Loss of miR-29a impairs decidualization of endometrial stromal cells by TET3 mediated demethylation of Col1A1 promoter

Summary A conceptual framework for understanding abnormal endometrial decidualization, with considerable significance for the diagnosis and treatment of abnormal decidualization-related changes in non-receptive endometrium in implantation failure during early pregnancy is very important. Here, we found the expression levels of miR-29a in endometrial tissues were associated with the menstrual phases and pregnancy outcome. Inhibition of miR-29a led to decreased decidualization of endometrial stromal cells (ESCs) in vitro, whereas Tet methylcytosine dioxygenase 3 (TET3) and its potential demethylation target, the collagen type I alpha 1 chain (Col1A1), were restored. The binding capacity of TET3 to the Col1A1 promoter could be enhanced by the inhibition of miR-29a. Finally, deletion of TET3 rescued the inhibitory effect of the miR-29a antagomir on the proliferation of decidualized ESCs in vitro and embryo implantation in vivo. Thus, loss of miR-29a causes implantation failure because of the limitation of ESCs decidualization-related changes in non-receptive endometrium during early pregnancy.


INTRODUCTION
Maternal-foetal communication is the basis for maintaining a successful and healthy pregnancy (Vento-Tormo et al., 2018). Endometrial stromal cells (ESCs) proliferate and differentiate significantly to achieve the states of decidualization during early pregnancy (Okada et al., 2018;Percharde et al., 2018). Dysregulated decidualization of ESCs impairs the invasion of the endometrium by trophoblast cells, which may lead to the development of a variety of pregnancy-associated issues, including infertility, early pregnancy loss (EPL), intrauterine growth retardation (IUGR), and preeclampsia Conrad et al., 2017;Kliman and Frankfurter, 2019). Previous studies have shown that homeobox A10 (HOXA10) or interleukin 6 (IL-6) regulate cell proliferation and decidualization of ESCs Piltonen et al., 2015). Steroid hormones and adhesion molecules are also reportedly involved in the regulation of ESC proliferation and decidualisation, which further affect embryo implantation and pregnancy (Salsano et al., 2019;Nallasamy et al., 2019). However, the underlying molecular mechanism of the decidualization defect remains unclear.
MicroRNAs (miRNAs) are a class of small, noncoding RNAs that negatively regulate gene expression by binding to the 3 0 -untranslated region (3 0 -UTR) of target mRNA (Saadatpour et al., 2016). Recent studies have found that miRNAs are differentially expressed in the endometrium during the process of decidualization and the window period of embryo implantation (Liang et al., 2017;Ferlita et al., 2018). It has been demonstrated that downregulation of miR-98 promotes proliferation of ESCs and inhibits apoptosis of ESCs by targeting Bcl-xl in the rat uterus during the receptive phase (Xia et al., 2014b). A recent study has found that miR-24 is downregulated in the decidual tissue from patients with recurrent miscarriage, thus affecting the pregnancy process by regulating the proliferation and apoptosis of ESCs . However, there are numerous unknown miRNAs that control the decidualization process of ESCs. MiR-29a reportedly induces trophoblast cell apoptosis and regulates the function of trophoblast cells in recurrent miscarriage and placenta accreta (Gu et al., 2016;Yang et al., 2018). It has been revealed that miR-29a promotes granulosa cell proliferation by reducing aromatase expression and oestradiol biosynthesis in polycystic ovary syndrome . Furthermore, it has been demonstrated that miR-29a-5p inhibits endometrial cancer-derived cell proliferation by targeting TPX2 (Jiang et al., 2018). Notably, another previous study has revealed that miR-29a inhibits apoptosis of ESCs by targeting apoptotic factors during embryo implantation (Xia et al., 2014a). However, the effects of miR-29a on the proliferation and decidualization of ESCs remain to be elucidated.
Here, we found aberrant expression of miR-29a in endometrial tissues from the secretory phase, proliferative phase, normal early pregnancy, and EPL. The inhibition of miR-29a by miR-29a antagomir treatment led to decreased decidualization and cell viability of ESCs in vitro. Previous studies have proved that there are many aberrant DNA methylation events during decidualization (Hong et al., 2020;Gao et al., 2012). TET family members, involved in DNA demethylation, reportedly play an important role in embryo implantation and development, and were reported to be potentially targeted by miR-29 (Kremer et al., 2018). Interestingly, TET3 is regulated by multiple miRNAs in the progress of epigenetic regulation (Dong et al., 2020). Therefore, we hypothesized the correlation between miR-29 and TET family molecules during decidualization. Here, we found TET3 negatively associated with miR-29a in decidual tissues from patients with EPL and decidualized ESCs in vitro. Using bioinformatics analysis, TET3 was predicted to be a potential direct downstream target of miR-29a, and this was further confirmed by luciferase reporter assay.
Col1A1, a component of type I collagen, is involved in the regulation of decidualization in ESCs. Collagen type I alpha 1 chain (Col1A1), an effective component of collagen fibers and bone marrow, participates in cell proliferation, infiltration, metastasis, and angiogenesis (Kim et al., 2019a(Kim et al., , 2019b. The aberrant transcriptional activation of Col1A1 causes a defect of decidualization in mice and in humans (Kim et al., 2019a(Kim et al., , 2019b. Col1A1 expression can be affected by DNA methylation events and promoter demethylation of COL1A1 is related to its expression and functional properties of human embryonic stem cells (Hewitt et al., 2011;Kim et al., 2019aKim et al., , 2019b. In addition, previous study showed that TET3 promotes multiple TGF-b pathway gene expression by demethylation function (Xu et al., 2020). Col1A1 was a key molecule of the TGF-b pathway and we found loss of miR-29a up-regulated the expression of Col1A1. However, whether the signaling axis miR-29 and Col1A1 participates in decidualization of ESCs via affecting DNA methylation events remains unclear. In this study, miR-29a could also inhibit the recruitment of TET3 to the Col1A1 promoter and could decrease the demethylation function of TET3. Deletion of TET3 was prevented by the inhibitory effect of the miR-29a antagomir on the proliferation of decidualized ESCs in vitro and embryo implantation in vivo. These results demonstrated that loss of miR-29a suppressed decidualization of ESCs through the regulation of the TET3-Col1A1 axis during embryo implantation.

