European Journal of Obstetrics & Gynecology and Reproductive Biology, 45 (1992) 113- 117 0 1992 Elsevier Science Publishers B.V. All rights reserved 0028-2243/92/$05.00

EUROBS

113

01360

Monoclonal enzyme immunoassay measurement of estradiol and progesterone receptors in in vitro fertilization and spontaneous cycles Juan Balasch a*b,Francisca Rivera b, Inmaculada C. JovC a and Juan A. Vanrell a Department of a Obstetrics and Gynecology and b Hormonal Laboratory, Faculty of Medicine, Hospital Clinic i Provincial, Barcelona, Spain Accepted

for publication

27 January

1992

Summary So far, the few studies performed on endometrial steroid receptors in in vitro fertilization (IVF) cycles have been carried out by the dextran-coated charcoal (DCC) method. Despite widespread efforts to standardize the DCC assay method, assay results still vary and thus, data obtained in IVF cycles are controversial. In the present article, late luteal estradiol (E,) and progesterone (P) receptor (R) levels were measured by a new monoclonal enzyme immunoassay (EIA) method in endometrial samples obtained from 21 patients in an IVF program who received no embryo transfer (ET) after ovarian stimulation with follicle-stimulating hormone/ human menopausal gonadotrophin/ human chorionic gonadotrophin under pituitary suppression with buserelin. Plasma levels of E,, P and prolactin (PRL) were measured in three blood samples collected in the midluteal phase. Results were compared with those obtained in a control group of 21 spontaneous cycles. E,, P and PRL were significantly higher in stimulated than in spontaneous cycles. The level of E,R was decreased in endometrium in IVF stimulated cycles, but PR remained unchanged. Monoclonal

enzyme

immunoassay;

Estradiol

receptor;

Progesterone

Introduction The establishment of pregnancy after in vitro fertilization (IVF) and embryo transfer (ET) depends on the generation of several good quality embryos and the existence of a receptive endometrium at the time of replacement and an

receptor;

In vitro fertilization

adequate steroid environment to prime and maintain the integrity of the endometrium following implantation. Ovarian stimulation, while providing adequate number of eggs for fertilization, influences the receptivity of an endometrium subjected to supraphysiological steroid levels consequent on the formation of multiple corpora lutea

HI. Correspondence to: Dr. Juan Balasch, Department of Obstetrics and Gynecology, Faculty of Medicine, Hospital Clinic i Provincial, C/Casanova 143, 08036-Barcelona, Spain.

Circulating steroid hormones mediate their effects on the target tissue endometrium through interaction with specific receptors resulting in new gene expression and changes in cell function.

114

Endornetrial receptors must therefore play a fundamental role in mediating the endometrial response to corpus luteum steroids. And thus, alterations of endometrial receptor dynamics may be involved in the low rate of success following ET. However, the literature on endometrial steroid hormone receptors in stimulated cycles for IVF is sparse [2-51. In all these previous studies on the subject, patients received clomiphene citrate and receptors were determined using the dextrancoated charcoal (DCC) method. To date, this is the most commonly used technique for determination of steroid receptors, but it is not devoid of problems due both to tritiated ligands and to receptor molecules. Thus, the presence of endogenous hormone is a limitation of the precise estimation of receptor content in the tissue by the DCC method and despite widespread efforts to standardize it, assay results still vary [6]. The recent preparation of specific monoclonal antibodies to steroid receptor of human origin provides a new approach for the detection of receptor molecules whether or not they are occupied with hormone [7-91. This new technique has greater sensitivity and allows more accurate classification of receptor status than the DCC method

[61. The current article reports the results of a study assessing endometrial estradiol (E,) and progesterone (P) receptors (R) by the newly available monoclonal enzyme immunoassay (EIA) technique, in patients receiving gonadotrophinreleasing hormone analogue (GnRH-A) and gonadotrophins for IVF. Material and Methods Twenty-one IVF patients who did not have ET and were undergoing hormonal and histological evaluation of the luteal phase were included in the study. All the women had a history of normal menstrual cycles, The mean age of these women was 34 years (range: 26-39). Patients’ indications for IVF included the following diagnoses: tubal infertility (8 patients), male factor (5 patients), endometriosis (5 patients) and unexplained infertility (3 patients).

