Uterine-specific Ezh2 deletion enhances stromal cell senescence and impairs placentation, resulting in pregnancy loss.

We previously reported that Ezh2 loss in uterus leads to infertility by 8 months of age. 10 To determine if normal fertilization/implantation occurred and the gestation stage at which Ezh2 deficiency resulted in pregnancy loss, mice were sacrificed at GD7-15 of pregnancy. We observed comparable numbers of implantation chambers and indistinguishable size of implantation sites at GD7 in both genotypes ( Figure 1 A). By GD10, the implantation chamber size/diameter was significantly reduced (p < 0.05) with early signs of hemorrhage near the fetus ( Figure 1 B). Fetal growth restriction was apparent at GD12 and 15, when the fetal-placental unit was reduced by up to half (p < 0.01), with extensive hemorrhaging in Ezh2 cKO compared to WT dams ( Figures 1 C and 1D). Figure 1 Mid-gestation pregnancy loss in Ezh2 cKO mice Upper panels: Embryo implantation sites in WT and Ezh2 cKO uterine horns on (A) GD7, (B) GD10, and (C) GD12. Black arrowheads denote hemorrhagic sites. Implantation site (IS) number and diameter in WT and Ezh2 cKO mice are shown in middle panels of Fig.1A (n = 5–8), B (n = 3) and C (n = 3–4). Lower panels: (D) Embryo implantation sites in WT and Ezh2 cKO uterine horns on GD15 (left panel) and quantitation of implantation site diameter (right panel; n = 3). Black arrowheads show hemorrhagic sites and fetal resorptions in Ezh2 cKO mice. (E) At GD15, in contrast to WT, implantation sites in Ezh2 cKO mice do not contain a recognizable placenta and fetus. All the images were captured at same magnification. (F) Pup number in primiparous litters of WT and Ezh2 cKO mice. n = 29 for WT, n = 12 for Ezh2 cKO. Data are presented as mean ± SEM and were analyzed using the Student’s t test. ∗∗p < 0.01 and ∗p < 0.05 vs. WT. Mid-gestation pregnancy loss in Ezh2 cKO mice Upper panels: Embryo implantation sites in WT and Ezh2 cKO uterine horns on (A) GD7, (B) GD10, and (C) GD12. Black arrowheads denote hemorrhagic sites. Implantation site (IS) number and diameter in WT and Ezh2 cKO mice are shown in middle panels of Fig.1A (n = 5–8), B (n = 3) and C (n = 3–4). Lower panels: (D) Embryo implantation sites in WT and Ezh2 cKO uterine horns on GD15 (left panel) and quantitation of implantation site diameter (right panel; n = 3). Black arrowheads show hemorrhagic sites and fetal resorptions in Ezh2 cKO mice. (E) At GD15, in contrast to WT, implantation sites in Ezh2 cKO mice do not contain a recognizable placenta and fetus. All the images were captured at same magnification. (F) Pup number in primiparous litters of WT and Ezh2 cKO mice. n = 29 for WT, n = 12 for Ezh2 cKO. Data are presented as mean ± SEM and were analyzed using the Student’s t test. ∗∗p < 0.01 and ∗p < 0.05 vs. WT. We then analyzed fetal-placental units on GD15 to determine whether the defect occurs in the fetus and/or placenta. In contrast to WT females, Ezh2 cKO dams lacked a recognizable placenta and fetus ( Figure 1 E). Since Ezh2 cKO females are infertile at around 8 months of age, we determined the impact of Ezh2 loss on the litter size of first pregnancy. Primiparous Ezh2 cKO females had smaller litters compared to WT ( Figure 1 F). Collectively, these results indicated that Ezh2 cKO dams showed embryo resorption and failed to carry pregnancy past mid-gestation, with extensive fetal hemorrhaging accompanying pregnancy loss. Although no apparent abnormality in gross morphology of implantation chambers was detected in pregnant Ezh2 cKO dams at GD7, we observed distinct signs of embryo abnormalities upon analysis of uterine sections stained with hematoxylin and eosin (H&E) ( Figure 2 A). Since stromal cell proliferation, differentiation, decidual angiogenesis, and proper trophoblast differentiation are critical for establishment of pregnancy, we next analyzed these processes by immunofluorescent staining. We observed comparable stromal cell proliferation at GD7 in Ezh2 cKO and WT mice, as shown by MKI67 staining ( Figure 2 B). Decidual angiogenesis as indicated by expression of an endothelial cell marker, CD31, was comparable in uterine sections of WT and Ezh2 cKO mice on GD7 ( Figure 2 C). However, cytokeratin 8 immunostaining suggested altered trophoblast differentiation in Ezh2 cKO dams in early pregnancy ( Figure 2 D). Since proper differentiation of the trophoblast cells is influenced by the decidual cells, we next examined decidualization in Ezh2 cKO mice on GD7 using known biomarkers of decidualization, such as prolactin family 8, subfamily a, member 2 ( Prl8a2 ), bone morphogenetic protein 2 ( Bmp2 ), and homeobox A10 ( Hoxa10 ) in GD7 uterine tissues. The mRNA expression for Prl8a2 and Bmp2 , but not Hoxa10 in GD7 implantation sites was significantly decreased in Ezh2 cKO compared to WT controls (p < 0.05) ( Figure 2 E). Figure 2 Ezh2 loss impairs trophoblast differentiation and decidualization during early pregnancy (A) GD7 implantation sites showing retarded growth in embryos (demarcated by dashed lines) from Ezh2 cKO dams. E, embryo; D, decidua; magnification bars, 100μm. (B) Immunofluorescence for MKI67 (green) in GD7 implantation sites. Nuclei were counterstained blue with DAPI. Magnification bars, 100μm. (C) Decidual angiogenesis analysis using CD31 immunofluorescence in GD7 implantation sites from WT and Ezh2 cKO mice. Magnification bars, 100μm. (D) Immunofluorescence showing co-localization of trophoblast cell marker cytokeratin 8 (KRT8; red) and MKI67 (green) in GD7 implantation sites. Magnification bars, 20μm. (E) mRNA levels of Prl8a2 , Bmp2 and Hoxa10 in GD7 implantation sites from WT and Ezh2 cKO mice. For comparisons, expression values were set to one in