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. 2017 Aug 17;23(9):646–653. doi: 10.1093/molehr/gax045

Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization

Alison J Camden 1,†, Maria M Szwarc 2,†, Sangappa B Chadchan 1, Francesco J DeMayo 3, Bert W O'Malley 2, John P Lydon 2, Ramakrishna Kommagani 1,*
PMCID: PMC5909854  PMID: 28911214

Abstract

STUDY QUESTION

Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization?

SUMMARY ANSWER

GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization.

WHAT IS KNOWN ALREADY

Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined.

STUDY DESIGN, SIZE, DURATION

Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8–12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs.

MAIN RESULTS AND THE ROLE OF CHANCE

Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively).

LARGE SCALE DATA

This study used the Nuclear Receptor Signaling Atlas.

LIMITATIONS, REASONS FOR CAUTION

Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models.

WIDER IMPLICATIONS OF THE FINDINGS

Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization.

STUDY FUNDING AND COMPETING INTEREST(S)

This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.

Keywords: progesterone, endometrium, decidualization, gene transcription, differentiation

Introduction

In early pregnancy, the uterus must become receptive to embryo invasion and implantation and then permit placentation to establish the maternal-fetal interface (Wilcox et al., 1999; Carson et al., 2000). These processes both require the fibroblast-like endometrial stromal cells lining the uterus to differentiate, or decidualize, into epithelial-like cells (Gellersen et al., 2007; Cha et al., 2012). Inadequate decidualization can lead to implantation failure or early embryo miscarriage of even a healthy blastocyst. In fact, miscarriage after implantation, which is likely due to decidualization defects, occurs in 30% of pregnancies (Salker et al., 2010, 2012; Teklenburg et al., 2010; Koot et al., 2012). Therefore, diagnosis and treatment of recurrent pregnancy loss will require a better understanding of this cellular transformation process.

Decidualization and establishment of a successful pregnancy rely on the steroid hormones estrogen and progesterone produced by the ovary (Wang and Dey, 2006). Estrogen supports embryo implantation by preparing the endometrium to be receptive to embryo invasion, whereas progesterone directs endometrial decidualization (Finn and Martin, 1970, 1972; Ma et al., 2003). The physiological actions of progesterone are mediated by the progesterone receptor (PR), a transcription factor that regulates gene expression through the ligand-dependent recruitment of accessory proteins known as coregulators (Lonard and O'Malley, 2012). Although studies with controlled hormone treatments of mice have revealed many of the cellular events by which progesterone controls initiation and progression of decidualization (Finn, 1982; Wang and Dey, 2006), the underlying mechanisms that direct these changes are unclear. Recent microarray-based transcriptional profiling on uterine tissues from ovariectomized mice acutely treated with progesterone revealed genes that are regulated by progesterone in the endometrium (Wetendorf and DeMayo, 2014). Herein, we sought to determine the role of one such gene, Growth Regulation by Estrogen in Breast Cancer 1 (GREB1), in the progesterone-dependent responses in the endometrium.

GREB1 was initially identified as an early estrogen-responsive gene in MCF-7 breast cancer cells (Ghosh et al., 2000), and its expression is strongly correlated with that of estrogen receptor in primary luminal-A breast cancers (Dunbier et al., 2010; Hnatyszyn et al., 2010). Additionally, GREB1 promotes estrogen-dependent proliferation of breast cancer cells (Rae et al., 2005, 2006), and overexpression of GREB1 stimulates colony formation and tamoxifen resistance in MCF-7 cells (Mohammed et al., 2013). GREB1 is also an androgen-responsive gene in LNCaP prostate cancer cells (Ghosh et al., 2000), and it promotes androgen-dependent proliferation of prostate cancer cells (Rae et al., 2005, 2006). Finally, GREB1 is upregulated in response to estrogen in ovarian cancer cells (Laviolette et al., 2014). Although these data suggest a pan-estrogen response in females, whether GREB1 responds to other closely related steroid hormones and their receptors is as yet unknown.

In this study, we define GREB1 as a novel progesterone-responsive gene in the uterus and demonstrate that it plays an important role in progesterone-driven human endometrial stromal cell (HESC) decidualization.

Materials and Methods

Ethical approval

Before endometrial tissue biopsy collection, volunteers provided written informed consent in accordance with an Institutional Review Board protocol from Baylor College of Medicine and the guidelines of the Declaration of Helsinki (‘WMA Declaration of Helsinki Serves as Guide to Physicians’ 1966).

