Endometrial aging is accompanied by H3K27ac and PGR loss

Wang, Yue; Zhou, Ping; Shan, Hongying; Liu, Xiyao; Cheng, Ming; Ye, Zhenhong; Chen, Xiunan; Liao, Baoying; Peng, Tianliu; Xiao, Chenxi; Huang, Ziying; Dong, Yunshu; Yu, Yang; Pan, Heng; Li, Rong

doi:10.1038/s43587-025-00859-5

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Published: 20 May 2025

Endometrial aging is accompanied by H3K27ac and PGR loss

Yue Wang ORCID: orcid.org/0000-0002-0737-6479^1,2,3,4^na1,
Ping Zhou^1,2,3,4^na1,
Hongying Shan^1,2,3,4^na1,
Xiyao Liu^1,2,3,4^na1,
Ming Cheng^1,2,3,4,
Zhenhong Ye^1,2,3,4,
Xiunan Chen^1,2,3,4,
Baoying Liao^1,2,3,4,
Tianliu Peng^1,2,3,4,
Chenxi Xiao^1,2,3,4,
Ziying Huang^1,2,3,4,
Yunshu Dong^1,2,3,4,
Yang Yu ORCID: orcid.org/0000-0002-4310-1966^1,2,3,4,5,
Heng Pan ORCID: orcid.org/0000-0002-5407-5687^1,2,3,4 &
…
Rong Li ORCID: orcid.org/0000-0003-0305-5579^1,2,3,4

Nature Aging volume 5, pages 816–830 (2025)Cite this article

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Abstract

Whether and how endometrial aging affects fertility remains unclear. In our in-house clinical cohort at the Center for Reproductive Medicine of Peking University Third Hospital (n = 1,149), we observed adverse pregnancy outcomes in the middle-aged group after excluding aneuploid embryos, implying the negative impact of endometrial aging on fertility. To understand endometrial aging, we performed comprehensive transcriptomic profiling of the mid-secretory endometrium of young (<35 years) and middle-aged (≥35 years) patients. This analysis revealed that H3K27ac loss is linked to impaired endometrial receptivity in the middle-aged group. We eliminated H3K27ac in young human endometrial stromal cells and observed reduced progesterone receptor (PGR), a critical regulator of endometrial receptivity. Lastly, we validated the association between H3K27ac/PGR loss and uterine aging in a mouse model. Our findings establish H3K27ac as a critical regulator of PGR and demonstrate that endometrial H3K27ac loss is associated with aging-related fertility decline. This work provides valuable insights into enhancing the safety and efficacy of assisted reproductive technologies in future clinical practices.

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Main

Due to the social advancement and lifestyle changes, there has been a rise in the number of persons conceiving in their late 30s, leading to frequent concerns about the impact of advanced maternal age (AMA) on pregnancies^1,2,3,4. Aging is associated with adverse pregnancy outcomes, including fertility decline, miscarriage, fetal growth restriction, stillbirth and preeclampsia^5,6,7,8. Although the aging-related decline in the quality of oocytes and embryos is a major causal factor of infertility^9,10, the effect of the aging endometrium remains controversial^10,11,12,13. Understanding the underlying mechanisms of endometrial aging and its impact on fertility is crucial for the effective treatment of aging-related female-factor infertility.

Embryo implantation is the most critical step of the reproductive process in many species¹⁴, requiring the sophisticated crosstalk between the implantation-competent blastocyst and receptive endometrium^15,16. Endometrial receptivity is a complex process that allows the embryo to attach, invade and develop in a specific period known as the window of implantation (WOI) or mid-secretory phase^17,18,19. The endometrium undergoes considerable structural and functional changes during the WOI²⁰. Growth factors, hormones, prostaglandins, adhesion molecules, nuclear receptors and epigenetic modifications tightly regulate endometrial receptivity^21,22,23,24. In addition to classical nuclear receptors, such as progesterone receptor (PGR) and estrogen receptor (ERα)²⁵, accurate histone modifications are also necessary for embryo implantation, enabling essential gene expression for decidualization, such as WNT4, ZBTB16, PROK1 and GREB1 (refs. ^24,26). As a histone modification linked to transcriptional activation, H3K27ac increases in human endometrial stromal cells during decidualization with the accumulation in the distal upstream region of IGFBP1, activating its expression^27,28. Nevertheless, the impact of H3K27ac on endometrial receptivity and its relevance to endometrial aging remain elusive.

In this study, we first confirmed the aging-related adverse impact of the endometrium on fertility based on pregnancy outcomes of a clinical cohort at the Center for Reproductive Medicine of Peking University Third Hospital (n = 1,149). Next, we characterized H3K27ac patterns in the mid-secretory endometrium of middle-aged patients, illustrating that H3K27ac loss is closely associated with PGR depletion and impaired endometrial receptivity, which was validated in a mouse model. Eliminating H3K27ac in young human endometrial stromal cells and young murine uteri reduced PGR expression. However, PGR inhibition did not affect H3K27ac in the mouse, indicating that H3K27ac is the upstream regulator of PGR. In summary, we identify H3K27ac loss as one of the hallmarks of endometrial aging, confirm the cooperatively regulatory roles of H3K27ac and PGR on genes and pathways relevant to endometrial receptivity and provide potential treatment strategies to improve reproductive outcomes in AMA pregnancies.

