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Endocrinology Vol. 139, No. 8 3512-3519
Copyright © 1998 by The Endocrine Society

ARTICLES

17ß-Estradiol Potentiates the Stimulatory Effects of Progesterone on Cadherin-11 Expression in Cultured Human Endometrial Stromal Cells1

George T. C. Chen, Spiro Getsios and Colin D. MacCalman

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Dr. Colin D. MacCalman, Department of Obstetrics and Gynecology, Faculty of Medicine, University of British Columbia, 2H30–4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: colinmac{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cadherin-11 (cad-11) is a novel member of the cadherin gene superfamily of calcium-dependent cell adhesion molecules. To date, the factors capable of regulating this cell adhesion molecule remain poorly characterized. We have recently determined that cad-11 expression in the human endometrium is tightly regulated during the menstrual cycle. The spatiotemporal expression of cad-11 in the stromal cells of the human endometrium during the menstrual cycle suggests that gonadal steroids regulate the expression of this endometrial cell adhesion molecule. In view of these observations, we have examined the ability of progestins, estrogens, and androgens, alone or in combination, to regulate cad-11 expression in isolated human endometrial stromal cells using Northern and Western blot analyses. In these studies, we have determined that progesterone, but not 17ß-estradiol or dihydrotestosterone, is capable of regulating cad-11 messenger RNA and protein expression levels in isolated endometrial stromal cells. In addition, 17ß-estradiol, but not dihydrotestosterone, was capable of potentiating the stimulatory effects of progesterone in a dose-dependent manner. Taken together, these observations suggest that both 17ß-estradiol and progesterone are required for maximal cad-11 expression in human endometrial stromal cells in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADAL steroids are key regulators of the cyclic remodeling processes that occur in the human endometrium during the menstrual cycle. It has been well established that 17ß-estradiol (E2) promotes cellular proliferation in the stroma and glandular epithelium of the endometrium (1, 2). Progesterone (P4) in turn, is believed to act upon the E2-primed endometrium, thereby initiating glandular secretion and the terminal differentiation of stromal cells into decidual cells (1). However, recent studies also suggest that the nonaromatizable androgen, dihydrotestosterone (DHT), is capable of mediating decidualization in human endometrial stromal cells in vitro (3, 4). The molecular and cellular mechanisms by which gonadal steroids regulate the differentiation of endometrial stromal cells into decidual cells remain poorly understood.

We have recently determined that cadherin-11 (cad-11), a novel member of the gene superfamily of calcium-dependent cell adhesion molecules known as the cadherins, is spatiotemporally expressed in the human endometrium (5). In particular, cad-11 is first detected in the endometrial stroma during the late secretory phase of the menstrual cycle when these cells are beginning to undergo decidualization. Maximum cad-11 levels are expressed in the decidua of early pregnancy (5, 6). Taken together, these observations suggest that cad-11 expression is associated with the terminal differentiation of endometrial stromal cells into decidual cells. To date, the factors capable of regulating cad-11 expression have not been identified.

The spatiotemporal expression of cad-11 in endometrial stromal cells during the menstrual cycle suggests that this cell adhesion molecule is hormonally regulated. We have previously demonstrated that gonadal steroids are key regulators of cadherin expression in murine tissues. For example, P4 and E2 were capable of increasing E-cadherin (E-cad) messenger RNA (mRNA) levels in the immature mouse uterus (7), whereas only E2 (but not P4, testosterone, or DHT) increased E-cad mRNA levels in the immature mouse ovary (8). Similarly, only E2 was capable of stimulating N-cadherin (N-cad) mRNA levels in the immature mouse ovary and testis in vivo (9). These observations have led us to hypothesize that the ability of steroids to regulate the developmental processes that occur in reproductive tissues may be mediated at least in part by their ability to modulate cadherin expression.

