SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization

  1. Pinxiu Huang
  2. Wenbo Deng
  3. Haili Bao
  4. Zhong Lin
  5. Mengying Liu
  6. Jinxiang Wu
  7. Xiaobo Zhou
  8. Manting Qiao
  9. Yihua Yang
  10. Han Cai
  11. Faiza Rao
  12. Jingsi Chen
  13. Dunjin Chen
  14. Jinhua Lu
  15. Haibin Wang  Is a corresponding author
  16. Aiping Qin  Is a corresponding author
  17. Shuangbo Kong  Is a corresponding author
  1. Department of Reproductive Medicine, The First Affiliated Hospital of Guangxi Medical University, China
  2. Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, China
  3. Department of Reproductive Medicine, Liuzhou Maternity and Child Health Hospital, China
  4. Affiliated Maternity Hospital and Affiliated Children’s Hospital of Guangxi University of Science and Technology, China
  5. Department of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, China

Abstract

The establishment of pregnancy in human necessitates appropriate decidualization of stromal cells, which involves steroids regulated periodic transformation of endometrial stromal cells during the menstrual cycle. However, the potential molecular regulatory mechanism underlying the initiation and maintenance of decidualization in humans is yet to be fully elucidated. In this investigation, we document that SOX4 is a key regulator of human endometrial stromal cells decidualization by directly regulating FOXO1 expression as revealed by whole genomic binding of SOX4 assay and RNA sequencing. Besides, our immunoprecipitation and mass spectrometry results unravel that SOX4 modulates progesterone receptor (PGR) stability through repressing E3 ubiquitin ligase HERC4-mediated degradation. More importantly, we provide evidence that dysregulated SOX4–HERC4–PGR axis is a potential cause of defective decidualization and recurrent implantation failure in in-vitro fertilization (IVF) patients. In summary, this study evidences that SOX4 is a new and critical regulator for human endometrial decidualization, and provides insightful information for the pathology of decidualization-related infertility and will pave the way for pregnancy improvement.

Editor's evaluation

The manuscript provides a novel mechanism of progesterone receptor stability mediated by SOX4 in human endometrial decidualization. The authors have addressed all the concerns raised by the reviewers and in addition provided the gels at better resolution, leading to a significantly improved paper.

https://doi.org/10.7554/eLife.72073.sa0

Introduction

Adequate crosstalk between implantation-competent embryo and receptive endometrium is prerequisite for successful pregnancy (Cha et al., 2012). The receptive endometrium in human requires remodeling of stromal cells under the regulation of rising progesterone and intracellular cyclic AMP to initiate the decidualization, which will undergo more extensive transformation after embryo implantation (Gellersen and Brosens, 2014). The human endometrial stromal compartment can decidualize during the progesterone-dominant early secretory phase of a nonconception cycle (Wang et al., 2020). During the pregnancy, the decidual process is poised to transit through distinct phenotypic phases that underpin endometrial receptivity, embryo selection, and ultimately resolution of pregnancy (Gellersen and Brosens, 2014). The decidual reaction plays a central role in the establishment of a pregnancy and continues throughout the pregnancy. Insufficient decidualization in endometrium is related to failed embryo implantation (Peter Durairaj et al., 2017), unexplained infertility, recurrent spontaneous abortion (Coulam, 2016), intrauterine growth retardation (Lefèvre et al., 2011), and preeclampsia (Garrido-Gomez et al., 2017). However, the underlying molecular mechanism governing the endometrial decidualization remains enigmatic (Okada et al., 2018).

A wide range of transcription factors crucial for stromal cell decidualization have been successively identified (Gellersen and Brosens, 2014). FOXO1, a member of FOXO fork-head transcription factors subfamily, is one of the earliest identified transcriptional factors in human endometrial stromal cells (HESCs) responding to decidualization stimulation (progesterone and cAMP) (Labied et al., 2006). Accumulative evidence has demonstrated that FOXO1 regulates transcription of PRL and IGFBP1 through direct binding to their promoters (Christian et al., 2002; Kim et al., 2005). Progesterone receptor (PGR), which is a critical master factors for the endometrial stromal cells (ESCs) decidualization, imparts endometrium receptivity through binding with P4 ligand and its nuclear translocation (Keller et al., 1979; Mulac-Jericevic et al., 2000). An array of PGR direct target genes had been unraveled by PGR ChIP-Seq in both human and mice (Rubel et al., 2012; Mazur et al., 2015; Chi et al., 2020). Abnormal PGR expression is closely relevant with unexplained infertility (Keller et al., 1979) and endometriosis (EMS) (Zhou et al., 2016; Pei et al., 2018). Although many transcription factors and downstream events in decidualization of both mice and human have been unraveled (DeMayo and Lydon, 2020), the precise mechanism orchestrating transcriptional regulatory network underpinning endometrial decidualization remained not fully explored.

SOX4 is a highly conserved transcription factor belonging to the SOX (SRY-box) family. Studies have shown that SOX4 is vital to a variety of biological processes, including embryogenesis, neural development, and differentiation (Moreno, 2020). Further, it has been noticed that SOX4 knockout mice die of cardiac malformation on the 14th day of pregnancy, suggesting the key role of SOX4 in embryonic development (Ya et al., 1998). In addition, an increasing number of reports indicate that SOX4 is related to tumor cell proliferation, metastasis, and epithelial–mesenchymal transformation (Li et al., 2020). Report also shows that SOX4 is highly expressed in breast cancer under the regulation of progesterone (Graham et al., 1999). Additionally, SOX17, another member of the SOX family, has also been observed to be a direct target of PGR in epithelium regulating IHH expression (Wang et al., 2018). While the significance and regulation of SOX4 in female pregnancy remain intangible.

Here, we provide evidence that SOX4, under the regulation of P4-PGR, guides human endometrium stromal cells (HESCs) decidualization by regulating FOXO1 expression as revealed by ChIP-Seq and RNA sequencing (RNA-Seq). Mechanism studies also unravel the importance of SOX4 in maintaining the protein stability of PGR by repressing ubiquitin E3 ligase HERC4. Moreover, both SOX4 and PGR have been demonstrated to be aberrantly downregulated in the endometrium of EMS patients suffering from implantation failure.

Results

SOX4 is dynamically expressed in human endometrial stromal cells regulated by P4-PGR signaling

To investigate the expression of transcription factors in HESCs, RNA-Seq was performed in normal endometrium stromal cells. We noted that among the transcription factors expressed in endometrium stromal cells, SOX4 was the 17th highest expressed (Figure 1A), and as the most abundant member in SOX family (Figure 1B). Meanwhile, previous RNA-Seq data in isolated mouse stromal and epithelial cells revealed that SOX4 expression was the most abundant SOX family in stromal cells (Figure 1—figure supplement 1A), while the expression of SOX17 was restricted to the epithelium (Deng et al., 2019). Thus, we speculated that the conserved expression of SOX4 in stroma was linked to decidualization. To explore whether SOX4 was under the regulation of dynamic change of estrogen and progesterone during the menstrual cycle, we first analyzed the expression of SOX4 in endometrial biopsy samples obtained from healthy, reproductive-aged volunteers with regular menstrual cycles. Albeit SOX4 was detected at a low level in the E2-dominant proliferative phase, its expression was progressively increased in the nucleus of stromal cellsin P4-dominant early, middle, late secretory phases and this high expression in stroma cell sustained in the decidual cells in early pregnancy (Figure 1C).

Figure 1 with 2 supplements see all
SOX4 is dynamically expressed in human ESCs.

Expression of all transcription factors (A) and SOX family genes (B) in human nondecidualized ESCs by RNA-Seq. The value in Y-axis indicated the RPKM (reads per kilobase per million mapped reads) in RNA-Seq data. (C) Immunohistochemical analysis of endometrial SOX4 protein expression in proliferative, secretory phases (early, middle, and late) of the menstrual cycle and early pregnancy (about 8 weeks). GE: gland epithelium; S: stroma. Scale bar: 100 μm. (D) Expression of SOX4 mRNA levels in decidualized stromal cells at different time points after the E2, MPA, and cAMP treatment. Results are presented as means ± standard error of the mean (SEM); n = 3; **p < 0.005; ***p < 0.0001. (E) Expression of SOX4 protein levels in decidualized stromal cells at different time points after the E2, MPA, and cAMP treatment. (F) Immunofluorescent detection of SOX4 protein localization in the undecidualized (D0) and decidualized (D4) human endometrial stromal cells (HESCs). Scale bar: 100 μm.

This upregulation of SOX4 in differentiated stromal cell in secretory phase was also substantiated in in vitro decidualized stromal cells induced by E2, MPA, and cAMP (EPC) cocktail, displaying gradually cumulated mRNA and protein levels in immortalized HESCs (Figure 1D, E) as well as in primary stromal cells (Figure 1—figure supplement 2A, B). Immunofluorescence also manifested that SOX4 was mainly localized in the nucleus of HESCs after EPC treatment (Figure 1F).

The intense expression of SOX4 in the stroma of secretory phase accompanied with rising P4 and inducted by EPC in in vitro stromal cells, suggesting that progesterone may be involved in the regulation of SOX4 expression. This assumption was supported by the observation that SOX4 expression was induced by 2 days treatment of MPA, a progesterone analog commonly used in decidualization induction, and abrogated in the presence of PGR antagonist RU486 (Figure 2A). Progesterone-induced SOX4 mRNA and protein levels were significantly attenuated with PGR knockdown in immortalized HESCs (Figure 2B–D). Thus, the P4-PGR signaling was critical for SOX4 expression in HESCs. Based on previous ATAC-Seq data from undecidualized and decidualized stromal cells and PGR ChIP-Seq from proliferative and secretory endometrium (Chi et al., 2020), we found that there was intensive potential PGR binding at SOX4 promoter (Figure 2E). This speculation was validated by PGR ChIP-qPCR at SOX4 promoter in decidualized stromal cells (Figure 2F). Furthermore, the binding of PGR on SOX4 promoter was also confirmed by luciferase reporter assay that overexpression of PGR increased the reporter activities in the presence of MPA in 293T cells (Figure 2G). These results indicated that PGR guided SOX4 expression via direct transcriptional regulation.

SOX4 is regulated by P4-progesterone receptor (PGR) signaling in human ESCs.

(A) Expression of SOX4 mRNA in the presence of MPA or MPA + RU486 for 2 days in immortalized human endometrial stromal cells (HESCs). Results are presented as means ± standard error of the mean (SEM); n = 3; ***p < 0.0001. (B) RNA level of PGR after siRNA-mediated knockdown. Results are presented as means ± SEM; n = 3; ***p < 0.0001. (C) Expression of SOX4 mRNA in the presence of MPA for 2 days with PGR knockdown. Results were presented as means ± SEM; n = 3; ***p < 0.0001. (D) Protein level of SOX4 and PGR after PGR knockdown in the presence of MPA for 2 days. Band quantification of PGR and SOX4 protein, relative to loading control GAPDH. *p < 0.05 (n = 3). (E) Visualization of PGR binding and chromatin accessibility on SOX4 locus. The chromatin accessibility is depicted in undecidualized and decidualized stromal cells and genome-wide PGR binding is generated from proliferated and middle secretory endometrium as revealed from previous reports. Undec: undecidualized HESCs; Dec: decidualized HESCs; Prolif: proliferative endometrium; Middle: middle phase of secretory endometrium. (F) ChIP assay of potential PGR binding on SOX4 as indicated from (E) in decidualized immortalized HESCs for 2 days. Data are plotted as mean ± SEM; n = 3; ***p < 0.0001. (G) Luciferase activity assay of SOX4 promotor in the presence of MPA and PGR in 293T cells. Results are presented as means ± SEM. n = 3; *p < 0.05; **p < 0.005; ***p < 0.0001.