Results

Aging endometrium exhibits impaired endometrial receptivity

To investigate the impact of endometrial aging on pregnancy outcomes, we collected the clinical information of 1,149 patients who had undergone pre-implantation genetic testing for aneuploidy (PGT-A) screening at the Center for Reproductive Medicine of Peking University Third Hospital (Fig. 1a and Supplementary Table 1). After excluding embryonic aneuploidy, the rates of biochemical pregnancy, clinical pregnancy and live birth were significantly lower in the middle-aged group (Fig. 1b), implying the adverse impact of endometrial aging on fertility.

Impaired endometrial receptivity is one of the hallmarks of endometrial disorder, featuring extensive morphological and molecular changes¹³. Changes in epithelial and stromal cells are necessary to establish endometrial receptivity and synchronize the endometrium with embryo implantation²⁹. We observed the abnormal proliferation of epithelial cells and dysregulated decidual function of stromal cells in middle-aged patients (Fig. 1c–f, Extended Data Fig. 1a,b and Supplementary Tables 2 and 3). Aging stromal cells failed to develop the typical morphology of decidual cells upon cyclic adenosine monophosphate (cAMP) and medroxyprogesterone acetate (MPA) treatment (Fig. 1d). Decidual markers, including PRL and IGFBP1, were significantly decreased in the middle-aged group during decidualization (Fig. 1e,f and Extended Data Fig. 1b).

We next focused on classic steroid hormone receptors PGR and ERα, which are central regulators of endometrial receptivity. PGR and ERα were significantly reduced in the mid-secretory endometrium of middle-aged patients (Fig. 1g–j and Extended Data Fig. 1c–e). Their reductions in the middle-aged group were also confirmed in isolated endometrial epithelial and stromal cells (Extended Data Fig. 1f–k). To determine the transcriptional signature of endometrial aging, we applied RNA sequencing (RNA-seq) to the mid-secretory endometrium of young (n = 10) and middle-aged (n = 8) patients (Supplementary Table 2). The endometrium of the middle-aged group showed a distinct gene expression profile compared to young participants, with a substantial downregulation of PGR and other positive regulators of endometrial receptivity (Fig. 1k,l and Extended Data Fig. 1l–p). Downregulated differentially expressed genes (DEGs) in the endometrium of middle-aged patients were enriched in the pathways of the mitotic cell cycle, cell cycle and G1/S transition (Fig. 1l). We also applied RNA-seq to isolated epithelial and stromal cells of the mid-secretory endometrium (n = 4 for each group) to characterize transcriptomic changes along with aging in the cell-type-specific manner (Supplementary Table 4). Gene expression profiles differed between the young and middle-aged groups in both epithelial and stromal cells (Extended Data Fig. 2). However, aging-related transcriptional changes varied between epithelial and stromal cells. Changes in stromal cells were more similar to the endometrium, including the downregulation of genes in the pathways of the mitotic cell cycle and cell cycle in the middle-aged group (Extended Data Fig. 2d), considering that most endometrial cells were stromal cells (>60%, estimated by CIBERSORTx)³⁰. Genes related to cell proliferation were upregulated in endometrial epithelial cells of the middle-aged group (Extended Data Fig. 2j), consistent with excessive epithelial proliferation.

Our observations revealed abnormal cell morphologies, hormonal responses and cell proliferation in the endometrium of middle-aged patients, which are associated with impaired endometrial receptivity.

Aging-related H3K27ac loss is associated with PGR reduction

We evaluated the overlap between aging-related DEGs and genes marked by different histone modifications to identify upstream regulators. Endometrial DEGs were significantly enriched in genes marked by H3K27ac or H3K27me3 (Fig. 2a). Unlike H3K27me3, the impact of H3K27ac on endometrial receptivity remains unclear³¹. Of note, genes encoding H3K27ac writers and readers were downregulated in the mid-secretory endometrium of middle-aged patients, whereas genes encoding erasers were upregulated (Fig. 2b), consistent with the reduced H3K27ac level in both aging epithelial and stromal cells (Fig. 2c–e). A significantly lower level of H3K27ac in the proliferative phase reassured that H3K27ac may primarily exert its function in the mid-secretory phase (Extended Data Fig. 3a–c). We, thus, speculate that H3K27ac loss is relevant to impaired endometrial receptivity in middle-aged patients.