WT group. Data are presented as mean ± SEM and were analyzed by Student’s t test, n = 3. Significant changes denoted by ∗p < 0.05 vs. WT. Ezh2 loss impairs trophoblast differentiation and decidualization during early pregnancy (A) GD7 implantation sites showing retarded growth in embryos (demarcated by dashed lines) from Ezh2 cKO dams. E, embryo; D, decidua; magnification bars, 100μm. (B) Immunofluorescence for MKI67 (green) in GD7 implantation sites. Nuclei were counterstained blue with DAPI. Magnification bars, 100μm. (C) Decidual angiogenesis analysis using CD31 immunofluorescence in GD7 implantation sites from WT and Ezh2 cKO mice. Magnification bars, 100μm. (D) Immunofluorescence showing co-localization of trophoblast cell marker cytokeratin 8 (KRT8; red) and MKI67 (green) in GD7 implantation sites. Magnification bars, 20μm. (E) mRNA levels of Prl8a2 , Bmp2 and Hoxa10 in GD7 implantation sites from WT and Ezh2 cKO mice. For comparisons, expression values were set to one in WT group. Data are presented as mean ± SEM and were analyzed by Student’s t test, n = 3. Significant changes denoted by ∗p < 0.05 vs. WT. In order to confirm that decidualization was suppressed in GD7 uteri, we utilized the oil-induced artificial decidualization model ( Figure 3 A). Control mice showed a robust decidual response 96 h after stimulation. In contrast, significant reductions in the weight of the stimulated uterine horn in Ezh2 cKO mice suggested that Ezh2 deletion reduced but did not completely abrogate decidualization (p < 0.05) ( Figures 3 B and 3C). Uteri in WT mice demonstrated fully differentiated stromal cells with characteristic large nuclei, while Ezh2 cKO mice had a reduced response with less differentiated stromal cells ( Figure 3 D). However, stromal cell proliferation was comparable in both genotypes ( Figure 3 E). These findings revealed that uterine Ezh2 deletion inhibited stromal decidualization, raising the possibility of subsequent placentation defects and intrauterine growth restriction of fetuses in Ezh2 cKO dams. Figure 3 Decidualization is compromised in Ezh2 cKO mice (A) Artificial decidualization protocol. (B) Gross morphology of unstimulated and oil-stimulated uterine horns from WT and Ezh2 cKO mice after 5 days of oil stimulation. (C) Ratio of uterine wet weight between oil-stimulated and unstimulated horns from WT (n = 3) and Ezh2 cKO (n = 5) mice. Data are presented as mean ± SEM and were analyzed by Student’s t test, ∗p < 0.05 vs. WT. (D) H&E staining of unstimulated and oil-stimulated uterine horns of WT and Ezh2 cKO mice. Ezh2 cKO stromal cells have relatively smaller nuclei and less differentiated stroma, characteristics of altered decidualization. (E) MKI67 immunohistochemistry in unstimulated and oil-stimulated uterine horns of WT and Ezh2 cKO mice. Comparable expression of MKI67 was observed in WT and Ezh2 cKO mice. Magnification bars, 100μm. Decidualization is compromised in Ezh2 cKO mice (A) Artificial decidualization protocol. (B) Gross morphology of unstimulated and oil-stimulated uterine horns from WT and Ezh2 cKO mice after 5 days of oil stimulation. (C) Ratio of uterine wet weight between oil-stimulated and unstimulated horns from WT (n = 3) and Ezh2 cKO (n = 5) mice. Data are presented as mean ± SEM and were analyzed by Student’s t test, ∗p < 0.05 vs. WT. (D) H&E staining of unstimulated and oil-stimulated uterine horns of WT and Ezh2 cKO mice. Ezh2 cKO stromal cells have relatively smaller nuclei and less differentiated stroma, characteristics of altered decidualization. (E) MKI67 immunohistochemistry in unstimulated and oil-stimulated uterine horns of WT and Ezh2 cKO mice. Comparable expression of MKI67 was observed in WT and Ezh2 cKO mice. Magnification bars, 100μm. Since stromal cell differentiation but not proliferation was compromised in Ezh2 cKO mice, we looked at stromal cell senescence, which has been reported to control decidualization and subsequent interaction with trophoblast cells. 27 , 28 , 29 Stromal cells isolated from WT and Ezh2 cKO GD4 uteri were 95% pure as shown by positive immunohistochemical staining for the stromal marker vimentin and negative staining for the epithelial marker cytokeratin (data are not shown). Following 72 h of decidualization, stromal cells showed reduced expression of mRNA for the decidualization marker Prl8a2 , consistent with our in vivo findings (p < 0.01) ( Figure 4 A). Stromal cells positive for senescence-associated β-galactosidase (SA-β-Gal) indicated enhanced senescence in stromal cells from Ezh2 cKO mice (p < 0.01) ( Figure 4 B). Figure 4 Increased stromal cell senescence in Ezh2- depleted stromal cells (A) qRT-PCR analysis of Prl8a2 expression. GD4 stromal cells from WT and Ezh2 cKO pregnant mice were decidualized in vitro then harvested for RNA isolation and qRT-PCR. Data are presented as mean ± SEM; n = 3 for each genotype; magnification bars, 100μm.; ∗∗p < 0.01 vs. WT. (B) SA-β-GAL staining in mouse primary stromal cells decidualized for 72h (left panel) and quantitation of SA-β-GAL-positive stromal cells (right panel). (C) Western blots (left) showing p21, p16, p53, H3K27me3, and pAKT levels in WT and Ezh2 cKO stromal cells decidualized for 72 h. Densitometric quantitation of protein expression levels are shown as fold change on the right. Data are presented as mean ± SEM and were analyzed using the Student’s t test. ∗∗p < 0.01 vs. WT; n = 3 for each group. Increased stromal cell senescence in Ezh2- depleted stromal cells (A) qRT-PCR analysis of Prl8a2 expression. GD4 stromal cells from WT and Ezh2 cKO pregnant mice were decidualized in vitro then harvested for RNA isolation and qRT-PCR. Data are presented as mean ± SEM; n = 3 for each genotype; magnification