SOX4 is required for human endometrial stromal cell decidualization

To depict the significance of SOX4 during decidualization, SOX4 was knockout by CRISPR/Cas9 which was ascertained by Sanger-sequencing in both alleles (Figure 3—figure supplement 1A) and further verified by western blot and immunofluorescence (Figure 3—figure supplement 1B, C). The expression of PRL and IGFBP1 was dramatically decreased in SOX4 knockout HESCs after decidualization for 2, 4, and 6 days compared with SOX4 intact cells (Figure 3A–C). Similar results were obtained in primary HESC after SOX4 knockdown by shRNA prior to EPC administration (Figure 3—figure supplement 2A–D). On the other side, overexpressing SOX4 in HESCs significantly augmented the expression of PRL and IGFBP1 in both immortalized (Figure 3D–F) and primary HESC (Figure 3—figure supplement 2A–C) decidualized for 4 days.

Figure 3 with 3 supplements see all
SOX4 regulated genes in decidualized human endometrial stromal cells (HESCs).

mRNA levels of PRL (A) and IGFBP1 (B) in undecidualized and decidualized immortalized HESCs after SOX4 knockout at indicated time points after the E2, MPA, and cAMP treatment. Results are presented as means ± standard error of the mean (SEM); n = 3; *p < 0.05; ***p < 0.0001. (C) Protein levels of IGFBP1 in undecidualized and decidualized HESCs after SOX4 knockout at indicated time points. (D, E) mRNA levels of PRL (C) and IGFBP1 (D) after SOX4 overexpression in undecidualized or decidualized HESCs for 4 days. n = 3; *p < 0.05. (F) Protein levels of SOX4 and IGFBP1 in decidualized HESCs for 4 days with control or SOX4 overexpression. Band quantification of SOX4 and IGFBP1 protein, relative to GAPDH. (G) Differential expressed genes detected by RNA sequencing (RNA-Seq) in immortalized HESCs decidualized for 2 days with control or SOX4 knockdown as visualized by volcano plot. (H) Heatmap of top differential expressed genes from RNA-Seq after SOX4 knockdown in decidualized HESCs. (I, J) KEGG (J) and Gene Ontology (GO) (K) analysis of the differentially expressed genes in RNA-Seq. Results were presented as means ± standard error of the mean (SEM). *p < 0.05; **p < 0.005; ***p < 0.0001. The above experiments were repeated three times.

Moreover, RNA-Seq was utilized in SOX4 intact or depleted immortalized HESCs treated with EPC for 2 days. The genes significantly downregulated after SOX4 knockdown included aforementioned IGFBP1 and PRL as well as other genes critical for decidualization, such as FOXO1, LEFTY2, WNT5A, WNT4, FOSL2, STAT3, LEFTY2, IL-11, and BMP2 (Figure 3G, H). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the differentially expressed genes were enriched in the FOXO signaling pathway, TGF-beta signaling pathway, and MAPK signaling pathway (Figure 3I), consistent with the observed defective decidualization in the absence of SOX4. The Gene Ontology (GO) annotation analysis showed that the differentially expressed genes were related to autophagy-associated pathway, insulin-like growth factor binding, collagen-containing extracellular matrix, and cell–substrate adhesion (Figure 3J). Together, these results revealed the critical role of SOX4 in HESCs decidualization.

Genome binding of SOX4 in decidualized stromal cells

To interrogate genes directly regulated by SOX4, we detected the chromatin-wide binding of SOX4 by ChIP-Seq. Our results showed that there were total of 23,709 SOX4-binding peaks with most of them enriched in promoters, distal intergenic and intron (Figure 4A). Peak enrichment indicated that SOX4 binding was primary closed to TSS as well as evidenced by heatmap of peak distribution (Figure 4B, C). We also noticed that SOX4 mainly binds to those genes with higher expression (RPKM ≥1) accompanied with few SOX4 binding on those less to no expression genes (RPKM <1) (Figure 4B). Motif analysis results revealed that the most significantly enriched motif was SOX4 consensus binding sites, further supported the reliability of this SOX4 ChIP-Seq. We also noticed that FRA2 and FOSL2, two critical members of AP1, were also highly enriched at SOX4-binding sites, indicating the potential cooperation between SOX4 and AP1 (Figure 4D). Considering the correlation between SOX4 binding and gene expression, we overlapped SOX4-binding genes with those downregulated genes after SOX4 knockdown and found that 389 genes with SOX4 binding decreased after SOX4 ablation, indicating the direct regulation of SOX4 on these genes (Figure 4E).

Figure 4 with 2 supplements see all
Genome wide binding of SOX4 in decidualized stromal cells.

(A) Distribution of SOX4-binding peaks as revealed from SOX4 ChIP-Seq in SOX4 overexpressed (HA-SOX4) immortalized human endometrial stromal cells (HESCs) decidualized for 2 days. (B) Distribution SOX4 binding in genebody. (C) Heatmap of SOX4-binding sites distribution. (D) Motif analysis of SOX4-binding sites. (E) Venn diagram of SOX4 directly binding peaks and SOX4 regulated genes after SOX4 knockdown. (F) SOX4-binding site in the STAT3, FOXO1, and FOSL2 with progesterone receptor (PGR) binding and chromatin accessibility in both primary stromal cell and stromal cell line. The chromatin accessibility is depicted in undecidualized and decidualized stromal cells and genome-wide PGR binding is generated from proliferated and middle secretory endometrium as revealed from previous reports. Undec: undecidualized HESCs; Dec: decidualized HESCs. (G–J) ChIP-qPCR assay of SOX4 binding on PRL, STAT3, FOXO1, and FOSL2 in HA-SOX4 overexpressed decidualized HESCs. Results were presented as means ± standard error of the mean (SEM); n = 3; *p < 0.05; **p < 0.005; ***p < 0.0001. (K) Read number of SOX4 binding in SOX4 regulated genes after SOX4 knockdown. (L) KEGG analysis of overlapped genes of SOX4 directly binding and SOX4 downregulated genes as well as nonoverlapped genes. (M) mRNA levels of FOXO1 in SOX4 knockout undecidualized and decidualized HESCs at indicated time points. Results were presented as means ± SEM; n = 3; **p < 0.005. (N) Protein levels of SOX4, FOXO1, and IGFBP1 in SOX4 knockout undecidualized and decidualized HESCs at indicated time points. Band quantification of indicated proteins, relative to loading control GAPDH. *p < 0.05; ***p < 0.0001. n = 3. (O) mRNA levels of FOXO1 after SOX4 overexpression in undecidualized or decidualized HESCs for 4 days. Results were presented as means ± SEM; n = 3; **p < 0.005. The above experiments were repeated three times.

It was gratifying that there were several latent SOX4-binding sites on the STAT3, FOXO1, and FOSL2 in both stromal cell line and primary cultured stromal cells (Figure 4F). ChIP-qPCR also confirmed the binding of SOX4 on these genes (Figure 4G–J). Those downregulated genes (900) after SOX4 knockdown possess more SOX4-binding reads compared with upregulated genes (1022) (Figure 4K), indicating the transcriptionalactivation effect of SOX4. Importantly, those SOX4 direct target genes also showed specific enrichment of the FOXO signaling pathway (Figure 4L). To further assess the regulation of SOX4 on FOXO1, SOX4 was knockout and the expression of FOXO1 was overtly decreased accompanied with compromised decidualization as marked by lessened IGFBP1 during the decidualization from days 2 to 6 (Figure 4M, N). On the other hand, FOXO1 mRNA and protein were upregulated in SOX4 overexpression stromal cell decidualized for 4 days, as well as IGFBP1 protein (Figure 4O, and Figure 4—figure supplement 1B). Moreover, the effects of SOX4 overexpression on decidualization were blocked when the FOXO1 was knockdown, suggested the FOXO1 as a crucial effector downstream of SOX4 (Figure 4—figure supplement 1A, B). Besides the downregulated genes, there were also 438 genes with SOX4-binding sites were upregulated and GO analysis revealed that cell cycle regulators were mostly enriched (Figure 4—figure supplement 2A, B). During the decidual induction, the stroma cell would stop proliferation and initiate the differentiation program, the aberrant cell mitotic division in the absence of SOX4 was consistent with the defective decidualization. In summary, these results suggested that SOX4 directly regulated transcription of transcription factor FOXO1, as well as other molecules conducive for human endometrial decidualization.

SOX4 stabilizes PGR through repressing ubiquitin–proteasome pathway

Previous studies showed that P4-PGR signaling was critical for both initiation and maintenance of decidualization (Gellersen and Brosens, 2014), which incited us to assess PGR expression in the absence of SOX4 during the decidualization. We were attracted by the finding that PGR protein levels, including both the PRA and PRB isoforms, were dramatically decreased in the absence of SOX4 on days 0, 2, 3, and 6 during the decidualization without overt transcription reduction (Figure 5A, B). Similar results were also observed in primary stromal cells, implying that SOX4 stabled PGR protein at post-transcriptional level (Figure 5—figure supplement 1A, B). Furthermore, treatment of cells with the proteasome inhibitor MG-132 for 6 hr efficiently attenuated the rapid degradation of PGR protein in SOX4-deficient cells (Figure 5C), but the autophagy inhibitor 3-methyladenine (3-MA) cannot rescue or attenuate the degradation of PGR protein (Figure 5—figure supplement 1C), suggested the PGR protein was mainly degraded through the proteasome in the absence of SOX4. Likewise, PGR protein degeneration was faster in SOX4-depleted HESCs 2 days after EPC treatment in the presence of the protein synthesis inhibitor cycloheximide (CHX) for 0, 3, 6, and 9 hr (Figure 5D, E). Thus, these results revealed that SOX4 knockdown led to shortened the half-life of PGR protein.

Figure 5 with 1 supplement see all
SOX4 stabilizes progesterone receptor (PGR) through repressing the ubiquitin–proteasome pathway.

(A) mRNA levels of PGR after SOX4 knockout in decidualized immortalized human endometrial stromal cells (HESCs) at indicated time points after the E2, MPA, and cAMP treatment. Results were presented as means ± standard error of the mean (SEM); n = 3. (B) Protein levels of PGR and SOX4 after SOX4 knockout in decidualized HESCs at indicated time points after the E2, MPA, and cAMP treatment. (C) Protein levels of PGR in the presence of shSOX4 or MG132. MG-132 is added 6 hr before collecting the immortalized cells. (D, E) Protein levels of PGR in the presence of protein synthesis inhibitor cycloheximide (CHX) in SOX4-knockdown cells. (F) PGR ubiquitination after immunoprecipitation with PGR antibody and blotted with ubiquitin antibody in SOX4-knockdown immortalized HESCs decidualized for 2 days. Cells were treated with MG-132 before cell lysis. The value for relative protein level is band quantification of indicated protein, relative to GAPDH. n=3. *p < 0.05; **p < 0.005; ***p < 0.001.

To further explore the regulatory apparatus of PGR protein stability, the ubiquitination status of PGR was determined. Immunoprecipitated PGR was blotted against ubiquitin antibody in decidualized immortalized HESCs pretreated with MG-132 for 6 hr to postpone protein degradation. Notably, PGR ubiquitination was increased after SOX4 depletion (Figure 5F). These results suggested that SOX4 knockdown reduced PGR protein stability through the polyubiquitin-mediated proteasome degradation pathway.