We next conducted CUT&Tag to explore the genomic distribution of H3K27ac in endometrial epithelial and stromal cells of young and middle-aged patients (n = 3 for each group) and to understand the association between H3K27ac loss and impaired endometrial receptivity (Extended Data Fig. 3d and Supplementary Table 4). H3K27ac peaks of biological replicates were merged, considering their consistency with each other (Extended Data Fig. 3e,f). In stromal cells, we observed thousands of differentially binding peaks, with H3K27ac loss in most peaks and genes in the middle-aged group (Fig. 2f,g and Extended Data Fig. 3g). As an important transcriptional activator^32,33, H3K27ac was prevalent at the transcription start site (TSS) compared to other genomic regions (Fig. 2h and Extended Data Fig. 3g). We observed fewer H3K27ac peaks in epithelial cells, and there were almost no differentially binding peaks between the two groups (Extended Data Fig. 3h–j). Therefore, we only focused on stromal cells for downstream analysis. In stromal cells, genes marked by H3K27ac exhibited enrichment in the Wnt and BMP signaling pathways (Extended Data Fig. 3k), which are crucial for stromal cell proliferation and differentiation during implantation^34,35,36,37. Aging-related H3K27ac loss happened around genes involved in signaling pathways relevant to endometrial receptivity (Fig. 2i,j). For instance, we observed eliminated H3K27ac in the promoter region of PGR and ESR1 (not the representative transcript of ESR1) in the middle-aged group (Fig. 2k). Compared to ERα, aging-related expression changes in PGR in endometrial stromal cells showed a greater magnitude (log₂ fold change, PGR versus ERα: qRT–PCR, −1.00 versus −0.70; RNA-seq, −1.99 versus −0.42; western blot, −0.48 versus −0.29). Thus, PGR was selected as the downstream target of H3K27ac for further exploration. To validate the causal relationship between H3K27ac and PGR, we applied different concentrations of A485 (an inhibitor of p300, a writer of H3K27ac) after 24 h of culture to eliminate H3K27ac in young endometrial stromal cells (n = 3 for each group). When treated with 2 μM A485, PGR was significantly downregulated upon H3K27ac reduction (Fig. 2l,m), confirming that eliminating H3K27ac in young endometrial stromal cells reduces PGR.

H3K27ac and PGR regulate endometrial receptivity together

We further explored the genomic distribution of PGR in human endometrial stromal cells. PGR CUT&Tag was applied to endometrial stromal cells of young and middle-aged patients (n = 3 for each group; Supplementary Table 4). PGR binding peaks of biological replicates were merged, considering their consistency with each other (Extended Data Fig. 4a). Consistent with lower PGR expression in aging endometrial stromal cells, we observed genome-wide depletion of PGR binding (Fig. 3a,b and Extended Data Fig. 4b). As an important transcription factor, PGR binding in the young group and PGR depletion in the middle-aged group were both prevalent around promoters (Fig. 3c and Extended Data Fig. 4b). Genes with PGR depletion in aging stromal cells were primarily enriched in pathways associated with endometrial receptivity, such as the Wnt signaling pathway^34,35 (Fig. 3d,e and Extended Data Fig. 4c,d). These observations revealed that aging-related abnormal PGR recruitment was relevant to impaired endometrial receptivity. To further investigate associations among PGR depletion, gene repression and impaired endometrial receptivity, we selected downregulated genes with simultaneous PGR depletion in aging stromal cells and performed pathway enrichment analysis (Fig. 3f,g). These genes were prevalent in pathways relevant to cell proliferation, differentiation and endometrial receptivity, such as the ECM–receptor interaction, and the Wnt, BMP and Hippo signaling pathways^34,38,39 (Fig. 3g). Dysregulated Wnt and ECM signaling pathways were reported to lead to abnormal proliferation and differentiation of endometrial cells^40,41,42. Representative genes of these pathways, such as FGF1, SOX4, PRC1, WNT2, WNT5A and ALDH1A1, showed gene repression and PGR depletion at the same time (Fig. 3h,i).

**Fig. 3: Aging endometrium shows genome-wide PGR depletion.**

Given that H3K27ac loss was associated with PGR depletion and abnormal endometrial receptivity, a new question is whether H3K27ac and PGR cooperatively regulate genes relevant to endometrial receptivity. We observed correlative genomic occupancies between PGR and H3K27ac (Fig. 4a–d). This co-localization was consistent along gene bodies and the entire genome (Fig. 4c,d), which was further confirmed by motif analysis and the co-immunoprecipitation (co-IP) experiment between PGR and p300 (Fig. 4e,f). Aging-related H3K27ac loss and PGR depletion also occurred at similar genomic regions (Fig. 4g), affecting key regulators of endometrial receptivity together, such as FOXO1, HOXA10 and HAND2 (Fig. 4h,i). These genes were significantly downregulated in the middle-aged group, along with H3K27ac and PGR loss (Fig. 4j,k and Extended Data Fig. 4e). Genes that exhibited simultaneous depletion of both PGR and H3K27ac in the middle-aged group were enriched in the Wnt and MAPK signaling pathways, revealing abnormal endometrial receptivity^35,43 (Fig. 4l,m). Our results indicated a collaborative relationship between H3K27ac and PGR, regulating genes related to endometrial receptivity cooperatively. This finding is unlikely due to technical artifacts because we also identified genomic regions that gain H3K27ac but lose PGR (Extended Data Fig. 4f).

**Fig. 4: H3K27ac and PGR exhibit correlated genomic occupancies and coordinated aging-related depletion.**

Eliminating H3K27ac impairs murine uterine receptivity

To functionally validate that aging-related endometrial H3K27ac loss impairs fertility by reducing PGR, we used an aging mouse model (10-month-old C57BL/6J mice). It was reported that the total number of pups and the average number of pups per litter in aging mice were significantly lower than in young mice^44,45,46, indicating impaired uterine receptivity. Consistently, our aging mice exhibited abnormal uterine receptivity with excessive proliferation of uterine luminal epithelial cells and decreased uterine H3K27ac on day 4 (Fig. 5a–c and Extended Data Fig. 5a,b). Aging murine uteri also showed distinct gene expression patterns compared to the young group (Fig. 5d and Extended Data Fig. 5c–f). Aging-related transcriptional changes were consistent between the human endometrium and the murine uterus (Fig. 5d–f). For instance, several pathways, including the cell cycle, G1/S transition and mitotic cell cycle, were enriched with aging-related downregulated genes in both humans and mice (Figs. 1l and 5d,e). Genes downregulated in the endometrium of middle-aged patients tended to be also downregulated in the aging murine uterus (Fig. 5f). These observations confirmed the reliability of using mice as the validation model.