Loss of miR-29a suppresses proliferation and decidualization of human ESCs
MiR-29a was reported to be activated in the process of embryo implantation and suppressed the apoptosis of ESCs by targeting the pro-apoptotic factors Bak1 and Bmf (Kim et al., 2019a). To investigate the potential role of miR-29a during endometrial tissue development, endometrial tissues from the proliferative phase or the secretory phase, as well as decidual tissues from patients with EPL or normal early pregnancy, were collected to determine the expression status of miR-29a by qRT-PCR. MiR-29a was preferentially expressed in the endometrial tissues from the secretory phase relative to those from the proliferative phase, whereas miR-29a was downregulated in the decidual tissues from EPL patients compared to those from normal early pregnancy donors ( Figure 1A). To study the significance of the abnormal expression levels of miR-29a, ESCs were isolated from human endometrial tissues and confirmed by IF staining of vimentin ( Figure 1B) and morphology analysis ( Figure 1C). To understand the mechanism by which loss of miR-29a contributed to decidualization and decreased viability of ECSs, oestradiol, progesterone, and cAMP were introduced to modulate the decidualization of human ESCs. The normal ESCs that did not undergo decidualization were used as control. As a result, miR-29a levels were significantly elevated in the decidualized ESCs compared with the control and inhibition of miR-29a significantly decreased the miR-29a levels in the decidualized ESCs compared with control antagomir, whereas miR-29a levels in miR-29a antagomir-treated ECSs were higher than those in the control group (Figure 1D). Cell viability was also increased in the decidualized ESCs compared to that observed in the initial state ( Figure 1E), whereas inhibition of miR-29a significantly attenuated the enhanced viability of decidualized ESCs ( Figure 1E). Interestingly, we found that the cell viability in miR-29a antagomir-treated ECSs was lower than that in the control group, which was not consistent with the miR-29a levels, suggesting that inhibition of miR-29a significantly impaired the viability of the decidualized ESCs, whereas upregulation of miR-29a led to a slight increase in cell viability of the decidualized ESCs. Additionally, levels of the decidualized biomarkers PRL and IGFBP1 were both elevated after decidualization but decreased when miR-29a expression was inhibited ( Figures 1F and 1G). iScience Article TET3 is a target of miR-29a TET3, involved in DNA demethylation, reportedly plays an important role in embryo implantation and development (Han et al., 2019). To define the association between miR-29a and the TET family, we (D) qRT-PCR analysis of miR-29a levels in decidualized ESCs transfected with Control/miR-29a antagomir relative to control. U6 served as an internal control.