SOX4 inhibits PGR degradation by regulating E3 ligase HERC4

Since ubiquitin E3 ligases were mainly responsible for protein ubiquitination (Rape, 2018), we next intended to identify the potential ubiquitin E3 ligase for PGR protein. Mass spectrometry (MS) was employed for PGR immunoprecipitated (IP) proteins in decidualized immortalized HESCs to estimate PGR-associated proteins (Figure 6—figure supplement 1A). Reassuringly, several E3 ubiquitination ligases were identified, including HERC4, RNF213, and UBR3 whose expressions were also upregulated according to RNA-Seq in SOX4-silenced HESCs (Figure 6A). Real-time PCR confirmed the abnormal upregulation of these E3 ligases when SOX4 was downregulated (Figure 6B, and Figure 6—figure supplement 1B, C). The protein level of HERC4 was consistently upregulated upon the ablation of SOX4 (Figure 6C). As HERC4 showed the most significant change after SOX4 knockdown and its interaction with PGR as a potential E3 ubiquitin ligase was also supported by UbiBrowser database (http://ubibrowser.ncpsb.org), we mainly focused on this E3 ligase in our following experiments.

Figure 6 with 1 supplement see all
SOX4 inhibits progesterone receptor (PGR) degradation by regulating E3 ligase HERC4.

(A) Venn diagram of overlapping genes between upregulated genes in SOX4 knockdown from RNA sequencing (RNA-Seq) and PGR-associated protein form IP-MS. mRNA and protein levels of HERC4 in the absence of SOX4 by qPCR (B) and western blot (C). Results were presented as means ± standard error of the mean (SEM); n = 3. (D) Protein interaction between PGR and HERC4 after overexpression of HA-PGR and Flag-HERC4 in 293T cells. (E) Protein interaction between PGR and HERC4 after overexpression of HA-PGR and Flag-HERC4 in decidualized immortalized human endometrial stromal cells (HESCs) for 2 days, D2: 2 days. (F) Localization of PGR and HERC4 after overexpression of PGR and HERC4 in decidualized HESCs for 2 days, scale bar: 100 μm. (G) PGR ubiquitination at the present of MG-132 after overexpression of HERC4 in decidualized HESCs for 2 days.(H) PGR ubiquitination at the present of MG-132 and ubiquitin with HERC4 overexpression or knockdown in 293T cells. (I) PGR ubiquitination at the present of MG-132, ubiquitin, and SOX4 with or without HERC4 in 293T cells. (J) Degeneration of PGR after HERC4 overexpression in 293T cells. (K) PGR protein levels at the present of HERC4 overexpression and in HERC4 knockdown decidualized immortalized HESCs for 2 days. (L) Protein levels of PGR, HERC4, and SOX4 after SOX4 and/or HERC4 knockdown in decidualized immortalized HESCs for 2 days. (M, N) The protein half-life of PGR after HERC4 knockdown at the present of protein synthesis inhibitor cycloheximide (CHX) in 2 days decidualized immortalized HESCs. (O, P) The protein half-life of PGR after SOX4 and/or HERC4 knockdown at the present of protein synthesis inhibitor CHX in 2 days decidualized immortalized HESCs. The value for relative protein level is band quantification of indicated protein, relative to GAPDH. The above experiments were repeated three times. n=3; *p < 0.05; **p < 0.005.

To verify whether HERC4 was a potential E3 ligase for PGR protein, we first detected the protein interaction between HERC4 and PGR. The coimmunoprecipitation result demonstrated a conserved physical interaction between endogenous HERC4 and PGR in decidualized stromal cells as well asexogenous expressed in 293T cells (Figure 6D, E). Immunofluorescence analysis showed the colocalization of HERC4 and PGR in decidualized HESCs (Figure 6F). To figure out whether HERC4 could induce endogenous ubiquitination of PGR, HERC4 was overexpressed in immortalized HESCs followed by decidualization for 2 days. The ubiquitination of PGR was higher in the presence of HERC4 than in control, as shown in Figure 6G, consistent with a reduced level of PGR protein in immortalized HESCs cells (Figure 6—figure supplement 1D). Conversely, HERC4 abolished by siRNA significantly reduced PGR ubiquitination in 293T cells (Figure 6H). The above studies demonstrated that HERC4 was a critical E3 ligase for PGR ubiquitination. Moreover, overexpression of HERC4 recused the decreasing PGR ubiquitination in the presence of SOX4 in 293T cells (Figure 6I). Hence, SOX4 affected PGR ubiquitination through E3 ubiquitin ligase HERC4.

Since HERC4 was a ubiquitin ligase of PGR and the increased ubiquitination of PGR was observed in the presence of HERC4, we next interrogated whether HERC4-mediated PGR degradation. This posit was underpinned by decreased PGR protein level with HERC4 overexpression in both 293T cells (Figure 6J) and immortalized HESCs (Figure 6K). PGR protein was also rescued by siRNA-mediated knockdown of HERC4 (Figure 6—figure supplement 1E) in SOX4 abolished decidualized immortalized HESCs (Figure 6L), and consistently, the IGFBP1 level was also rescued (Figure 6—figure supplement 1F). Likewise, PGR half-life increased in both normal and SOX4 abolished immortalized HESCs after HERC4 repression (Figure 6M, N). In a word, these studies demonstrated that SOX4-mediated PGR degradation by modulating E3 ligase HERC4.

Since there were 41 lysine residues in the PGR protein, UbiBrowser (http://ubibrowser.ncpsb.org) was applied to predict the potential domain recognized by E3 ligase HERC4 (Figure 7—figure supplement 1A). As the latent recognition domains were located in DBD and LBD domains of PGR, PGR was divided into four fragments (F1, F2, F3, and F4) accordingly (Figure 7A). Only F3 (DBD domains of PGR) exhibited protein degradation after cotransfected with HERC4 in 293T cells (Figure 7B). Point mutation of all Lysine (K) to arginine (R) in DBD region (Figure 7C) showed that K588, K613, K617, and K638 were critical for PGR degradation (Figure 7D), which was further sustained by incapable PGR ubiquitination mediated by HERC4 in these mutant forms when compared with WT or K565R in 293T cells (Figure 7E). Collective, these results suggested that K588, K613, K617, and K638 were vital for PGR ubiquitination by HERC4.

Figure 7 with 1 supplement see all
HERC4 mediates progesterone receptor (PGR) ubiquitination at K588, K613, K617, and K638.

(A) Schematic diagram of PGR structure. F1–F4 represents four different functional domains, respectively. (B) Protein levels of different fragments of PGR (Myc-tagged F1–F4) at the present of HERC4 in 293T cells. (C) Lysine (K) sites in PGR DBD domain. (D) Protein levels of PGR after Lysine mutant to Arginine in DBD with HERC4 overexpression in 293T cells.The value for relative protein level is band quantification of indicated protein, relative to GAPDH. (E) PGR ubiquitination after Lysine mutant to Arginine in DBD at the present of HERC4, ubiquitin and MG-132 in 293T cells. n=3; *p < 0.05; **p < 0.005.

Aberrantly decreased endometrial SOX4 expression is associated with recurrent implantation failure undergoing IVF treatment

There was considerable evidence indicating that HESCs decidualization was severely impaired in patients withthe EMS, both in eutopic and ectopic lesions (Klemmt et al., 2006). Recurrent implantation failure (RIF) has also been reported to be associated with compromised decidualization (Zhou et al., 2019). We next analyzed the expression levels of SOX4, PGR, FOXO1, HERC4, IGFBP1, and PRL in the mid-secretory endometrium of healthy women (control, n = 12) versus women who suffered RIF due to endometriosis (EMS-RIF, n = 12). The expressions of SOX4, FOXO1, IGFBP1, and PRL were significantly decreased in EMS-RIF group with increased HERC4 compared with the control, but the mRNA level of PGR was comparable in both groups (Figure 8A–F). At protein level, a large portion of endometrial samples from women with EMS-RIF showed reduced expression of SOX4, FOXO1, and IGFBP1. Although the PGR mRNA levels were comparable between healthy and RIF samples, PGR protein was significantly decreased accompanied by increased HERC4 in EMS-RIF group (Figure 8G, H). Immunostaining analysis further revealed significantly reduced PGR, SOX4, IGFBP1, and FOXO1 expression and increased HERC4 in stromal cells in women with EMS-RIF (Figure 8I).

Figure 8 with 1 supplement see all
Endometrial SOX4 expression in RIF of women with EMS undergoing IVF treatment.

(AF) Expression of SOX4, FOXO1, IGFBP1, PRL, PGR, and HERC4 in mid-secretory endometrium from control (n = 12) and EMS-RIF (n = 12). Results were presented as means ± standard error of the mean (SEM). *p < 0.05. (G, H) Protein levels of SOX4, FOXO1, IGFBP1, PGR, and HERC4 in mid-secretory endometrium from control (n = 12) and EMS-RIF (n = 12). C1–C12 and R1–R12 represent tissues from different patient. (I) Localization of SOX4, FOXO1, IGFBP1, PGR, and HERC4 in control (n = 3) and EMS-RIF groups as detected by immunostaining. C4, C7, C11, R4, R5, and R12 represent tissues from different patient. GE: gland epithelium; S: stroma. Scale bar: 100 μm. (J) Protein levels of SOX4, IGFBP1, FOXO1, and PGR in primary human endometrial stromal cells (HESCs) of control and EMS by western blot. (K) Protein levels of IGFBP1, FOXO1, PGR, and SOX4 in decidualized primary endometrial stromal cells from EMS-3 after overexpression of SOX4 and/or PGR.

Additionally, primary HESCs were obtained from the proliferative phase endometrium of three healthy and three EMS-RIF patients (Figure 8—figure supplement 1A). The expression levels SOX4, FOXO1, PGR, and IGFBP1 were lower in the primary HESCs of EMS-RIF cultured with EPC for 4 days compared to the control (Figure 8J). Moreover, overexpression of SOX4 and/or PGR at least partially restored FOXO1 and IGFBP1 expression in EMS-RIF stromal cells (Figure 8K). Collectively, our in vivo and in vitro evidence strongly suggested the crucial role of SOX4 in decidualization and female fertility.

Discussion

Here, we find that SOX4 is the most abundant SOX family member in HESCs and plays a vital role in decidualization. Previous studies have demonstrated that the SOX family controls cell fate and differentiation in various developmental processes (She and Yang, 2015; Angelozzi and Lefebvre, 2019). A previous study has confirmed that epithelial SOX17, one of SOX transcription factors, is indispensable for uterine receptivity and embryo implantation by regulating IHH expression (Wang et al., 2018). The embryo adhesion of implantation process occurred between the embryo and epithelia, and the proper differentiation of epithelia is required for this event (Ye, 2020). Moreover, for the endometrial receptivity establishment, the stromal compartment also needs to differentiate in response to the endocrine hormone progesterone and estrogen that is known as decidualization, and the crosstalk between the stroma and epithelia has been widely approved to participate to the establishment of endometrial receptivity (Li et al., 2011).

In the present study, we uncover the critical role of SOX4 in stromal cell decidualization by RNA-Seq and ChIP-Seq. Several previously unappreciated genes of SOX4 are unveiled through RNA-Seq. FOXO1 is indispensable in the process of decidualization account for the widely overlapping of binding peaks with PGR (Vasquez et al., 2015). In this investigation, FOXO1 is regulated by SOX4 at transcriptional level, indicating the essential role of SOX4 in decidualization. We are also very surprised to notice that the direct regulation of SOX4 on FOSL2, an important part of AP1 complex involving inflammation (Renoux et al., 2020), expression directly. Moreover, we also find that FOSL2 and FRA2 motifs are significantly enriched in SOX4-binding sites, suggesting the complex interplay between these factors. The regulation of SOX4 on FOLS2 implicates an alternative role of SOX4 regulating decidualization, which deserves further investigation.