We observed high H3K27ac in both uterine epithelial and stromal cells during the peri-implantation stage (day 4), when the uterus is in the receptive state, suggesting that H3K27ac is closely associated with endometrial receptivity and embryo implantation (Fig. 5g). To investigate whether H3K27ac loss impairs uterine receptivity, we injected 5 μl of 100 μM A485 into the uterine horn on day 3 of the pregnancy, and H3K27ac was effectively eliminated on day 4 (Fig. 5h,i). A485 mice exhibited implantation failure on day 5, along with abnormal proliferation of epithelial cells and uterine dysfunction (a defective luminal closure characterized by increased luminal epithelial branches), but without observed blastocyst alterations (Fig. 5j–m and Extended Data Fig. 6a–e). Blastocysts that failed to implant were observed in the uterine lumen in the A485 group (Fig. 5j). PGR expression was decreased in the A485 group, whereas ERα remained unaffected (Fig. 5n–p and Extended Data Fig. 6f,g). Next, RNA-seq was performed on the control and A485 uterine samples on day 4. We observed distinct transcriptional profiles between the control and A485 groups, resembling the aging process (Extended Data Fig. 6h–l). Progesterone-responsive genes, such as Hoxa10, Hand2 and Ihh, were downregulated in the A485 group along with PGR depletion (Fig. 5p,q). Estrogen-responsive genes Ltf and Muc1 were upregulated even though ERα was unaffected (Fig. 5p,q), which might be due to the imbalance between progesterone and estrogen caused by PGR depletion. The impact of H3K27ac loss on PGR was further evaluated in uterine stromal cells specifically. Upon A485 treatment, murine uterine stromal cells showed decreased Pgr expression, increased apoptosis rate and defective decidualization (Fig. 5r–t). It was reported that abnormal stromal cell proliferation and apoptosis impair endometrial receptivity^47,48,49. Of note, decidualization is different between humans and mice. The human decidua is formed routinely and is shed off in the absence of an embryo⁵⁰. However, in mice, decidualization of stromal cells occurs after successful embryo implantation^50,51. Nonetheless, we still identified a conserved mechanism between humans and mice: H3K27ac regulates PGR expression in stromal cells and, thus, affects endometrial/uterine receptivity.

To further characterize the relationship between PGR and H3K27ac, antiprogesterone RU486 was injected into the uterine horn on day 3, whereas the control group was administered with DPBS. Progesterone-responsive genes, such as Hand2, Hoxa10 and Ihh, were significantly decreased in the RU486 group, confirming the effective interference of PGR (Extended Data Fig. 7a). PGR disruption also resulted in dramatic transcriptional changes (Extended Data Fig. 7b–f), similar to the aging process. However, those aging-like changes were not accompanied by H3K27ac loss or significant expression changes of H3K27ac writers and erasers (Fig. 5u,v and Extended Data Fig. 7g,h). Thus, we conclude that H3K27ac is an upstream factor regulating PGR in the receptive murine uterus, whereas PGR cannot affect H3K27ac reversely.

Lastly, we comprehensively compared transcriptional changes among aging, H3K27ac loss and PGR inhibition by an integrated analysis of all the RNA-seq data. Uterine samples in the aging, A485 and RU486 groups shared similar transcriptional signatures, which were distinct from the young and control groups (Fig. 5w). Inhibition of either H3K27ac or PGR would result in aging-like transcriptional changes (Extended Data Fig. 8a–f), dysregulating genes and pathways closely related to uterine receptivity (Extended Data Fig. 8g,h). Our findings suggest that eliminating H3K27ac resembles the aging process and has similar transcriptional effects as PGR inhibition, although some genes and pathways were regulated by H3K27ac only (Extended Data Fig. 8f,g). These results collectively demonstrate that H3K27ac can regulate genes and pathways related to endometrial receptivity by itself or via PGR (Supplementary Fig. 1).

Discussion

In 1983, the association between AMA and potential adverse pregnancy outcomes was reported⁵². Fertility heavily declines with increasing reproductive age^53,54,55, accompanied by elevated risks of certain diseases, including early miscarriages, late miscarriages, diabetes and chromosomal abnormalities^3,5,56,57. Retrospective cohort studies have suggested that donor age is crucial to successful pregnancy in oocyte donation^10,58. Unlike comprehensive studies focusing on the impact of aging-related abnormalities in oocytes and embryos, the effect of endometrial aging on pregnancy outcomes remains controversial^{9,11,12,13,59,60}. These studies were limited by either small sample sizes or the interference of potential confounders. It is critical to understand the relationship between endometrial disorders and adverse pregnancy outcomes in middle-aged patients. To address this challenge, we characterized pregnancy outcomes of patients over and under 35 years of age who underwent PGT-A at our hospital. This PGT-A cohort presumably controlled the potential impacts of embryonic aneuploidy. We observed significantly lower pregnancy rates in the middle-aged group, implying that endometrial aging adversely affects pregnancy. Our in-house cohort study provides several advantages that support the reliability of our findings: (1) a large cohort (n = 1,149) in the same reproductive center; (2) matched oocyte and endometrium age while using PGT-A to control the embryonic aneuploidy; and (3) exclusion of diseases and factors that could interfere with endometrial receptivity. Even though PGT-A cannot rule out a non-aneuploidy aging effect on embryos, our results still represent a concrete effort to confirm the endometrial factor contributing to aging-related infertility, as embryonic aneuploidy is the leading cause of AMA pregnancy loss^61,62.

