Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor β (TGFβ) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFβ signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFβ family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings unveil a previously unidentified dysfunction in BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.


INTRODUCTION
Endometriosis is a debilitating disease affecting ~ 190 million women of reproductive age globally 1 .
De ned as the occurrence of endometrial glands and stromal compartments outside of the uterine cavity, endometriosis leads to and in ammatory state, not only locally within the lesion sites and pelvic cavity, but systemically as well 2,3 .Lesions are typically located within the pelvic areas but can also involve distant sites 4 .Patients with endometriosis often suffer from chronic pelvic pain, severe dysmenorrhea, or infertility, which signi cantly decrease the quality of life of the affected patients.Currently, there is no de nite explanation for the pathogenesis of endometriosis; however, several theories have been proposed to explain the disease, including retrograde menstruation, 5 recruitment and transformation of mesenchymal and hematopoietic stem cells, 6 müllerian duct remnants, 7 and the coelomic metaplasia theories 8 .Regardless of their initial pathogenesis, the main symptomatic process involves increased production of in ammatory cytokines and pain mediators, as well as dysfunction of sympathetic nerve bers [9][10][11] .Treatment options for endometriosis are limited to empirical nonsteroidal anti-in ammatory drugs (NSAIDs), hormonal therapies, or surgery 12 .
In addition to causing pain and in ammation, endometriosis often leads to infertility [13][14][15] .Around 40 percent of women with endometriosis are estimated to have infertility 16,17 , and of women with infertility, 25 to 50 percent are estimated to also suffer from endometriosis 18,19 .Endometriosis affects fecundity by impairing ovarian functions, inducing chronic intraperitoneal in ammation and through a state of progesterone resistance 14,20 .Patients with endometriosis present with an abnormally prolonged follicular phase 21 , which further leads to dysfunctional folliculogenesis and granulosa cell cycle kinetics 22,23 .As a key feature of endometriosis, chronic intraperitoneal in ammation stems from increased levels of in ammatory cytokines, chemokines as well as prostaglandins.Such in ammatory processes can lead to infertility by decreasing intrafollicular estrogen level 24 , oocyte quality 25 and sperm motility 25 .
The normal function of the eutopic endometrium is also compromised in patients with endometriosis, as demonstrated by progesterone resistance that is characterized by declined expression of progesterone receptor (PR) and coactivators 26,27 .De cient progesterone signaling pathways likely lead to impaired decidualization, defective embryo implantation and increased infertility rates in patients with endometriosis 28 .During pregnancy, individuals with endometriosis also experience higher rates of gestational complications, including preterm birth and antepartum hemorrhage 29,30 .These defects which arise during later pregnancy, are likely fueled by defects during early pregnancy and decidualization, which negatively affect immune cell in ltration and the degree of spiral artery remodeling 30 .Additional signaling defects have been identi ed in the endometrium of women with endometriosis, including defective mesenchymal stem cell differentiation 31,32 , increased decidual senescence and elevated proin ammatory stress 33 .Despite this progress, actionable therapeutic targets to improve the fertility outcomes in individuals with endometriosis are lacking.Uncovering the mechanisms and pathways involved that negatively impact the eutopic endometrium in patients with endometriosis is critical to help optimize chances for successful pregnancy.
Our studies aim to uncover the molecular underpinning of infertility associated with endometriosis by focusing on transcriptomic signatures of the eutopic endometrium from individuals with endometriosis.Here, we use patient-derived stromal cells and state-of-the-art endometrial stromal and epithelial assembloids to de ne key signaling alterations during decidualization in patients with endometriosis.

Transcriptomic pro ling in endometrial stromal cells from individuals with and without endometriosis reveals activation of key pathways during early and late in vitro decidualization
The human endometrium undergoes spontaneous decidualization in response to the rising levels of progesterone [34][35][36] .The concerted action of estrogen and progesterone transforms the endometrium from a non-receptive state into a receptive state, subsequently allowing embryo implantation and development to occur.Because patients with endometriosis can have decreased fecundity due in part, to defective endometrial function, 30 we aimed to systematically determine the transcriptomic differences between the two groups during in vitro stromal cell decidualization.Previous analyses were performed to determine the decidualization potential of endometrial stromal cells from normal patients during the early and late decidualization phases 37,38 .These studies found unique transcriptional signatures that were activated at each phase, indicating that the process of decidualization is a transcriptionally active process that requires long-term remodeling of chromatin and subsequent changes in gene expression 37,38 .Other studies compared the decidualization potential of endometrial stromal cells obtained from individuals with and without endometriosis, however these studies focused only on the late phases of decidualization 39 .To identify markers and pathways that are differentially controlled in the eutopic endometrium of individuals without endometriosis, we performed transcriptomic analyses of stromal cells from individuals with and without endometriosis that were induced to decidualize in vitro during early and late phases.
We used a well-characterized method 36, [40][41][42] to induce endometrial stromal cell decidualization and to compare differentially expressed genes between normal and endometriosis samples from the early (2 days) to late (8 days) stages of decidualization.Patient-derived endometrial stromal cells from patients with and without endometriosis were cultured in vitro and treated with estrogen (E2), medroxyprogesterone acetate (MPA) and 8-bromo cyclic adenosine monophosphate (cAMP) (EPC) to induce decidualization in vitro, and then collected at 2, 4, 6, 8 days after EPC cocktail treatment to pro le transcriptomic changes using RNA sequencing (Fig. 1A).We performed a time course comparison between the differentially expressed genes at each timepoint respectively.In total, 334 transcripts changed signi cantly during the EPC treatment in the cells derived from individuals without endometriosis (n = 3, normal), with 152 transcripts showing an increase and 182 showing a decrease by day 8 of EPC treatment (Supplementary Table 1, > 1.4, < 0.4-fold change, FDR < 0.05).In stromal cells from individuals with endometriosis (n = 4), 878 transcripts showed a signi cant change after EPC treatment, with 464 being increased and 414 decreased by Day 8 of EPC treatment (Supplementary Table 1, > 1.4, < 0.4-fold change, FDR < 0.05).Among these, only 122 transcripts (12.4%) were shared between normal and endometriosis upregulated genes, and 105 (10.7%) shared genes were conserved in the downregulated genes between the two groups (Supplementary Fig. 1A).These results indicate that endometrial stromal cells arising from individuals with endometriosis display a unique transcriptomic response to decidualization treatment in vitro that is different to stromal cells derived from individuals without endometriosis.