HESC isolation

Endometrial biopsies were obtained from healthy women of reproductive age during the proliferative phase (Days 8–12) of their menstrual cycle. HESCs were isolated as described previously (Kommagani et al., 2013), cultured, and propagated in DMEM/F-12 media containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Experiments were carried out using early passage (no more than four passages) HESCs isolated from at least three subjects, all of which decidualized under decidualization conditions. Data in figures are from cells from one representative patient, which showed an intermediate level of decidualization. Results are shown as mean ± SE from three biological replicates of one representative patient-derived primary endometrial cell line. Transformed HESCs were obtained from ATCC (ATCC CRL-4003) (Krikun et al., 2004). Transformed HESCs were maintained in phenol-red-free DMEM/F-12 medium with 3.1 g/l glucose and 1 mM sodium pyruvate supplemented with 10% charcoal/dextran-treated FBS, 1% ITS (insulin, transferrin, sodium selenite + Premix, 1.5 g/l sodium bicarbonate, and 500 ng/ml puromycin [all from Life Technologies, Carlsbad, USA]).

HESC siRNA Transfection and Decidualization

For transfections, HESCs were plated in 1× Opti-MEM I reduced-serum media (Invitrogen Corporation, Carlsbad, USA) in six-well culture plates and treated in triplicate with Lipofectamine 2000 reagent (Invitrogen Corporation, Carlsbad, USA) and 60 pmol of the following siRNAs: non-targeting siRNA (D-001810-10-05) or siRNAs targeting PR (L-006763-00-0005), SRC-2 (NCOA-2 [L-020159-00-0005]), or GREB1 (L-008187-01-0005) (GE Healthcare Dharmacon Inc., Lafayette, CO). After 48 h, decidualization was initiated by treating HESCs with 100 nM estrogen, 10 μM Medroxyprogesterone acetate (MPA) (Sigma-Aldrich), and 50 μM cyclic adenosine monophosphate (Sigma-Aldrich) in 1× in Opti-MEM I reduced-serum media containing 2% FBS. Decidualization medium was changed every two days until Day 3 or 6, depending on the experiment. Cells were then harvested, and the RNeasy total RNA isolation kit (Qiagen Inc., Valencia, CA) was used to isolate RNA as described previously (Kommagani et al., 2016). Quantitative PCR analyses were performed by using appropriate primers (Supplementary Data Table S2) and TaqMan 2× master mix (Applied Biosystems/Life Technologies, Grand Island, NY). Ribosomal RNA (18 S) was used as an internal control. Transcript levels of decidualization markers PRL and IGFBP-1 were used as an indication of HESC decidualization (Brosens et al., 1999).

Mice and hormone treatments

CD1 wild-type mice were housed at the mouse facility and maintained on a 12-h light: 12-h dark cycle. Mice were treated humanely, and surgical procedures were performed according to an animal protocol approved by the Institutional Animal Care and Use Committee. PR knockout (PRKO) mice and isogenic wild-type siblings were described previously (Lydon et al., 1995). To assess uterine progesterone responses, six-week-old mice were ovariectomized, rested for two weeks to allow endogenous levels of ovarian-derived steroid hormones to diminish, and then subcutaneously injected with 100 μl sesame oil (vehicle control) or 1 mg progesterone (Sigma-Aldrich, St. Louis, MO) in 100 μl sesame oil as indicated in figure legends. At the indicated times after injections, mice were euthanized, whole uterine tissues were collected, and RNA was isolated with the RNeasy total RNA isolation kit.

Statistical analysis

A two-tailed paired student t-test was used for statistical significance testing. Results were considered statistically significant with a P-value < 0.05. Asterisks represent the level of significance: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Results

Although GREB1 responds to estrogen and androgen in breast and prostate cancer cells, respectively (Rae et al., 2005, 2006), its regulation by other steroid hormones is unknown. To assess the range of hormone pathways by which GREB1 expression might be regulated, we searched for it in the Nuclear Receptor Signaling Atlas (Ochsner et al., 2012). We used the Transcriptomine tool to identify data points from existing expression profile datasets, revealing that GREB1 expression is affected by numerous hormones/nuclear receptors, including progesterone (Supplementary Data Fig. S1). In-depth mining of this tool revealed that acute (4 or 6 h) treatment with progesterone induced multiple Greb1 probes in two different microarray datasets from mouse uteri (Fig. 1A and Supplementary Data Table S1).

Figure 1.