To further understand endometrial aging, we systematically characterized aging human endometrial epithelial and stromal cells at molecular, cellular and histological levels. We identified that H3K27ac loss is closely associated with aging-related transcriptional changes and PGR depletion. Using RNA-seq and CUT&Tag data, we determined H3K27ac targets and correlated aging-related H3K27ac loss to impaired endometrial receptivity. We confirmed H3K27ac as an upstream regulator of PGR because eliminating H3K27ac in young human endometrial stromal cells substantially reduced PGR. Moreover, H3K27ac and PGR exhibited correlated genomic occupancies, and their simultaneous depletion was prevalent in genes relevant to endometrial receptivity. We extended our investigation using an aging mouse model to validate initial observations. Inhibiting H3K27ac in the uterus of young mice induced aging-like phenotypes, including decreased PGR, extensive transcriptional changes, excessive epithelial proliferation, increased stromal apoptosis and impaired stromal decidualization, indicating defective uterine receptivity. We also compared transcriptional changes induced by H3K27ac and PGR inhibitors. H3K27ac inhibition resembled transcriptional changes caused by PGR inhibition but with hundreds of genes affected by H3K27ac loss only. Therefore, H3K27ac may function as a critical epigenetic regulator of uterine gene expression in mice through PGR-dependent and PGR-independent manners.

H3K27ac is a well-established chromatin marker of active enhancers and promoters. It is critical to reproductive events, such as zygotic genome activation and early embryonic development^63,64,65. However, the association between H3K27ac and endometrial aging has not been clarified yet. In the present study, we identified H3K27ac loss as a hallmark of endometrial/uterine aging in humans and mice. Further studies are needed to elucidate the causal factors of aging-related H3K27ac loss. Acetyl-CoA is a substrate of histone acetyltransferases⁶⁶. The consistent ACS5 expression in the endometrial epithelium throughout the menstrual cycle suggests the functional role of acyl-CoA synthesis⁶⁷. In aging mice, acetyl-CoA declines in the brain⁶⁸. Increasing acetyl-CoA in the brain may help maintain mitochondrial homeostasis and mitigate aging-related metabolic deficits in Alzheimer’s disease⁶⁸. We, thus, speculate that aging-related disruption in acetyl-CoA metabolism may lead to H3K27ac loss.

PGR dysfunction was previously identified in the aging murine uterus⁶⁹. Our work contributes to determining and characterizing an upstream regulator of PGR. However, due to the lack of the region-specific inhibitor of H3K27ac, our conclusions about the impact of H3K27ac and PGR loss on endometrial receptivity have not been fully confirmed⁷⁰. It has been reported that H3K27ac regulates chromatin state and affects the expression and binding of transcription factors^71,72,73. We cannot distinguish the direct regulatory function of H3K27ac from its effects mediated through PGR. Whether H3K27ac could affect endometrial receptivity independent of PGR remains an open question. Moreover, H3K27ac is not the only epigenetic regulator of PGR and genes relevant to endometrial receptivity. For instance, the causal relationship between DNA methylation and reproductive aging was also reported in mice⁷⁴. DNA methylation, RNA methylation and histone modifications may collectively regulate PGR and endometrial receptivity. The dynamic interactions and regulatory functions of these epigenetic factors are worthy of extra exploration in the future.

In the present study, we confirmed that endometrial aging is one of the critical factors leading to adverse pregnancy outcomes. We further elaborate on relationships among H3K27ac loss, PGR depletion and endometrial aging. H3K27ac extends beyond direct modulation of PGR expression to cooperatively interact with PGR in coordinating the transcriptional network of endometrial receptivity. Our work provides a new theoretical basis for the underlying mechanism of impaired endometrial receptivity in middle-aged patients. H3K27ac and PGR loss could serve as potential biomarkers and therapeutic targets for endometrial aging, potentially improving the fertility of middle-aged patients.

Methods

Clinical data collection

The current study was conducted at the Center for Reproductive Medicine of Peking University Third Hospital. A real-world retrospective PGT-A cohort study was performed to evaluate the effects of AMA on endometrial receptivity and pregnancy outcomes. Subfertile couples who were referred to the Center for Reproductive Medicine of Peking University Third Hospital for a PGT-A procedure from January 2018 to December 2022 were potentially eligible if they had at least one autologous euploid blastocyst (no mosaics) destined for frozen-thawed embryo transfer (FET). None of the participants had been diagnosed with uterine adhesion, immune system disease, hyperprolactinemia or thyroid dysfunction before the recruitment. Additional exclusion criteria were as follows: (1) couples either did not have a developed embryo for biopsy or did not have a euploid blastocyst for transfer; and (2) couples had any disease that would interfere with endometrial receptivity, including endometriosis, adenomyosis, fibroids, polyps and polycystic ovary syndrome. Single blastocyst FET was performed each time until a live birth or exhaustion of all euploid embryos occurred. Some patients experienced repeated transfers using surplus euploids. Reproductive outcomes were compared between patients who were younger than 35 years of age or 35 years of age or older at their last transfer. Biochemical pregnancy was determined by a positive serum β-hCG test result. The clinical pregnancy was defined as an ultrasonographic visible gestational sac inside the uterine cavity, and ectopic pregnancy was defined as outside the uterus. Early pregnancy loss was defined as pregnancy loss that occurred during the first trimester. Live birth was defined as the delivery of any viable neonate with gestational age ≥24 weeks. The sex of the newborn was counted after delivery.