Gene ontology classi cation and upstream factor analyses reveal enrichment of TGFβ signaling and oxidative stress response in endometriosis
To understand the pathways that were overrepresented among the differentially expressed genes, we performed a gene ontology analysis of all the differentially regulated genes (> 1.4, < 0.4-fold change, FDR < 0.05) in the normal or endometriosis datasets (Supplementary Table 2).Among the top 10 categories in the normal patients, we found that cytosolic tRNA aminoacylation, interleukin-2 signaling, TGFβregulation of extracellular matrix and BDNF signaling pathways were overrepresented among the differentially expressed genes at day 8 of EPC relative to baseline (Day 0) (Supplementary Fig. 1B).Enriched pathways in the endometriosis dataset included categories such as, TGFβ-regulation of extracellular matrix, interleukin-2 signaling, cardiomyocyte hypertrophy, FSH regulation of apoptosis, and Leptin signaling pathway (Supplementary Fig. 1C).Our results suggest that while the cells from both groups displayed uniquely regulated genes and signaling networks, several of these key pathways, such as TGFβ signaling and interleukin-1 signaling were shared between the two groups.
We performed an upstream transcription factor analysis of the differentially regulated genes in the endometriosis or normal stromal cells to identify master regulatory networks driving the differential transcriptional response (Supplementary Table 3).By mining the consensus gene targets in the ENCODE and ChEA Transcription Factor Targets dataset 43,44 , we identi ed that genes regulated by the CCAAT Enhancer Binding Protein Beta (CEBP/β) and Transcription Factor 3 (TCF3) were enriched in the normal stromal cells (Fig. 1B).CEBP/β has been shown to be a key factor in endometrial stromal cell decidualization that controls the transcription of the PR 45,46 TCF3 is also shown to control endometrial stromal cell proliferation and decidualization 47 .
On the other hand, regulation of genes by the NFE2 Like BZIP Transcription Factor 2 (NFE2L2) and SMAD4 transcription factors was highly enriched in the endometriosis dataset (Fig. 1C).NFE2L2, encodes NRF2, and is an important regulator of oxidative stress response that controls the expression of genes that contribute to ferroptosis resistance [48][49][50] .SMAD4 is the downstream activated transcription factor controlling expression of genes downstream of bone morphogenetic proteins (BMPs, through SMAD1 and SMAD5) or TGFβ/activin ligands (through SMAD2 and SMAD3) 51 .Some of these differential responses could also be observed at the gene level (Fig. 1D), as indicated by the expression of IGFBP1 52 , ZBTB16 53 (decidualization markers), SLC40A1, GPX3, PTGS2 54,55 (markers of oxidative stress and ferroptosis), or LEFTY2, SMAD1 56 (BMP/SMAD-signaling pathways).
In summary, we observed that conventional transcriptional programs driving decidualization were found to be overrepresented in the endometrial stromal cells from individuals without endometriosis, suggesting a normal decidualization response.However, endometrial stromal cells from individuals with endometriosis displayed an impaired response to oxidative stress and defective BMP/TGFβ signaling pathways.

Identi cation of perturbed BMP/TGFβ signaling pathways in the decidualizing stromal cells from individuals with endometriosis
We examined the dynamic pro les in the early and late decidual cell transcriptomes and visualized the differentially expressed genes between individuals with and without endometriosis in volcano plots using a > 2 or < ½ fold-change and FDR < 0.05 (Fig. 2A-D).At baseline, we observed that 393 transcripts were differentially expressed between the normal and endometriosis groups, with 166 being upregulated and 227 downregulated in the endometriosis group compared to the normal group (Supplementary Table 4).Two days after EPC administration, 400 genes were down regulated in the endometriosis samples compared to the normal counterparts.We then observed 216, 95 and 99 genes to be down regulated on Day 4, Day 6, and Day 8, in endometriosis samples compared to the normal samples, respectively.Meanwhile, 323, 138, 64 and 43 genes were up regulated in endometriosis compared to normal at Day 2, Day 4, Day 6, and Day 8, respectively (Supplementary Table 4).
To understand the pathway dependent differences in the endometriosis groups, we implemented Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in the differentially expressed genes spanning from Day 0 to Day 8 timepoints between normal and endometriosis donors (Fig. 2E, and Supplementary Table 4).Our enrichment analysis showed distinct enrichment patterns at each of the different time points we assessed.Of notice, the TGFβ signaling pathway was the only category shared in the timepoints during decidualization.Alterations in this category included mostly the ligands of the TGFβ family, such as BMP4, BMP2, LEFTY2, GDF6, INHBA, and LTBP1, in which BMPs typically signal through the SMAD1/5/4 transcription factors while TGFβ and activins signal through the SMAD2/3/4 transcription factors 56 .We also observed persistent downregulation of the classical BMP/SMAD1/5/4 target genes, ID2 and ID3, suggesting that impaired BMP signaling was resulting in decreased transcriptional activation by SMAD1/5/4 in stromal cells derived from individuals with endometriosis.
After overlapping the differentially expressed genes from different time points, we identi ed that 48 genes were consistently down-regulated and that 20 genes were consistently up-regulated regardless of the EPC treatment length (Supplementary Fig. 2A-B).Further enrichment for genes associated with human diseases by DisGeNET 57 revealed that the 48 consistently down-regulated genes in the individuals with endometriosis were signi cantly associated with fertility complications such as early pregnancy loss, miscarriage, and spontaneous abortion (Supplementary Fig. 2C, Supplementary Table 4).
We also observed that genes related to retinoic acid synthesis and metabolism were persistently decreased in the endometriosis group compared to the normal group during decidualization (Supplementary Table 4).For example, the retinoic acid receptor responder 1 (RARRES1) was signi cantly decreased in endometriosis across all the time points.Aldehyde dehydrogenase 1 family member B1 (ALDH1B1) was also signi cantly decreased in endometriosis relative to normal decreased across all time points.Retinoic acid receptor beta (RARB) was decreased on days 2 and 4 of EPC treatment in endometriosis relative to normal.Reprogramming of the endometrium by retinoic acid signaling is critical for endometrial decidualization and early pregnancy 58,59 .Furthermore, altered retinoic acid metabolism also affects endometriotic stromal cell decidualization 60 .Therefore, our results are in line with previous observations, and support the hypothesis that alterations in retinoic acid metabolism drive fertility defects in individuals with endometriosis.
We also observed that the GATA protein binding 6 (GATA6) was signi cantly decreased in the endometriosis group relative to normal cells on days 0, 2, 4, and 6 of EPC treatment.GATA6 is a PR direct target gene 61 and is upregulated in ectopic endometriotic stromal cells from lesions due to methylation defects 62,63 .Overall, the time course decidualization analysis between normal and endometriosis donors highlighted various pathways that are differentially expressed between the two groups, providing new therapeutic or diagnostic opportunities for endometriosis-associated infertility.Furthermore, results from our transcriptomic pro ling emphasized the critical roles of the BMP signaling pathways in driving the decidualization processes of the normal endometrium.