Previous studies have demonstrated that gonadal steroids can induce cellular differentiation in endometrial stromal cells in vitro (3, 10, 11). In view of these observations, we have examined the ability of estrogens, progestins, and androgens, alone or in combination, to regulate cad-11 expression in isolated endometrial stromal cells using Northern and Western blot analyses. In these studies, we have determined that P4, but not E2 or DHT, is capable of regulating cad-11 expression in human endometrial stromal cells. However, maximum levels of cad-11 mRNA and protein levels were detected in stromal cells cultured in the presence of E2 and P4, suggesting that E2 enhances the P4-mediated increase in stromal cad-11 expression. In contrast, DHT was not capable of potentiating the stimulatory effects of P4 on stromal cad-11 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues
Endometrial tissue biopsy specimens (n = 15) were obtained from women of reproductive age in accordance with a protocol for the use of human tissues approved by the committee for ethical review of research involving human subjects, University of British Columbia. All patients had normal menstrual cycles and had not received hormones for at least 3 months before the collection of the tissues. The stage of the menstrual cycle was determined by the last menses and was confirmed by histological evaluation according to the criteria of Noyes et al. (1). Tissues used in this study were obtained during the midsecretory phase of the menstrual cycle.

Cell preparation and culture
The endometrial stromal cells were separated from the glandular epithelium by enzymatic digestion and mechanical dissociation using a protocol modified from that reported by Shiokawa et al. (12). Briefly, the endometrial biopsy specimens were minced and subjected to 0.1% collagenase (type IA, Sigma Chemical Co., St. Louis, MO) and 0.1% hyaluronidase (type I-S, Sigma Chemical Co.) digestion in a shaking water bath at 37 C for 1 h. The cell digest was then passed through a nylon sieve (38 µm). The isolated glands were retained on the sieve, and the eluate containing the stromal cells was collected in a 50-ml tube. The stromal cells were pelleted by centrifugation at 800 x g for 10 min at room temperature. The cell pellet was washed once in phenol red-free DMEM containing 10% charcoal-stripped FBS before being resuspended and plated in phenol red-free DMEM containing 25 mM glucose, 25 mM HEPES, 1% (wt/vol) L-glutamine, antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone) and supplemented with 10% charcoal-stripped FBS. The culture medium was replaced 30 min after plating to reduce epithelial cell contamination. The purity of the cell cultures was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin, and factor VIII (data not shown). These cellular markers have been previously used to determine the purity of human endometrial cell cultures (11). As defined by these criteria, the endometrial stromal cell cultures used in these studies contained less than 1% of endometrial epithelial or vascular cells.

Hormone treatments
The stromal cells (passage 2) were grown to confluence, washed with PBS, and cultured in phenol red-free DMEM supplemented with 10% charcoal-stripped FBS and containing P4 (1 µM), E2 (30 nM), DHT (0.1 µM) or vehicle (0.1% ethanol). The concentrations of hormones used in these experiments were selected on the basis of previous studies (4, 11, 13). The cells were cultured in the presence or absence of the steroids for 0–96 h before being harvested for Northern or Western blot analysis. In these and the following studies, the culture medium was changed every 24 h.

To determine whether a combination of steroids was required for maximal cad-11 expression in endometrial stromal cells, the cells were cultured in the presence of P4 (1 µM) plus E2 (30 nM) or P4 (1 µM) plus DHT (0.1 µM) for 0–96 h before being harvested for Northern or Western blot analysis.

Finally, to determine whether the ability of E2 to potentiate the effects of P4 on stromal cad-11 expression was dose dependent, the cells were cultured in the presence of vehicle (0.1% ethanol), E2 (30 nM), P4 (1 µM) or P4 (1 µM) plus varying doses of E2 (0.5–100 nM) for 96 h. The cells were then harvested for Northern or Western blot analysis.

Northern blot analysis
Total RNA was prepared from the cultured stromal cells by the phenol-chloroform method of Chomczynski and Sacchi (14). The RNA species were resolved by electrophoresis in 1% agarose gels containing 3.7% formaldehyde. Approximately 20 µg total RNA were loaded per lane. The fractionated RNA species were then transferred onto charged nylon membranes.