PGR is a master regulator of the decidualization process since decidualization is mainly influenced by the progesterone–PGR signaling. After dimerization and nuclear translocation, PGR protein interacts with the following proteins to synergistically direct the differentiation program: FOXO1, C/EBPβ, STAT3, STAT5, HOXA10, and HOXA11 (Gellersen and Brosens, 2014). An array of modulators regulates the functional plasticity of PGR, including subcellular distribution, protein modification, and interaction with coregulators (Wu et al., 2018). In this study, we were intrigued by the finding that SOX4 affects PGR protein stability rather than its transcription.

During the menstrual cycle, estrogen–ER signaling induced endometrial PGR mRNA in the proliferative phase, recapitulating the regulation manner of PGR in mouse uteri (Tan et al., 1999; McKinnon et al., 2018). Here, we provided evidence that PGR protein is further stabilized by increased SOX4 at post-transcriptional regulation in the later secretory phase. Previous studies have validated that SOX4 regulates P53 stability through E3 ligase Mdm2 (Pan et al., 2009). In breast cancer cells, E3 ligase BRCA1 (Calvo and Beato, 2011) and CUEDC2 (Zhang et al., 2007) have been shown to regulate PGR protein stability. Multiple evidence in our research has verified that the E3 ligase HERC4 functions as a ubiquitin-modified enzyme for the PGR protein. Further exploration revealed that lysine residual in the PGR DNA-binding domain possess modified sites for ubiquitination. In the endometrial cells, there are very limited reports regarding PGR protein modification. Our previous study proved that Bmi1 facilitates the PGR ubiquitination through E3 ligase E6AP, which promotes PGR transcriptional activity instead of protein degradation (Xin et al., 2018). Besides, SUMOlation of PGR has been reported to occur at the K388 site and fine-tunes the transcriptional activity of PGR (Jones et al., 2006). The Lys-388 is a key site not only for PGR-B SUMOlation, but also a key site for CUEDC2-mediated ubiquitination in proteasome in breast cancer (Zhang et al., 2007). Here, we characterized that K588, K613, K617, and K638 were critical sites for E3 ligase HERC4, which mediated PGR ubiquitination and degradation. There were other E3 ligases interacting with PGR as revealed by our mass spectrum, whether these E3 ligases also mediated PGR ubiquitination remains largely unknown. Multiple specific E3 ligases may exist for PGR ubiquitination rely on individual lysine residues.

Here, we report a previously unrecognized PGR ubiquitination and degradation modified by the ubiquitin E3 ligase HERC4 regulated by SOX4 in endometrial stromal cells. There are several potential postulations concerning the underlying mechanism by which SOX4 inhibits HERC4 expression in stromal cells. SOX4 has been reported to interact with repressive histone modifiers, such as H3K27 trimethylation enzyme EZH2, to directly repress the target gene transcription (Koumangoye et al., 2015). On the other hand, SOX4 indirectly represses the target gene expression through regulating EZH2 expression (Tiwari et al., 2013). Since no direct SOX4 binding on HERC4 is observed, the detailed mechanism by which SOX4 regulates HERC4 transcription in endometrial stroma cells requires further exploration.

Considering the critical role of progesterone during the pregnancy establishment and maintenances, both natural and synthetic progestogens have been widely used to improve endometrial function in women with a history of RIF and unexplained recurrent pregnancy loss. However, some of these patients still suffered from RIF and recurrent miscarriages due to progesterone resistance. The causes of progesterone resistance may be related to defects in the PGR signaling and its molecular chaperones as well as decreasing PGR transcriptional activity (McKinnon et al., 2018). PGR functional deficiency is related to abnormal PGR mRNA levels, post-transcriptional modifications, post-translational modifications, and protein stability (Xin et al., 2016; McKinnon et al., 2018). However, the specific causes of PGR defects are, at present, poorly explored and understood.

EMS is frequently accompanied by progesterone resistance and infertility. Several transcription factors, including the factors in GATA and HOX family can regulate the progesterone signaling, and the epigenetic differential methylation of PR-B, HOX, and GATA family transcription factor can lead to their decreased expression, leading to the disturbed progesterone signaling in the EMS (Longo et al., 2020). It is interesting that lower SOX4 expression is strongly relevant with reduced PGR protein expression in endometrial stromal cells of EMS who experience RIF, and the level of PGR is restored to some extent when SOX4 is overexpressed in these stromal cells. It is conceivable that PGR defects caused by insufficient SOX4 may potentially result in implantation failures, even with high doses of progestogens supplementation during IVF. In our previous study using human endometrial samples from different patients with recurrent spontaneous abortion cohort, we also observed progesterone resistance as revealed by defective PGR signaling, with normal PGR protein level but diminished transcriptional PGR activity (Xin et al., 2018). This implies that any disturbances in the transduction of P4-PGR signaling pathway will severely influence the endometrial cell responsiveness to progesterone and contribute to infertility.

Collectively, our investigation provides compelling evidence that SOX4 plays a key role in HESCs decidualization through the transcriptional regulation of critical factors related to decidualization. Meanwhile, SOX4 endows human stromal cells appropriate progesterone responsiveness by fine-tuning PGR protein stability. Aberrant SOX4 expression is strongly associated with decreased PGR and FOXO1 expression in the endometrium of women who have experienced RIF with EMS, implying the high clinic relevance of SOX4 in female fertility and pregnancy maintenance. A better understanding of the regulatory network of SOX4 will facilitate the development of therapeutic strategy for the clinical treatment of RIF in EMS.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (H. sapiens)T HESCsATCCCRL-4003RRID: CVCL_C464
AntibodyAnti-SOX4(Rabbit polyclonal)AbcamCat # ab80261 RRID:AB_1658989WB (1:600)ICC (1:1000)IF (1:300)
AntibodyAnti-SOX4(Rabbit polyclonal)DiagenodeCat # C15310129IP (1:100)
AntibodyAnti-PGR(Rabbit monoclonal)CSTCat # 8757 RRID:AB_2797144WB (1:500)ICC (1:3000)IF (1:200)IP (1:50)ChIP (1:50)
AntibodyAnti-HERC4(Rabbit polyclonal)Protein techCat # 13691-1-AP RRID:AB_10596480WB (1:1000)ICC (1:1000)IF (1:300)
AntibodyAnti-FOXO1(Rabbit polyclonal)AbcamCat # ab39670 RRID:AB_732421WB (1:1000)ICC (1:3000)
AntibodyAnti-IGFBP1(Rabbit polyclonal)AbcamCat # ab228741WB (1:1000)IHC (1:500)
AntibodyAnti-Ub(Mouse monoclonal)CSTCat # 3936 RRID:AB_331292WB (1:1000)
AntibodyAnti-HA(Rabbit monoclonal)CSTCat # C29F4WB (1:1000)IP (1:100)ChIP (1:100)
AntibodyAnti-Myc(Mouse monoclonal)CSTCat # 2276 RRID:AB_331783WB (1:1000)
AntibodyAnti-Flag(Mouse monoclonal)SigmaCat # F9291 RRID:AB_439698WB (1:1000)
AntibodyAnti-GAPDH(Rabbit polyclonal)AbmartCat # P30008WB (1:2000)
Chemical compound, drugE2SigmaE8875Final concentration: 10 nM
Chemical compound, drugMPASigmaM1629Final concentration: 1 μM
Chemical compound, drugcAMPMCEHY-B0764Final concentration: 0.5 mM
Chemical compound, drugMG132SelleckS2619Final concentration: 20 μM
Chemical compound, drug3-MASelleckS2767Final concentration: 20 mM
Chemical compound, drugCHXMedChemExpressHY-12320Final concentration: 20 μg/ml
Chemical compound, drugInsulin–transferrin–seleniumThermo Fisher51500-056Final concentration: 1%
Chemical compound, drugPuromycinMedChemExpressHY-15695Final concentration: 500 ng/ml
Chemical compound, drugRNAi MAXThermo Scientific137780307.5 μl/6-well
Chemical compound, drugLipofectamine 2000Thermo Scientific116680305 μl/6-well/2500 ng DNA
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaE1910
Commercial assay or kitChIP-IT high SensitivityActive Motif53,040
Commercial assay or kitKAPA DNA HyperPrep KitRocheKK8502

Sample of clinical cases

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Healthy women who had already gave birth and infertile women with RIF due to EMS were recruited from Liuzhou Maternity and Child Health Care Hospital in China from March 2018 to December 2020. The study had been approved by hospital ethics committee and all participants signed informed consent. The age of participates is between 20 and 38 years of age with body mass index between 18 and 23. The thickness of the endometrium on ovulation days was between 8 and 16 mm with menstrual cycle between 28 ± 7 days and no steroid treatment or other medication for at least 2–3 months before biopsy. Detailed information of patients is listed in Supplementary file 1. Patients with polycystic ovary syndrome, endometrial polyps, chronic endometritis, and hydrosalpinges were excluded. Only endometrial tissue with no apparent pathology assessed by a pathologist was kept for further experiments. Decidual tissue in early pregnancy was collected from the women who underwent legal termination of an apparently normal early pregnancy (about 8 weeks). RIF referred to the inability to achieve a clinical pregnancy after transferring at least four good-quality embryos in a minimum of three fresh or frozen cycles in a woman under 40 years old (Coughlan et al., 2014).

Isolation and culturing of primary endometrial stromal cells

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Primary HESCs were obtained from healthy, reproductive-aged volunteers with regular menstrual cycles or EMS stage IV patients with infertility. An endometrial biopsy was performed during the proliferative phase of the menstrual cycle. These participants were recruited from The First Affiliated Hospital of Xiamen University in China from July 2019 to October 2020. Staging of EMS was according to rAFS (The American Fertility Society, 1985). Detailed information of patients is listed in Supplementary file 1, and pathology of pelvic EMS is shown in Figure 8—figure supplement 1A. The study had been approved by hospital ethics committee and all participants signed informed consent. Participants were documented not under hormone treatments for at least 3 months before surgery. Only endometrial tissue with no apparent pathology assessed by a pathologist was kept for further experiments. Primary HESCs of EMS women were isolated by laparoscopy, and the isolation and culturing of primary endometrial stromal cells were performed as follows. The endometrial tissues were first cut into pieces as small as possible and subjected to type IV collagenase (2% concentration) digestion for 1 hr. Two hours after cell seeding, culture medium was changed to remove the floating cells.

Establishment of SOX4 knockout cell lines

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Immortalized HESCs cell lines were purchased from ATCC Corporation (American Type Culture Collection, ATCC crl-4003). SOX4 knockout was generated by CRISPR/Cas9 approach as previously described (Zhang et al., 2019). (1) sgRNAs targeting SOX4 gene (NM_003107.3) were designed on the website (https://zlab.bio/guide-design-resources) and subcloned into the pL-CRISPR.EFS.GFP vector (Addgene plasmid #57818). (2) Cas9 plasmid was packaged with lentivirus. After infecting stromal cells, Cas9 positive cells were sorted and were plated into a 96-well plate with one cell per well. (3) Knockout efficiency was verified by DNA sequencing and western blot after single-cell-derived clone expansion.