Altered BMP signaling impairs decidualization in the endometrium of individuals with endometriosis
Our time course transcriptomic analyses revealed that BMP ligands and SMAD1/5/4 transcriptional targets, ID2 and ID3, were consistently downregulated in stromal cells from individuals with endometriosis during in vitro decidualization (Fig. 2).Previous studies in mouse models indicate that BMP signaling via the SMAD1/5 transcription factors is essential for endometrial receptivity, embryo implantation and decidualization [64][65][66][67] .To explore the speci c roles of BMP signaling pathways in the decidualization defects observed in individuals with endometriosis, we rst assessed the activation of BMP signaling pathways at different time points during time course decidualization.As shown in Fig. 3A, in the samples from individuals without endometriosis (n = 5), SMAD1/5, the signal transducers of the BMPs, were activated in a time-dependent manner.Upon EPC stimulation, phosphorylated SMAD1/5 (pSMAD1/5) gradually increased along with the EPC treatment length.However, in the samples derived from individuals with endometriosis (n = 4), activation of the BMP signaling pathway was impaired, as manifested by the decreased levels of pSMAD1/5 during the time course EPC stimulation (Fig. 3B-C).Gene expression analysis using RT-qPCR demonstrated decidual markers such as BMP2 and IGFBP1 showed an increasing trend throughout Day 2 to Day 8 EPC treatment in the stromal cells with blunted induction in the endometriosis cells (Fig. 3D-E).These results indicate that while normal decidualizing stromal cells successfully engage BMP/SMAD1/5 signaling, stromal cells from endometriosis did not induce BMP/SMAD1/5 signaling and failed to decidualize.
The discrepancy in response to the EPC treatment observed between individuals with and without endometriosis was also identi ed from the RNA-seq data.Figure 3F-G shows exemplary genes that present contrasting trends during our time course decidualization analysis.Similar to BMP2, the ID1 and ID3 genes were progressively increased over the time course treatment in the samples derived from donors without endometriosis while they were conversely down regulated in the endometriosis cohort.
Inhibitor of DNA-binding (ID) genes are not only known downstream BMP responsive genes 68,69 but also are important for endometrial remodeling and decidua formation [70][71][72] .Such inverted trends substantiated the dysfunctional BMP signaling pathways in the endometriosis groups.Our data indicated that in individuals with endometriosis, an impaired BMP signaling pathway is accompanied by dysfunctional endometrial decidualization.