The Northern blots were hybridized with a radiolabeled complementary DNA probe specific for human cad-11 according to the methods of MacCalman et al. (15). The blots were then washed twice with 2 x SSPE (20 x SSPE consists of 0.2 M sodium phosphate, pH 7.4, containing 25 mM EDTA and 3 M NaCl) at room temperature, twice with 2 x SSPE containing 1% SDS at 55 C, and twice with 0.2 x SSPE at room temperature. To standardize the amounts of total RNA in each lane, the blots were probed with a radiolabeled synthetic oligonucleotide specific for 18S ribosomal RNA (rRNA) as described by MacCalman et al. (15). The blots were again subjected to autoradiography to detect the hybridization of the radiolabeled probe to the 18S rRNA. The autoradiograms were then scanned using an LKB laser densitometer (LKB, Rockville, MD). The absorbance values obtained for the cad-11 mRNA transcript were normalized relative to the corresponding 18S rRNA absorbance value.

Western blot analysis
For Western blot analysis, the stromal cells were washed with PBS and incubated in 100 µl chilled cell lysis buffer (Tris-HCl, pH 7.5, containing 0.5% Nonidet P-40, 0.5 mM CaCl2, and 1.0 mM PMSF) at 4 C for 30 min on a rocking platform. The cell lysates were centrifuged at 10,000 x g for 20 min, and the supernatant was used in the Western blot analyses. Aliquots (20 µg) were subjected to SDS-PAGE under reducing conditions, as described by Laemmli (16). The stacking gels contained 5% acrylamide, and the separating gels were composed of 7.5% acrylamide. The proteins were electrophoretically transferred from the gels onto nitrocellulose paper according to the procedures of Towbin et al. (17). The nitrocellulose blots were probed with a mouse monoclonal antibody (C11-113H) directed against human cad-11 (ICOS Corp., Bothell, WA). The Amersham ECL system (Amersham, Arlington Heights, IL) was used to detect antibody bound to antigen. The autoradiograms were then scanned using an LKB laser densitometer.

Statistical analysis
The results are presented as the mean relative absorbance (±SE) for at least three independent experiments. Statistical differences between time points and treatments were assessed by ANOVA. Differences were considered significant for P < 0.05. Significant differences between the means were determined using the least significant test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of gonadal steroids on stromal cad-11 mRNA and protein levels
A single cad-11 mRNA transcript of 4.4 kb was detected in all of the total RNA extracts prepared from the cultured endometrial stromal cells. The addition of vehicle (0.1% ethanol) to the culture medium had no significant effect on the levels of the cad-11 mRNA transcript present in these endometrial stromal cell cultures (Fig. 1AGo). In contrast, P4 caused a significant increase in the stromal cad-11 mRNA levels after 24 h of culture in the presence of this steroid (Fig. 1BGo). The levels of the cad-11 mRNA transcript continued to increase until the end of these studies at 96 h. E2, or DHT alone did not significantly increase cad-11 mRNA levels at any of the time points examined in these studies (Fig. 1Go, C and D, respectively).



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  		Figure 1. Autoradiograms of Northern blots containing total RNA extracted from isolated stromal cells cultured in the presence of vehicle (A), 1 µM P4 (B), 30 nM E2 (C), or 0.1 µM DHT (D). The cells were harvested 0, 6, 12, 24, 48, 72, or 96 h after treatment (lanes a–g, respectively). The blots were probed for cad-11 (top) or 18S rRNA (bottom). The autoradiograms were scanned using a laser densitometer. The absorbance values obtained for the cad-11 mRNA transcript were then normalized to the values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (autoradiograms not shown) were standardized to the 0 h control and are represented (mean ± SEM; n = 3) in the bar graphs. *, P < 0.05.

 
Western blot analysis, using extracts prepared from endometrial stromal cells cultured in the presence or absence of gonadal steroids and a mouse monoclonal antibody directed against human cad-11, revealed a single cad-11 protein species (Mr, 125 kDa) in all of the cellular extracts. In agreement with the Northern blot analysis, the addition of vehicle to the culture medium did not significantly alter cad-11 expression levels in the endometrial cell cultures (Fig. 2AGo). Similarly, P4 caused an increase in cad-11 expression after 24 h of culture in the presence of this steroid (Fig. 2BGo). The expression levels of cad-11 continued to increase until the end of these experiments at 96 h. In addition, we failed to detect a significant increase in cad-11 expression levels in endometrial stromal cells cultured in the presence of E2 or DHT at any of the time points examined in these studies (Fig. 2Go, C and D, respectively).



