Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology

FERTILITY AND STERILITY

Vol. 51, No.2, February 1989

Copyright© 1989 The American Fertility Society

Printed in U.S.A.

Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology Robert G. Forman, M.D. Bernard Eychenne, M.Sc. Catherine Nessmann, M.D.

Rime Frydman, M.D. Paul Robel, M.D.

Hopital Antoine Beclere, Clamart, and Institut National de la Sante et de la Recherche Medicate (INSERM) Unite 133, Bicetre Cedex, France

The corpus luteum-endometrial unit was investigated in in vitro fertilization (IVF) cycles using endocrine, morphologic, and biochemical measurements on the day normally scheduled for embryo transfer (day 16), in 12 stimulated and 4 natural cycles. Advanced endometrial histologic maturity was recorded in 9 of the 12 stimulated cycles. No in-phase endometria were seen when the preovulatory plasma estradiol (E 2 ) was >500 pg/ml or the day 16 plasma progesterone (P) > 10 ng/ml in natural or stimulated cycles. Significant negative correlations were noted between both preovulatory E 2 and day 16 P and the concentration of cytosolic progesterone receptor (PRe). Advanced endometrial maturity tended to be associated with low concentrations of PRe. Regardless of endometrial maturity, the natural cycle was characterized by low cytosolic E 2 receptors (ERe) and high PRe, whereas the concentration of both receptors was usually greatly reduced in stimulated cycles. It is concluded that the advanced endometrial maturation observed in stimulated IVF cycles is a consequence of the production of supraphysiologic levels of sex steroids by the corpus luteum that cause profound modifications of endometrial receptor dynamics. Fertil Steril51:310, 1989

The assessment of the luteal phase has proven to be one of the most controversial areas in reproductive medicine. Over the years, several methods have been proposed, including analysis of basal body temperature, serial measurements of circulating progesterone (P) concentration, and morphologic examination of the endometrium. 1- 3 In in vitro fertilization (IVF) cycles, the situation is further complicated by the multitude of variables associated with this form of treatment. The most Received May 6, 1988; revised and accepted September 29, 1988. * See de Gynecologie-Obstetrique, Hopital Antoine Beclere. t Reprint requests and present address: Robert G. Forman, M.D., Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, England. INSERM Unite 133, Lab Hormones. § See Anatomopathologie, Hopital Antoine Beclere.

*

310

Forman et al. Assessing the early luteal phase in IVF

accurate method for monitoring luteal phase quality after IVF remains the pregnancy rate after embryo transfer (ET). This is a very insensitive measure, however, as large numbers of pregnancies are required to achieve statistically significant results. 4 Unfortunately, the interpretation of more easily quantifiable markers of luteal phase physiology is difficult. In one study, the endometrium at the time of ET was reported as being at an advanced stage of maturational development, 5 but it is uncertain whether this has a positive or negative influence on implantation. Furthermore, although plasma P concentrations are sometimes used to assess the luteal phase in IVF, we have recently reported that pregnancies can occur with equal frequency across a wide range of luteal phase P values. 6 It has been appreciated for several years that circulating steroid hormones can only act after binding to their specific receptors in the target orFertility and Sterility

gan(s). 7 Endometrial receptors must therefore play a pivotal role in mediating the endometrial response to corpus luteum steroids. This study was proposed to examine these relationships in the context of human IVF, and in particular, to adopt a multidisciplinary approach to studying the corpus luteum-endometrial unit on the day normally scheduled for ET. MATERIALS AND METHODS Patients and Stimulation