Genome-wide binding of SMAD4 reveals differential binding patterns in the endometrium of individuals with and without endometriosis
Upon ligand binding, canonical BMP signaling pathways use SMAD1/5/4 proteins to initiate nuclear transcriptional control.SMAD1/5 forms heterodimers and translocate into the nucleus together with common SMAD4 56 .Given that BMP/SMAD1/5/4 signaling is essential for implantation and decidualization [64][65][66][67]72,73 , our goal was to investigate the mechanisms that underpin defective BMP signaling in individuals with endometriosis during decidualization at the transcriptional level. To doso, we utilized the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) method to pro le the genome-wide SMAD4 binding sites in the EPC-treated (4 days) endometrial stromal cells derived from both individuals with and without endometriosis.We observed a distinct pattern of SMAD4 binding activities between the two groups (Supplementary Fig. 3A-B).We exempli ed the binding activities by showing the Integrative Genomics Viewer (IGV) track view of the ID1 and ID3 loci.We observed that SMAD4 binding was diminished in the endometriosis groups at the ID1 and ID3 loci (Fig. 4A).In total, we identi ed 2060 peaks showing differences in signal intensity between normal and endometriosis groups.Among these, 1190 peaks showed decreased enrichment in the endometriosis group, while 870 peaks had increased enrichment in the endometriosis group.
Peak annotation revealed that the majority of these peaks were located within the ± 3 kb promoter region (72.29%) (Fig. 4B, Supplementary Table 5).Additionally, we performed the Reactome pathway enrichment 74 for the genes that were differentially bound by SMAD4 in the endometriosis group (Fig. 4C, Supplementary Table 5).We found that categories related to 'signaling by TGFβ family members', 'signaling by TGFβ receptor complexes', and 'TGFβ activated SMADs' were enriched.These ndings agreed with our transcriptomic results and indicate that a defective TGFβ/SMAD4 signaling program is abnormal in the endometrium of individuals with endometriosis.Because SMAD4 is the "common" SMAD that forms a complex with both BMP-SMAD1/5 and TGFβ/activin-SMAD2/3, our CUT&RUN experiments cannot speci cally differentiate between the two pathways or differentiate between SMAD1/5 and SMAD2/3-mediated signaling.However, given that SMAD4 enrichment was decreased at the ID1 and ID3 promoter regions, and this corresponds with decreased ID gene expression, it was likely that BMP signaling pathways also impair SMAD1/5/4 binding activities in the stromal cells from individuals with endometriosis.
Interestingly, apart from TGFβ related categories, pathways involved in 'chromatin modifying enzymes', 'signaling by NTRKs,' and 'NGF-stimulated transcription,' were also among the top enriched categories (Fig. 4C).Neurotrophic tyrosine receptor kinases (NTRKs) are well-documented for their roles in pain and in ammation in endometriosis and are elevated in the endometriotic lesions of affected patients [75][76][77] .
Nerve growth factors 78 signal through the NTRKs and were recently shown to be associated with endometriosis through genome wide association studies 79 .Hence, our studies suggest that abnormal NTRK signaling may also impact the eutopic endometrium and affect receptivity in patients with endometriosis.
To further delineate the chromatin level differences between the normal and endometriosis groups, we pro led the depositions of histone mark H3K27 acetylation (H3K27ac) in the EPC-treated stromal cells (Supplementary Table 6).H3K27ac modi cation on the chromatin has been well-de ned in the enhancer and promoter regions and is usually accompanied by active transcription activities [80][81][82] .Similar to SMAD4 binding patterns, H3K27ac marks also showed distinct patterns between the normal and endometriosis groups following a 4-day EPC treatment (Fig. 4D, Supplementary Fig. 4A).We identi ed 1439 peaks that had increased enrichment in the normal group and 1122 peaks that had more enrichment in the endometriosis group (Supplementary Table 6).
For the genes that preferentially have more H3K27ac peaks in the endometriosis group, Gene Ontology (GO) enrichment analysis indicated positive regulation of cell adhesion being the most enriched category (Supplementary Fig. 4B, Supplementary Table 6).This includes genes such as vascular cell adhesion molecule 1 (VCAM1), CD44 molecule (CD44), and Wnt family member 5A (WNT5A) (Supplementary Table 6).Higher levels of VCAM1 are found in ectopic endometriotic lesions and in the eutopic endometrium of individuals with endometriosis and may contribute to disease establishment and progression 83 .CD44 was previously shown to be elevated in the eutopic endometrium of patients with endometriosis and is involved in the attachment and invasion of endometrial cells into the peritoneum 84,85 .WNT5A controls endometrial mesenchymal stem cell renewal by activating WNT/βcatenin signaling 86 .Overall, these results corroborate previous studies indicating that the increased cell adhesion abilities in the eutopic endometrium of those with endometriosis facilitate lesion establishment at ectopic sites 87 (Supplementary Fig. 4B, and Supplementary Table 6).
GO enrichment on the genes that have less H3K27ac peaks in the endometriosis group indicated that categories involving transcription factor binding, extracellular matrix structural constituent and transcription corepressor activity were de cient during decidualization in the endometrium of individuals with endometriosis (Fig. 4E, Supplementary Table 6).Additionally, genes in the SMAD binding category, such as SMAD3, SMAD6, the SMAD speci c E3 ubiquitin protein ligase 2 (SMURF2), and the transforming growth factor beta receptor 1 (TGFBR1/ALK5), had fewer H3K27ac peaks in the endometrial stromal cells from individuals with endometriosis, corroborating our previous transcriptomic results in which a dysfunctional TGFβ/BMP signaling pathway was identi ed in the decidualizing stromal cells from individuals with endometriosis (Fig. 4E).Additional defects in H3K27ac deposition in individuals with endometriosis were observed in the well-known progesterone-responsive genes RARB and CEBPA loci 88,89 (Supplementary Fig. 4C).These results show that defective endometrial transcriptional responses driven by TGFβ and BMP signaling in individuals with endometriosis are detected at the chromatin level, as evidenced by different genome wide SMAD4 and H3K27ac binding patterns in normal versus endometriosis groups.

Silencing of SMAD1 and SMAD5 perturbs endometrial stromal cell decidualization
Previously published studies in mouse models indicate that the BMP/SMAD1/5 signaling pathways are critical for decidualization and endometrial receptivity [64][65][66][67]72,73 . In ou present study, we found that the decreased decidualization potential of stromal cells from individuals with endometriosis correlated with defective BMP/SMAD1/5 activation.To functionally examine the role of BMP signaling pathway in mediating decidualization, we perturbed the SMAD1/5 complex using small interfering RNA (siRNA) in endometrial stromal cells from individuals without endometriosis and treated them with EPC to induce in vitro decidualization (Supplementary Fig. 5A).The knockdown effect was validated at the transcript and protein level (Fig. 5A, Supplementary Fig. 5B, Supplementary Table 7).Upon the knockdown of SMAD1/5, we observed that canonical decidualization markers such as IGFBP1 and WNT4 were signi cantly down regulated (Fig. 5A).KEGG pathway enrichment on the differentially expressed genes revealed that the TGFβ and FOXO signaling pathways were also enriched within the downregulated group of genes (Fig. 5B).FOXO family plays a critical role in regulating progesterone-dependent differentiation and decidualization 90 and is indispensable for implantation and decidua formation 91 .We highlighted several key genes changes in the heatmap format to visualize the effect of SMAD1/5 knockdown (Fig. 5C).
To further map the direct target genes and potential co-factors of SMAD1/5 during decidualization, we used Binding and expression target analysis (BETA) 92 to consolidate our genomic pro ling of SMAD4 and the transcriptomic pro ling of SMAD1/5 perturbation.Among the direct targets that were activated by SMAD1/5 (which were down regulated upon SMAD1/5 perturbation and were bound by SMAD4, labeled as Down-targets), were the TGFβ signaling pathway and pathways regulating the pluripotency of stem cells (Fig. 5D).We also performed motif analysis on the direct target genes to provide mechanistic insight to the SMAD1/5 mediated gene expression during decidualization.We uncovered potential SMAD1/5 co-repressors such as NFATC2 and T-box family (TBX1/TBX15).NFATC2 is involved in cGMP-PKG signaling pathways and has a role in regulating immune, in ammatory responses 93 , it is also reported to be elevated in the thin-endometrium patients who usually have de cient implantation and lower pregnancy rate 94 .Acting mainly as repressors, TBX family genes are crucial for embryonic development and tissue differentiation and formation 95 .A recent study has shown that TBX15 was elevated in patients with adenomyosis 96 .As for transcriptional co-activators, apart from the canonical pan-tissue co-activator NFIC 97 , the PR motif was enriched in the SMAD1/5 direct target genes, con rming our previous nding that SMAD1/5 may regulate progesterone-responsive genes at the transcriptional level (Fig. 5E).
We also identi ed two genes, MALAT1 and HDAC4, that contained a SMAD4 binding site and were decreased by SMAD1/5 siRNA knockdown in EPC-treated stromal cells, suggesting that they are direct target genes that are activated by BMP/SMAD1/5/4.The direct SMAD4 binding activities in MALAT1 and HDAC4 loci were visualized as genome track views in Supplementary Fig. 5C-D.MALAT1 and HDAC4 are both involved in facilitating decidualization and in the pathogenesis of endometriosis [98][99][100][101][102][103] .In summary, our studies combine datasets from SMAD1/5 siRNA-mediated knockdowns with SMAD4 binding studies in endometrial stromal cells during decidualization.The results from these not only validated the indispensable roles of SMAD1/5 during human decidualization, but also provided additional layers of regulation in the downstream networks of SMAD1/5 mediated BMP signaling pathways.