bars, 100μm.; ∗∗p < 0.01 vs. WT. (B) SA-β-GAL staining in mouse primary stromal cells decidualized for 72h (left panel) and quantitation of SA-β-GAL-positive stromal cells (right panel). (C) Western blots (left) showing p21, p16, p53, H3K27me3, and pAKT levels in WT and Ezh2 cKO stromal cells decidualized for 72 h. Densitometric quantitation of protein expression levels are shown as fold change on the right. Data are presented as mean ± SEM and were analyzed using the Student’s t test. ∗∗p < 0.01 vs. WT; n = 3 for each group. To confirm the increase in senescent stromal cells of Ezh2 cKO uteri, we performed western blotting for the senescence markers p16 and p21. In Ezh2 -deficient stromal cells, p16 and p21 expression was significantly upregulated (p < 0.01) ( Figure 4 C). Studies using the human decidualization model suggested that withdrawal of decidualization stimuli downregulates p53, but upregulates the phosphorylation and activation of AKT. In addition, p -AKT can also stabilize p21 to promote senescence. We detected suppressed p53 expression in Ezh2 cKO stromal cells, whereas p -AKT levels were upregulated compared to WT controls (p < 0.01) ( Figure 4 C). Since histone methylation regulates DNA damage-independent senescence by upregulating p16 expression and EZH2 is the master regulator of histone methylation, we measured the H3K27me3 mark in stromal cells. H3K27me3 expression was strongly suppressed in Ezh2 cKO stromal cells (p < 0.01), suggesting that Ezh2 loss promotes senescence through p16 upregulation. As pregnancy progress, trophoblast cells undergo a differentiation program to form placenta. To gauge the impact of uterine Ezh2 loss on placental development, we investigated placental architecture at GD12. The WT GD12 placentas ( Figure 5 A) consisted of three well-organized layers: the labyrinth (L), the junctional zone (JZ) consisting of spongiotrophoblast and glycogen-rich trophoblast cells, and a layer of parietal trophoblast giant cells bordering the maternally derived decidua (D). However, placentas from Ezh2 cKO mice exhibited various alterations in these placental layers. While some placentas were similar to WT, we observed a significantly smaller L layer and expanded JZ in some placentas from Ezh2 cKO mice ( Figure 5 B). Figure 5 Placentation defects in Ezh2 cKO mice (A) Architectural defects in various layers of GD12 placenta from Ezh2 cKO dams compared to WT. Labyrinth, L; Junctional zone, JZ; Decidua, D; magnification bars, 50μm. (B) Immunofluorescence for placental proteins TPBPA, p57, CD31, and KRT8 in GD12 placenta from WT and Ezh2 cKO dams. Nuclei were counterstained with DAPI. Reduced numbers of TPBPA-positive spongiotrophoblast cells are localized in the junctional zone, with aberrant infiltration in labyrinth layer of placenta from Ezh2 cKO dams. In comparison to WT placenta, attenuated p57 expression occurs in both the junctional zone and labyrinth layer in placentas from Ezh2cKO dams. Suppressed CD31 expression indicates an altered vascular network in Ezh2 cKO placenta. Co-localization of CD31 and KRT8 are also shown in yellow. n = 3; magnification bars, 50 and 100μm. Placentation defects in Ezh2 cKO mice (A) Architectural defects in various layers of GD12 placenta from Ezh2 cKO dams compared to WT. Labyrinth, L; Junctional zone, JZ; Decidua, D; magnification bars, 50μm. (B) Immunofluorescence for placental proteins TPBPA, p57, CD31, and KRT8 in GD12 placenta from WT and Ezh2 cKO dams. Nuclei were counterstained with DAPI. Reduced numbers of TPBPA-positive spongiotrophoblast cells are localized in the junctional zone, with aberrant infiltration in labyrinth layer of placenta from Ezh2 cKO dams. In comparison to WT placenta, attenuated p57 expression occurs in both the junctional zone and labyrinth layer in placentas from Ezh2cKO dams. Suppressed CD31 expression indicates an altered vascular network in Ezh2 cKO placenta. Co-localization of CD31 and KRT8 are also shown in yellow. n = 3; magnification bars, 50 and 100μm. To further investigate this abnormal placental morphology, placental samples from WT and Ezh2 cKO were probed for trophoblast-specific protein alpha (TPBPA) using immunofluorescence. In WT mice, the TPBPA-positive spongiotrophoblast cells were localized exclusively in the JZ ( Figure 5 B). In contrast, in Ezh2 cKO mice TPBPA immunostaining was significantly reduced in JZ spongiotrophoblast cells and aberrant infiltration of spongiotrophoblast cells occurred in the labyrinth layer ( Figure 5 B), indicating impaired placental development. Trophoblast differentiation is essential for establishing cell lineages that regulate placental development, maintenance, and function. p57, a cyclin-dependent kinase (CDK) inhibitor, plays an important role in development of labyrinth trophoblasts and spongiotrophoblasts in humans and mice. 30 , 31 To investigate whether trophoblast differentiation is affected in Ezh2 cKO mice, we monitored p57 expression in GD12 placental samples. Placentas of WT mice abundantly expressed p57 in the JZ and labyrinth layer, but p57 was markedly reduced in Ezh2 KO placentas ( Figure 5 B), indicating compromised trophoblast differentiation. Decreased p57 expression in the labyrinth layer of Ezh2 cKO placentas raised the possibility of impaired placental vasculature. Indeed, the WT placenta abundantly expresses the endothelial cell marker CD31 ( Figure 5 B), reflecting the widespread labyrinth vascular network. Labyrinth CD31 expression was severely attenuated in Ezh2 cKO placentas, indicating defective vascularization ( Figure 5 B). Collectively, these results indicated that uterine Ezh2 ablation interferes with trophoblast differentiation, causing defective placentation and fetal loss.