OPEN ACCESS
iScience 24, 103065, September 24, 2021 3 iScience Article examined the levels of TET1, TET2, and TET3 mRNA following decidualization of ESCs and miR-29a inhibition. Interestingly, ESC decidualization had no effect on TET1 levels but significantly increased TET2 levels, whereas it downregulated TET3 expression levels (Figures 2A-2C, respectively). However, loss of miR-29a in decidualized ESCs led to a significant decrease in TET2 levels, whereas TET1 and TET3 levels were both elevated (Figures 2A-2C, respectively). Using the miRNA target prediction program analysis, miR-29a was predicted to target the 3 0 UTR of TET3 ( Figure 2D). Moreover, luciferase activity of the TET3 3 0 UTR WT luciferase construct was increased significantly in the absence of miR-29a, whereas it remained unchanged in the control or TET3 3 0 UTR MUT constructs, indicating that miR-29a might target the 3 0 -UTR of iScience Article TET3 in ESCs ( Figure 2E). Furthermore, we found that TET3 protein levels were significantly downregulated in the decidual tissues from early pregnancy compared with the endometrial tissues from patients in either proliferative phase or secretory phase, whereas TET3 expression was remarkably upregulated in the decidual tissues from patients with EPL compared with the decidual tissues from early pregnancy (Figures 2F and 2G), suggesting that the expression of TET3 was negatively associated with miR-29a expression in different stages of endometrial and decidual tissues.

Loss of miR-29a induces interaction of TET3 and Col1A1 promoter in human ESCs
Col1A1, a component of type I collagen, is involved in the regulation of decidualization in ESCs (Kim et al., 2019b). In contrast, promoter demethylation of Col1A1 is related to its expression and functional properties of human embryonic stem cells (Hewitt et al., 2011). Based on the role of TET3 in DNA demethylation during embryo implantation and development, we hypothesized that Col1A1 was a downstream factor of TET3. First, we found that inhibition of miR-29a significantly upregulated Col1A1 expression in the decidualized ESCs ( Figures 3A and 3B), which might be the consequence of demethylation of TET3. To confirm this hypothesis, ChIP analysis was performed to determine the binding capacity of TET3 with the promoter region of Col1A1 in decidualized ESCs. The fold enrichment of TET3 occupancy on the Col1A1 promoter region was attenuated in the decidualized ESCs, whereas it was restored following transfection with miR-29a antagomir, suggesting that the loss of miR-29a might induce the recruitment of TET3 to the Col1A1 promoter in the human ESCs ( Figures 3C and 3D). To further prove this hypothesis, Col1A1 promoter-luciferase reporter plasmids were constructed. Compared with the control reporter, TET3 overexpression did not affect the relative luciferase activity of the pGL3 empty vector or MUT Col1A1 promoter-luciferase reporter, while significantly inhibiting the WT Col1A1 promoter-luciferase activity, indicating that TET3 could specifically bind to the Col1A1 promoter region in ESCs ( Figure 3E). Subsequently, hMeDIP-qPCR was performed to determine whether inhibition of miR-29a could affect the enrichment of 5-hmC at the Col1A1 promoter.
The results showed that the 5-hmC level of the Col1A1 promoter decreased significantly in the decidualized ESCs; however, it was restored by addition of miR-29a antagomir ( Figure 3F), suggesting that the inhibition of miR-29 promoted the demethylation function of TET3 to suppress the promoter methylation of Col1A1, and leading to enhanced Col1A1 expression. Taken together, it can be inferred miR-29a may play a role in targeting TET3 and inhibiting TET3 from binding to the Col1A1 promoter region, thus methylating the promoter and suppressing the transcription of Col1A1.