Twelve consecutive patients undergoing IVF in whom oocytes were obtained, but fertilization was unsuccessful because of sperm factors, were ineluded in the trial after counseling and· informed consent. All patients were stimulated according to the protocol of programmed oocyte retrieval as previously described. 8 Briefly, this included a preliminary phase of pretreatment with a progestogen, norethisterone (N) 10 mg/day (Norluten, Smith, Kline and French, Paris, France), or a contraceptive pill, Triella (T), containing ethinylestradiol and norethisterone (Cilag, Paris, France). This pretreatment was discontinued on a preselected day that was arbitrarily designated day 0, and the following day was day 1 of the IVF cycle. A fixed schedule ovulation stimulation regimen then was administered, and ovulation systematically induced on day 11 using 5000 units of human chorionic gonadotropin (hCG; Organon, St Denis, France). Three different fixed stimulation regimens were given during the period of this study: 1. FCH2: Follicle-stimulating hormone (FSH, Metrodine, Serono, Levallois, France) 300 IU a day on days 0 and 1, Clomiphene citrate (CC) 100 mg days 2 to 6 and human menopausal gonadotropin (hMG; Neopergonal, Serono, Levallois, France) 150 IU FSH/150 IU luteinizing hormone (LH) on days 2, 4, 6, 8, and 10. 2. CH3: Clomiphene citrate 100 mg days 2 to 6 and hMG 150 IU FSH/150 IU LH on days 2 to 10 inclusive. 3. H4: hMG 225 IU FSH/225 IU LH on days 2, 3, and 4; hMG 150 IU FSH/150 IU LH on days 5 to 10 inclusive. Dydrogesterone (Duphaston, Duphar, Villeurbanne, France), 30 mg a day, was routinely commenced in all patients on the evening of oocyte retrieval, in accordance with our policy for luteal phase support. In addition to these 12 stimulated IVF cycles, 4 Vol. 51, No.2, February 1989

natural cycles were included in the study. These were patients scheduled for transfer of thawed frozen embryos in a spontaneous menstrual cycle. Daily monitoring of plasma estradiol (E 2 ) and LH was begun at 8 A.M. on cycle day 9 and continued until a spontaneous LH surge commenced in plasma, defined as an LH concentration 180% that of the mean of the previous values. Embryo transfer was planned for the 4th day after LH rise. In the four patients included in this study, the embryo(s) did not survive the thawing procedure. Contrary to the programmed oocyte retrieval cycles, dydrogesterone was not administered in the days before endometrial biopsy. Collection of Samples

In the 12 IVF patients, plasma was collected at the time of ovulation induction with hCG and analyzed for E 2 and LH. The remaining plasma was stored at -20"C. On the day normally scheduled for ET (48 hours after oocyte retrieval), a further 10 ml of blood was collected and, after separation, the plasma was frozen at -20"C. Estradiol and progesterone (P) were subsequently measured in both samples in the same assay, using commercially available radioimmunoassay (RIA) kits. On the day scheduled for ET (normalized to day 16), an endometrial biopsy was obtained from the receptor-rich fundal area in all16 patients, using a Novak curette. No anesthesia was required, and all patients tolerated the procedure with minimal discomfort. The biopsy specimen was immediately divided into two approximately equal portions, one of which was placed in liquid nitrogen for receptor studies and the other preserved in Bouin's medium containing formalin. These latter specimens were histologically dated by a pathologist (C.N.) as described below. The pathologist was unaware of the cycle day or treatment. The frozen biopsies were stored until four or five such specimens were obtained. These were then assayed in a batch for cytosolic and nuclear E 2 and P receptor concentrations as described below. Measurement of Endometrial Estrogen and Progesterone Receptor Concentrations

The assay of estrogen receptor (ER) and progesterone receptor (PR) was based on the method described by Bayard et al. 9 and modified by Levy et al. 10 The endometrium was homogenized and nuclear (800 X g pellet) and cytoplasmic (105,000 X g supernatant) preparations were obtained. For the assay of cytosolic ER (ERe), triple aliForman et al. Assessing the early luteal phase in IVF