Miscarriage occurs in ∼15% of clinically confirmed pregnancies and results in ∼23 million pregnancy losses/year worldwide, and miscarriage rises with maternal age. 32 , 33 Although embryonic chromosomal abnormalities are a major cause of miscarriage, abnormalities in uterine structure and/or function are also associated with pregnancy loss. 33 Successful uterine remodeling immediately before and during early pregnancy are necessary to allow normal embryo implantation, stromal cell decidualization, trophoblast differentiation, and subsequent placental development required for normal pregnancy progression. Alterations of these processes at any stage may result in a loss of pregnancy. 16 Despite several uterine factors known to be associated with miscarriages, underlying molecular mechanisms by which a dysregulated uterine microenvironment induces miscarriage remain unclear. The histomethyltransferase EZH2 can alter gene expression by trimethylating lysine-27 in histone 3 (H3K27me3) to repress gene transcription. EZH2 plays important roles in cell proliferation, apoptosis, and senescence in normal cells, and is also involved in neoplastic development and progression. Conditional uterine Ezh2 deletion produces epithelial cell stratification and constitutive epithelial proliferation and alters uterine adenogenesis, resulting in infertility. 9 , 10 , 34 Despite defects in uterine development, Ezh2 cKO mice become pregnant at 2–4 months. However, the pups per litter and pup survival were significantly reduced in Ezh2 cKO dams at even 2 months of age. 10 Furthermore, Ezh2 cKO mice exhibit normal ovarian functions, such as estrus cycle and the presence of normal numbers of corpora lutea, indicating normal ovarian function. This raises the possibility that uterine abnormalities in Ezh2 cKO mice result in infertility. 10 The present study sought to determine the cause(s) of this infertility in Ezh2 cKO mice. Our findings indicated that Ezh2 loss does not affect either fertilization or embryo implantation as indicated by the comparable number of implantation chambers in WT and Ezh2 cKO mice. However, compared to WT mice, Ezh2 cKO dams had reduced numbers of viable fetuses, suggesting pregnancy loss later in gestation. This accounts for the decreased primiparous litter size in Ezh2 cKO mice, and the progressive infertility in these animals suggests that uterine dysfunctions affecting pregnancy outcome may be progressive with advancing age. Stromal cell decidualization is necessary for pregnancy establishment as decidual cells supply nutrients and provide immune protection for the conceptus by suppressing maternal immune response. Decidualization defects are a major factor in recurrent pregnancy loss in women. 14 , 24 , 35 A recent study suggested that Ezh2 -deficient stromal cells show fibroblast activation and peri-embryonic collagen deposition, which leads to a wound healing response at the maternal-fetal interface, resulting in pregnancy loss. 36 Alterations in stromal cell proliferation and differentiation are hallmarks of defective decidualization in mice. PRL8A2 deficiency disrupts the pregnancy-associated uterine adaptations to hypoxia and results in pregnancy loss in mice. 37 , 38 In addition, BMP2 promotes differentiation through WNT4 activation in mouse and human endometrial stromal cells. 39 Although there was no significant change in stromal cell proliferation in GD7 uteri of Ezh2 cKO mice, expression of classical decidualization markers such as Prl8a2 and Bmp2 were significantly reduced and their expression patterns were consistent with other previously used mouse models lacking uterine Ezh2 . 9 , 34 The artificial decidualization model is used to study in vivo decidualization in the absence of embryonic factors. The attenuated decidual response in Ezh2 cKO mice following artificial decidualization further confirmed that observed decidualization defects result from altered uterine maternal factors, rather than from the embryo. Comparable stromal cell proliferation in WT and Ezh2 cKO uteri suggested that altered cell multiplication does not account for impaired decidualization in Ezh2 cKO uteri and that the compromised differentiation of stromal cells reflects intrinsic uterine factors rather than any fetal contribution. Cellular senescence results in permanent cell-cycle arrest, but is not accompanied by cell death. 40 In recent years, enhanced decidual cell senescence has been reported to be associated with impaired decidualization and recurrent pregnancy loss. 27 , 28 , 29 , 41 , 42 Decidualization of stromal cells results in emergence of a large population of mature decidual cells and induces acute senescence in a subpopulation of stromal cells referred to as senescent decidual cells. Senescent decidual cells create a transient pro-inflammatory environment required for uterine remodeling to facilitate embryo implantation. 27 However, these senescent decidual cells must be eliminated after a short period of time by uterine natural killer (uNK) cells to return the uterine environment to normal. 43 In contrast, senescent decidual cells that persist in the uterus as chronic senescent cells can induce senescence in neighboring cells though paracrine signaling. 