Figure 1

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Greb1 is rapidly induced by progesterone in the mouse and human endometrium. (A) Graph depicting the Greb1 probes found to be induced by progesterone in expression profiling data sets in the Nuclear Receptor Signaling Atlas Transcriptomine database. (B) Greb1 transcript levels in uteri from ovariectomized mice treated with vehicle or progesterone (P4) (1 mg) for 6, 12 or 24 h. Results represent the mean ± SE; n = 3 mice/group. (C) Relative levels of GREB1 and FOXO1A (positive control) transcripts from transformed HESCs treated with vehicle (control) or MPA (1 μM) for indicated time points. Results represent the mean ± SE from three biological replicates; **P < 0.01.

To validate the in silico finding, we ovariectomized wild-type mice, treated them with progesterone, and then examined Greb1 transcript levels in the uterus. We found that Greb1 expression was elevated ~5-fold (5.6 ± 0.81) at 6 h after treatment and was back down to control levels at 12 and 24 h (Fig. 1B). Similarly, treatment of transformed HESCs with MPA led to a significant induction (5.2 ± 0.26) in GREB1 transcript levels within 4 h (Fig. 1C). As anticipated, expression of the well-established progesterone-responsive gene Forkhead Box Protein O1A (FOXO1A) was also increased in MPA-treated cells (Fig. 1C). These results suggest that GREB1 is an early progesterone-responsive gene in the human and mouse endometrium.

Because the PR is required for progesterone-driven transcription, we next wondered whether PR was required for Greb1 induction by progesterone. To test this, we ovariectomized control and PR knockout (PRKO) mice, treated them with progesterone for 6 h, and then measured Greb1 transcript levels in the uterus. As shown in Fig. 2A, progesterone significantly induced (6.0 ± 0.25) Greb1 transcript levels in uteri from control mice but not in those from PRKO mice. As expected, uterine expression of the PR-responsive gene Amphiregulin (Areg) was also increased upon progesterone treatment in control but not in PRKO mice. Further, in silico analysis of a PR chromatin immunoprecipitation-sequencing data set (Rubel et al., 2012) revealed multiple PR-binding sites in the Greb1 locus (Fig. 2B).

Figure 2.

Figure 2

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Greb1 is induced by progesterone and PR in the mouse uterus and HESCs. (A) Relative levels of Greb1 and Areg (positive control) transcripts in uteri from ovariectomized wild-type (WT) and PR knockout (PRKO) mice treated for 6 h with P4 (1 mg). Results represent the mean ± SE; n = 3 mice/group. (B) Distribution of binding sites for PR (orange arrows) on Greb1 locus from murine uterine tissues treated for 6 h with P4 (1 mg) or vehicle (oil). (C) Relative transcript levels of GREB1 and FOXO1A from transformed HESCs (three biological replicates) treated with vehicle (control), MPA or MPA plus RU486 (1 μM) for 4 h. Note: The RU486 was added 1 h before MPA. (D) Relative transcript levels of GREB1 and PR from transformed HESCs (three biological replicates) transfected with control siRNA or PGR siRNA treated with vehicle or MPA (1 μM) for 4 h. Results represent the mean ± SE; *P < 0.05 and **P < 0.01. HESC, human endometrial stromal cell; PR, progesterone receptor.

To determine whether PR was required for MPA-mediated induction of GREB1 in HESCs, we performed two assays. First, we demonstrated that whereas MPA treatment led to induction (2.5 ± 0.04) of GREB1 transcript levels by 4 h, co-administration of RU468 (a PR antagonist) significantly attenuated this induction (Fig. 2C). Second, we used siRNAs to knock down PR expression, resulting in attenuated (2.0 ± 0.10) MPA-induced GREB1 expression (Fig. 2D). Collectively, these results strongly indicate that PR is required for rapid induction of Greb1 transcript levels by progesterone in the murine uterus and human endometrial stromal cells.

Next, to determine whether PR was required for GREB1 expression during in vitro human endometrial decidualization, we examined GREB1 transcript levels in decidualizing primary HESCs and decidualizing HESCs in which PR was knocked down. GREB1 transcript levels were significantly induced by over 150-fold during HESC decidualization (Fig. 3A), but levels of GREB1 transcripts were significantly reduced in HESCs transfected with PR siRNA (Fig. 3A). Recently, SRC-2 was identified as an upstream transcriptional coactivator of PR in mediating HESC decidualization (Kommagani et al., 2013). Consistent with this, GREB1 induction was significantly lower in decidualizing HESCs in which SRC-2 was knocked down (Fig. 3B). Together, these results suggest that a progesterone-PR-SRC-2 signaling axis activates GREB1 transcription in decidualizing HESCs.

Figure 3.