Clinical sample collection

All endometrial samples were obtained from normally cycling patients undergoing endometrial biopsy in our center due to infertility combined with an uneven internal echo of the uterine cavity detected by transvaginal ultrasound. All patients of the well-characterized samples were normo-ovulatory, with regular cycles (21–35 d), and had not been on steroid hormone medications within 3 months before the sampling. The endometrium was collected 7–10 d after the serum luteinizing hormone (LH) peak, or the endometrium was paraffin embedded and stained with hematoxylin and eosin (H&E), and the mid-secretory phase was determined by two senior pathologists. Patients with any pathological findings that invade the endometrial cavity, as previously detected by transvaginal ultrasound, pelvic magnetic resonance imaging, endometrial biopsy or serum cancer antigen (CA) 125 determination, such as endometriosis, submucosal myomas, intramural myomas >4 cm and hydrosalpinx, were further excluded. The definition of middle-aged patients in this study is older than 35 years of age. Specimens of patients aged 24–32 and 38–45 were eventually selected for tissue collection. The menstrual cycle was mainly categorized into proliferative, mid-secretory or other phases based on histopathologic criteria and were retrospectively assigned back to samples.

This study was approved by the Ethics Committee of Reproductive Medicine of the Peking University Third Hospital (no. 20195Z-067). All procedures were performed following the principles stated in the Declaration of Helsinki. Written informed consent was obtained from all patients.

Human primary endometrial stromal cell isolation, culture and treatment

Endometrial tissues of young and middle-aged patients were rinsed with DPBS to remove mucus and blood contaminants and then minced into 8–10-mm³ fragments. The remaining tissues were digested with collagenase I and DNase I for 1 h, and cell suspensions were filtered through 100-µm and 40-µm wire gauze in sequence to remove excess epithelial cells. The filtered cell suspension was centrifuged to collect stromal cells. Approximately 8 × 10⁵ viable stromal cells were seeded into six-well plates and cultured in phenol red-free DMEM/F-12 (Gibco) with 10% charcoal-stripped FBS (CS-FBS; Vistech). After 4 h, the culture medium was changed to remove the floating cells.

After being passaged once, isolated stromal cells were used to induce decidualization. When the cell density reached 50–60%, the medium was replaced by phenol red-free DMEM/F-12 with 1% CS-FBS supplemented with 10 nM estradiol (Sigma-Aldrich, 613967) and 1 μM progesterone (Sigma-Aldrich, P0130) for different days.

Animal feeding and treatments

This study involves the use of animals and was reviewed and approved by our institutional animal ethics committee (A2023030). The experiments adhere to the guidelines set by the committee and are conducted following the principles of the 3Rs (Replacement, Reduction and Refinement) to ensure the ethical treatment of animals in research. All efforts have been made to minimize animal suffering and to use the minimum number of animals necessary to achieve the scientific objectives of the study.

Eight-week-old and 10-month-old C57BL/6J mice were acquired from Beijing Vital River Laboratory Animal Technology. All mice were kept in a controlled environment with a 12-h light/dark cycle, a room temperature of 20–25 °C and humidity of 55% ± 10% and had access to food and water at all times.

Female mice were mated with male mice, and the day of detection of vaginal plug was considered day 1 of the pregnancy. On day 3 of the pregnancy, mice in the case group received injections of 5 μl of 100 μM A485 (Selleck Chemicals, S8740) or RU486 (Selleck Chemicals, S2606) on each side of the uterine horn, whereas mice in the control group were given 5 μl of DPBS on each side of the uterine horn. On day 4 of the pregnancy, endometrial samples were obtained for molecular testing. On day 5 of the pregnancy, implantation sites were identified with an intravenous injection of Chicago blue dye solution (Sigma-Aldrich, C8679), and the number of implantation sites, marked by distinct blue bands, was recorded.

Western blotting

Western blotting was performed as previously described⁷⁵. Samples were homogenized in the lysis buffer, followed by the separation of proteins on a 12% SDS-PAGE gel. After this, the proteins were transferred to PVDF membranes (Millipore) and blocked with 5% non-fat dry milk. Specific primary and secondary antibodies were then incubated on the membranes in sequence. Antibodies against H3K27ac, IGFBP1, PGR, ERα, p300, FoxO1, HOXA10 and HAND2 were used. GAPDH served as control. Detailed information for antibodies is provided in Supplementary Table 5.

qRT–PCR

Total RNA was extracted from uterine tissues or cells using TRIzol reagent (Thermo Fisher Scientific). cDNA was made by HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech, R323-01). qRT–PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad, 172-5201) on a QuantStudio 12K Flex (Applied Biosystems). Each PCR experiment was repeated at least three times, and relative expression levels were determined by the ΔCT method with normalization to GAPDH. All PCR primers are listed in Supplementary Table 6.