28 , 44 Accumulation of these chronic senescent cells leads to reduced uterine plasticity and impaired decidualization. To better characterize factors involved in the impaired decidualization in Ezh2 cKO mice, we utilized an in vitro model of this process. Reduced expression of the decidualization marker Prl8a2 mRNA in Ezh2 cKO decidual cells indicates abnormal decidualization in vitro . The increased senescent stromal cell population in Ezh2 cKO uteri suggests that EZH2 normally inhibits decidual senescence. Interestingly, uNK cells and genes associated with their recruitment and differentiation appear to be significantly reduced in GD8 decidua in Ezh2 cKO mice. 9 , 36 Based on our findings of enhanced decidual senescence in Ezh2 cKO stromal cells, we posit that impaired immune clearance of senescent stromal cells by uNK cells in Ezh2 cKO uteri results in accumulation of senescent cells that contribute to defective decidualization. Mechanistically, EZH2 downregulation induces cellular senescence by either methylation-independent activation of the DNA damage response or loss of methylation marks that result in elevation of the CDK inhibitors p21 and p16, critical for cell-cycle arrest and senescence. 45 Reduction in H3K27me3 levels and elevated p21 and p16 protein expression in stromal cells in Ezh2 cKO uteri supports the hypothesis that Ezh2 loss could reduce H3K27me3 levels and promote senescence through elevating p21 and p16. In human endometrial stromal cells, withdrawal of the decidualization stimulus resulted in dedifferentiation, disappearance of p53, and increased AKT activation. 46 Furthermore, loss of uterine p53 results in premature decidual senescence and promotes preterm birth through the COX2/PGF synthase pathway. 47 Decidual progression requires inactivation of AKT signaling in human endometrial stromal cells and increased activation compromises decidualization in patients with endometriosis. 48 , 49 , 50 We recently reported that Ezh2 cKO mice show high estrogen-independent uterine p -AKT expression. 13 Our results also indicate that reduced p53 and elevated p -AKT expression in Ezh2 -deleted stromal cells could be responsible for senescence-mediated defects during decidualization. The placenta is critical for fetal growth and development and the majority of fetal growth restrictions result from abnormal placental development and function, which affects 3–8% of pregnancies worldwide. 51 As pregnancy progress, trophoblast cells directly interact with the decidualized stroma and its extracellular matrix (ECM) to form placenta. 52 , 53 , 54 Recent studies indicate that senescent human endometrial cells have degraded ECM compared to well-organized ECM in non-senescent cells. Inadequate interaction of trophoblastic cells with senescent endometrial cells could cause defective placentation in human. 29 Increased senescence in decidua of Ezh2 cKO mice may be positively correlated with the altered trophoblast differentiation. Some studies suggested that EZH2 expression is significantly decreased in placental villi of women who had recurrent miscarriages and its silencing in trophoblast cells attenuated trophoblast invasion. 55 , 56 To further characterize uterine abnormalities affecting placenta development, we studied GD12 placentas in Ezh2 cKO mice. Placentation defects included perturbations in trophoblast differentiation, size of the labyrinth and JZ, mislocalization of spongiotrophoblast cells, and reduced vascularization. 51 CDK p57 knockout mice have defective placenta development, leading to perinatal lethality. 30 , 31 Decreased p57 expression in Ezh2 cKO mice indicated poor trophoblast differentiation in placenta. We found an attenuated labyrinth layer with aberrant infiltration of TPBPA-positive spongiotrophoblast cells and a less intricate vascular network, which caused placental insufficiency in Ezh2 cKO mice. In addition to labyrinth defects, an extended JZ with reduced TPBPA-positive spongiotrophoblast cells indicated impaired placentation in these mice. In summary, our results show that mice with a conditional uterine Ezh2 deletion ovulate, and fertilization and embryo implantation subsequently occur normally. In contrast, infertility in Ezh2 cKO mice is associated with defective decidualization and enhanced decidual senescence. Furthermore, these mice have altered trophoblast differentiation, resulting in placentation defects that culminate in mid-gestation pregnancy loss; these placental defects may be crucial for infertility in Ezh2 cKO mice. Since the embryos in our studies were normal, the lack of Ezh2 in the maternal uteri is the cause of pregnancy loss. This study provides important mechanistic insights to understand EZH2-mediated decidual senescence and unravel the role of maternal EZH2 loss in post-implantation placentation defects. Thus, our findings suggest that EZH2 loss or downregulation in pregnant women may cause increased miscarriages and infertility. In this study, we show the impact of uterine EZH2 loss on pregnancy progression and its molecular mechanism in regulation of stromal cell decidualization and trophoblast differentiation in an Ezh2 cKO mice model. However, there are some limitations of this study. We have utilized mouse models in this study and results must be validated in humans to correlate our findings for their clinical relevance. In addition, viable fetuses from Ezh2 cKO dams need to be studied further to determine if there are any intrauterine growth restrictions and they are small for gestational age.