Figure 3

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PR and its coactivator SRC-2 regulate GREB1 expression in decidualizing HESCs. (A) Levels of GREB1 and PR transcripts in primary HESCs (three biological replicates) transfected with control or PR siRNAs and induced to decidualize for the indicated lengths of time. (B) Levels of GREB1 and SRC-2 transcripts in primary HESCs transfected with control or SRC-2 siRNAs and induced to decidualize for the indicated lengths of time. Representative data from three replicates from one patient sample are shown as mean ± SE; *P < 0.05 and **P < 0.01.

We next asked whether GREB1 was required for progesterone-driven human endometrial decidualization. As a proof-of-principle, we first used telomerase-transformed HESCs as a model for in vitro decidualization. As shown in Fig. 4A, GREB1 transcript levels were significantly induced in a time-dependent manner as the decidualization program advanced (Fig. 4A). Induction of the well-established decidual marker prolactin (PRL) confirmed that the transformed HESCs effectively decidualized (Fig. 4A). When transformed HESCs were transfected with siRNAs targeting GREB1 before initiating decidualization, PRL expression was significantly reduced (2.0 ± 0.06) (Fig. 4B). Consistent with this finding, the level of PRL secreted into the culture media was significantly reduced by GREB1 knockdown (Supplementary Fig. 2).

Figure 4.

Figure 4

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GREB1 is required for decidualization of immortalized human endometrial stromal cells. (A) Levels of GREB1 and PRL transcripts from immortalized human endometrial stromal cells induced to decidualize for the indicated lengths of time. (B) Levels of GREB1 and PRL transcripts in immortalized human endometrial stromal cells transfected with control or GREB1 siRNAs treated and induced to decidualize for the indicated lengths of time. Results stated as the mean ± SE from three biological replicates from a representative experiment (experiment repeated three times); *P < 0.05, **P < 0.01, and ***P < 0.001.

To confirm that GREB1 was essential for hormone-driven endometrial decidualization, we knocked down GREB1 in primary HESCs and then initiated in vitro decidualization (Fig. 5). Whereas control cells transformed from a fibroblastic to an epithelioid cell morphology, HESCs in which GREB1 was knocked down failed to undergo this cellular transformation process (Fig. 5A). Consistent with this finding, induction of the decidual markers insulin-like growth factor-binding protein-1 (IGFBP-1) and PRL was abrogated by GREB1 knockdown (Fig. 5B). Together, these results indicate that GREB1 is indispensable for progesterone-dependent HESC decidualization.

Figure 5.

Figure 5

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GREB1 is essential for primary human endometrial stromal cell decidualization. HESCs were transfected with negative control siRNA or GREB1 siRNA as indicated. (A) Left, morphology of HESCs transfected with control or GREB1 siRNA after zero or six days of culture in decidualization conditions. Right, levels of GREB1 transcripts confirm the effective knockdown of GREB1 in HESCs. (B) Levels of IGFBP-1 and PRL transcripts in HESCs transfected with control or GREB1 siRNAs and induced to decidualize for the indicated lengths of time. from three replicates from one patient sample are shown as mean ± SE; **P < 0.01.

Finally, to determine the mechanism by which GREB1 contributed to decidualization, we assessed expression of the PR target genes FOXO1A and WNT4, which promote endometrial decidualization (Li et al., 2013; Vasquez et al., 2015). As shown in Fig. 6, induction of FOXO1A and WNT4 was significantly reduced in GREB1-knockdown HESCs than in control HESCs during decidualization. In contrast, levels of PR transcript were not affected by GREB1 knockdown. These results suggest that GREB1 modulates PR-mediated transcription during endometrial decidualization. In conclusion, we demonstrate that GREB1 is a novel progesterone-responsive gene in the mouse uterus and HESCs and reveal GREB1 as a critical mediator of the progesterone-dependent endometrial stromal cell decidualization program.

Figure 6.

Figure 6

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GREB1 is required for the induction of key decidual genes in decidualizing HESCs. HESC cells were transfected with negative control siRNA or GREB1 siRNA as indicated. Transcript levels of FOXO1A, WNT4 (P4 targets) and PR (estrogen target) in HESCs transfected with control or GREB1 siRNAs and induced to decidualize for the indicated lengths of time. Representative data from three replicates from one patient sample are shown as mean ± SE; *P < 0.05 and **P < 0.01.

Discussion

Herein, our results demonstrate that GREB1 is a progesterone target gene that functions to control decidualization in human endometrial stromal cells. Rapid induction of GREB1 by progesterone depends on the PR coactivator SRC-2, which we recently showed is essential for progesterone-dependent endometrial stromal cell decidualization (Kommagani et al., 2013). In addition, GREB1 mediates transcription of the PR targets WNT4 and FOXOA1 and thus is a key molecular signal controlling cell fate in progesterone-dependent endometrial decidualization. Collectively, our results demonstrate that GREB1 is induced rapidly by progesterone in endometrial stromal cells and that GREB1 in turn acts as a downstream mediator of progesterone-dependent transcriptional responses that are essential for decidualization. This conceptual advance places GREB1 in a tightly controlled feed-forward regulatory loop that is essential for HESC decidualization.

