Gelprinter Plus; TDI, Madrid, Spain). Pvu II restriction sites in the cdna products were determined by using Sequaid II software (Rhoads and Ronfa, 1989; Molecular Genetics Laboratory, Kansas State University, Kansas City, KS). Embryo Adhesion Assay The embryonic adhesion to polarized endometrial epithelial cells was checked at day 7 of embryonic development (5 days after day-2 embryos were transferred or 48 hours after blastocysts were transferred). Blastocyst attachment and trophoblast outgrowth were assessed as described elsewhere (19 21). Briefly, two investigators counted the number of embryos adhered to the epithelial monolayer in each group by using a mechanical assay in which the incubation dishes are moved 3 cm of diameter for 10 seconds; embryos floating in the medium are considered not attached, and those that were not floating were considered to be attached. The ratio of attached blastocysts to total blastocysts cultured corresponds to the embryo adhesion rate. Statistical Analysis Data are expressed as mean SE. Adhesion rates among groups were compared by using 2 and 2 for trend tests when possible. RESULTS By using RT-PCR, the presence of ER mrna was documented in polarized endometrial epithelial cells as a 386 base pair band at various E 2 concentrations (Fig. 1). 964 Valbuena et al. Effect of estradiol on embryonic adhesion Vol. 76, No. 5, November 2001

4 FIGURE 2 Effect of E 2 on embryo adhesion in treated polarized endometrial epithelial cells and blastocysts (n 235). *P.05, 2 test and 2 test for trend. Dose-Response Experiments Increasing E 2 concentrations resulted in progressive reduction of embryonic adhesion, which was statistically significant at E 2 concentrations 10 6 M. Adhesion rates were 70% in the control experiment, 73.7% in 10 8 M, 71.8% in 10 7 M, 53.8% in 10 6 M, 50% in 10 5 M, and 25.6% in 10 4 M (Fig. 2). These results suggest that increasing concentrations of E 2 had a deleterious effect on embryo adhesion in this in vitro model, similar to in vivo observations. To distinguish whether E 2 was adversely affecting the embryo or the endometrium, we designed a second study in which polarized endometrial epithelial cells, with and without endometrial stromal cells were treated with increasing doses of E 2 for 3 days, cultured under physiologic concentrations, and had blastocysts added. In embryos cultured in pretreated polarized endometrial epithelial cells (n 410), rates of embryonic adhesion were significantly decreased only at maximal E 2 concentrations (10 4 M). Adhesion rates were 71.6% in the control experiment, 75% in 10 8 M, 61.9% in 10 7 M, 56.2% in 10 6 M, 56.5% in 10 5 M, and 47.8% in 10 4 M). Embryos cultured with pretreated polarized endometrial epithelial cells plus endometrial stromal cells (n 414) had reduced adhesion rates at E 2 concentrations 10 5 M (82.6% in control experiments, 82.6% in 10 8 M, 81.2% in 10 7 M, 76.8% in 10 6 M, 65.2% in 10 5 M, and 49.3% in 10 4 M), and the test for trend was significant. These results suggest that although E 2 affects the endometrium and reduces the adhesion rate, this effect is not as important as in the previous experiment, when both endometrium and embryo were treated. Embryos alone were treated with increasing E 2 concentrations from day 2 to day 5, and embryo development at blastocyst stage was checked. Blastocysts were then transferred to untreated polarized endometrial epithelial cells until day 7. Estradiol concentrations 10 6 clearly affected embryo development in terms of blastocyst formation rate and number of degenerated embryos (Fig. 3). All embryos in E 2 concentrations of 10 4 M degenerated. Adhesion of blastocysts (n 400) was also affected in a dose-dependent manner; E 2 concentrations 10 7 M were found to be deleterious (68.8% in the control experiment, 74% in 10 8 M, 58.6% in 10 7 M, 43.4% in 10 6 M, and 28.9% in 10 5 M). Time Course Experiments To further demonstrate the toxic effect of increasing concentrations of E 2 on embryonic development, we performed the adhesion assay after embryos were exposed to E 2 for 5 days (early transfer) versus 2 days (late transfer). In the first experiment, embryos were co-cultured with polarized endometrial epithelial cells from day 2 to day 7 in increasing E 2 concentrations. In the second experiment, embryos were grown in vitro to the blastocyst stage and were cultured from day 5 to 7 with polarized endometrial epithelial cells under increasing E 2 concentrations. In the early transfer group (n 495), the rate of hatched FERTILITY & STERILITY 965