Epigenetic regulation modulates gene expression in an organism without altering the DNA sequence and is responsible for transcriptional activation or suppression of genes to produce a unique pattern of a spatial-temporal gene activity. Enhancer of zeste homolog 2 (EZH2) is the rate-limiting catalytic subunit of the polycomb repressive complex 2 (PRC2), a histone methyltransferase critical for histone modifications that maintain gene repression. 1 PRC2 represses gene transcription by trimethylating histone 3 at lysine 27 (H3K27me3), which induces transcriptional silencing. 1 Ezh2 overexpression or gain of function mutations occur in endometrial 2 , 3 , 4 ; breast 5 , 6 and prostate 7 cancer, as well as in cancers of non-reproductive tissues. 8 Loss of function mutations in Ezh2 are also associated with abnormal growth/cell proliferation. 8 Some evidence suggests that EZH2 plays an important role in normal uterine physiology, as well as uterine pathologies such as endometrial cancer. We and others have reported that mice lacking uterine Ezh2 are infertile by 8 months of age. 9 , 10 Uterine development was also affected and Ezh2 cKO mice had increased numbers of uterine glands and age-dependent endometrial hyperplasia that led to organomegaly. 9 , 10 Moreover, in contrast to wild-type (WT) littermates, uterine epithelium of ovariectomized Ezh2 cKO mice constitutively proliferates despite minimal circulating estrogen levels. 10 , 11 , 12 A luminal epithelial stratification and endometrial hyperplasia at later stages of uterine development has also been reported in Ezh2 cKO mice. 9 Our recent study showed that uterine EZH2 loss is accompanied by high estrogen-independent expression of the protein kinase p -AKT, 13 but underlying molecular mechanisms responsible for aberrant epithelial proliferation and fertility defects in Ezh2 cKO mice remain largely unknown. Uterine preparation for pregnancy depends on an ordered progression of events involving uterine luminal and glandular epithelial proliferation followed by stromal cell proliferation and differentiation. 14 , 15 In mice, luminal and glandular epithelium proliferate rapidly at days 1 and 2 of pregnancy. As pregnancy proceeds, these cells exit the cell cycle and differentiate to become receptive for implantation. Implantation is initiated when the embryo attaches to the receptive uterine epithelium on day 4 of pregnancy. This is followed by invasion of the embryo into the underlying stroma and decidualization, the enlargement and differentiation of the fibroblastic stromal cells into secretory decidual cells, ensues. 16 , 17 , 18 A major function of decidual tissue is to protect the embryo and regulate its growth and development prior to placentation. The decidua secretes paracrine factors that promote angiogenesis, ensuring adequate oxygen and nutrient supplies to the embryo. Proper trophoblast differentiation is critical for normal placentation and is also influenced by secretion of unknown factors from differentiating stromal cells. 19 , 20 Pregnancy loss or miscarriage is the most common pregnancy complication in the clinical setting, 21 and results from maternal uterine deficits, as well as fetal factors. 22 , 23 For example, decidualization facilitates embryo implantation and subsequent development, 16 and abnormal stromal cell decidualization is a major factor in recurrent miscarriages, 24 but the mechanism of this effect is poorly understood. Initial work indicated that down-regulation of EZH2 is critical for human uterine stromal cell decidualization. 25 In contrast, elevated EZH2 expression following embryo implantation in mouse decidual stromal cells suggests a potential role in the decidualization process. 26 However, the precise role and mechanisms of action of EZH2 in uterine decidualization remain unclear. In this study, using genetic and cell biological approaches, we demonstrate that EZH2 regulates decidual senescence, trophoblast differentiation and placental development, and that uterine loss of EZH2 results in fetal resorption and pregnancy loss in mid-gestation. This study provides important insights into EZH2-mediated decidual function critical for placental development and establishment of pregnancy.

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-MKI67 Abcam Cat# ab16667; RRID: AB_302459 Rat monoclonal anti-KRT8 Developmental Studies Hybridoma Bank Cat# TROMA-I; RRID: AB_531826 Rabbit polyclonal anti-CD31 Abcam Cat# ab124432; RRID: AB_2802125 Mouse monoclonal anti-p21 Santa Cruz Biotechnology Cat# sc-6246; RRID: AB_628073 Rabbit monoclonal anti-p16 Cell Signaling Technology Cat# 80772; RRID: AB_2799960 Mouse monoclonal anti-p53 Cell Signaling Technology Cat# 2524; RRID: AB_331743 Mouse monoclonal anti-GAPDH Proteintech Cat# 60004-1-Ig; RRID: AB_2107436 Rabbit monoclonal anti-H3K27me3 Cell Signaling Technology Cat# 9733; RRID: AB_2616029 Mouse monoclonal anti-H3 Cell Signaling Technology Cat# 14269; RRID: AB_2756816 Rabbit monoclonal anti p -AKT Cell Signaling Technology Cat# 4060; RRID: AB_2315049 Rabbit monoclonal anti-AKT Cell Signaling Technology Cat# 4685; RRID: AB_2225340 Rabbit polyclonal anti-TPBPA Abcam Cat# ab104401; RRID: AB_10901888 Rabbit polyclonal anti-p57 Santa Cruz Biotechnology Cat# sc-8298; RRID: AB_2078155 Goat Anti-Mouse IgG (H + L)-HRP Conjugate Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat Anti-Rabbit IgG (H + L)-HRP Conjugate Bio-Rad Cat# 1706515; RRID: AB_11125142 Alexa Fluor® 488 AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Labs Cat# 711-545-152; RRID: AB_2313584 Alexa Fluor® 488 AffiniPure Donkey Anti-Rat IgG Jackson ImmunoResearch Labs Cat# 712-545-150; RRID: AB_2340683 Rhodamine (TRITC) AffiniPure Donkey Anti-Rat IgG Jackson ImmunoResearch Labs Cat# 712-025-153; RRID: AB_2340636 Chemicals, peptides, and