Loss of miR-29a suppresses proliferation and decidualization by activation of TET3/Col1A1 signaling
To determine whether miR-29a regulated ESC proliferation and decidualization through TET3, we introduced three small, interfering RNAs targeting TET3, and the knockdown efficiency was confirmed by western blotting ( Figure 4A). The most effective RNA was selected for the subsequent experiments. Knockdown of TET3 reversed the inhibitory effect of miR-29a antagomir on the proliferation of decidualized ESCs (Figure 4B). In addition, PRL and IGFBP1 protein levels were also partially restored in the absence of TET3 . The elevated mRNA level of Col1A1 in the presence of the miR-29a antagomir in decidualized ESCs was abrogated by TET3 knockdown, indicating that TET3 was a key effector in miR-29a-associated proliferation and decidualization of human ESCs ( Figure 4F).

Loss of miR-29a impairs embryo implantation in vivo
Intravenous injections with either miR-29a inhibitor (antagomir) or adenovirus containing TET3 shRNA were administered to 6-to 8-week-old C57BL/6 female mice, resulting in different embryo implantation on gestation day 8. Implantation sites were visualised by using the Chicago Sky Blue 6B reaction and were represented as blue spots on gestation day 8. As shown in Figure 5A, embryos were observed in the uterine horns of the pregnant mice. Compared with the pregnant mice treated with inhibitor NC adenovirus vectors, the number of embryos implanted in the uterus and the weight of the uterus were significantly reduced in the pregnant mice treated with the miR-29a inhibitor-containing adenovirus vectors ( Figures 5B and 5C). Moreover, the number of uterine embryo implantation events and the weight of the uterus remained unchanged in the pregnant mice treated with control adenovirus vectors (scramble) compared with the pregnant mice treated with miR-29a inhibitor-containing adenovirus vectors ( Figures 5B and 5C). In addition, the number of uterine embryo implantation events and uterine weight recovered in the absence of TET3 ( Figures 5B and  5C), thereby demonstrating that TET3 acted as a downstream factor of miR-29a in the embryo implantation process. The expression levels of miR-29a in the endometrial tissue were significantly increased in the normal pregnancy mice compared with the non-pregnant control group, whereas they were downregulated ll OPEN ACCESS iScience 24, 103065, September 24, 2021 5 iScience Article in the endometrial tissue of the pregnant mice injected with miR-29a inhibitor-containing adenovirus vectors ( Figure 5D). Furthermore, downregulation of TET3 did not significantly change the expression levels of miR-29a in the endometrium of pregnant mice compared with miR-29a inhibitor-containing adenovirus vector-treated pregnant mice ( Figure 5D). These results showed that miR-29a inhibitor-containing adenovirus vectors could significantly inhibit the increase in miR-29a content, whereas downregulation of TET3 demonstrated a less remarkable effect on the content of miR-29a in the endometrial tissue of pregnant mice.

Loss of miR-29a suppresses pinopodes of endometrium during embryo implantation
To determine the effect of miR-29a inhibitor on the histological morphology of the endometrium during embryo implantation, the decidua tissues of the uterus were collected from mice subjected to different  iScience Article treatments. The nucleus was round or oval with larger nucleoli and darker staining of cytoplasm was observed in samples obtained from the pregnant mice injected with inhibitor NC-containing adenovirus vectors compared to that observed in samples in the non-pregnant control group (Figures 6A and 6B). Moreover, the nucleus was smaller and cytoplasmic staining was less intense in miR-29a inhibitor-containing adenovirus vector-treated pregnant mice than that observed in the inhibitor NC-containing adenovirus vector-treated mice ( Figures 6A and 6B). Furthermore, the morphology of decidual tissues showed vacuolization, and the cell arrangement was disordered in the pregnant mice injected with miR-29a iScience Article inhibitor-containing adenovirus vectors compared with the inhibitor NC-containing adenovirus vectortreated mice ( Figures 6A and 6B). However, we found that downregulation of TET3 improved the miR-29a inhibitor-associated detrimental effects on histological morphology of decidual tissue in the pregnant mice ( Figures 6A and 6B). The number of pinocytes in the endometrium obtained from different groups was observed by scanning electron microscopy. As shown in Figure 6C, the pinopodes were in the development stage, and few had short microvilli in their endometrial surface of the non-pregnant control group. The number of pinocytes in the normal pregnancy group was significantly higher compared to that in the non-pregnant control group, whereas the miR-29a inhibitor treatment significantly decreased the number of pinocytes in the endometrium compared to the normal pregnancy group ( Figure 6C). Interestingly, the downregulation of TET3 expression partially rescued the miR-29a inhibitor-associated effects and decreased the number of pinocytes on the endometrial surface ( Figure 6C).