311

quots of 90 p.l of cytosol were incubated with 50 nmol of 3H-E 2 and 1 p.mol of dihydrotestosterone (DHT) with or without a 100-fold excess of nonlabeled E 2 for the measurement of total and nonspecific binding, respectively. The DHT was added to selectively inhibit the binding of 3H-E 2 to sex-hormone-binding globulin. For the assay of cytosolic PR (PRe), triple aliquots of 90 p.l of charcoal-stripped cytosol were incubated with 100 nmol of 3H-P and 5 p.mol of cortisol with or without a 100-fold excess of nonlabeled P for the measurement of total and nonspecific binding, respectively. The cortisol was added to selectively inhibit the binding of 3 H-P to corticosterone-binding globulin. The incubations were performed for 3 hours at 3o·c for E 2 and o·c for P. Following the incubation, the free and loosely bound hormone was removed using charcoal dextran buffer, and after centrifugation, 100 p.l aliquots of supernatant were removed for measurement of radioactivity bound to the cytosolic components. The nuclear receptor assay was performed using the glass fiber filter system described by Levy et al. 10 Triple aliquots of 90 p.l of nuclear suspension were incubated in the same conditions described for the cytoplasmic assays. The nuclear membranes were dissociated using buffer containing 7% ethanol and triton-X100. Bound radioactivity was measured in a liquid scintillation counter (Packard Tricarb Series 4600, Chicago, IL). The counting efficiency was approximately 50% for tritiated steroid in aqueous medium. Deoxyribonucleic acid (DNA) was measured using the technique described by Groyer and Robel, 11 involving the dissociation of nucleoprotein complex by heparin followed by measurement of DNA using mithramycin fluorescence. Receptor concentrations for E 2 and P are expressed in pmol receptor/mg DNA. Hormone Assays

Estradiol and P were measured in plasma using commercially available radioimmunoassay kits (BioMerieux, Charbonnieres-les-Bains, Lyon, France). The intra-assay variability for E 2 ranged from 20.3% at a mean E 2 concentration of 20 pg/ml to 3.6% at 4000 pg/ml. The lower limit of assay sensitivity was taken as 60 pg/ml. Interassay variability was 8.2% at a mean E 2 of 230 pg/ml, and 6.4% at 1851 pg/ml. The intra-assay variability for P was 14.0% at a mean P concentration of 0.22 ng/ml and 6.3% at 39.40 ng/ml. Interassay variability was 312

Forman et al. Assessing the early luteal phase in IVF

7.4% at a mean P concentration of 1.65 ng/ml, and 6. 7% at 35.20 ng/ml. The lower limit of assay sensitivity was 0.1 ng/ml. Luteinizing hormone was measured by immunoradiometric assay (Bio-Merieux, Charbonnieres-les-Bains, Lyon, France). The intra-assay variability was 2.0% at a mean LH concentration of9.8 IU/1 and 1.8% at 40.8 IU/l. lnterassay variability was 8.4% at a mean LH concentration of 3.8 IU/1 and 3.4% at 33.8 IU/1. The lower limit of assay sensitivity was 1 IU /l. Endometrial Dating

Paraffin embedded sections were cut at 5 p.m intervals and stained with hematoxylin and eosin and periodic acid-Schiff. The endometrium was dated according to Noyes criteria based on glandular, stromal, and vascular development. 12 RESULTS

Table 1 summarizes the results in the 16 cycles. All receptor assays were performed in triplicate. Receptor concentrations were not ascertained for patients 1, 2, and 3. In several patients, there was insufficient material to perform the nuclear receptor assays for E 2 and/or P (wet weight of endometrial sample <40 mg or <50 p.g of DNA in the nuclear fraction). These cases are indicated in Table 1. Relationship of Follicular to Luteal Phase Hormone Concentrations

There was a good correlation between preovulatory plasma E 2 (estimated from plasma obtained at the time of hCG administration) and the plasma concentration of E 2 and P on day 16 (the day of scheduled ET). The correlation coefficients (r) for E 2 and P were 0.645 (P < 0.02) and 0. 790 (P < 0.01), respectively. The regression equation for E 2 on day 16 was y = -15 - 0.449x and for P, y = 0.466 - 0.018x, where x is the preovulatory plasma E 2 concentration. Steroid Hormone Receptor Concentrations

The correlation between plasma hormone and receptor concentrations is shown in Table 2. Significant negative correlations were observed between preovulatory E 2 and PRe (r = -0.649, P < 0.02) and between plasma P on day 16 and PRe (r = -0.684, P < 0.02). There was no correlation between any of the hormonal parameters and cytosolic estrogen receptor (ERe) or total ER and PR Fertility and Sterility

Table 1

Summary of Histological, Hormone and Receptor Results in the Luteal Phase Plasma hormone concentration Preovulatory

Patient

Treatment

Histology date

5 6 7 8 1 3 10 2 4 9 12 11 15 16 13 14

Natural Natural Natural Natural TFCH2 TFCH2 TFCH2 NFCH2 NFCH2 NFCH2 NFCH2 NCH3 NCH3 NCH3 NH4 NH4