In vitro culture of HESCs andd decidualization

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Immortalized HESCs were maintained in DMEM/F12 without phenolic red (Gibco) in the presence of 10% charcoal stripped fetal bovine serum (CS-FBS, Biological Industries), glucose (3.1 g/l), sodium pyruvate (1 mM, Sigma); sodium bicarbonate (1.5 g/l, Sigma), penicillin–streptomycin (50 mg/ml, Solarbio); insulin–transferrin–selenium (1%, Thermo Fisher) and puromycin (500 ng/ml). The primary cultured cells were maintained in DMEM/F12 (Gibco) without phenolic red and 10% CS-FBS. For decidualization, HESCs were cultured in medium at the present of estrogen (E2, 10 nM, Sigma), medroxyprogesterone acetate (MPA, 1 mM, Sigma), and dibutyl cyclophospsinoside (db-cAMP, 0.5 mM, MCE) in 2% CS-FBS with different days. All the cells were cultured in 5% CO2, 95% air, 100% humidity at 37℃ and culture medium was replaced every 48 hr. The endometrial stromal cell line was purchased from ATCC and authenticated using STR profiling by ATCC, and tested to be free from mycoplasma contamination.

Plasmid construction and siRNA transfection

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The overexpressed plasmids of SOX4 (pLVML-FLAG-SOX4-IRES-puro and pLVX-HA-IRES-ZSgrenn-SOX4), HERC4 (pLVX-FLAG-HERC4-IRES-Zsgreen, pENTER-FLAG-HERC4, and HA-PCMV-HERC4), PGR (pLVX-MYC-PGR-IRES-Zsgreen and HA-pCMV-PGR), and ubiquitin (pRK5-HA-Ubiquitin-WT and MYC-Ubiquitin-WT) were purchased from Wu Han Miao Ling Company or homemade. The list for plasmid used in this study is shown in Supplementary file 3. HESCs were transfected with these plasmids by Lipofectamine 2000 transfection reagent (Invitrogen) or infected with the lentivirus, and 48 hr later, HESCs were decidualized with EPC treatment. ShSOX4 lentivirus was purchased from Shanghai Ji Man Company. The ShSOX4 lentivirus and the control lentivirus were placed in the HESCs medium, 48 hr after infection, HESCs were decidualized with EPC.siRNAs targeting SOX4, HERC4, PGR, and FOXO1 were purchased from Guangzhou RiboBio Biological Company (see the specific sequence for details in Supplementary file 2). RNA interference was carried out according to the manufacturer’s instructions. Briefly, 10 mM siRNA was transfected into HESC with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, USA). For the knockdown and overexpression experiment, the silencing and overexpression were first performed without differentiation stimulus, and 24 hr later hESCs were decidualization for indicated time.

ChIP-Seq and ChIP-qPCR

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ChIP-Seq was performed according to the manual of ChIP-IT high Sensitivity (Active motif, catalog no. 53040). Briefly, both immortalized HESCs and primary cultured endometrial stroma cells (treated with EPC to induced decidualization for 2 days) with exogenous expressed HA-SOX4 were crosslinked and immunoprecipitated withHA (CST, c29F4) and PGR (CST, 8757) antibodies. Immunoprecipitated and input DNA were quantified using Qubit 4.0 fluorometer. Libraries were prepared using the KAPA DNA HyperPrep Kit (KK8502) and sequenced with an Illumina Nova-PE150. ChIP-qPCR was performed as ChIP-Seq with antibodies HA (CST, c29F4) and PGR (CST, 8757) and the immunoprecipitated DNA was detected by QPCR. All the PCR primers used in ChIP-qPCR are listed in Supplementary file 2.

Immunoprecipitation

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For protein interaction, IP experiments were performed as previously described (Xin et al., 2018). Anti-PGR (CST, 8757), Anti-HA (CST, c29F4), SOX4 (Diagenode, C15310129), and mouse IgG or rabbit IgG Mouse Anti-Rabbit IgG (Conformation Specific, L27A9 mAb) were used for IP. The immunoprecipitants were washed four times in lysis buffer, resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and immunoblotted with corresponding antibodies.

RNA-Seq

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Scramble or siSOX4 RNA were transfected into HESCs at 50% confluence with RNAi MAX (Invitrogen). Immortalized HESCs were decidualized for 2 days after transfection and RNAs were collected for RNA-Seq of BGISEQ (China, BGI). Purified RNA was prepared and subjected to 50 bp single-end RNA-Seq.

IP MS

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Immortalized HESCs were used to prepare IP samples after treated with EPC 2 days. After Co-IP with PGR-conjugated agarose beads, the immunoprecipitants were resolved with SDS–PAGE and visualized using Coomassie brilliant blue stain. The discrete bands between PGR and IgG control were isolated, digested, purified, and subjected to liquid chromatograph (LC)–MS in School of Life Sciences, Xiamen University.

Dual-luciferase reporter assay

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Construction of luciferase reporter was performed as previously described (Jiang et al., 2015). The promoter regions of SOX4 were amplified from genomic DNA and subcloned into pGL3 plasmid. All constructs were transiently transfected into 293T cells using Lipofectamine 2000. Total cell lysates were prepared 36 hr after transfection, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized by Renilla luciferase activity.

PGR ubiquitination assay

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The ubiquitination assays were performed as previously described (Xin et al., 2018). MG-132 (Sigma M8699, 20 μM) was added to the cultured medium 6 hr before cells collection. Cell lysis was immunoprecipitated with antibodies against PGR and HA, respectively. Proteins were released from the beads by boiling in SDS–PAGE sample buffer, and ubiquitination was analyzed by immunoblotting with different antibodies.

Protein stability experiment and degradation assay

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To determine the protein stability of PGR protein, SOX4-knockdown and control immortalized HESCs decidualized for 2 days followed by treatment with CHX (MedChemExpress, 20 μg/ml), a protein synthesis inhibitor, for 0, 3, 6, 9 hr or 0, 4, 8, 12 hr. The protein degradation assay of PGR, the immortalized HESCs with 50% confluency were induced decidualization for 2 days, followed by treatment with the proteasome inhibitor MG132 (Selleck, 20 μM, 6 hr), and the autophagy inhibitor 3-MA Selleck, 20 mM, 6 hr. A group treated with Dimethylsulfoxide (DMSO) served as the negative control group.

Immunostaining

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For healthy endometrial tissue, proliferative phase samples were timed based on the patient’s cycle day, and luteal phase samples were timed using the subject’s urinary luteinizing hormone surge. All the samples from the biopsy were fixed in formalin and embedded in paraffin for section. After deparaffinization and hydration, formalin-fixed paraffin embedded endometrial sections (5 μm) were subjected to antigen retrieval by autoclaving in 10 mM sodium citrate solution (pH = 6.0) for 10 min. A diaminobenzidine (Sigma) solution was used to visualize antigens. Sections were counterstained with hematoxylin. In immunofluorescence studies, formalin-fixed HESCs cells were blocked with 5% Bovine Serum Albumin (BSA) in PBS and immune stained by antibodies for SOX4 (Abcam,ab80261), PGR (CST, 8757), FOXO1 (Abcam, ab39670), IGFBP1 (Abcam, ab228741) and HERC4 (Proteintech, 13691-1-AP). Signals were visualized by secondary antibody conjugated with Cy2 or Cy3 fluorophore (Jackson Immunoresearch). Sections were counterstained with Hoechst 33,342 (2 μg/ml, Life Technology).

Real-time PCR

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Quantitative real-time PCR was performed as described previously (Jiang et al., 2015). Total RNA was extracted from HESCs or endometrium using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The quality of RNA was determined by electrophoresis and concentration of RNA was measured by Nanodrop. A total of 1 μg RNA was used to synthesize cDNA. Quantitative real-time PCR was performed with SYBR Green (Takara) on an ABIQ5 system. All expression values were normalized against GAPDH. All PCR primers are listed in Supplementary file 2.

Western blot analysis

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Western blot analysis was performed as described previously (Zhang et al., 2019). Antibodies against SOX4, IGFBP1, FOXO1, PGR, HERC4, FOSL2, HA, Myc, Flag, and ubiquitin were used. GAPDH served as a loading control. The value for relative protein level represents the quantification of band intensity of indicated protein with the loading control GAPDH using Image J.

Bioinformatic analysis

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The softwares for RNA-Seq and ChIP-Seq analysis including STAR (2.7.3a) for alignment, MACS2 (2.2.7.1) for peakcall of ChIP-Seq, ggplot2 (3.3.5) for visualization, ngs.plot.r (2.61) for peak heatmap of ChIP-Seq and the packages of edgeR (3.9), Complex heatmap (2.4.3), ChIPseeker (1.24.0) in R (4.1). For ChIP-Seq, after filter the raw data to remove adapter from read by Trimgalore, the clean reads were aligned to the human genome (Hg38) by HISAT2. Only uniquely aligned reads were kept for downstream analysis. MACS2 was applied for peak call using default parameters. The enrichment of SOX4 binding on specific gene was visualized in Integrative Genomics Viewer (IGV). RNA-Seq raw data were initially filtered to obtain clean data after quality control by Trimgalore. Clean data were aligned to the human genome (Hg38) by HISAT2. RPKM value of each gene was calculated by EdgeR package in R.

Statistics

Statistical analysis was performed with GraphPad Prism. The data were shown as the mean ± standard error of the mean. Statistical analyses were performed using two-tailed Student’s t-test or analysis of variance. All experiments were repeat at least three times and p values less than 0.05 were considered statistically significant.

Data availability

The sequencing data generated in this study have been deposited in the Gene Expression Omnibus database under accession code GSE146280 (RNA-Seq), GSE174602 (ChIP-Seq in stromal cell line) and GSE174602 (ChIP-Seq in primary cultured stromal cell).

The following data sets were generated
    1. Deng W
    (2020) NCBI Gene Expression Omnibus
    ID GSE146280. Gene expression in human endometrium cells (ATCC 4003) after Sox4 was abolished by siRNA.
    1. Deng W
    (2022) NCBI Gene Expression Omnibus
    ID GSE174602. Gene expression in human endometrium cells (ATCC 4003) after Sox4 was abolished by siRNA (ChIP-Seq in stromal cell line).
The following previously published data sets were used
    1. Deng W
    2. Dey S
    (2019) NCBI Gene Expression Omnibus
    ID GSE116096. Gene expression in human endometrium cells (ATCC 4003) after Sox4 was abolished by siRNA (ChIP-Seq in primary cultured stromal cell).

References

    1. Xin QL
    2. Qiu JT
    3. Cui S
    4. Xia GL
    5. Wang HB
    (2016)
    Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation
    Sheng Li Xue 4:435–454.

Decision letter

  1. T Rajendra Kumar
    Reviewing Editor; University of Colorado, United States
  2. Mone Zaidi
    Senior Editor; Icahn School of Medicine at Mount Sinai, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Your manuscript has been evaluated by 3 independent experts. All of them have agreed that your manuscript is very interesting and provides new knowledge to the field. They also raised several concerns including the use of immortalized cells for ChipSeq studies instead of native stromal cells, lack of methodological details for some of the experiments, lack of full-description of differentially regulated genes, incomplete clinical data and a clear rationale for the reported studies.

Based on your manuscript, the reviews and your responses, we invite you to submit a revised version incorporating the revisions as outlined in your response to the reviews.

Some Essential Revisions that require your careful attention are listed below:

– Expand Materials and methods section to provide more experimental details.

– Provide Rationale for Sox4 ChIP-seq using immortalized cells and provide details of timing for ChIP-seq studies.

– Provide data on knock down of SOX4 and HERC4 to rescue decidualization gene expression.

– Indicate unregulated genes in the absence of SOX4 in ChIP-seq experiments.

– Clarify discussion on receptivity and decidualization.

– Provide more clinical data including sample size, characteristics and sample collection periods.

– Numerous methodological details need to be provided as suggested by Reviewer 3.

Additional detailed comments by the Reviewers are listed below. Please address them point-by-point in your Revised manuscript.