Characteristics of ovarian stimulation and luteal phase evaluation in these patients have been previously reported [lo]. Briefly, ovarian stimulation was carried out with follicle-stimulating hormone (FSH) (Fertinorm, Serono S.A., Spain) and human menopausal gonadotrophins (HMG) (Pergonal, Serono S.A., Spain) under pituitary suppression with buserelin (Suprefact, Hoechst A.G., Germany) (long protocol). Gvulation was induced with 5000 IU human chorionic gonadotrophin (HCG) (Physex, Leo S.A., Spain) and buserelin was discontinued on the day of HCG administration. Oocyte aspiration was performed by vaginal ultrasonography 35-36 h after HCG injection. The reasons that these women did not undergo an ET in their IVF cycle, and who thus qualified for this study and had an endometrial biopsy taken, were the following: failure to recover oocytes (7 patients), failure of fertilization after oocyte recovery (12 patients) and failure of cleavage 48 h after fertilization (2 patients). Additional doses of 5000, 2500 and 2500 IU HCG were given on days of follicular aspiration and 2 and 5 days later, respectively, to support the luteal phase in all patients. Luteal phase assessment included midluteal plasma P, E, and prolactin (PRL) radioimmunoassay measurements, and late secretory endometrial biopsies (within 3 days of anticipated menses and 11-13 days after basal body temperature-BBT-dip). For assessment of midluteal plasma P levels, three blood samples were obtained between the 5th and 10th postovulatory days according to BBT, and the highest value obtained was used to evaluate results as the best correspondent to the P peak. Plasma E, and PRL were quantitated simultaneously with the highest P sampling. Endometrial samples were obtained using a Semm suction-curette (Wisap, Germany) from the receptor-rich fundal area. One strip of tissue was immediately frozen in carbon dioxide ice and stored at -70pC until assayed (average wet weight 49.7 mg; range: 24102), and another was fixed for histologic examination. As previously reported [lo], 2 of 21 patients had out-of-phase endometrial biopsies according to the pathologic criteria of Noyes et al. [ll]. Thus, as control group we included the last

115

21 infertile normally menstruating women (mean age 32; range: 24-38 years) candidates for artificial insemination with donor sperm, who underwent the same luteal phase evaluation as did IVF patients and comprised 19 normal and 2 out-ofphase endometrial biopsies. The protocol was approved by the Investigation and Ethics Committee of our hospital. Receptor assays

Steroid receptor determinations were carried out by a new standardized EIA technique developed by Abott Laboratories (Chicago, IL) which has been recently shown to be useful for routine use in the clinical laboratory [9,12]. This solid phase EIA is based on the ‘sandwich’ principle. In the first step, beads coated with one monoclonal antibody to human E,P or PR are incubated with specimens (tissue cytosols), the appropriate standards and controls. During this incubation E,R or PR present in the specimens, standards and controls bind to the beads and unbound material is then removed by aspiration and washing. In the next step, a second monoclonal anti-E,R or anti-PR antibody conjugated with horseradish peroxidase is incubated with the beads to measure the amount of bound steroid receptor. After aspiration of the excess conjugate and washing, the beads are incubated with the enzyme substrate solution (hydrogen peroxide and O-phenylenediamine * 2HCl). The intensity of the color developed under this condition, which is proportional to the amount of E,R or PR in the sample, is read with a spectrophotometer at 492 nm. A straight line is obtained by plotting the absorbance of the standards versus their E,R or PR concentrations; the receptor concentration of the specimens and controls can easily be determined from this standard curve. The EIA method was performed as recommended by the manufacturers on the cytosols used for the DCC assay. All procedures were carried out at 0-4°C. The frozen endometrium was pulverized and homogenized in 5 vol. of Tris homogenization buffer (10 mM Tris-HC1/1.5 mM EDTA/lO% glycerol/O.5 mM dithiothreitol, pH 7.4). Cytosol was obtained by centrifugation of the homogenate for 60 min at 105,000 x g. The

TABLE I Luteal hormonal levels a and receptor concentrations ’ in the hvo groups studied

E, @g/ml) P (ng/ml) PRL (ng/ml) ER (fmol/mg) PR (fmoI/ng)

IVF cycles

Spontaneous cycles

P

1311k859 153+ 95 19+ 8 43 (16-199) 193 (5-744)

156 i 86 19+ 5 9rf. 5 79 (44-281) 202 (9-480)

< 0.001 < 0.001 < 0.001 < 0.001 0.2

a Mean+ SD. b Median (range).

protein concentration was determined and adjusted to 1-2 mg cytosol protein/ml according to the method of Bradford [13]. Then, aliquots of 100 ~1 were used to perform the E,R and PR EIA determination according to the principle described above. Significant differences in hormonal levels were determined by Student’s t-test. The Mann-Whitney’s U-test was used to compare receptor concentrations (fmol/mg cytosol protein). Remits Table I summarizes the hormonal findings and E,R and PR concentrations in endometrium in IVF and spontaneous control cycles. Plasma levels of E,, P and PRL were significantly higher after ovarian stimulation than in spontaneous cycles. Concentration of E,R in spontaneous cycles was significantly higher than in cycles after GnRH-A/ FSH/ HMG/ HCG. There was no significant difference between the two groups in PR in luteal endometrium. Discussion Implantation failure following ET is a major problem in IVF cycles. It is not known what role the female sex steroid receptors might play in implantation. However, their central position in the mechanism of hormone action suggests that they have an important influence on the endometrial state at the time of implantation, and it has been shown that early pregnancy endometrium is