BMP2 supplementation enhances the decidualization potential in stromal cells and endometrial assembloids of individuals with endometriosis
We consistently observed alterations in the BMP/SMAD1/5/4 signaling pathway in the decidualizing stromal cells from individuals with endometriosis, suggesting that inherent defects in the activation of this pathway were present in the affected individuals.We also identi ed that BMP/SMAD1/5/4 signaling networks are essential for decidualization in the normal eutopic endometrium.To test whether the addition of recombinant BMP2 supplementation could restore the decidual response in the endometrium from individuals with endometriosis, we added BMP2 to endometrial stromal cell cultures and to 3dimensional endometrial stromal/epithelial co-cultures, or "assembloids" (Fig. 6).For the stromal cell experiments, cells derived from the eutopic endometrium of patients with endometriosis were treated as shown in Fig. 6A with the EPC cocktail or EPC + BMP2 for a total of 4 days.The extent of decidualization was examined using qPCR analysis of the decidual markers, FOXO1, PRL, SPP1 and IGFBP1 (Fig. 6B-E).FOXO1, PRL and IGFBP1 all showed an increased trend in expression following EPC + BMP2 treatment versus EPC treatment alone.However, only the expression of SPP1 changed signi cantly after BMP2 supplementation (Fig. 6B-E).Correspondingly, we observed that the combined addition of EPC + BMP2 synergized the expression of pSMAD1/5 relative to BMP2 or EPC treatment alone (Fig. 6F-G).
To test the impact of BMP2 supplementation on the decidualization potential of endometrial stromal and epithelial assembloids we generated co-cultures as previously described using the strategy outlined in Fig. 6H 104 .Individual cultures of endometrial stromal cells and epithelial organoids were established.Four days after initial establishment, the co-cultures were created by encapsulating stromal cells and epithelial organoids in the collagen matrix cultured in expansion medium (ExM) supplement with E2 for 2 days in a shaking system.The culturing medium was then switched to a minimal decidualization media (MDM) containing the decidualization cocktail (EPC) +/-BMP2 for an additional 4 days.Live assembloid cultures were visualized using phase microscopy (Fig. 6I) and using histology or uorescence microscopy after xation and staining (Fig. 6J-L).qPCR analysis of the treated assembloids showed that the BMP2 + EPC supplementation signi cantly increased FOXO1 expression and caused an increased trend in the expression of decidual markers WNT4 and IGFBP1 relative to EPC treatment alone (Fig. 6M-O).We also observed fewer FOXJ1-positive ciliated cells in the endometrial assembloids following EPC + BMP2 treatment, compared with EPC treatment alone (Supplementary Fig. 6).These results con rm that BMP2 supplementation increases decidual gene expression in endometrial stromal or in 3D assembloid cultures from individuals with endometriosis.

Ethics statement and endometrial sample collection
All patient specimens were collected following informed patient consent approved under protocol H-21138 and through the Human Tissue and Pathology Core at Baylor College of Medicine (BCM), following guidelines approved by the Institutional Review Board at BCM.Samples are maintained using de-identi ed codes to preserve con dentiality.Endometrial samples were obtained from women with con rmed endometriosis (n = 7, mean age, 36.7 +/-6.9) or from women without endometriosis (n = 7, mean age, 38.4 +/-5.3)undergoing endometrial biopsies or hysterectomies.Samples categorized in the normal group were free of endometriosis, according to pathology examination reports.

Histological assessment of endometrial assembloids
Assembloids were xed in 4% PFA at room temperature for 15 mins and then immobilized in the Histogel (Thermo Fisher, Cat #22-110-678).After processing the assembloids in Histogel, assembloid blocks were dehydrated through a series of ethanol washes and processed for para n embedding at the Human Tissue Acquisition and Pathology Core at Baylor College of Medicine.Para n blocks were sectioned using 5 µm thick sections.Assembloid sections were depara nized in Histoclear and rehydrated in a series of 100%, 95%, 80%, and 70% ethanol washes, followed by washing in dH20.For identifying the morphological structures, assembloid sections were sectioned and stained with hematoxylin and eosin.
For immuno uorescence staining, assembloid sections were heated in boiling 10mM sodium citrate, pH 6.0 for 20 minutes for antigen retrieval and quenched in 3% hydrogen peroxide for 10 minutes.After blocking with 3% BSA for 1 hour, assembloid sections were incubated with primary and uorescent secondary antibodies according to the manufacturer's instructions and nuclei were stained with 1mg/ml DAPI (1:100-dilution, ThermoFisher, Cat # D1306).The stained slides were mounted in VECTASHIELD antifade mounting medium (Vector Laboratories #H-1000-10).Fluorescence images were taken on a Zeiss LSM780 confocal microscope at the Optical and Vital Microscopy Core at Baylor College of Medicine.