DISCUSSION
MiRNAs can participate in the post-transcriptional regulation of multiple physiological and pathological processes, including gene expression, modification, transcription, and translation (Bhaskaran and Mohan, 2014). MiR-29a is a key regulator of cell differentiation in diverse cell types, including bone marrow cells, T cells, hematopoietic stem cells, and myoblasts (Hu et al., 2015;Galimov et al., 2016;Xuan et al., 2017).
A previous study has shown that highly expressed miR-29a is necessary for establishing early pregnancy in the rat uterus during embryo implantation (Xia et al., 2014a). Our study demonstrated that the expression of miR-29a in decidual tissues from early pregnant women was higher than that in non-pregnant women, whereas miR-29a expression was remarkably downregulated in the decidual tissues from patients with iScience Article EPL compared to those from normal controls, suggesting that miR-29a might be involved in the decidualization of the endometrium during early pregnancy. The important prerequisite for embryo implantation is decidualization of the endometrium when the uterus enters a receptive state (Lessey and Kim, 2017). The number of embryos implanted in the uterus was reduced in the miR-29a inhibitor treated pregnant mice, which indicated that miR-29a exerted an important role in embryo implantation in mice in vivo. Moreover, we showed inhibition of miR-29a impeded the pinopodes of endometrium in pregnant mice, which suggested that loss of miR-29a might be associated with impaired endometrial decidualization as well as miR-29a was critical to the receptive endometrium in pregnant mice.
Decidualization of ESCs is a complicated biological mechanism involving crosstalk between multiple genes involved in a myriad cellular processes, including ESC proliferation and differentiation, which affects embryo implantation, maintenance of pregnancy, placental development, and fetal growth (Okada et al., 2014(Okada et al., , 2018. Although miR-29a is highly expressed in the rat uterus during early pregnancy and targets the pro-apoptotic factor genes Bak1 and Bmf in ESCs (Xia et al., 2014a), whether miR-29a can regulate proliferation and decidualization of ESCs and the underlying molecular mechanism remain unknown. In this study, we found that the expression of miR-29a was significantly elevated in decidualized ESCs and that inhibition of miR-29a significantly attenuated the proliferation of ESCs and the decidualized biomarkers PRL and IGFBP1, which suggested that miR-29a expression was required for the proliferation and decidualization of ESCs.
DNA methylation and demethylation are critical for the regulation of pregnancy as a pathway of epigenetic modification (Guo et al., 2014). A study has revealed that DNA methylation of Chromobox homolog 4 initiates stromal cell decidualization during early pregnancy (Gao et al., 2012). DNA demethylation is a chemical process which results in the removal of a methyl group (CH3) from a methylated variant of cytosine, 5-hydroxymethylcytosine (5-hmC), to generate 5-methylcytosine (5-mC). It has been demonstrated that demethylation modifications of H3K27 regulate the states of decidualization in uterine stromal cells during early and late pregnancy stages (Nancy et al., 2018). Previous studies have shown that TET-mediated demethylation is essential for normal embryo implantation and development (Khoueiry et al., 2017;Blaschke et al., 2013). In addition, another study has demonstrated that TET levels are implicated in embryo development during pregnancy (Vasconcelos et al., 2019). In this study, we found that the expression of TET3, but not TET1 and TET2, was negatively regulated by miR-29a in decidualized ESCs. The luciferase activity of the 3 0 -UTR of wild-type TET3 was significantly inhibited by the treatment with miR-29a agomir, suggesting that TET3 was a potential target of miR-29a in ESCs. Moreover, we showed that TET3 was significantly upregulated in the decidual tissues of patients with EPL, in contrast to the expression status of miR-29a. A previous study has shown that miR-29a may regulate the methylation level of mouse embryos by targeting Dnmt3a and Dnmt3b (Movahed et al., 2019). Our results revealed that downregulation of miR-29a increased the 5-hmC level of Col1A1 in the decidualized ESCs, indicating that miR-29a might inhibit the demethylation capacity of TET3 in decidualized ESCs. A recent study has shown that TET3-mediated demethylation promotes embryo development by regulating the pluripotency gene levels (Cheng et al., 2019). Interestingly, we found that downregulation of miR-29a decreased PRL and IGFBP1 protein expression levels, whereas it reversed the loss of TET3, suggesting that miR-29a-mediated negative regulation of TET3 demethylation might be associated with the process of ESC decidualization, which contributed to the pathophysiology of impaired decidualization in women with EPL.