5 FIGURE 3 Effect of E 2 on embryo development when mouse embryos (n 609) were treated with increasing E 2 concentrations. P.05 by 2 test and 2 ;; test for trend. ;; hatched blastocyst; hatching blastocyst; extended blastocyst; cavitated blastocyst; early blastocyst; degenerated blastocyst. and hatching blastocysts at day 5 was decreased at E 2 concentrations 10 7 M (66.7% in the control experiment, 63.9% in 10 8 M, 42.2% in 10 7 M, 42.2% in 10 6 M, 38.9% in 10 5 M, and 7.9% in 10 4 M). The adhesion rate significantly decreased in the early transfer group compared with the late transfer group at E 2 concentrations 10 7 M (82.1% vs. 83.1% in the control experiment, 82.6% vs. 84.5% in 10 8 M, 59.2% vs. 74.6% in 10 7 M, 43.8% vs. 70.4% in 10 6 M, 35.5% vs. 66.2% in 10 5 M, and 26.7% vs. 36.6% in 10 4 M) (Fig. 4). DISCUSSION The effect of supraphysiologic levels of E 2 on embryonic implantation is controversial; different clinical studies have produced discrepant results. To clarify this issue, we used an established in vitro model for embryonic adhesion (19 21) to perform E 2 dose-response and time course experiments. We sought to establish whether the effect of E 2 is directed to the embryo or the endometrium and to test whether late embryo transfer may minimize the unwanted effects of high E 2 concentrations on embryo implantation. ER has been described in mouse embryos (26) and human oocytes (27). To ensure that the endometrial monolayer was also responsive to E 2,wefirst demonstrated that polarized endometrial epithelial cells possess ER at the mrna level when treated with different E 2 concentrations. Using this in vitro model, we demonstrated that E 2 concentrations 10 6 M have a deleterious effect on the embryo adhesion phase, similar to that reported in vivo (1 7). Of note, when polarized endometrial epithelial cells were pretreated, embryonic adhesion was decreased only at maximal E 2 concentrations (10 4 M), and when polarized endometrial epithelial cells plus endometrial stromal cells were pretreated, embryonic adhesion was altered at E 2 concentrations 10 5 M. However, E 2 concentrations 10 6 M clearly affected embryo development in terms of blastocyst rate and degenerated embryos, and lower E 2 concentrations ( 10 7 M) affected adhesion of treated blastocysts in a dose-dependent manner. These results suggest that at the endometrial embryonic interface, E 2 affects embryonic adhesion, and the probable target of E 2 is the embryo. The endometrium is also affected, but at higher E 2 concentrations. We and other investigators have consistently found lower implantation rates per embryo replaced in high responders (1 7), and we had thought that this effect was due to an alteration of endometrial receptivity. There were several reasons for this assumption. First, oocyte quality, fertilization rate, and embryo development until day 2 were similar to that in normal responders (3). Second, observations were made in oocyte donation that pregnancy and implantation rates in recipients of embryos derived from high responders were similar to those in normal responders (3). Finally, 966 Valbuena et al. Effect of estradiol on embryonic adhesion Vol. 76, No. 5, November 2001

6 FIGURE 4 Effect of E 2 on embryo adhesion in early (n 391) versus late (n 426) embryo transfer with increasing E 2 concentrations. P.05 by 2 test and 2 ;;; ;;; test for trend. ;;; early transfer; late transfer. embryo quality and implantation seem to be normal in subsequent frozen-thawed embryo transfers (7). However, in all these cases, oocytes and embryos were developed in vitro without the influence of high E 2 concentrations and were transferred to recipients with physiologic E 2 levels. Therefore, the effect of E 2 on the embryo was not assessed. We performed time course experiments to ascertain that increasing concentrations of E 2 primarily affected the embryo. In the adhesion assay, embryos were exposed to E 2 for 5 days (from day 2 to day 7 of development) in an attempt to reproduce the events of a day-2 transfer. A second group of embryos was grown in vitro to the blastocyst stage and was then cultured from day 5 to 7 with polarized endometrial epithelial cells under increasing E 2 concentrations (mimicking a blastocyst transfer). The embryonic adhesion rate was decreased significantly in the early transfer group compared with the late transfer at E 2 concentrations 10 7 M (Fig. 4). This finding suggests that reduction of embryonic exposure to E 2 in the late embryo transfer may offset the toxic effect of E 2 on embryonic implantation. Because of ethical and legal issues, we used an in vitro model to elucidate the effect of E 2 on embryonic adhesion. Our results should be considered in light of the limitations of an in vitro model. In addition, we used mouse embryos rather than human embryos. Nevertheless, the assay that we used has been useful in validating paracrine interactions at the human endometrial embryonic interface (20, 21). Finally, the E 2 concentrations that we used (2.1 pg/ml as control, 10 8 M [2.724 pg/ml], 10 7 M [27.24 pg/ml], 10 6 M [272.4 pg/ml],10 5 M [2,724 pg/ml], and 10 4 M [27,240 pg/ml]) cannot be extrapolated to the serum E 2 levels routinely achieved in COH, since we tested E 2 concentrations at the maternal embryonic interface. In conclusion, we found that higher E 2 concentration affects embryonic adhesion. Although E 2 reduces the receptivity of the endometrium, the probable target is the embryo. Strategies to reduce E 2 levels (5) or to reduce the time of exposure of the embryo at the cleavage stage may improve implantation rates in high responders. References 1. Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil Steril 1990;53: Pellicer A, Ruiz A, Castellví RM, Calatayud C, Ruiz M, Tarín JJ, et al. Is the retrieval of high numbers of oocytes desirable in patients treated with gonadotrophin-releasing hormone analogues (GnRHa) and gonadotrophins? Hum Reprod 1989;4: Simón C, Cano F, Valbuena D, Remohí J, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum estradiol levels in high and normal responder patients. Hum Reprod 1995;10: Pellicer A, Valbuena D, Cano F, Remohí J, Simón C. Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertil Steril 1996; 65: Simón C, García-Velasco J, Valbuena D, Peinado J, Moreno C, Remohí J, et al. Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil Steril 1998; 70: Gidley-Baird AA, O Neil C, Sinosich MJ, Porter RN, Pike IL, Saunders DM. Failure of implantation in human in vitro fertilization and embryo transfers patients: The effects of altered progesterone/estrogen ratios in humans and mice. Fertil Steril 1986;45: NG EHY, Yeung WSB, Lau EYL, So WWK, Ho PC. High serum oestradiol concentration in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent frozen-thawed embryo transfer cycles. Hum Reprod 2000;15: Fossum GT, Davidson A, Paulson RJ. Ovarian hyperstimulation inhibits embryo implantation in the mouse. J In Vitro Fertil Embryo Transfer 1989;6: Chenette PE, Sauer MV, Paulson RJ. Very high serum estradiol levels are not detrimental to clinical outcome of in vitro fertilization. Fertil Steril 1990;54: FERTILITY & STERILITY 967

7 10. Sharara FI, McClamrock HD. High estradiol levels and high oocyte yield are not detrimental to in vitro fertilization outcome. Fertil Steril 1999;72: Sharara FI, McClamrock HD. Ratio of oestradiol concentration on the day of human chorionic gonadotrophin administration to mid-luteal oestradiol concentration is predictive of in-vitro fertilization outcome. Hum Reprod 1999;14: Haning RV Jr, Boehnlein LM, Carlson IH, Zweibel WJ. Diagnosisspecific serum17 estradiol (E 2 ) upper limits for treatment with menotrophins using 125 I direct E 2 assay. Fertil Steril 1984;42: Testart J, Belaisch-Allart J, Forman R, Gazengel A, Hazout A, Strubb N, et al. Influence of different stimulation treatments on oocyte characteristics and in vitro fertilizing ability. Hum Reprod 1989;4: Toner J, Brzyski R, Oehninger S, Veeck L, Simonetti S, Muasher S. Combined impact of the number of preovulatory oocytes and cryopreservation on IVF outcome. Hum Reprod 1991;6: García JE, Acosta AA, Hsiu JG, Jones HW. Advanced endometrial maturation after ovulation induction with human menopausal gonadotropin/human chorionic gonadotropin for in vitro fertilization. Fertil Steril 1984;41: Bladford AJ, Najmabadi S, Paulson RJ. Ultrastructural characteristics of the luteal phase endometrium in donors undergoing controlled ovarian hyperstimulation. Fertil Steril 1997;67: Fauser BCJM, Devroey P, Yen SSC, Gosden R, Crowley WF, Baird DT, et al. Minimal ovarian stimulation for IVF: appraisal of potential benefits and drawbacks. Hum Reprod 1999;14: Simón C, Mercader A, García-Velasco J, Nikas G, Moreno C, Remohí J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999;84: Shiotani M, Noda Y, Mori T. Embryo-dependent induction of uterine receptivity assessed by an In vitro model of implantation in mice. Biol Reprod 1993;49: Martin JC, Jasper M, Valbuena D, Meseguer M, Remohí J, Pellicer A, et al. Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression. Biol Reprod 2000;63: Galán A, O Connor E, Valbuena D, Herrer R, Remohí J, Pampfer S, et al. The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion. Biol Reprod 2000;63: Noyes RW, Herting AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1: Simón C, Piquette G, Frances A, Polan ML. Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab. 1993;77: Simón C, Gimeno MJ, Mercader A, O Connor JE, Remohí J, Polan ML, et al. Embryonic regulation of integrins b 3, a 4 and a 1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab. 1997;82: Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162: Hiroi H, Mommoeda M, Inoue S, Tsuchiya F, Matsumi H, Tsutsumi O, et al. Stage-specific expression of estrogen receptor subtypes and estrogen responsive finger protein in preimplantational mouse embryos. Endocrinol J 1999;46: Wu TCJ, Wang L, Wan YJY. Detection of estrogen receptor messenger ribonucleic acid in human oocyte and cumulus-oocyte complexes using reverse transcriptase-polymerase chain reaction. Fertil Steril 1993;59: Valbuena et al. Effect of estradiol on embryonic adhesion Vol. 76, No. 5, November 2001