In addition to progesterone, other hormones also participate in endometrial decidualization. For example, MPA can act as an agonist for the androgen receptor, which plays a role in human endometrial stromal cell decidualization (Bentel et al., 1999; Gibson et al., 2016; Walters et al., 2016). Additionally, locally produced estrogen supports murine endometrial decidualization (Das et al., 2009), and human endometrial decidualization results in increased estrogen biosynthesis (Gibson et al., 2013). Androgen, estrogen, and progesterone all exert their biological actions through their cognate nuclear receptors belonging to nuclear receptor subfamily 3 (Mangelsdorf et al., 1995). Given the observation that GREB1 is required for estrogen- and androgen-mediated proliferation of breast and prostate cancer cells, respectively (Ghosh et al., 2000), GREB1 might function as a common downstream mediator of this nuclear receptor subclass in human endometrial decidualization. Future investigation should be directed at defining the mechanisms by which androgen, estrogen and progesterone differentially regulate GREB1 expression to mediate a wide array of physiological responses such as endometrial decidualization.

Although GREB1 was shown to modulate cell cycle progression in multiple cancer cell lines, our findings are the first to reveal a role for GREB1 in a normal physiological process regulated by a steroid hormone. Because endometrial decidualization is associated with cell cycle arrest initially at the G0/G1 phase, and later at the G2/M phase (Logan et al., 2012), it will be intriguing to test whether GREB1 governs cell cycle progression in this process. Our finding that GREB1 knockdown led to decreased levels of the PR targets FOXO1A and WNT4 suggests that GREB1 acts as a PR transcriptional cofactor in the endometrium, similar to its reported action as an estrogen receptor cofactor mediating estrogen responses in breast cancer cells (Mohammed et al., 2013).

Our findings provide important information for addressing infertility. Whereas the probability of establishing a successful pregnancy within one menstrual cycle (termed the monthly fecundity rate) is very high in other mammals, the rate is only 20–30% in humans (Foote and Carney, 1988; Chard, 1991; Stevens, 1997). Despite recent advances, only ~30% of women using assisted reproductive technologies in the United States have a live birth (Centers for Disease Control and Prevention (http://www.cdc.gov/art/reports/index.html)). Approximately 50% of embryos implant, and up to half of these embryos are lost soon after implantation (Rinehart, 2007). This high attrition rate can be attributed to poor embryo quality, endometrial dysfunction (Macklon and Brosens, 2014) and inadequate endometrial decidualization (Brosens et al., 2014). Identification of key molecular mediators of decidualization such as GREB1 will hopefully lead to improved reproductive outcomes in women with peri-implantation loss.

Our results may also have implications for treatment of endometriosis, as GREB1 expression is elevated in both epithelial and stromal cells in human endometriotic lesions (Pellegrini et al., 2012). Given the results presented here and the established role of GREB1 in estrogen responses, we speculate that GREB1 enables progesterone-dependent proliferation and differentiation of HESCs into decidual cells, thereby generating a receptive endometrium for embryo implantation. However, dysregulation of GREB1 accelerates the estrogen-driven cellular proliferation that leads to ectopic endometriotic lesion expansion. These normal and pathological roles of GREB1 are clinically linked in that a non-receptive endometrium and endometriosis both result from derailment of normal endometrial responses to estrogen and progesterone, which in turn can lead to subfertility or infertility in reproductive-age women. Future work on GREB1 should thus improve our ability to diagnose and manage fertility-associated disorders such as recurrent pregnancy loss and endometriosis.

Supplementary Material

Supplementary Data
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Acknowledgement

We thank Deborah J. Frank in the Department of Obstetrics and Gynecology, Washington University School of Medicine, for assistance with manuscript editing.

Supplementary data

Supplementary data are available at Molecular Human Reproduction online.

Authors' roles

A.J.C., M.M.S., S.B.C. and R.K. conducted most of the studies and wrote the manuscript draft. M.M.S. and R.K. collected human stromal cells. F.J.D. provided reagents and reviewed the final draft of the manuscript. B.W.O. and J.P.L. discussed experimental design and provided resources and funding. R.K. discussed experimental design and provided resources and funding.

Funding

National Institutes of Health (NIH)/National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O., and a NIH/NICHD grant (R01 HD-042311) to J.P.L.

Conflict of interest

None declared.

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