RNA extraction and quantitative PCR from endometrial stromal cells and endometrial assembloids
The RNAs of endometrial stromal cells were extracted by using QIAGEN RNeasy micro kit (QIAGEN, Cat #74004) according to the manufacturer's instruction.The assembloids were lysed in Trizol reagent (Life Tech, Cat #10296010) and the RNAs were extracted by using Direct-zol RNA microprep kit (Zymo Research, Cat #R2062).A total of 50-200ng of RNA from each sample was transcribed into cDNA by using qScript cDNA supermix (Quantabio, Cat #95048-100).Real time quantitative PCR was performed on Bio-Rad CFX384 Touch Real-Time PCR Detection System.Fold changes of target genes were calculated using delta delta Ct method and normalized GAPDH 106 .Primer sequences are listed in Supplementary Table 8.

Protein extraction and western blotting
Cells were washed with ice-cold 1×DPBS and lysed in M-PER mammalian protein extraction reagent (ThermoFisher, Prod#78505) supplement with protease inhibitor cocktail (ThermoFisher, Cat #78437) and phosphatase inhibitor cocktail (ThermoFisher, Cat #78426).Protein concentrations were quanti ed by using Pierce BCA protein assay kit (ThermoFisher, Cat #23225).A total of 20 µg of protein lysate was loaded onto 4-12% Bis-Tris Plus Mini protein gels (ThermoFisher, Cat # NW04122BOX) and transferred onto nitrocellulose membranes (Bio-Rad, Cat #1704270).The membranes were blocked with 5% non-fat milk in TBST buffer for an hour at room temperature and then incubated with primary antibodies at 4°C overnight.Antibody information is listed in Supplementary Table 9.The next day, the membranes were probed with HRP-conjugated secondary antibodies (Jackson ImmunoResearch) for two hours at room temperature and protein bands were visualized by using SuperSignal West chemiluminescent substrate (Pierce) on Bio-Rad Chemidoc Touch Imaging system.Protein bands were quanti ed by using Image Lab software (Rio-Rad).
Gene expression pro ling using RNA sequencing All sequencing data are available in the NCBI Gene Expression Ominibus under SuperSeries GSE243158.The secure token for reviewer access is wfolcuqovnwxnkd.

Time course EPC studies
Endometrial stromal cells from 4 normal and 4 endometriosis samples were treated with 35nM estradiol, 1µM medroxyprogesterone acetate and 1µM 8-Br-cyclic AMP for 0, 2, 4, 6, or 8 days.RNA expression pro les were obtained at each timepoint using RNA sequencing analyses (20-30 million paired-end reads using NovaSeq System from Novogene Corporation Inc. Reads were trimmed with fastp v0.23.2 and aligned using STAR 2.7.10a to human genome assembly GRCh38.p13.Differentially expressed genes between normal patients and endometriosis EPC-treated cells were obtained by comparing to the baseline samples (Day 0).Signi cantly changed genes during the time course treatment were obtained using an ANOVA F-test using an FDR < 0.05 from patients without (n = 3) and with endometriosis (n = 4).All DEGs are presented in Supplementary Table 1.EnrichR [107][108][109] was used to identify the gene ontology classi cations (Supplementary Table 2), as well as ENCODE and ChEA consensus transcription factors known to regulate differentially expressed genes in the normal and endometriosis EPC-treated stromal cells (Supplementary Table 3).Differentially expressed genes between normal and endometriosis stromal cells were obtained by comparing transcripts at each time point of treatment.Differentially expressed genes between normal (n = 4) and endometriosis (n = 3) were identi ed using a Wald test with a cutoff values of fold-change > 2 or < 1/2 and FDR < 0.05 (Supplementary Table 4).Gene and pathway enrichment analysis was conducted using R package Cluster Pro ler 110 .
RNAs were isolated by using QIAGEN RNeasy micro kit and subjected to RNA sequencing analysis to identify differentially regulated transcripts.Samples were normalized through effective library sizes and DESeq2 was used identify differentially expressed genes between the siCTL and siSMAD1/5 EPC-treated cells using an FDR < 0.05 (Supplementary Table 7).Gene and pathway enrichment analysis was conducted using R package Cluster Pro ler 110 .

SMAD4 and H3K27 genome-wide binding studies using CUT & RUN
CUT&RUN experiments were performed following a previously published protocol 111 .After endometrial stromal cells were treated with EPC for 4 days, they were collected by digesting with 0.25% Trypsin (ThermoFisher, Cat #25200056) for 3 min.After the digestion, cells were pelleted down at 300 x g for 3 min and viably frozen down in the freezing medium (90% FBS with 10% DMSO) until experiment day.On the day of the experiment, cell vials were quickly thawed and washed 3 times with washing buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 X Protease Inhibitor).For each reaction, 1.3 x 10 6 cells were used for the subsequent Concanavalin A bead binding step.After 10 min incubation with Concanavalin A beads, bead-cell complexes were resuspended in 100 µl antibody buffer (washing buffer supplemented with 0.01% digitonin, and 2mM EDTA) per reaction.1 µl of IgG antibody (Sigma, Cat #I5006), H3K27ac (Cell Signaling, Cat #8173) and SMAD4 antibody (Abcam, Cat #ab40759) were added to each reaction respectively.After overnight incubation at 4°C, bead-cell complexes were washed twice with 200 µl cold dig-washing buffer (washing buffer supplemented with 0.01% digitonin) and resuspended in 50 µl cold dig-washing buffer with 1 µl pAG-MNase (EpiCypher, Cat #15-1016).After incubation at room temperature for 10 min, bead-cell complexes were washed twice with 200 µl cold digwashing buffer and resuspended in 50 µl cold dig-washing buffer, then 1 µl 100 mM CaCl 2 was added to each reaction.The mixture was incubated at 4°C for 2 hours and the reaction was stopped by adding 50 µl stop buffer (340mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 100 ug/mL RNase A, 50 mg/mL glycogen, 0.5 ng E. coli DNA Spike-in (EpiCypher, Cat #18-1401) and incubated at 37°C for 10 min.The supernatant was collected and subjected to DNA puri cation with phenol-chloroform and ethanol precipitation.Sequencing libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, Cat #E7645) following manufacture's protocol.Paired-end 150 bp sequencing was performed on a NEXTSeq550 (Illumina) platform and each sample was targeted for 10 million reads.
Sequencing raw data were de-multiplexed by bcl2fastq v2.20 with fastqc for quality control and then mapped to reference genome hg19 by Bowtie2, with parameters of --end-to-end --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700.For Spike-in mapping, reads were mapped to E. coli genome U00096.3.Spike-in normalization was achieved through multiply primary genome coverage by scale factor (100000 / fragments mapped to E. coli genome).CUT&RUN peaks were called by Model-based Analysis of ChIP-Seq (MACS/2.0.10) 112 with the parameters of -f BAMPE -g 1.87e9 -q 0.05 (H3K27ac) orq 0.1 (SMAD4).Track visualization was done by bedGraphToBigWig20, bigwig les were imported to Integrative Genomics Viewer for visualization.For peak annotation, genomic coordinates were annotated by ChIPseeker 113 .Differential binding analysis and clustering were conducted using DiffBind 114 .Direct targets motif analysis was conducted through Binding and Expression Target Analysis (BETA) 92 with parameter BETA plus -p -e -k LIM -g hg19 --gs hg19.fa--bl.Gene and pathway enrichment analysis was conducted using R package Cluster Pro ler 110 .Annotated peak les were included in Supplemental