17 15 16 17 16 17 15 17 16 17 17 17 17 17 17

E2

p

pg/ml

550 385 370 95 805 415 700 450 970 345 710 730 1555 800 1165 1755

Receptor concentration

LH

E2

p

ng/ml

IU/1

pg/ml

ng/ml

0.7 0.4 0.4 0.3 <0.1 0.1 0.1 0.2 0.1 <0.1 0.1 0.1 0.2 0.1 0.1 0.1

57 19.5 11 17 2.2 2.8 1.5 3.6 1.4 2.0 3.4 6.0 4.8 2.0 4.5 3.1

110 115 100 125

2.3 1.8 3 0.2

7.58 4.42 7.64 2.57

455 65 165 165 235 295 590

10.2 10.5 20 10 15 25.8

2.41

1290 555

21.8 23 25.5

PRe

PRn

ERe

ERn

pmol/~ng DNA

0 7.01 3.10 4.46 0.31 0.85 0.62 1.15

0 3.90

xb

0.40

xb xb xb 7.30 5.20

xb

2.21 1.53 2.09

0.36 0.63 2.33 0 5.94 1.79 7.04 1.87 0.43 0.24 0.73 0.34 0

xb xb xb 0

xb xb 14.6 0.89 3.32

xb

0.73 0.61 1.09

bX, Insufficient tissue to perform assay.

• -,No result available.

(cytosolic plus nuclear receptor). There was a significant positive correlation between plasma P on day 16 and the ratio of nuclear to cytosolic PR (PRn/PRc) (r = 0.758, P < 0.05). Endometrial Dating

The results of the histologic examination of the endometrial biopsies are shown in Table 1. Biopsies dated 15 and 16 were grouped together and are designated "in phase." Specimens dated day 17 are referred to as "advanced." All dates are relative to ovulation normalized to day 14. Figure 1 relates endometrial histology to preovulatory E 2 and day 16 P. All the in phase endometria were associated with low E 2 • Advanced endometrium was only found in cycles with preovulatory E 2 concentrations >500 pg/ml. Although cycles programmed with CH3 and H4 (daily hMG) always Table 2

Day16

produced advanced endometria, FCH2 cycles were associated with both endometrial patterns. Advanced endometrial maturity could be seen in association with a wide range of day 16 P concentrations, although no in-phase endometria occurred with a P concentration > 10 ng/ml. Figure 2 compares the histologic findings to the concentrations of PRe and ERe on day 16. There was a tendency for advanced endometria to be associated with lower receptor concentrations, notably for PRe. It is interesting to observe that, regardless of endometrial maturity, the natural cycles were associated with high PRe and low ERe, whereas for cycles with daily hMG (CH3 and H4), the concentrations of both receptors were very low. Influence of Treatment

The treatments used for follicular stimulation can be conveniently grouped into three categories:

Correlation Coefficients (r) Between Hormone and Receptor Concentrations Concentration of Receptor

Concentration of hormone

ERcc

PRcc

PRtc

ERtc

-0.327 (8) -0.693 (7) -0.173 (8)

-0.361 (7) -0.249 (6) -0.257 (7)

pmol/mgDNA

Preovulatory E 2 (pg/ml) Day 16 E 2 (pg/ml) Day 16 P (ng/ml) • Significant correlation P < 0.02. b Significant correlation P < 0.05. Vol. 51, No.2, February 1989

-0.649 (13)" -0.555 (12) -0.621 (12)b

-0.326 (13) -0.090 (12) -0.178 (12) c

Number of cycles analyzed is indicated in parenthesis.