Immunostaining

Human endometrial and mouse uterine tissues were fixed in 4% paraformaldehyde. Five-micrometer paraffin-embedded sections were used for immunohistochemistry staining. Rehydrated sections were microwaved in sodium citrate buffer for 20 min to repair antigens and then blocked with 0.5% BSA-PBS for 1 h. After this, the primary antibodies PGR, ERα and Ki67 were incubated at 4 °C overnight. Signals were visualized by horseradish peroxidase (HRP)-conjugated secondary antibodies. In immunofluorescence experiments, paraffin-embedded sections and cells were incubated with H3K27ac, MUC1 and phalloidin primary antibodies. Signals were visualized by secondary antibodies labeled with Alexa Fluor 488 (anti-rabbit; Invitrogen), and the cell nuclei were stained with DAPI. Detailed information for antibodies is provided in Supplementary Table 5.

Co-IP

The human endometrium was used for co-IP. Diluted p300 and IgG antibodies were added to magnetic beads, which were then fully suspended and incubated in a flip mixer for 30 min at room temperature, followed by 2 h at 4 °C. After rinsing the magnetic beads (MCE, HY-K0202) with PBST, samples were added and incubated overnight at 4 °C in a flip mixer. The magnetic beads were then separated and resolved by SDS-PAGE. Magnetic beads were isolated and redissolved by 1× SDS-PAGE loading buffer, and subsequent immunoblot experiments were performed using antibodies against PGR and ERα.

RNA isolation and library preparation

Total RNA was extracted from human endometrium and mouse uteri using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). Then, the libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. Each group was performed with at least three biological replicates.

RNA-seq and analysis

The libraries were sequenced on an Illumina NovaSeq 6000 platform, and 150-bp paired-end reads were generated. Approximately 50 million raw reads were generated for each sample. Raw reads in FASTQ format were first processed using fastp⁷⁶, and low-quality reads were removed to obtain clean reads. Approximately 48 million clean reads were retained for each sample. Clean reads were then mapped to the reference genome using HISAT2 (ref. ⁷⁷). Human reference genome hg38 was used for the human data, and mouse reference genome mm39 was used for the murine data. Read counts of each gene were obtained by HTSeq-count⁷⁸. Differential expression analysis was performed by DESeq2 (ref. ⁷⁹). DEGs were determined by adjusted P < 0.05 and absolute log₂ fold change > 1. The enrichment analysis was performed by Metascape⁸⁰. Aging-related DEGs in the human endometrium were compared to published chromatin immunoprecipitation sequencing (ChIP-seq) data of histone modifications^{81,82,83,84,85,86}.

CUT&Tag library construction

Cell nuclei extracted from the mid-secretory endometrium of young and middle-aged groups were used for CUT&Tag assay after being isolated using a cell nuclear isolation kit (Bioyou, 52201-10). CUT&Tag assay was performed using a Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme Biotech, TD903) according to the manufacturer’s instructions⁸⁷. Initially, concanavalin A–coated magnetic beads (ConA beads) were added to resuspended cell nuclei and incubated at room temperature to bind the cell nuclei. Cell membrane permeabilization was achieved using the non-ionic detergent Digitonin. The cell nuclei were then bound by ConA beads. Subsequently, primary antibodies for H3K27ac and PGR, along with secondary antibodies and the Hyperactive pA-Tn5 Transposase, were incubated with the nuclei. The Hyperactive pA-Tn5 Transposase precisely cleaved the DNA fragments bound to the target proteins. The cut DNA fragments were ligated with P5 and P7 adaptors by Tn5 transposase, and the libraries were amplified by PCR using the P5 and P7 primers. The purified PCR products were assessed using the Agilent 2100 Bioanalyzer. Finally, the libraries were sequenced on the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads for subsequent analysis. Each group was performed with three biological replicates.

CUT&Tag analysis

The raw sequence data were first trimmed by fastp⁷⁶ to obtain clean reads. Clean reads were aligned to the human reference genome hg38 using Bowtie 2 (ref. ⁸⁸). Peak calling was performed by MACS2 with the ‘narrowPeak’ parameter⁸⁹. Normalization was performed with the RPGC method. Peak annotation was performed by ChIPseeker⁹⁰. The motif analysis was conducted by Homer⁹¹. Differentially binding peaks were determined by DiffBind⁹², with adjusted P < 0.05 and absolute log₂ fold change > 0.75.

Statistics and reproducibility

For the cohort study, categorical variables were shown in ratio and percentage, and differences between groups were compared by Fisher’s exact test or chi-square test, as specified in the legend. Continuous variables were reported as mean ± s.d. and compared by the Mann–Whitney U rank-sum test owing to the non-normality of data. All experiments were repeated at least three times. Data are presented as mean ± s.d. in a combo chart with columns and scatter plots. Statistical analysis was performed by two-sided unpaired Student’s t-test or Mann–Whitney U rank-sum test (when data did not follow a normal distribution). Statistical analysis and visualization were performed using Prism for Windows (version 9.5.1; GraphPad Software). A two-tailed P value less than 0.05 was considered statistically significant. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications^93,94,95.