DISCUSSION
Our study, which performs a time course analysis of decidualization in stromal cells from individuals with and without endometriosis, reveals that dysregulated TGFβ and BMP signaling is prevalent in the endometrium of individuals with endometriosis and may underlie the fertility defects experienced by this group.As such, our results present transcriptomic evidence supporting the hypothesis that patients with endometriosis display abnormal decidualization programs that can partially explain the elevated infertility rates within that population.Previous studies analyzed the time-course gene expression pro les of in vitro decidualized normal endometrial stromal cells 37,38 , while others have analyzed the expression differences between normal and endometriosis stromal cells in the late decidualization phase 39 .Our data, on the other hand, present a comprehensive transcriptomic analysis of the decidualization pro les of stromal cells derived from individuals without and with endometriosis during early and late phases.We rst performed a time-course transcriptomic analysis of the datasets to identify the genes that are expressed within each group as they undergo in vitro decidualization and identi ed that stromal cells from patients with endometriosis displayed gene expression signatures that were controlled by NFE2L2, a marker of oxidative stress, and SMAD4, the effector of TGFβ and BMP signaling.In the second analysis of the datasets, we directly compared the transcriptomes of stromal cells from individuals without endometriosis to those from individuals with endometriosis as they underwent in vitro decidualization.
From these studies, we found that TGFβ/BMP signaling pathways emerged once again as a consistently enriched pathway during each decidualization time point.Hence, while we highlight several signaling pathways that are differentially expressed between the two groups during decidualization, we focused on characterizing the TGFβ/BMP signaling differences in the endometrium of individuals with and without endometriosis.
By examining the time-dependent endometrial response to hormones in individuals with and without endometriosis, we showed that some key pathways were conserved between the two groups, such as interleukin-2 signaling and TGFβ regulation of extracellular matrix, while others were unique to each group, such as BDNF signaling in normal endometrium and cardiomyocyte hypertrophy in endometriosis.
Accordingly, in ammatory-related genes that are controlled by IL2 signaling, such as IL6, IL24, and IL1R1, showed similar trends in the normal and endometriosis groups during the time-course decidualization analyses.However, others also showed different activation patterns when comparing endometrial stromal cells from individuals with and without endometriosis.These included NFE2L1, which increased in the endometriosis group but decreased in the normal group, and RGS5, which decreased more extensively in the normal group when compared to endometriosis.Decidualization induces extensive genetic and epigenetic remodeling programs in the endometrium that result in morphological and functional specialization of the tissue.Our results suggest that while the endometrial stromal cells from individuals with endometriosis engage similar transcriptional responses to those from individuals without, alterations in other key pathways may compromise their complete decidualization potential.
To identify the regulatory factors that could be driving the different transcriptional responses between the endometrial stromal cells from individuals with or without endometriosis, we explored consensus gene targets in the ENCODE and ChEA Transcription Factor Targets datasets 43,44 .This analysis indicated that CEBP/β is a major transcription factor controlling the transcriptional response to decidualization in the normal endometrium, controlling genes such as FBXO32, YARS, and MMP19.CEBP/β has also been shown to be a master regulator of human endometrial cell decidualization, which controls the expression of PGR by directly binding to its promoter 45,46 .Analysis of endometrial stromal cells from individuals with endometriosis identi ed NFE2L2 and SMAD4 as the top two transcription factors controlling gene expression during decidualization.NFE2L2 is a central factor controlling the intracellular response to stress and was previously shown to be activated in the endometrial epithelial cells of cows exposed to heat stress 115 .NFE2L2 (also known as NRF2) controls the expression of antioxidant genes in the cell by binding to DNA antioxidant response elements (or "AREs") 116 .One class of genes controlled by NFE2L2 are the glutathione peroxidase genes (i.e., GPX3 and GPX4), which play key roles in the control of cellular oxidative stress damage 54 .Hence, our data supports theories regarding the altered response to oxidative stress in the endometrium of individuals with endometriosis as a possible leading cause for impaired decidualization 117 .Others have also suggested that impaired response to oxidative stress through defective iron metabolism is an underlying factor in women with recurrent pregnancy loss 118 .
The ENCODE and ChEA Transcription Factor gene target analysis also identi ed SMAD4 as a major regulatory factor controlling transcription in endometriosis.The TGFβ signaling pathway was also notably altered in the endometrium of individuals with endometriosis when we directly compared the genes that were differentially regulated between normal and endometriosis groups at each time point during decidualization (Fig. 2E).SMAD4 is the common SMAD that can transmit BMP signaling (via SMAD1/5) or activin/TGFβ signaling 56 .Our previous studies, which show that BMP/SMAD1/5 signaling and TGFβ/SMAD2/3 signaling are critical for decidualization and fertility, are in line with these results 65- 67,119−121 .We focused on the roles of BMP/SMAD1/5 signaling given that we observed altered expression of BMP ligands (BMP4, BMP6) as well as decreased expression of canonical SMAD1/SMAD5 targets in the decidualizing stromal cells from individuals with endometriosis.The canonical SMAD1/5 target genes, ID2 and ID3, were signi cantly decreased during days 4 and 6 of EPC treatment in the endometrial stromal cells derived from individuals with endometriosis relative to those without.GREMLIN2, which is a secreted antagonist of the BMPs was increased in the endometrial stromal cells from individuals with endometriosis on Day 0. The expression of FST was decreased in the endometriosis group after 2, 4, 6 and 8 days of EPC treatment.Thus, using gene ontology analysis and upstream regulatory factor analyses, we concluded that the transcriptional control by BMP/SMAD signaling was a key pathway controlling decidualization the endometrium of individuals without endometriosis that was perturbed in individuals with endometriosis.However, this does not exclude potential defects in the activin/TGFβ/SMAD2/3 signaling axis in these patients.