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  		Figure 2. Western blot analysis of cad-11 expression levels in isolated stromal cells cultured in the presence of vehicle (A), 1 µM P4 (B), 30 nM E2 (C), or 0.1 µM DHT (D). Twenty micrograms of protein extracted from endometrial stromal cells cultured for 0, 6, 12, 24, 48, 72, or 96 h in the presence or absence of steroids were loaded in each lane (lanes a–g, respectively). Western blot analysis was performed using a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The autoradiograms were then scanned using an LKB laser densitometer. The results derived from this analysis as well as those from three other studies (autoradiograms not shown) were standardized to the 0 h control and are represented (mean ± SEM; n = 4) in the bar graphs. *, P < 0.05.

 
Effects of P4 plus E2 or DHT on cad-11 mRNA and protein levels in endometrial stromal cells
There was a significant increase in cad-11 mRNA and protein expression levels in endometrial stromal cells cultured in the presence of E2 plus P4 for 12 h (Fig. 3AGo). Similarly, stromal cad-11 protein expression levels were significantly increased after 12 h of culture under these conditions (Fig. 3BGo). Cad-11 mRNA and protein expression levels continued to increase until the end of these studies at 96 h (Fig. 3Go, A and B, respectively). The cad-11 mRNA and protein levels detected in the endometrial stromal cells cultured in the presence of E2 plus P4 for 12–96 h were significantly greater than those observed in cells cultured in P4 for the same periods of time (P < 0.05).



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  		Figure 3. The effects of E2 on the P4-mediated increase in stromal cad-11 mRNA levels (A) or protein expression levels (B). Stromal cells were cultured in the presence of 1 µM P4 plus 30 nM E2 for 0, 6, 12, 24, 48, 72, or 96 h (lanes a–g) before being harvested for Northern or Western blot analysis. A, Autoradiograms of a Northern blot containing total RNA extracted from treated endometrial stromal cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study as well as those from two other studies (autoradiograms not shown) were standardized to the 0 h control and are represented (mean ± SEM; n = 3) in the bar graphs. *, P < 0.05. B, Autoradiogram of a Western blot containing protein extracted from the treated endometrial stromal cells and probed with a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The absorbance values obtained from this study as well as those from three other studies (autoradiograms not shown) were standardized to the 0 h control and are represented (mean ± SEM; n = 4) in the bar graphs. *, P < 0.05.

 
In contrast, there was no significant difference between the cad-11 mRNA and protein levels observed in cells cultured in the presence of P4 plus DHT and those detected in cells cultured in P4 alone at any time point examined in these studies (P < 0.05; Figs 4Go, A and B, respectively).



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  		Figure 4. Effects of DHT on the P4-mediated increase in stromal cad-11 mRNA levels (A) or protein expression levels (B). Stromal cells were cultured in the presence of 1 µM P4 plus 0.1 µM DHT for 0, 6, 12, 24, 48, 72, or 96 h (lanes a–g) before being harvested for Northern or Western blot analysis. A, Autoradiograms of a Northern blot containing total RNA extracted from the treated endometrial stromal cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study as well as those from two other studies (autoradiograms not shown) were standardized to the 0 h control and are represented (mean ± SEM; n = 3) in the bar graphs. *, P < 0.05. B, Autoradiogram of a Western blot containing 20 µg protein extracted from the treated endometrial stromal cells and probed with a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The absorbance values obtained from this study as well as those from three other studies (autoradiograms not shown) were standardized to the 0 h control and are represented (mean ± SEM; n = 4) in the bar graphs. *, P < 0.05.

 
Effects of varying doses of E2 to potentiate the P4-mediated increase in stromal cad-11 mRNA and protein levels
To determine whether the ability of E2 to potentiate the P4-mediated increase in cad-11 expression was dose dependent, the stromal cells were cultured in the presence of P4 plus varying doses of E2 (0.5–100 nM; Fig. 5Go). Increasing doses of E2 progressively enhanced the effects of P4 on stromal cad-11 mRNA and protein levels (Fig. 5Go, A and B, respectively). Maximum cad-11 mRNA and protein expression levels were observed in cells cultured in the presence of 30 nM E2. There was no further enhancement in stromal cad-11 mRNA and protein expression levels when the concentration of E2 was increased to 100 nM (Fig. 5Go, A and B, respectively).