Reviewer #1 (Recommendations for the authors):

(1) An overarching issue is that the Results and Figure legends are not entirely descriptive about the experiments conducted to generate the data provided in the figures. The reader must delve into the Materials and methods to figure out how the experiment was conducted in terms of the approach. This issue can be rectified by providing more detail on the experimental approach for each major investigation and result. The Materials and methods need to be very clear on how experiments were conducted and analyzed.

(2) Line 188: What is the rationale for not using a SOX4 antibody to conduct the ChIP-seq studies in non-immortalized cells as opposed to overexpression of using a transfection approach? More detail needs to be presented on the treatments and timing of SOX4 ChIP-seq.

(3) Figures 6M and 6N: The figure legend indicates that this experiment was repeated three times, but the graphs do not provide error bars. Was the data for each Western blot quantified with respect to PGR protein and normalized to GAPDH? If so, why are error bars not present on the graphs?

(4) Supplemental Figure 4: The authors contend that HERC4 overexpression decreases PGR abundance, but the level of PGR protein is not assessed, and the PGR immunolocalization data is not very convincing.

(5) Functional studies should be conducted to rescue decidualization gene expression with knockdown of HERC4 and SOX4. Alternatively, the authors can express a HERC4 resistant form of PGR and investigate the impact of SOX4 on decidualization.

(6) Figure 8: The data on EMS-3 are very intriguing but incomplete. If IGFBP1 is enhanced by SOX4 and PGR overexpression, then FOXO1 should also be investigated as well as PRL and other SOX4 and/or PGR responsive genes. In essence, the endometriosis angle for this patient is incomplete as presented.

(7) The manuscript would benefit from editing to correct problems with English grammar and usage.

(8) Line 356: HOX010 and HOXO11 should be HOXA and not HOXO.

(9) Discussion: The fact that different patients with endometriosis-associated RIF have varying defects in several transcription factors should be highlighted in the Discussion. In essence, the underlying variability in several of the key transcription factors can all lead to the syndrome of progesterone resistance.

Reviewer #2 (Recommendations for the authors):

1) In Figure 1, the authors have examined the SOX4 expression during the menstrual cycle, how about its expression in the decidual tissue during the pregnancy?

2) In Figure 4 The author showed the overlap the down-regulated genes in the absence of SOX with the ChIP data, how about the up-regulated genes?

3) In Figure 4 the motif analysis for SOX4 ChIP-seq suggested the potential co-binding of regulatory region between the SOX4 and other proteins such as FOSL2, did the author tried to test the protein physical interaction during the decidualization?

4) In Figure 5, while the proteasome inhibitor MG-132 could almost rescue the PR protein degradation in the SOX4 shRNA treatment, did the authors consider whether other protein degradation system, such as the lysosome mediated protein degradation may also be involved? This may deserve some discussions.

Other suggestions:

1. The "Eichment" in all the label in the ChIP-PCR data should be changed into "Enrichment".

2. In the manuscript, the authors used "human endometrial stromal cells (hESCs)" while the hESCs is usually short for "human Embryonic Stem Cells", I suggest to make a little change into "human endometrial stromal cells (HESCs)", which may reduce potential confusion.

Reviewer #3 (Recommendations for the authors):

1. The scientific background should be more clear and straightforward, it would be interesting that the authors discuss it in the manuscript.

a. The connection between receptivity and decidualization is confused. Decidualization is a differentiation process that regulates mainly the stromal compartment regulating embryo invasion, the next step of embryo adhesion, which is mainly govern by endometrial receptivity affecting the epithelial compartment. So, why the authors linked these processes and what is the connection?

b. Intro (lines 59-61): The receptive endometrium in human requires remodeling of stromal cells under the regulation of rising progesterone and intracellular cyclic AMP, which will undergo more extensive transformation driven by factors secreted from embryos. Which reference support this affirmation?

c. In the introduction ´Insufficient decidualization in endometrium is related to failed embryo implantation, unexplained infertility, recurrent spontaneous abortion (Coulam 2016), intrauterine growth retardation (Lefevre, Palin et al., 2011), and preeclampsia (Garrido-Gomez, Dominguez et al., 2017). However, the underlying molecular mechanism governing the endometrial decidualization remains enigmatic (Okada, Tsuzuki et al., 2018)´. There is lack of references to relate insufficient decidualization with failed embryo implantation and insufficient decidualization with unexplained infertility in this paragraph. The fact is that deficient decidualization relates to defects in embryo invasion and subsequent placentation and pregnancy complications. But the authors are trying to connect receptivity with insufficient decidualization and infertility. This is unclear and the authors need to be more precise that the scientific background that is exposed in this manuscript.

d. Intro: "both SOX4 and PGR have been demonstrated to be aberrantly downregulated in the endometrium of endometriosis (EMS) patients suffering from implantation failure". It is confused the focus of this study connecting role of SOX4 in decidualization with patients with implantation failure. If the authors want to relate SOX4 with implantation failure should demonstrate the connection with endometrial receptivity and embryo adhesion.

2. The clinical data included in this manuscript is very concise and need to be completed.

a. The authors described that some experiment was performed with normal samples, clinically what means ´normal´? The authors should define the clinical characteristics, the sample size and when the samples were collected.

b. Is the Table S2 (line 933) the equivalent to supplementary Table 1 (line 999)? please review it. This table include the clinical information about the patients analyzed in the last part of the article, but the authors should include another table including the clinical data of the patients that were recruited to collect the endometrial samples that are the source of primary hESCs used in the rest of paper. Also, this table include abbreviations that need to be defined and the history of previous pregnancies of controls to prove fertile status.

3. There are some general aspects that must be improved in the drafting of the results to improve the comprehensibility, much more details in some experiments are needed.

a. The nomenclature of cell types needs to be clearer, sometimes it is confusing to know whether you are using cell lines or primary endometrial stromal cells isolated from biopsies (e.g. In line 120 "endometrial stromal cells"), they are cell line or primary cells? it must be specified also in figure 1 legend, in line 129 "in endometrial biopsy samples obtained from healthy reproductive-aged volunteers" linked with Figure 1C, it would be better to specify which type of cells are you using in figure 1 because it seems that there are 2 type of cells. The authors explained results using immortalized cells and 293T cells, this are same cells? Please detail.

b. Another nomenclature issue to improve is the use of different terms for non-decidualized and decidualized cells "undifferentiated and differentiated stromal cells"; "undecidualized and decidualized"; "non-decidualized and decidualized". The authors should use unified nomenclature.

c. Authors expose results analyzing decidualized and non-decidualized cells, but the figures do not show decidualized vs non-decidualized instead of this they shown D0 and D4. Why do they not use decidualized and non-decidualized cells at same time of treatment (D4)? This comparison let us to know what is happening with cells under hormonal treatment compared with cells growing at the same time without decidualization inductors. The author should explain how they performed the experiments and why they decided to make this experimental design.

d. I have missed the times at which some treatments are performed, such as the decidualization treatments in Figure 2 A and the silencing and overexpression treatments. It should be specified for how long the silencing and overexpression is performed as well as decidualization. Additionally, in some treatments you must specify the dose of the drug you are using.

e. Why did you performed an RNA-Seq only 2 days after decidualization when in all of you experiments you show that decidualization markers increase their value from day four (e.g. Figure 3 B and C)? This may be somewhat confusing; the authors should explain this decision.

4. Some experimental part of the manuscript could be improved it:

a. Western Blot in general you have to specify in which day you have performed it, in some cases it has not been mentioned (e.g. Figure 3A). Also, the authors need to well-defined the double band that found in the PGR protein experiment, as well as, to improve some GAPDH western blot where the bands are undistinguished between conditions (example figure 2D and Figure 3G). Furthermore, graphic including the band quantification and statistical analysis in each western blot could help supporting the findings.

b. Immunostaining should be more described along the manuscript explaining that are based in the analysis of endometrial tissue and to specify details such as day of sample collection.

5. The authors need to improve details on methodology.

a. RNAseq data need to be explain in detail. Figure 1A-B: "(A) Expression of all transcription factors in human non-decidualized ESCs by RNA-Seq. (B) Expression of SOX family genes in human non-decidualized ESCs by RNA-Seq" is based in the normal endometrium stromal cells analysis and figure legend define that are non-decidualized cells. Which cells are analysed in RNAseq analysis? Please, do not use the term normal to clinically define type of sample. Which experimental design was used? Sample size (N)? Raw and normalized counts? Filtered and quality control process? Which statistics were used? Please improve the results showed for RNAseq analysis.

b. Versions of software and bioinformatic tools need to be included.

c. New sections in M and M need to be included for RNA extraction and quality evaluation (specifying RIN threshold consider), gene ontology enrichment, and real-time PCR.

d. How batch effect was evaluated, and variables tested for controlling confounding effects.

e. What function was used in edgeR for identify differentially expressed genes (e.g. exactTest), p-value adjustment and threshold for consider a transcript significantly deregulated, the fold change obtained and if there was some threshold to select genes to be included in subsequent analysis.

f. Antibody concentrations used in immunoprecipitation and immunostaining must be included. Also, reagent concentrations for transfection including lipofectamine and times followed to procced such as medium change during transfection. We suggest creating a Key Resource Table with all the chemical compounds, kits, antibodies, plasmids, and software used with the references and concentrations with the aim of being more transparent and increasing the reproducibility of experiments.

6. Regarding the discussion there are some aspects to be commented:

a. Line 409: The paper of SOX4 in decidualization linked with implantation failure in endometriosis could be improved. In this sense I suggest a proposal about how the authors think about the mechanism underlying with these two biological processes.

b. PGR isoforms A and B have different roles during the menstrual cycle, being the isoform B the predominant in decidualization. Authors should recognize it is a limitation not included this differentiation in the manuscript.

https://doi.org/10.7554/eLife.72073.sa1

Author response

Some Essential Revisions that require your careful attention are listed below:

– Expand Materials and methods section to provide more experimental details.

We have made the corresponding modification in the “Materials and methods” section in the revision as suggested.

– Provide Rationale for Sox4 ChIP-seq using immortalized cells and provide details of timing for ChIP-seq studies.

We conducted new ChIP-seq experiment using the primary endometrial stroma cells and got the similar results as in the immortalized cells (See revised Figure 4F) and the data have been deposited in the Gene Expression Omnibus database. The details of the timing are provided in the revised manuscript (See line 553-555).

– Provide data on knock down of SOX4 and HERC4 to rescue decidualization gene expression.

We conducted new experiments. The data on knock down of SOX4 and HERC4 to rescue decidualization gene expression is included in Figure 6—figure supplement 1F and also described in the revised manuscript (See line 297-298).

– Indicate unregulated genes in the absence of SOX4 in ChIP-seq experiments.

The data for upregulated genes in the absence of SOX4 is shown in the revised Supplementary Figure 4, and we also described this in the revised manuscript (See line 220-225).

– Clarify discussion on receptivity and decidualization.

We added the discussion on receptivity and decidualization in the revised manuscript (See line 54-61 and line 352-359).

– Provide more clinical data including sample size, characteristics and sample collection periods.

The detailed information for clinical data was provided in the Supplementary file 1. Clinical information of all patients and controls.

– Numerous methodological details need to be provided as suggested by Reviewer 3.

We provided the methodological details as reviewer suggested in the revised manuscript (See “Materials and methods” section).

Reviewer #1 (Recommendations for the authors):

(1) An overarching issue is that the Results and Figure legends are not entirely descriptive about the experiments conducted to generate the data provided in the figures. The reader must delve into the Materials and methods to figure out how the experiment was conducted in terms of the approach. This issue can be rectified by providing more detail on the experimental approach for each major investigation and result. The Materials and methods need to be very clear on how experiments were conducted and analyzed.