Col1A1, a component of type I collagen, is reportedly targeted by histone deacetylase 3 (HDAC3), which regulates decidualization of ESCs (Kim et al., 2019b). Here, we demonstrated that Col1A1 expression was significantly upregulated following downregulation of miR-29a expression in the decidualized ESCs, suggesting that downregulation of miR-29a enhanced the demethylation function of TET3 to suppress the promoter methylation of Col1A1, leading to an enhanced Col1A1 expression. Loss of HDAC3 causes a defect in decidualization through interaction with Col1A1 (Kim et al., 2019b), indicating that Col1A1 affects decidualization and may be involved in the interaction with HDAC3. However, miR-29a could not directly target the 3 0 -UTR of Col1A1 ( Figures S1A and S1B). Interestingly, we found that loss of miR-29a enhanced the interaction of TET3 and Col1A1 and reversed the inhibitory effect of the miR-29a antagomir on the proliferative activity and the expression of decidualized biomarkers in the decidualized ESCs. In addition, inhibition of the TET3 downregulated miR-29a antagomir enhanced the expression of Col1A1 in the decidualized ESCs, which was involved in the loss of TET3 demethylation function. Thus, the binding of TET3 to Col1A1 functioned as a critical switch during miR-29a-mediated decidualization. Promoter demethylation ll OPEN ACCESS iScience 24, 103065, September 24, 2021 iScience Article of COL1A1 is observed during the differentiation of human embryonic stem cells (Hewitt et al., 2011). As described previously, TET3 demethylation might be associated with the process of ESC decidualization. Thus, a miR-29a downregulation-mediated increased expression of Col1A1 might depend on the function of TET3 demethylation. In fact, the hMeDIP-qPCR assay confirmed that TET3 demethylated the Col1A1 promoter in decidualized ESCs, which showed a more pronounced effect in the absence of miR-29a. These results suggested that the loss of miR-29a suppressed proliferation and decidualization of ESCs by activation of TET3/Col1A1 signaling. Embryo implantation in vivo revealed that inhibition of miR-29a significantly reduced the number of uterine embryo implantation and uterine weight compared with the NC group, whereas knockdown of TET3 restored the number of uterine embryo implantation and uterine weight, suggesting that TET3 was a downstream factor of miR-29a and downregulation of TET3 could reverse miR-29a inhibitor impaired embryo implantation in mice. Interestingly, loss of miR-29a impaired the histological morphology and decreased the number of pinocytes, whereas downregulation of TET3 could restore the effect of miR-29a inhibitor on the histological morphology and pinopodes in the endometrium during embryo implantation in mice, which suggested that downregulation of TET3 could also reverse miR-29a inhibitor impaired endometrial decidualization and receptive state in pregnant mice. These results indicated that the miR-29a/TET3 signaling was a key regulator in endometrial decidualization and embryo implantation in mice.
In conclusion, our study found that loss of miR-29a could suppress proliferation and decidualization of ESCs by enhancing the function of TET3 demethylation and the interaction of TET3 and Col1A1 in vitro, and that it impaired embryo implantation in vivo, which elucidated the important role of miR-29a/TET3/Col1A1 in regulating the process of decidualization and maintenance of decidual function during early pregnancy. These results suggest that the loss of miR-29a causes implantation failure because of the limitation of ESC decidualization-related changes in non-receptive endometrium during early pregnancy. Thus, our results provide a conceptual framework for understanding abnormal endometrial decidualization, with considerable significance for the diagnosis and treatment of abnormal decidualization-related changes in non-receptive endometrium in implantation failure during early pregnancy.