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  		Figure 5. Effects of varying concentrations of E2 on the P4-mediated increase in stromal cad-11 mRNA levels (A) or protein expression levels (B). Stromal cells were cultured in the presence of vehicle, 30 nM E2, 1 µM P4, or 1 µM P4 plus 0.5, 1, 5, 10, 30, or 100 nM E2 (lanes a–i, respectively) for 96 h before being harvested for Northern or Western blot analysis. A, Autoradiograms of Northern blots containing total RNA extracted from the treated stromal cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study as well as those from two other studies (autoradiograms not shown) were standardized to the vehicle control and are represented (mean ± SEM; n = 3) in the bar graphs. *, P < 0.05. B, Autoradiograms of Western blots containing 20 µg protein extracted from the treated stromal cells and probed with a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The absorbance values obtained from this study as well as those from three other studies (autoradiograms not shown) were standardized to the vehicle control and are represented (mean ± SEM; n = 4) in the bar graphs. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A single cad-11 mRNA transcript of 4.4 kb was detected in all of the total RNA extracts prepared from the cultured endometrial stromal cells. This cad-11 mRNA transcript has been previously detected in RNA extracts prepared from a range of human tissues, including endometrium, placenta, and ovary (5, 6, 18). Similarly, the single cad-11 protein species (Mr, 125 kDa) detected in the stromal cell extracts using Western blot analysis has been observed in total cellular extracts prepared from choriocarcinoma cells (5). The present studies demonstrate that P4, but not E2 or DHT, is capable of regulating cad-11 mRNA and protein levels in endometrial stromal cells in vitro. In addition, the P4-mediated increase in stromal cad-11 mRNA and protein expression levels can be further enhanced by the addition of E2 to the culture medium. These observations indicate that both gonadal steroids are required for maximal cad-11 expression in human endometrial stromal cells.

Long term culture of endometrial stromal cells in the presence of P4 has been shown to modulate the expression of endometrial proteins associated with the remodeling processes that occur in the endometrium during the late secretory phase of the menstrual cycle. For example, P4 has been shown to stimulate fibronectin production in endometrial stromal cells (19), a key component of the decidual extracellular matrix (20), and suppress the expression of endometrial metalloproteinases, which are believed to play a key role in the breakdown of this tissue during menstruation (21, 22, 23). In these studies, P4 increased cad-11 mRNA and protein expression levels in the isolated endometrial stromal cells within 24 h, suggesting that this gonadal steroid is a key regulator of this endometrial cell adhesion molecule. In addition, we have previously determined that cad-11 is first expressed in endometrial stromal cells beginning to undergo decidualization during the late secretory phase of the menstrual cycle when P4 is the predominant steroid (5). Collectively, these observations suggest that a P4-mediated increase in stromal cad-11 expression may serve as a useful marker for the early cellular events involved in the process of decidualization in vivo and in vitro.

We failed to detect a significant increase in cad-11 expression in endometrial stromal cells cultured in the presence of the nonaromatizable androgen, DHT. Furthermore, DHT was not capable of enhancing the P4-mediated increase in cad-11 expression in endometrial stromal cells. Collectively, these results demonstrate that androgens are unable to increase stromal cad-11 mRNA or protein expression levels in either a direct or an indirect manner. Although androgen receptors have been detected in the human endometrium and decidua (3), it is still unclear whether androgens play a direct role in the process of decidualization in the human. For example, recent studies have demonstrated that DHT alone can induce PRL secretion in isolated endometrial stromal cells and potentiate the effects of P4 on the secretion of this endometrial protein in vitro (4). In addition, pharmacological doses of DHT were able to maintain, but not initiate, decidualization in the stromal cells of the mouse uterus (24). However, the actions of DHT on the rodent endometrium could be suppressed by the anti-progestin, RU486, suggesting that the actions of this androgen on the murine decidua were mediated by its ability to interact with the P4 receptor (24).