Many thanks for this concern. We provided detailed information on the experimental approach for each major investigation and result. We also provided entire description in the figure legend.

(2) Line 188: What is the rationale for not using a SOX4 antibody to conduct the ChIP-seq studies in non-immortalized cells as opposed to overexpression of using a transfection approach? More detail needs to be presented on the treatments and timing of SOX4 ChIP-seq.

Indeed, we have tried commercially available antibodies as much as possible (Abcam, ab86809; Abcam, ab80261; Santa Cruz, sc-518016; Diagenode, C15310129). But none of them work well for ChIP-seq assay in the stromal cells, so we choose the exogenously expressed HA-Tagged SOX4 to explore the genome-wide binding sites of SOX4, and the exogenously expressed level was controlled at a comparable level to endogenous SOX4. Moreover, during the revision, the ChIP-seq experiment in the primary stromal cells recapitulated the results in non-immortalized cells (See revised Figure 4F), and the data have been deposited in the Gene Expression Omnibus database.

(3) Figures 6M and 6N: The figure legend indicates that this experiment was repeated three times, but the graphs do not provide error bars. Was the data for each Western blot quantified with respect to PGR protein and normalized to GAPDH? If so, why are error bars not present on the graphs?

Thanks for this concern. The statistical analysis of quantification of PGR expression was calculated in three independent experiments after normalized to GAPDH. In the revised figures, the error bars were present on the graphs.

(4) Supplemental Figure 4: The authors contend that HERC4 overexpression decreases PGR abundance, but the level of PGR protein is not assessed, and the PGR immunolocalization data is not very convincing.

Thanks for this concern. Besides the immunolocalization data, the level of PGR protein was also assessed by immunoblot and PGR abundance was decreased in the presence of HERC4 overexpression (See revised Figure 6K).

(5) Functional studies should be conducted to rescue decidualization gene expression with knockdown of HERC4 and SOX4. Alternatively, the authors can express a HERC4 resistant form of PGR and investigate the impact of SOX4 on decidualization.

Many thanks for this concern to strength our manuscript. We conducted new experiment to knockdown both HERC4 and SOX4 to explore the decidualization status and uncovered that HERC4 knockdown partially rescued IGFBP1 expression caused by the SOX4 deficiency (See revised Figure 6—figure supplement 1F).

(6) Figure 8: The data on EMS-3 are very intriguing but incomplete. If IGFBP1 is enhanced by SOX4 and PGR overexpression, then FOXO1 should also be investigated as well as PRL and other SOX4 and/or PGR responsive genes. In essence, the endometriosis angle for this patient is incomplete as presented.

As suggested, we conducted the new experiment to investigate FOXO1 expression when SOX4 and PGR were overexpressed, and found that FOXO1 was upregulated (See revised Figure 8K).

(7) The manuscript would benefit from editing to correct problems with English grammar and usage.

We corrected the problems with English grammar and usage.

(8) Line 356: HOX010 and HOXO11 should be HOXA and not HOXO.

Many thanks for this suggestion to strength our manuscript and we have made correction.

(9) Discussion: The fact that different patients with endometriosis-associated RIF have varying defects in several transcription factors should be highlighted in the Discussion. In essence, the underlying variability in several of the key transcription factors can all lead to the syndrome of progesterone resistance.

Thanks for this valuable concern, and we added the discussion for several transcription factors, such as factors in GATA and HOX family, which are reported to be associated endometriosis and progesterone resistance (See line 431-435).

Reviewer #2 (Recommendations for the authors):

1) In Figure 1, the authors have examined the SOX4 expression during the menstrual cycle, how about its expression in the decidual tissue during the pregnancy?

Thanks for concerns to strengthen our manuscript. We detected the expression of SOX4 in the decidual tissue during the early pregnancy and found that the SOX4 was also highly expressed in the decidual cells during the pregnancy (See revised Figure 1C).

2) In Figure 4 The author showed the overlap the down-regulated genes in the absence of SOX with the ChIP data, how about the up-regulated genes?

We also provided the information for the up-regulated genes in the revised Supplementary Figure 4, and added the description in the revised manuscript (See line 220-225).

3) In Figure 4 the motif analysis for SOX4 ChIP-seq suggested the potential co-binding of regulatory region between the SOX4 and other proteins such as FOSL2, did the author tried to test the protein physical interaction during the decidualization?

Thanks for this thoughtful concern. We conducted the co-immunoprecipitation experiment as the reviewer suggested. The results showed that the there was no interaction between the SOX4 and FOSL2 in our co-IP system (See Author response image 1), suggesting SOX4 cannot strongly interact with the FOSL2.

Author response image 1
HA antibody was used to immunoprecipitant HA-tagged SOX4 in stromal cells, the immunoprecipitated proteins was blotted with the indicated antibodies.

4) In Figure 5, while the proteasome inhibitor MG-132 could almost rescue the PR protein degradation in the SOX4 shRNA treatment, did the authors consider whether other protein degradation system, such as the lysosome mediated protein degradation may also be involved? This may deserve some discussions.

Thanks for this concern. We conducted the experiment and found that the inhibitor of lysosome cannot rescue the protein degradation (See revised Figure 5—figure supplement 1C and line 242-244).

Other suggestions:

1. The "Eichment" in all the label in the ChIP-PCR data should be changed into "Enrichment".

Many thanks for this suggestion and we have corrected this mistake in the revised figures.

2. In the manuscript, the authors used "human endometrial stromal cells (hESCs)" while the hESCs is usually short for "human Embryonic Stem Cells", I suggest to make a little change into "human endometrial stromal cells (HESCs)", which may reduce potential confusion.

As suggested, we have corrected it.

Reviewer #3 (Recommendations for the authors):

1. The scientific background should be more clear and straightforward, it would be interesting that the authors discuss it in the manuscript.

a. The connection between receptivity and decidualization is confused. Decidualization is a differentiation process that regulates mainly the stromal compartment regulating embryo invasion, the next step of embryo adhesion, which is mainly govern by endometrial receptivity affecting the epithelial compartment. So, why the authors linked these processes and what is the connection?

Different from the scenario in the rodent models in which the decidualization occurred after the embryo attachment, decidualization of the human endometrium does not require embryo adhesion. The human endometrial stromal compartment can initiate the decidualize during the progesterone-dominant early secretory phase of menstrual cycle. When the endometrium entered into the receptive state mainly dominating by the endocrine progesterone and estrogen, the stromal compartment has initiated the decidualization, which has also been demonstrated by the recent single cell RNA-Seq data (Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle. Nat Med. 2020 Oct;26(10):1644-1653. PMID: 32929266). The implantation window open, which accompanied with the receptive endometrium, accompanied with a widespread decidualization feature in the stromal fibroblasts, which was confirmed by our data about the decidualization marker IGFBP1 immunostaining (Figure 8I). Just as the reviewer stated, the embryo adhesion occurred between the blastocyst and the endometrial epithelium, but the proper differentiation of epithelium preparing for adhesion with the embryo is also regulated the stromal compartment, which have been demonstrated by mouse models. Only when the epithelium and stroma compartment properly differentiated under the influence of endocrine progesterone and estrogen, the endometrial receptivity can be established well, and the differentiation process for stromal compartment is the decidualization. In this study, we focus the decidualization, which means the stromal cell differentiation, directed by the progestogen signal for preparing the endometrial receptivity.

b. Intro (lines 59-61): The receptive endometrium in human requires remodeling of stromal cells under the regulation of rising progesterone and intracellular cyclic AMP, which will undergo more extensive transformation driven by factors secreted from embryos. Which reference support this affirmation?

Thanks for this concern. We have provided references for this description and reconstruct this sentence:

“The receptive endometrium in human requires remodeling of stromal cells under the regulation of rising progesterone and intracellular cyclic AMP, which will undergo more extensive transformation after embryo implantation”.

c. In the introduction ´Insufficient decidualization in endometrium is related to failed embryo implantation, unexplained infertility, recurrent spontaneous abortion (Coulam 2016), intrauterine growth retardation (Lefevre, Palin et al., 2011), and preeclampsia (Garrido-Gomez, Dominguez et al., 2017). However, the underlying molecular mechanism governing the endometrial decidualization remains enigmatic (Okada, Tsuzuki et al., 2018)´. There is lack of references to relate insufficient decidualization with failed embryo implantation and insufficient decidualization with unexplained infertility in this paragraph. The fact is that deficient decidualization relates to defects in embryo invasion and subsequent placentation and pregnancy complications. But the authors are trying to connect receptivity with insufficient decidualization and infertility. This is unclear and the authors need to be more precise that the scientific background that is exposed in this manuscript.

Thanks very much for this suggestion. We have added the reference demonstrating the relationship between the decidualization and uterine receptivity (See line 62). Moreover, we also provided the scientific background for relating insufficient decidualization with failed embryo implantation (See line 352-359 in “Discussion” section).

d. Intro: "both SOX4 and PGR have been demonstrated to be aberrantly downregulated in the endometrium of endometriosis (EMS) patients suffering from implantation failure". It is confused the focus of this study connecting role of SOX4 in decidualization with patients with implantation failure. If the authors want to relate SOX4 with implantation failure should demonstrate the connection with endometrial receptivity and embryo adhesion.

Thanks very much for this suggestion. The similar concern for the relation between the decidualization and endometrial receptivity. We have added some discussion for the decidualization and uterine receptivity, including the embryo adhesion (See line 352-359).

2. The clinical data included in this manuscript is very concise and need to be completed.

a. The authors described that some experiment was performed with normal samples, clinically what means ´normal´? The authors should define the clinical characteristics, the sample size and when the samples were collected.

Thanks for this concern. The normal samples mean that the endometrial samples were collected from the women who have given birth, and we have changed the ´normal ´with ´healthy´. We also provided a table about clinical information for the sample collection (See Supplementary file 1. Clinical information of all patients and controls).

b. Is the Table S2 (line 933) the equivalent to supplementary Table 1 (line 999)? please review it. This table include the clinical information about the patients analyzed in the last part of the article, but the authors should include another table including the clinical data of the patients that were recruited to collect the endometrial samples that are the source of primary hESCs used in the rest of paper. Also, this table include abbreviations that need to be defined and the history of previous pregnancies of controls to prove fertile status.

Thanks for this concern to strength our manuscript. We have provided the new Supplementary file 1. Clinical information of all patients and controls. for the clinical sample which was used for isolating the primary cultured cell.

3. There are some general aspects that must be improved in the drafting of the results to improve the comprehensibility, much more details in some experiments are needed.

a. The nomenclature of cell types needs to be clearer, sometimes it is confusing to know whether you are using cell lines or primary endometrial stromal cells isolated from biopsies (e.g. In line 120 "endometrial stromal cells"), they are cell line or primary cells? it must be specified also in figure 1 legend, in line 129 "in endometrial biopsy samples obtained from healthy reproductive-aged volunteers" linked with Figure 1C, it would be better to specify which type of cells are you using in figure 1 because it seems that there are 2 type of cells. The authors explained results using immortalized cells and 293T cells, this are same cells? Please detail.

Thanks for this suggestion. We have added the detailed information in the revised manuscript and figure legends. 293T cells was utilized in this section mainly due to lower transfection efficiency in immortalized stromal cells. Similar results in both cell types also corroborate the regulation of PR protein stability by the ubiquitin E3 ligase HERC4.

b. Another nomenclature issue to improve is the use of different terms for non-decidualized and decidualized cells "undifferentiated and differentiated stromal cells"; "undecidualized and decidualized"; "non-decidualized and decidualized". The authors should use unified nomenclature.