Forman et al. Assessing the early luteal phase in IVF

313

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Figure 1 Maturational age of endometrium related to plasma concentrations of E 2 and P. Open circles represent natural cycles, cross-hatched circles represent stimulated FCH2 cycles (alternate day hMG), and closed circles represent stimulated CH3/ H4 cycles (daily hMG).

firms the work of Garcia et al. 5 in IVF cycles. These authors indicated that the maturity of the endometrium was related to the serum progesterone concentration at the time of biopsy. The present work extends these observations, and shows that both the endometrial maturity and the P concentration at the time of biopsy are directly related to the preovulatory E 2, and therefore, the response to stimulation. Furthermore, the morphologic changes seen in stimulated cycles are associated with marked alterations of endometrial steroid hormone receptors compared with spontaneous cycles. The literature on endometrial steroid hormone receptors in stimulated cycles is sparse. Investigators have assessed endometrial receptor kinetics in the natural menstrual eye le m h umans91314 · · an d primates. 15 Although there is great variability in the results of these studies, there is general agreement that bothER and PR increase during the fol. . duc t"1ve act"10n of E 2· 13•15 hcular p h ase due toth em In the luteal phase, progesterone exerts an antagonistic effect on its own receptor arid that of E 2.16•17 The present study permitted a detailed analysis of the hormonal influence on receptor levels on 1 particular day of the luteal phase. The following schema can be proposed to explain the results: Ovarian stimulation causes supraphysiologic levels of E 2 at the end of the follicular phase, leading to the induction of a large number of E 2 and P receptors. Following administration of the ovulation inducing stimulus, hCG, the corpus luteum produces supraphysiologic amounts of P (the degree of P production correlating with the preovulatory E2 concentration). Progesterone binds to the available PR in the endometrium to initiate the stromal and glandular changes characteristic of the secretory phase. In stimulated cycles, high concentrations of P are prematurely obtained compared with the natural cycle and this, associated with an abun0

natural cycles, programmed cycles stimulated with hMG on alternate days (FCH2), and programmed cycles in which daily hMG was administered (CH3 and H4). Although significance testing was not performed because of the small sample sizes, it appeared as if there was a progressive increase in follicular steroidogenesis, as evidenced by the preovulatory E 2 concentration, as the dose of hMG in the stimulation was increased (Table 3). There was a concomitant increase in luteal phase concentrations of E 2and P. When comparing hormone receptor concentrations with treatment groups, there was a clear trend for a decrease in PRe as the hMG dose increased (Table 3). There was no apparent relationship between ERe and the treatment group. DISCUSSION

This study investigated the corpus luteum-endometrial unit in IVF cycles on the day on which embryos would normally be transferred into the uterus. The data demonstrates the profound hormonal, cellular, and molecular modifications resulting from stimulated folliculogenesis. A significant correlation was observed between the steroidogenic response to stimulation, assessed by the preovulatory E 2 concentration, and the corpus luteum production of E 2 and P. This agrees with our previous observations in over 200 cycles. 6 The concentration of E 2 at the time of ovulation induction with hMG was related to the dose of hMG administered in the follicular stimulation regimen. The high circulating steroid hormone concentrations on the day of ET were associated with advanced endometrial histologic maturity. This con314

Forman et al. Assessing the early luteal phase in IVF

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10.0

7.5

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7.5

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Figure 2 Maturational age of endometrium related to the concentration of cytosolic E 2 and P receptor. Open circles represent natural cycles, cross-hatched circles represent stimulated FCH2 cycles (alternate day hMG), and closed circles represent stimulated CH3/H4 cycles (daily hMG). Fertility and Sterility

Table 3

Plasma Hormone and Cytosolic Receptor Concentrations in the 3 Treatment Groups 4 Stimulated cycles Alternate day hMG (FCH2)b

Natural cycleb Preovulatory E 2 (pg/ml) Day 16 E 2 (pg/ml) Day 16 P (ng/ml) PRe (pmol/mg DNA) ERe (pmol/mg DNA)

a Results are expressed as means ± standard error of the mean.