E2 did not increase cad-11 mRNA or protein levels in isolated endometrial stromal cells, suggesting that this gonadal steroid does not have a direct effect on stromal cad-11 expression. Similarly, previous studies have failed to demonstrate a direct effect of this gonadal steroid on the production of the two decidual cell markers, PRL and insulin-like growth factor-binding protein-1 (10), or the secretion of metalloproteinases by endometrial stromal cells in vitro (21). Furthermore, although E2 is essential for the synthesis of specific proteins in the endometrium, including P4 and E2 receptors, depleted levels of this gonadal steroid during the luteal phase of the menstrual cycle do not appear to effect endometrial development in vivo (25). To date, the role(s) of E2 in the differentiation of endometrial stromal cells in vivo and in vitro remain poorly understood.

E2 was capable of potentiating the stimulatory effects of P4 on cad-11 mRNA and protein levels in isolated endometrial stromal cells, suggesting that this gonadal steroid has an indirect effect on stromal cad-11 expression. Maximum levels of insulin-like growth factor-binding protein-1 and PRL have also been detected in endometrial stromal cells cultured in the presence of both E2 and P4 (11). However, Grosskinsky et al. (26) failed to detect an increase in the expression of integrin subunits in endometrial stromal cells cultured in the presence of these two gonadal steroids despite observing an increase in PRL production in these cell cultures. As the differential expression of several integrin subunits in the human endometrium during the secretory phase of the menstrual cycle appears to be required for successful implantation (27), these observations indicate that the process of decidualization is a complex series of hormonally dependent and independent events. To date, the cellular mechanisms involved at the different stages of this developmental process remain poorly defined.

The mechanism(s) by which E2 enhances the effects of P4 on endometrial stromal cell differentiation have not been determined. However, several mechanisms have been recently proposed. For example, as E2 has been shown to induce P4 receptors in human endometrial stromal cells in vivo and in vitro (28), the effects of E2 on the terminal differentiation of endometrial stromal cells may be mediated by an increase in P4 availability. Although this proposed mechanism could explain the ability of E2 to enhance the P4-mediated increase in stromal cad-11, the effects of gonadal steroids are also believed to be mediated through growth factors (29). In particular, the effects of E2 on endometrial cells have been shown to be mediated at least in part by an increase in the levels of epidermal growth factor. Furthermore, Somkuti et al. (30) have recently suggested that a combination of gonadal steroids and epidermal growth factor is required for the spatiotemporal expression of integrin subunits in human endometrial cells. The ability of growth factors, alone or in combination with gonadal steroids, to regulate cad-11 in human endometrial cells has not been determined. In view of these observations, we are currently examining potential mechanisms by which E2 enhances the effects of P4 on cad-11 expression in isolated endometrial stromal cells.

In summary, our findings demonstrate that P4, but not E2 or DHT, is capable of regulating cad-11 mRNA and protein expression levels in isolated endometrial stromal cells. However, E2 in conjunction with P4 appears to be necessary to achieve maximal cad-11 expression in these cells. In view of these observations, it is tempting to speculate that the ability of steroids to regulate the terminal differentiation of endometrial stromal cells into decidual cells is mediated at least in part by their ability to regulate cad-11 expression.


   Acknowledgments
 
The authors thank Dr. M. D. Stephenson, Department of Obstetrics and Gynecology, University of British Columbia, for providing the endometrial biopsy specimens and the ICOS Corp. (Bothell, WA) for their kind gift of the monoclonal antibody used in these studies. We are grateful to Dr. Riaz Farookhi, Department of Obstetrics and Gynecology, McGill University (Montreal, Canada) for carefully reading this manuscript and for his helpful comments.


   Footnotes
 
1 This work was supported by a grant from the Medical Research Council of Canada (to C.D.M.), a scholarship from the Medical Research Council of Canada (to C.D.M.), and a Graduate Fellowship from the University of British Columbia (to S.G.). Research on human subjects was approved by the Committee for Ethical Review of Research Involving Human Subjects, University of British Columbia, Vancouver, Canada. All subjects provided informed consent for these studies. Back

Received January 22, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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