Thanks for this concern to strength our manuscript and we have uniformly changed into the “undecidualized and decidualized".

c. Authors expose results analyzing decidualized and non-decidualized cells, but the figures do not show decidualized vs non-decidualized instead of this they shown D0 and D4. Why do they not use decidualized and non-decidualized cells at same time of treatment (D4)? This comparison let us to know what is happening with cells under hormonal treatment compared with cells growing at the same time without decidualization inductors. The author should explain how they performed the experiments and why they decided to make this experimental design.

The main purpose of this experiment is to determine the gene expression during the decidualization process, that is from the beginning of treatment (D0) throughout to D6. The similar approach has been used in the published paper (Recurrent pregnancy loss is associated with a pro-senescent decidual response during the peri-implantation window. Commun Biol. 2020 Jan 21;3(1):37. PMID: 31965050).

d. I have missed the times at which some treatments are performed, such as the decidualization treatments in Figure 2 A and the silencing and overexpression treatments. It should be specified for how long the silencing and overexpression is performed as well as decidualization. Additionally, in some treatments you must specify the dose of the drug you are using.

Thanks for this concern to make our description clearer. HESCs were cultured with differentiation medium for two days, since the expression of PR increased significantly on the second day of HESC differentiation. For the knockdown and overexpression experiment, the silencing and overexpression was first performed without differentiation stimulus, 24 hours later hESCs were decidualization for another 2 days. This information had been added in the revised manuscript (See line 546-549). The information including the doses of drug have been included in the Key resource table.

e. Why did you performed an RNA-Seq only 2 days after decidualization when in all of you experiments you show that decidualization markers increase their value from day four (e.g. Figure 3 B and C)? This may be somewhat confusing; the authors should explain this decision.

Thanks for this concern. During the process of decidualization, SOX4 was significantly induced as early as 2 days after decidualization, and the expression of decidualization markers PRL and IGFBP1 was obvious increased at the later time, such on D4 and D6. To explore the direct effect of SOX4 on the regulation for decidualization, the time when SOX4 was induced was chose for the RNA-Seq to minimize the secondary effects. The decidualization status as determined by the marker genes expression was on D4, when the marker gene expression increased to a high level.

4. Some experimental part of the manuscript could be improved it:

a. Western Blot in general you have to specify in which day you have performed it, in some cases it has not been mentioned (e.g. Figure 3A). Also, the authors need to well-defined the double band that found in the PGR protein experiment, as well as, to improve some GAPDH western blot where the bands are undistinguished between conditions (example figure 2D and Figure 3G). Furthermore, graphic including the band quantification and statistical analysis in each western blot could help supporting the findings.

Thanks for this concern. We added the information about the timing in the revised figure legend. The double band in the WB data for PR indicate PRA and PRB respectively and we have added this information in the revised manuscript (See line 235). As suggested by the reviewer, we improved the bands of GAPDH by rerunning the WB and also provided the data for band quantification and statistical analysis (See revised Figure 2 and 3).

b. Immunostaining should be more described along the manuscript explaining that are based in the analysis of endometrial tissue and to specify details such as day of sample collection.

Thanks for this concern. The detailed information for Immunostaining has been provided as reviewer suggested in the revised manuscript (See line 618-621).

5. The authors need to improve details on methodology.

a. RNAseq data need to be explain in detail. Figure 1A-B: "(A) Expression of all transcription factors in human non-decidualized ESCs by RNA-Seq. (B) Expression of SOX family genes in human non-decidualized ESCs by RNA-Seq" is based in the normal endometrium stromal cells analysis and figure legend define that are non-decidualized cells. Which cells are analysed in RNAseq analysis? Please, do not use the term normal to clinically define type of sample. Which experimental design was used? Sample size (N)? Raw and normalized counts? Filtered and quality control process? Which statistics were used? Please improve the results showed for RNAseq analysis.

Thanks very much for the reviewer’s suggestion. The term “normal” was changed to “non-decidualized ESCs” cell in the revised manuscript. RNA-Seq was perform in non-decidualized ESCs for Figure 1A-B. The average FPKM values (N = 3) of all transcription factors were used for these plots. Quality scores of reads above 30 was kept for alignment and only uniquely mapped reads were used for downstream quantification analysis by exact negative binomial test in edgeR package of R. The results of RNA-Seq of Figure 1A and 1B were improved per suggestion.

b. Versions of software and bioinformatic tools need to be included.

The softwares for RNA-Seq and ChIP-seq analysis including STAR (2.7.3a) for alignment, MACS2 (2.2.7.1) for peakcall of ChIP-seq, ggplot2 (3.3.5) for visualization, ngs.plot.r (2.61) for peak heatmap of ChIP-seq and the packages of edgeR (3.9), Complexheatmap (2.4.3), ChIPseeker (1.24.0) in R (4.1).

c. New sections in M and M need to be included for RNA extraction and quality evaluation (specifying RIN threshold consider), gene ontology enrichment, and real-time PCR.

The description about RNA extraction and quality evaluation, gene ontology enrichment, and real-time PCR had been modified or added as reviewer’s suggestion.

d. How batch effect was evaluated, and variables tested for controlling confounding effects.

To reduce the batch effect, the libraries of all RNA samples were prepared at the same time. The pooled RNA libraries were sequenced in a same flow cell. After sequencing, the sequencing depth and principal component analysis of all samples were evaluated. After library size calculation, sample dispersion evaluation and normalization, gene expression was calculated as FPKM (Fragments per kilobase of transcript per million mapped reads).

e. What function was used in edgeR for identify differentially expressed genes (e.g. exactTest), p-value adjustment and threshold for consider a transcript significantly deregulated, the fold change obtained and if there was some threshold to select genes to be included in subsequent analysis.

The function of exactTest in edgeR was applied for p-value calculation. Fold changed above 2 or less than 0.5 and p-Value < 0.05 were the significance threshold for consideration of differential expression. Only those differentially expressed genes (DEG) met the significance threshold were selected for downstream analysis.

f. Antibody concentrations used in immunoprecipitation and immunostaining must be included. Also, reagent concentrations for transfection including lipofectamine and times followed to procced such as medium change during transfection. We suggest creating a Key Resource Table with all the chemical compounds, kits, antibodies, plasmids, and software used with the references and concentrations with the aim of being more transparent and increasing the reproducibility of experiments.

Thanks for this concern. We have provided more tables for all the chemical compounds, kits, antibodies, and plasmids (See Key resources table and Supplementary file 3. The plasmids used in this study).

6. Regarding the discussion there are some aspects to be commented:

a. Line 409: The paper of SOX4 in decidualization linked with implantation failure in endometriosis could be improved. In this sense I suggest a proposal about how the authors think about the mechanism underlying with these two biological processes.

Thanks very much for this concern. We narrow down the statement about the connection between decidualization and implantation to prevent the disruption the focus of this investigation.

b. PGR isoforms A and B have different roles during the menstrual cycle, being the isoform B the predominant in decidualization. Authors should recognize it is a limitation not included this differentiation in the manuscript.

Thanks for this concern. As suggested by the reviewer, both PRA and PRB were expressed in the stromal cells, and PRB played predominant role in decidualization, we also observe the expression of PRA and PRB were both influenced by the SOX4 and since the PRB was more critical for decidualization, the regulation of HERC4 on PR was mainly focused on PRB.

https://doi.org/10.7554/eLife.72073.sa2

Article and author information

Author details

  1. Pinxiu Huang

    1. Department of Reproductive Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
    2. Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    3. Department of Reproductive Medicine, Liuzhou Maternity and Child Health Hospital, Liuzhou, China
    4. Affiliated Maternity Hospital and Affiliated Children’s Hospital of Guangxi University of Science and Technology, Liuzhou, China
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing – original draft
    Contributed equally with
    Wenbo Deng
    Competing interests
    No competing interests declared
  2. Wenbo Deng

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Formal analysis, Funding acquisition, Resources, Software, Writing - review and editing
    Contributed equally with
    Pinxiu Huang
    Competing interests
    No competing interests declared
  3. Haili Bao

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Zhong Lin

    Department of Reproductive Medicine, Liuzhou Maternity and Child Health Hospital, Liuzhou, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  5. Mengying Liu

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5987-1287
  6. Jinxiang Wu

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  7. Xiaobo Zhou

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Manting Qiao

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  9. Yihua Yang

    Department of Reproductive Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  10. Han Cai

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  11. Faiza Rao

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Writing - review and editing
    Competing interests
    No competing interests declared
  12. Jingsi Chen

    Department of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  13. Dunjin Chen

    Department of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  14. Jinhua Lu

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
  15. Haibin Wang

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing - review and editing
    For correspondence
    haibin.wang@vip.163.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9865-324X
  16. Aiping Qin

    Department of Reproductive Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
    Contribution
    Conceptualization, Funding acquisition, Project administration, Resources
    For correspondence
    qinaiping@gxmu.edu.cn
    Competing interests
    No competing interests declared
  17. Shuangbo Kong

    Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing - review and editing
    For correspondence
    shuangbo_kong@163.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7513-4041

Funding

National Key R&D program of China (2021YFC2700302)

  • Haibin Wang

National Key R&D program of China (2018YFC1004404)

  • Shuangbo Kong

National Natural Science Foundation of China (81830045)

  • Haibin Wang

National Natural Science Foundation of China (82030040)

  • Haibin Wang

National Natural Science Foundation of China (81960280)

  • Aiping Qin

National Natural Science Foundation of China (82001553)

  • Pinxiu Huang

National Natural Science Foundation of China (81701483)

  • Wenbo Deng

National Natural Science Foundation of China (81971419)

  • Wenbo Deng

Fundamental Research Funds of the Central Universities (20720190073)

  • Wenbo Deng

Guangxi Natural Science Foundation project (2019JJB140179)

  • Pinxiu Huang

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by National Key R&D program of China (2021YFC2700302 to H.W., 2018YFC1004404 to S.K.), National Natural Science Foundation of China (81830045 and 82030040 to H.W., 81960280 to A.Q., 82001553 to P.H., 81701483 and 81971419 to W.D.), Fundamental Research Funds for the Central Universities (20720190073 to W.D.), and Guangxi Natural Science Foundation Project (2019JJB140179 to P.H.). Thanks to Zhixiong Huang and Qionghua Chen for their helpful assistant during collecting the endometrial sample from patients with endometriosis.

Ethics

The studies for collecting the endometrial tissue were approved by The Ethical Committee of the Faculty of the Liuzhou Maternity and Child Health Hospital (2020-074). The studies for isolating the primary endometrial cells were approved by The Ethical Committee of The First Affiliated Hospital of Xiamen University (XMYY-2021KYSB044). All participants signed informed consent.

Senior Editor

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Reviewing Editor

  1. T Rajendra Kumar, University of Colorado, United States

Publication history

  1. Received: July 9, 2021
  2. Preprint posted: July 19, 2021 (view preprint)
  3. Accepted: March 3, 2022
  4. Accepted Manuscript published: March 4, 2022 (version 1)
  5. Version of Record published: March 15, 2022 (version 2)

Copyright

© 2022, Huang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Pinxiu Huang
  2. Wenbo Deng
  3. Haili Bao
  4. Zhong Lin
  5. Mengying Liu
  6. Jinxiang Wu
  7. Xiaobo Zhou
  8. Manting Qiao
  9. Yihua Yang
  10. Han Cai
  11. Faiza Rao
  12. Jingsi Chen
  13. Dunjin Chen
  14. Jinhua Lu
  15. Haibin Wang
  16. Aiping Qin
  17. Shuangbo Kong
(2022)
SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization
eLife 11:e72073.
https://doi.org/10.7554/eLife.72073

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