To further characterize the genome-wide distribution of the downstream effectors of the BMPs, we used CUT&RUN to detect SMAD4 binding events in stromal cells from individuals with and without endometriosis after EPC treatment.To detect the chromatin-level changes between the two cohorts, we also mapped H3K27ac marks in the endometrial stromal cells from the normal and endometriosis groups.The binding studies showed that there were notable changes in the distribution of both SMAD4 and H3K27ac between the endometrial stromal cells of individuals with and without endometriosis, suggesting that the gene expression changes were a result of altered transcription factor binding events.This was further con rmed by intersecting the SMAD4 binding events with differentially expressed genes following SMAD1/SMAD5 siRNA-mediated knockdown in endometrial stromal cells treated with EPC to induce in vitro decidualization.We rst identi ed that the double knockdown of SMAD1 and SMAD5 blunted the decidualization capacity of endometrial stromal cells, as evidenced by the decrease of the canonical decidualization markers, IGFBP1 and WNT4.Merging of the SMAD4 binding peaks and downregulated genes after SMAD1/5 knockdown showed enrichment of genes with consensus sequences related to transcription by NFIX, SOX10, and PR.Progesterone receptor is the master regulator of decidualization 122,123 , suggesting that impaired BMP/SMAD1/5 signaling perturbs transcriptional activation of PR, blunting endometrial cell reprogramming.Furthermore, we identi ed direct SMAD4 binding sites on the genes of the MALAT1 and HDAC4 genes, which are critical for endometrial stromal cell decidualization [98][99][100][101][102][103] .Our results show molecular evidence that impaired BMP/SMAD signaling underlies the decidualization defects in the endometrium of individuals with endometriosis.
To verify the ndings that an impaired BMP/SMAD1/5 signaling pathway was driving decidualization defects in endometriosis, we tested whether the addition of recombinant human BMP2 to decidualizing cultures of endometrium could increase endometrial decidualization markers in individuals with endometriosis.Previous studies have shown that ectopic expression of BMP2 in endometrial stromal cells could potentiate decidualization in the normal endometrium 73 .We used both 2D endometrial stromal cells as well as 3D epithelial/stromal cocultures or "assembloids" to recapitulate paracrine signaling events between the two cell types.We found that relative to EPC treatment alone, the addition of BMP2 + EPC increased the expression of canonical decidual genes in both the stromal cell and assembloid cultures of endometrium from individuals with endometriosis.Our ndings indicate that BMP2 and the downstream activated signaling pathways are defective in the endometrium of patients with endometriosis and that BMP2 supplementation may correct the defect.
BMPs are subgroups of the TGFβ ligand family and BMP signaling pathways are indispensable in the female reproductive tract, especially during early pregnancy establishment 56,121 .Upon binding of BMP ligands, the serine-threonine kinase receptors (ALK1/ALK2/ALK3/ALK6 and BMPR2/ACVR2A/ACVR2B) will subsequently phosphorylate the signal transducers, SMAD1 and SMAD5 and phosphorylated SMAD1/5 will then form homodimers and translocate into the nucleus together with a common SMAD4 protein to initiate transcriptional programming 56 .BMP signaling pathways are key in transforming the maternal endometrium into a receptive environment for further support embryo implantation.From the uterine-speci c knockout mouse models, BMP ligands 64,66 , kinase receptors and SMAD signal transducers 45,65,67,124,125 are essential in decidualization and implantation, which are prerequisites for the establishment of a healthy pregnancy.Apart from regulating the decidualization and implantation process, BMP signaling pathways are also involved in immunomodulation in the endometrium.
Conditional deletion of Bmpr2 in the mouse uterus diminishes the uterine natural killer cell populations, which regulate the immune response in the endometrium, preventing the rejection of the embryo as a foreign entity.Such an immune-privileged microenvironment is crucial in the early stages of pregnancy 125 .
Indeed, the essential roles that BMP signaling pathways play in cell differentiation, proliferation and antiin ammation potentiates its signi cance in the context of endometriosis.Interestingly, BMP2 levels were decreased in the peritoneal uid of women with endometriosis 126 .Recent large-scale genome-wide association studies (GWAS) also identi ed BMPR2 as one of the endometriosis risk loci 79 .In women with recurrent implantation loss, BMP7 was identi ed to harbor a deleterious mutation that was predicted to be disease-causing 127 .Our studies highlight that the BMP signaling pathway is abnormal in the endometrium of individuals with endometriosis and may underlie the fertility defects in that population of patients.
Using transcriptomic and genome-wide binding analyses in patient-derived 2D and 3D-endometrial cultures, we show that abnormal BMP signaling pathways affect fertility in individuals with endometriosis by directly affecting the decidualization process of the endometrium.Our ndings presented here corroborate previous studies that noted the abnormal endometrial response to hormones in individuals with endometriosis [31][32][33] .However, they also reveal alterations in new pathways, such as the BMP/SMAD signaling pathways, oxidative stress responses, and retinoic acid signaling pathways, opening new potential avenues for the development of biomarkers or therapeutics for endometriosisassociated infertility.

Declarations
Figures    (i.e., were downregulated by SMAD1/5 and have a SMAD4 binding site).E) Motif analysis was performed on the group genes identi ed to be SMAD1/5/4 direct targets and displayed as "uptargets" (genes that were increased after SMAD1/5 knockdown and had a SMAD4 peak) or as "downtargets" (genes that were downregulated after SMAD1/5 knockdown and had a SMAD4 peak).