recombinant proteins Estradiol Sigma-Aldrich Cat# E1024 Progesterone Sigma-Aldrich Cat# P0130 Dimethyl sulfoxide Sigma-Aldrich Cat# D4540 Hydrogen Peroxide, 30% Fisher Scientific Cat# H325-500 Methanol Fisher Scientific Cat# A452-4 Hematoxylin Solution, Gill No. 2 Sigma-Aldrich Cat# GHS216 Sodium Dodecyl Sulfate Fisher Scientific Cat# BP166 SuperSignal™ West Pico PLUS Chemiluminescent Substrate Thermo Fisher Scientific Cat# 34577 Corn oil Sigma-Aldrich Cat# C8267 Normal Goat Serum Blocking Solution Vector Laboratories Cat# S-1000 Hank’s Balanced Salt Solution CORNING Life Sciences Cat# 20-021-CVR DMEM/Hams F-12 50/50 Mix CORNING Life Sciences Cat# 10-090-CV Fetal Bovine Serum, charcoal stripped Gibco Cat# A3382101 DAPI Electron Microscopy Sciences Cat# 17984-24 Sodium Citrate Dihydrate Fisher Scientific Cat# BP327 0.25% Trypsin-EDTA CORNING Life Sciences Cat# 25-053 Antibiotic-Antimycotic Gibco Cat# 15240062 Halt™ Protease Inhibitor Cocktail (100X) Thermo Scientific™ Cat# 87786 TRIzol™ Reagent Invitrogen™ Cat# 15596026 Collagenase Sigma-Aldrich Cat# C2674 Pancreatin Sigma-Aldrich Cat# P3292 Dispase Sigma-Aldrich Cat# D4693 2-Mercaptoethanol Bio-Rad Cat# 1610710 Neutral buffered formalin Fisher Scientific Cat# SF100 Critical commercial assays Vectastain® Elite ABC-HRP Kit Vector Laboratories Cat# PK-4000; RRID: AB_2336820 PrimeScript™ RT reagent kit Takara Bio Cat #RR037A PrimeScript™ perfect real-time TB green Takara Bio Cat# RR0420A DAB Substrate Kit (3,3′-diaminobenzidine) Vector Laboratories Cat# SK-4100; RRID: AB_2336382 Senescence-associated β-galactosidase staining kit Cell Signaling Technology Cat# 9860 Pierce™ BCA protein assay kit Thermo Scientific™ Cat# 23250 Experimental models: Organisms/strains Ezh2 flox/flox mice Jackson Laboratories Stock # 022616 Pgr wt/Cre mice Gift of Drs, John Lydon (Baylor College of Medicine, Houston TX) and Franco DeMayo (NIEHS, Research Triangle Park NC) N/A Oligonucleotides Full list of qRT-PCR primers, see Tables S1 and S2 This paper N/A Software and algorithms ImageJ National Institutes of Health https://imagej.nih.gov/ij/ GraphPad Prism GraphPad Software www.graphpad.com Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Paul S. Cooke ( [email protected] ). This study did not generate new unique reagents. Adult female Ezh2 cKO mice with a conditional deletion of uterine Ezh2 were generated using mice that were homozygous for floxed Ezh2 and also expressed Cre recombinase driven by the progesterone receptor gene. Mice were housed in standard polycarbonate/polysulfone cages at 25°C with 12L:12D cycles and given water and a standard rodent diet ad libitum . All animal experiments were approved by the IACUC of the University of Florida and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Genotyping of transgenic mice was performed by multiplex PCR on genomic DNA ( Table S1 ). Uterine stromal decidualization was experimentally induced in adult ovariectomized hormone-primed mice as described previously. 19 Briefly, adult female WT and Ezh2 cKO mice were ovariectomized. Two weeks later, mice were injected with 100 ng of 17β-estradiol (E2) in 0.1 mL of corn oil subcutaneously (sc) every 24 h for three consecutive days. After two days of rest, sc hormone injections containing 1 mg progesterone (P4) and 10 ng E2 in 0.1 mL vehicle were given daily for three consecutive days. Decidualization was induced in one horn by injecting 20 μL of oil into the lumen, while the other horn was left unstimulated as an internal control. Mice were treated with additional daily E2 + P4 injections for up to 96 h post-stimulus. Mice were euthanized, uterine horns were collected and each uterine horn was weighed separately. Unstimulated and stimulated uterine horns were fixed in formalin and processed for histology. Fertility of Ezh2 cKO mice was assessed up to 8–9 months of age, as we have described previously. 10 Pup number in the first litter was also counted. For timed mating experiments, WT and Ezh2 cKO mice were mated with fertile wild-type males. Gestation day (GD) 1 was defined as the day the vaginal plug was observed. To examine implantation sites throughout gestation, pregnant mice were sacrificed on the morning of GD7, 10, 12 and 15. Number and diameter of implantation sites were recorded using Vernier calipers. Implantation sites were collected in 10% neutral buffered formalin and paraffin embedded for immunohistochemistry and immunofluorescence. For RNA experiments, embryos were removed from implantation sites using fine forceps and uteri were frozen at −80°C until further processing. Uterine tissues were processed and subjected to immunofluorescence as described previously. 20 Briefly, paraffin-embedded tissues were sectioned at 5 μm and mounted on microscopic slides. Sections were deparaffinized in xylene, rehydrated through a series of ethanol washes, and rinsed in water. Antigen retrieval was performed by immersing the slides in 0.1M citrate buffer solution, pH 6.0, followed by microwave heating for 25 min. The slides were allowed to cool and endogenous peroxidase activity was blocked by incubating sections in 0.3% hydrogen peroxide in methanol for 15 min at room temperature. After washing with PBS for 15 min, the slides were incubated in a goat serum blocking solution for 1 h. This was followed by incubation overnight at 4°C with antibodies specific for cytokeratin 8 (KRT8, 1:50, Developmental Studies Hybridoma Bank, TROMA-1), platelet/endothelial cell adhesion molecule 1 (PECAM1/CD31, 1:250, Abcam), and MKI67 (1:500, Abcam). Placental sections were also subjected to immunofluorescence using these primary antibodies: cytokeratin 8, PECAM1/CD31, trophoblast specific protein alpha, (1:500, Abcam), and p57 (1:100, Santa Cruz Biotechnology). Secondary antibodies including fluorescent-tagged