Limitations of the study
Despite miR-29a/TET3 signaling was a key regulator in endometrial decidualization and embryo implantation in mice, the changes and role of Col1A1 in uterine embryo implantation in miR-29a-or TET3-shRNA challenged mice remains to be explored by more research. Second, the role of miR-29a/TET3/Col1A1 in regulating the process of decidualization and maintenance of decidual function during early pregnancy will be further validated in the clinic.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.

Data and code availability
All relevant data and analyses are included in the present manuscript and its supplemental information. This study did not generate original code. Any additional information required to reanalyze the data reported in this work paper is available from the Lead Contact upon request.

Sample collection
Human endometrial tissues of proliferative-phase women (10 cases) and secretory-phase women (15 cases), as well as human decidual tissues of EPL patients (23 cases) and normal early pregnancy women (22 cases) with the age of 24 to 38 years old, were collected from December 2018 to December 2019 at the Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China. The EPL diagnosis was conducted based on medical history, physical examination, and transvaginal ultrasound scanning (detection of embryo sac without foetal heart activity in the uterine cavity). Inclusion criteria included presence of embryo at 8-10 gestational weeks at the time of diagnosis, with no maternal history of recurrent spontaneous abortion, chromosomal abnormalities, endocrine disease, anatomical abnormalities of the genital tract, immunological disease, infection, or trauma. All samples were washed in normal saline to remove excess blood. Samples were immediately snap-frozen in liquid nitrogen and stored at À80 C. Informed consent was obtained from all participants prior to sample collection. This study was performed in accordance with the ethical standards of the Declaration of Helsinki. This study protocol was approved by the Ethical Committee of the Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China (file no. 20180192).

Mice
C57BL/6 mice (female=30, male=15; age: 6-to 8-week-old) were bred at a room temperature of approximately 22 C and a 12L:12D photoperiod. The handling of all animals complied with the relevant regulations for experimental animals. Animal care followed the guidelines recommended by the Animal Care and Use Committee (ACUC) of the School of Medicine, Zhejiang University.

In vivo mice embryo implantation
Female and male mice were placed in cages at a ratio of 2:1, and the day of observation of the first vaginal plug was recorded as the first day of pregnancy (Day 1). Mice were randomly divided into five animals per group. Mice were injected with control, miR-29a inhibitor (antagomir), and TET3 shRNA adenovirus vectors (1310 9 pfu per mouse in a 100 mL volume) through the tail vein for two consecutive days from Day 1. Animals were sacrificed on Day 8, and uterus samples were acquired for subsequent treatment. On Day 8, the uterus was photographed, and the number of embryonic implants and the weight of the uterus were determined.

RNA isolation and RT-PCR
RNA was isolated using TRIzol following the manufacturer's instructions. In total, 20 mL template, 175 ng cDNA, 400 nM primers, and 10 mL of the RT-PCR Master Mix (2X) were gently mixed together. RT-qPCR was performed according to the manufacturer's instructions using the SYBR Green PCR kit. Primers used in the present study are listed in Supplementary tables. U6 and GAPDH served as internal miRNAs and mRNA controls, respectively. All relative expressions were calculated using the 2 -DDCt method and reported as fold change relative to the internal control and control groups.

Isolation, culture, and identification of ESCs
ESCs were isolated from the normal endometrium. Briefly, endometrial tissue was rinsed twice with sterile phosphate-buffered saline (PBS) and cut into 0.5-1-mm 3 fragments with ophthalmic scissors, and fragments were then digested in collagenase solutions for 1 h at 37 C. The digestion reactions were terminated using DMEM/F12 containing 10% fetal bovine serum (FBS), and the samples were filtered through wire sieves with various pore sizes to remove cell impurities and epithelial cells. After performing centrifugation at 800 g for 5 min, ESCs were resuspended in DMEM/F12 containing 10% FBS and incubated in a 5% CO2 incubator at 37 C. Immunofluorescence (IF) staining was performed to determine the purity of isolated ESCs using monoclonal anti-Vimentin. To induce decidualisation, ESCs were cultured with DMEM/F12 containing 2% FBS in the presence of 10 nM oestradiol, 1 mM progesterone, and 0.5 mM cAMP for 5 consecutive days, as per methods previously reported (Le et al., 2017). decidualization was confirmed by the induction of IGFBP-1 and PRL protein expression, which are specific markers of decidualization (Grasso et al., 2018).