116

characterized by a large concentration of PR, exceeding those of any period of the menstrual cycle [14]. IVF stimulated cycles are usually associated to supraphysiologic E, and P luteal phase levels [4,15,16]. Accordingly, our IVF patients showed steroid and PRL levels significantly higher than those found in spontaneous cycles. It has been suggested that these high levels of circulating sex steroids may cause profound modifications of endometrial receptor dynamics and alter the receptivity of the endometrium [1,4,16]. However, data in the literature on endometrial steroid receptors in IVF-stimulated cycles are sparse and controversial (Table II). Both E,R and PR levels in secretory endometrium have been found to be unchanged or decreased. These discrepancies may be explained in part by differences in ovarian stimulation treatment, the moment of the luteal phase studied, and the technique used for detection of receptors. Thus, E,R was found decreased in those studies in which clomiphene citrate was used for superovulation and endometria were sampled in the very early luteal phase [3,4]. It is possible that this drug may lead to decreased E,R at the time of embryo replacement due to its half-life [17], the long-lasting nuclear location of the E,R [181, and the dose-response relationship between the dose of clomiphene citrate and endometrial E,R concentrations in IVF patients [3]. Our patients received a GnRH-A until the day of HCG injection. Very recently, studies have been initiated to test GnRH-A in women with endometrial carcinoma and low-affinity GnRH binding sites have been

shown in neoplastic endometria [19]; so this implies that there may be some direct effects of analogues on E,R. Three of the four previous studies found a significant decrease in luteal phase endometrial PR concentrations in IVF stimulated cycles (Table II). It is well established that in the luteal phase, P exerts an antagonistic effect on its own receptor and that of E, [14]. Thus, the decrease in PR concentration has been attributed to an acute P effect [3,4]. Molina et al. [5] confirm the short-age of PR in endometrium after ovarian stimulation but emphasize that the reasons for low RP levels are uncertain. Levy et al. [14] noted that the early luteal phase reduction in PR was entirely due to the decrease in the cytosolic components as the nuclear receptor increased. Previous studies included in Table II used the DCC assay method which does not take into account the level of occupied steroid receptor; hence, the possibility that changes occur in the distribution of RP levels with increased nuclear RP and decreased cytoplasmic PR levels cannot be excluded. This may be in agreement with our results showing a lack of decrease in PR when measured by an EIA technique. Since the EIA method is based upon the recognition of the receptor molecule itself rather than receptorligand binding, it is independent of the presence of both endogenous hormones in the patients’ tissue and proteins that nonspecifically bind steroids. Recent studies using different techniques have indicated that the unoccupied steroid receptor as well as the occupied forms are nuclear proteins, and that the apparent cytoplasmic

TABLE II Luteal phase endometrial steroid receptors in IVF stimulated cycles Authors

No. of patients

Ovarian stimulation a

(year)

Day of endometrial sampling

Estrogen receptors

Progesterone receptors

Salat-Baroux et al. (1984) Riinnberg et al. (1985) Forman et al. (1989) Molina et al. (1989) Present study

26 10 12 7 21

CC + HMG CC + HMG CC + FSH + HMG CC + HMG GnRH-A + FSH + HMG

21 13-15 16 22-25 24-25

Unchanged Decreased Decreased Unchanged Decreased

Unchanged Decreased Decreased Decreased Unchanged

a CC = clomiphene citrate; HMG = human menopausal gonadotrophin; agonist.

FSH = follicle stimulating hormone; GnRH-A = GnRH

117

location of the unoccupied receptor represented extraction of loosely bound receptor from the nucleus into the cytosol when cells were homogenized for subcellular fractionation [20,211. Also immunocytochemistry with monoclonal antibodies has shown that the major portion of steroid receptor that contributes to immunocytochemical staining is that portion detectable in the cytosol after tissue homogenization [22]. The EIA method may therefore be more accurate than the DCC method. Thus, further studies with the monoclonal EIA method would be desirable to clarify the matter of steroid endometrial receptors in IVF cycles. Acknowledgement The authors thank Ms. Leonor her expert technical assistance.