dance of PR, combines to induce an advancement of endometrial maturity. The elevated P concentration also suppresses the production of ER and PR and induces the synthesis of the E 2 dehydrogenase enzyme complex, which acts in the endometrium to promote the local conversion of E 2 to the less biologically active estrone. 18 As a consequence, circulating E 2 is prevented from inducing new ER and PR synthesis, and P acts unopposed to downregulate both receptors. Levy et al. 13 noted that the early luteal phase reduction in progesterone receptor was entirely due to the decrease in the cytosolic component as the nuclear receptor increased. The nuclear transfer of receptor following hormone binding can be deduced from our data by the strong positive correlation between plasma P and the ratio of PRn/PRc. In the traditional two-step theory of receptor action, the cytosolic receptor is the protein in its native form, which, after binding to the ligand, translocates to the nucleus and initiates gene transcription. 7 Evidence accumulating over the past 3 years, however, has seriously questioned the validity of this two-step theory. In particular, the results of experiments using cell enucleation techniques 19 and immunocytochemical studies with monoclonal antibodies raised against ER and PR20·21 show that the majority of receptor is located within the nu~ cleus even in its native form. The majority opinion at present holds that it is possible to reconcile traditional and new interpretations of receptor action. The unoccupied receptor normally is located in the nucleus, although it is suggested that this protein, which was recovered in the cytosolic fraction of a tissue homogenate, represents a receptor that is loosely bound to nuclear components. Binding of the steroid to the receptor results in tighter nuclear association. 22 The more recent theories are still compatible with the two-step theory in that inactive native receptor is activated after binding to the steroid and that the steroid receptor complexes Vol. 51, No.2, February 1989

628 ± 217 ± 13.1 ± 3.1 ± 4.2 ±

350 ±94 (4) 113 ± 5.2 (4) 1.8 ± 0.6 (4) 5.6 ± 1.3 (4) 0.8 ± 0.5 (4) b

87 (7) 65 (5) 1.95 (5) 1.5 (4) 1.4 (4)

DailyhMG (CH3 and H4)b 1201 ± 202 (5) 682 ± 213 (4) 24 ± 6.8 (4) 1.5 ± 0.8 (5) 0.4 ± 0.1 (4)

Number of cycles analyzed are indicated in parenthesis.

regulate nuclear function. In fact, using immunocytochemical techniques, progesterone-induced down-regulation of ER and PR in the luteal phase of normal women has been recently confirmed.23 We feel that a multidisciplinary approach to the investigation of the luteal phase may provide a more accurate assessment of corpus luteum-endometrial function than isolated hormonal or histologic measurements. Receptor disturbances have been noted in patients with luteal phase inadequacy.13•24 The fertility implications of a precociously mature endometrium are uncertain. It has been hypothesized that the advanced endometrial maturity in IVF cycles might be beneficial5 as the embryo is transferred into the uterus 24 hours in advance of its arrival after in vivo conception. In clinical practice, however, no significant difference in pregnancy rates was recorded after the transfer of a single embryo in natural or stimulated cycles. 25 Ultrastructural observations have confirmed the advanced endometrial maturity in stimulated cycles compared with the natural menstrual cycle. In particular, scanning electron microscopy in humans has shown the formation of apical protrusions or pinopodes, which in rats are associated with the period of uterine receptivity to the embryos, 6 days after ovulation in natural cycles and 2 days after ovulation in stimulated cycles. 26 In stimulated cycles in humans, pinopode formation was greatly reduced 6 days after ovulation. One of the unavoidable methologic problems with this study was that patients in the stimulated IVF cycles received exogenous progestogens before biopsy whereas this did not occur in the natural cycles. Although it is conceivable that this may have biased the results, there are several reasons to doubt this. Dydrogesterone does not modify circulating P levels4 and, as the spatial configuration of the molecule and its metabolites (9{j; lOa-pregna4-6 diene) is so different from that of progesterone, Forman et al. Assessing the early luteal phase in IVF

315

immunologic cross-reactivity in the RIA can be excluded.27 The possibility of dydrogesterone itself provoking the advancement of endometrial maturity also must be considered. We think this is unlikely, as dydrogesterone was administered in all of the stimulated cycles, but cycles remained in phase when the endocrine response to stimulation was poor. However, it is not possible to totally exclude an adjuvant effect of exogenous progestogen administration. In conclusion, this study investigated endocrine, morphologic, and biochemical parameters to assess the luteal phase in IVF cycles. The data confirms that endometrial maturity is advanced on the day of ET in stimulated cycles, and that this is related to the magnitude of the respose to stimulation. Study of endometrial steroid hormone receptors demonstrated that P down-regulates the available PR. More detailed studies of the luteal phase using a multidisciplinary approach may be useful in identifying the period of uterine receptivity and help to define the "implantation window."

11.

12. 13.

14.

15.

16.

17.

18.

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