rhodamine donkey anti-rabbit, 488 donkey anti-mouse, 488 donkey anti-rabbit, 488 donkey anti-goat were purchased from Jackson Immuno Research. Fluoromount-G with DAPI was from eBiosciences. Images were captured using the Olympus BX51 microscope equipped for fluorescence and connected to a Jenoptik ProgRes C14 digital camera with c-mount interface containing a 1.4 Megapixel CCD sensor. Fluorescent images were processed and merged using Adobe Photoshop Extended CS6 (Adobe Systems). Total RNA was isolated from uterine tissues and cultured mouse endometrial stromal cells (MESCs) using TRIzol as per the manufacturer’s protocol. Total RNA concentration was determined using a NanoDrop-200 spectrophotometer (Thermo). RNA was transcribed to cDNA using the PrimeScript RT reagent kit (Takara Bio #RR037A). For mRNA expression, PrimeScript perfect real-time TB green (Takara Bio #RR0420A) was used and amplification was performed in a CFX96 thermal cycler (Bio-Rad). Threshold cycle values (CT) were exported as an excel file and the ΔΔCt method was utilized for relative RNA expression analysis. Target gene expression was normalized against a housekeeping gene, Rplp0 . Experiments were performed in triplicate with three different animals or cell cultures in each set. Graphs were plotted as mean fold-change +SEM relative to WT control. Primer sequences are listed in Table S2 . Sections (5–6 μm) of paraffin-embedded implantation sites were cut with a microtome. Immunohistochemistry was performed using the Vectastain Elite ABC-HRP Kit (Vector Laboratories), with minor modifications. Uterine sections were deparaffinized and rehydrated. Antigen retrieval was performed in boiling sodium citrate buffer and endogenous peroxidase activity was blocked using hydrogen peroxide, as described. 13 Slides were incubated with primary antibody against MKI67 (Abcam, dilution 1:500) at 4°C overnight. The chromogen substrate 3, 3′-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA) was used to detect positively stained cells. Slides were counterstained using Gill hematoxylin (Sigma-Aldrich) for 15 s and mounted with Permount (Fisher Scientific). Images were captured using an Olympus BH-2 microscope under bright field illumination with a 25X lens. The MESCs were isolated from GD4 uteri, as previously described. 19 In brief, uteri from Ezh2 cKO and WT mice cleaned of fat were washed and cut open longitudinally, and digested by incubating with 6 mg/mL dispase (Sigma, D4693) and 25 mg/mL pancreatin (Sigma, P3292) in a 1x Hanks Balanced Salt Solution (HBSS) for 45 min at room temperature, followed by 15 min at 37°C. Following incubation, supernatant containing epithelial cells was removed by aspiration and the enzymatic reaction was stopped by adding heat-inactivated fetal bovine serum (FBS) to remaining tissue pieces. Uterine fragments were washed twice with HBSS to remove the serum. Uterine fragments were digested in 5 mL HBSS solution containing 0.15 g/L collagenase for 1 h at 37°C in a shaker incubator. After digestion, 10% FBS in 5 mL of HBSS was added to stop enzymatic digestion. Endometrial stromal cells were separated as described previously 19 and resuspended in DMEM/F12 supplemented with an antibiotic and antimycotic solution, 2% (v/v) charcoal stripped FBS, 10 nM E2 and 1 μM P4. The MESC were seeded in T-25 culture flasks at an initial plating density of 4 × 10 5 cells. After 72 h incubation in E2-and P4-supplemented DMEM/F12, cells were detached from the flasks and lysed either in SDS buffer for western blotting or in Trizol for RNA extraction. Three independent replicates derived from WT or Ezh2 cKO uteri were utilized for each group. Senescence-associated β-galactosidase (SA-β-Gal) staining was performed using the senescence β-galactosidase staining kit (Cell Signaling Technology #9860) according to manufacturer’s instructions. In brief, MESCs were seeded in 6-well plate and decidualized for 72h as described above. Following treatment, cells were washed with PBS, placed in the fixative provided for 10 min and then washed twice with PBS. Fixed cells were then incubated in a staining solution (pH 6) in a 37°C incubator without CO 2 for at least 16h. Images of random fields were captured using bright-field phase contrast microscopy. The MESCs were lysed, homogenized in 1% SDS lysis buffer supplemented with Halt protease inhibitor cocktail (Thermo Scientific) and kept for 10 min at 95°C. Lysates were centrifuged at 1800 x g for 5 min to remove cell debris. Protein content of the lysate was estimated using the Pierce BCA protein assay kit (ThermoFisher Scientific). Equal amounts of protein lysates were separated by SDS-PAGE gel electrophoresis (Bio-Rad) and transferred to Immobilon-P PVDF membrane (Millipore). Non-specific binding was blocked using 5% nonfat dry milk for 1 h at room temperature and then the membrane was incubated with a primary antibody against p21 (Santa Cruz Biotechnology sc-6246, 1:250), p16 (Cell Signaling Technology #80772, 1:1000), p53 (Cell Signaling Technology #2524, 1:1000), H3K27me3 (Cell Signaling Technology #9733, 1:1000) p -AKT or AKT (Cell Signaling Technology #4060, 1:2000 or #4685, 1:1000, respectively) or GAPDH (Proteintech 60004-1-Ig, 1:5000) at 4°C overnight. The next day, membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for 1h. Antibody binding was visualized with SuperSignal west pico PLUS chemiluminescent substrate (Thermo Scientific) and images were captured by C-DiGit blot scanner (LI-COR Biosciences). Band intensities were quantified with ImageJ software. Data are presented as mean ± SEM and were analyzed using the Student’s t test. Differences between groups were considered significant at p < 0.05. Statistical analysis was performed using Graph Pad Prism 9.0 (Graph Pad Software, Inc., San Diego, CA).