Sanchez for

References 1 Macnamee MC, Edwards RG, Howles CM. The influence of stimulation regimes and luteal phase support on the outcome of IVF. Hum Reprod 1988;3(suppl):43s-52s. 2 Salat-Baroux J, Giacomini P, Cornet D. Study of the luteal phase after ovulation and in vitro fertilization. Fertil Steril 1984;41(suppl):16s. 3 Ronnberg L, Isotalo H, Kauppila A, Martikainen H, Vihko R. Clomiphene-induced changes in endometrial receptor kinetics on the day of ovum collection after ovarian stimulation: a study on cytosol and nuclear estrogen and progestin receptors and 17/3_hydroxysteroid dehydrogenase. Ann NY Acad Sci 1985;442:408-415. 4 Forman RG, Eychenne B, Nessmann C, Frydman R, Robel P. Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology. Fertil Steril 1989;51:310-316. 5 Molina R, Castilla JA, Vergara F, Perez M, Garrido F, Herruzo AJ. Luteal cytoplasmic estradiol and progesterone receptors in human endometrium: in vitro fertilization and normal cycles. Fertil Steril 1989;51:976-979. Thorpe SM. Monoclonal antibody technique for detection of estrogen receptors in human breast cancer: greater sensitivity and more accurate classification of receptor status than the dextran-coated charcoal method. Cancer Res 1987;47:6572-6575. Greene GL, Jensen EV. Monoclonal antibodies as probes for estrogen receptor detection and characterization. J Steroid Biochem 1982;16:353-359. Hendler FJ, Yuan D. Relationship of monoclonal antibody binding to estrogen and progesterone receptor content in breast cancer. Cancer Res 1985;45:421-429.

9 Goussard J, Lechevrel Ch, Martin PM, Roussel G. Comparison of monoclonal antibodies and tritiated ligands for estrogen receptors assays in 241 breast cancer cytosols. Cancer Res 1986;46(suppll:4282s-4287s. 10 Balasch J, Jove I, Marquez M, Vanrell JA. Hormonal and histological evaluation of the luteal phase after combined GnRH-agonist/gonadotrophin treatment for superovulation and luteal phase support in IVF. Hum Reprod 1991;6:914-917. 11 Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:3-25. 12 Leclercq G, Bojar H, Goussard J, Nicholson RI, Pichon MF, Piffanelli A. Pusette A, Thorpe S, Lonsdorfer M. Abbot monoclonal enzyme immunoassay measurement of estrogen receptors in human breast cancer: a European multicenter study. Cancer Res 1986;46(suppl):4233s-4236s. 13 Bradford MM. A rapid and sensitive method for the quantitation of pg quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248254. 14 Levy C, Robe1 P, Gautray JP, De Brux J, Verma U, Descomps 8, Baulieu EE, Eychenne B. Estradiol and progesterone receptors in human endometrium: normal and abnormal menstrual cycles and early pregnancy. Am J Obstet Gynecol 1980;136:646-651. 15 Cohen JJ, Debache C, Pigeau F, Mandelbaum J, Plachot M, de Brux J. Sequential use of clomiphene citrate, human menopausal gonadotrophin, and human chorionic gonadotrophin in human in vitro fertilization. II. Study of luteal phase adequacy following aspiration of the preovulatory follicles. Fertil Steril 1984;42:360-365. 16 Vargyas J, Kletzky 0, Marrs RP. The effect of laparoscopic follicular aspiration on ovarian steroidogenesis during the early preimplantation period. Fertil Steril 1986;45: 221-22s. Schulz KD, Holzel F, Bettendorf G. The uptake and distribution of 14Cclomiphene citrate in different organs of newborn females guinea pigs. Acta Endocrinol 1971;68:605-613. Clark JH, Anderson JN, Peck EJ. Estrogen receptor-antiestrogen complex: Atypical binding by uterine nuclei and effects on uterine growth. Steroids 1973;22:707-718. Kiesel L. The use of GnRH analogues in the treatment of cancer of the breast and the reproductive organs. In Lunenfeld B and Insler V (eds.), The Current Status of GnRH Analogues. The Parthenon Publishing Group, Casterton Hall, Carnforth, Lanes, 1991;77-88. 20 Welshons WV, Cornier EM, Wolf MF, Williams PO, Jordan VC. Estrogen receptor distribution in enucleated breast cancer cell lines. Endocrinology 1988;122:23792386. 21 King WJ, Greene GL. Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature 1984;307:745-747. 22 West NB, McClellan MC, Sternfeld MD, Brenner RM. Immunocytochemistty versus binding assays of the estrogen receptor in the reproductive tract of spayed and hormone-treated macaques. Endocrinology 1987;121: 1789-1800.