No data were excluded from the analyses. Data collection and analysis were not performed blinded to the conditions of the experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the study’s findings are provided in the Source Data and Supplementary Information. Human RNA-seq and CUT&Tag data have been uploaded to the Genome Sequence Archive (accession number HRA007501). Murine RNA-seq data have been uploaded to the Genome Sequence Archive (accession number CRA022502). BED files of CUT&Tag data have been uploaded to the Open Archive for Miscellaneous Data (accession number OMIX008964). The human reference genome hg38 can be accessed in the National Center for Biotechnology Information (NCBI) (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.39_GRCh38.p13/). The mouse reference genome mm39 can be accessed in the NCBI (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/635/GCF_000001635.27_GRCm39/). Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Project of China (2021YFC2700303 and 2022YFC2702500), the National Natural Science Foundation of China (81925013, 82288102, 82225019, 82192873, 82301888 and 82271699), the Key Clinical Projects of Peking University Third Hospital (BYSYZD2023028), the Beijing Nova Program (20220484073), the Special Project on Capital Clinical Diagnosis and Treatment Technology Research and Transformation Application (Z211100002921054) and Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJCPI20250032). We want to thank all the patients and staff of the Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital for their contributions. We thank CapitalBio Corporation for technical assistance.

Author information

These authors contributed equally: Yue Wang, Ping Zhou, Hongying Shan, Xiyao Liu.

Authors and Affiliations

State Key Laboratory of Female Fertility Promotion, Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
Yue Wang, Ping Zhou, Hongying Shan, Xiyao Liu, Ming Cheng, Zhenhong Ye, Xiunan Chen, Baoying Liao, Tianliu Peng, Chenxi Xiao, Ziying Huang, Yunshu Dong, Yang Yu, Heng Pan & Rong Li
National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
Yue Wang, Ping Zhou, Hongying Shan, Xiyao Liu, Ming Cheng, Zhenhong Ye, Xiunan Chen, Baoying Liao, Tianliu Peng, Chenxi Xiao, Ziying Huang, Yunshu Dong, Yang Yu, Heng Pan & Rong Li
Key Laboratory of Assisted Reproduction, Peking University, Ministry of Education, Beijing, China
Yue Wang, Ping Zhou, Hongying Shan, Xiyao Liu, Ming Cheng, Zhenhong Ye, Xiunan Chen, Baoying Liao, Tianliu Peng, Chenxi Xiao, Ziying Huang, Yunshu Dong, Yang Yu, Heng Pan & Rong Li
Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
Yue Wang, Ping Zhou, Hongying Shan, Xiyao Liu, Ming Cheng, Zhenhong Ye, Xiunan Chen, Baoying Liao, Tianliu Peng, Chenxi Xiao, Ziying Huang, Yunshu Dong, Yang Yu, Heng Pan & Rong Li
Beijing Advanced Center of Cellular Homeostasis and Aging-Related Diseases, Institute of Advanced Clinical Medicine, Peking University, Beijing, China
Yang Yu

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Yue Wang
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Ping Zhou
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Hongying Shan
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Xiyao Liu
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Ming Cheng
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Xiunan Chen
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Baoying Liao
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Chenxi Xiao
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Contributions

Concept and design: R.L., H.P., Y.Y. and Y.W. Acquisition, analysis or interpretation of data: M.C. and Y.W. Drafting of the paper: Y.W., P.Z. and H.S. The experiments were performed and data were analyzed by, and critical revision of the paper for important intellectual content was performed by, H.S., X.L., M.C., Z.Y., X.C., B.L., T.P., C.X., Z.H. and Y.D. Obtained funding: R.L., H.P., Y.Y. and Y.W. Administrative, technical or material support: R.L., H.P. and Y.Y. Supervision: R.L. All authors approved the final version for submission.

Corresponding authors

Correspondence to Yang Yu, Heng Pan or Rong Li.

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Nature Aging thanks Francesco Demayo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The mid-secretory endometrium exhibits different phenotypes and gene expression between young and middle-aged patients.

a, The percentage of Ki67-positive cells in endometrial luminal epithelial and stromal cells in the mid-secretory phase (n = 3). b, Relative protein levels of IGFBP1 in human endometrial stromal cells during induced decidualization (n = 3). c,d, Histochemical scoring assessment (H-score) of PGR and ERα in the mid-secretory endometrium (n = 3). e, Relative protein levels of PGR and ERα in the mid-secretory endometrium (n = 5). f,g, Protein levels of PGR and ERα in human mid-secretory endometrial stromal cells (n = 4). h, Relative mRNA levels of PGR and ESR1 in human mid-secretory endometrial stromal cells (n = 4). i,j, Protein levels of PGR and ERα in human mid-secretory endometrial epithelial cells (n = 4). k, Relative mRNA levels of PGR and ESR1 in human mid-secretory endometrial epithelial cells (n = 4). l, PCA plot of human mid-secretory endometrial RNA-seq data. m, Volcano plot illustrating gene expression changes between the young and aging mid-secretory endometrium (|log2FC| > 1, P.adj < 0.05). n, Heatmap showing gene expression of differentially expressed genes (DEGs) between the young and aging mid-secretory endometrium. o, FPKM of LIF, SOX4, FGF1, WNT2, IL7, IHH in the human mid-secretory endometrium (n = 10 and n = 8 for the young and middle-aged groups, respectively). The adjusted P value was determined by DESeq2. Whiskers represent upper quartile + 1.5 interquartile range (IQR) and lower quartile − 1.5 IQR. p, Pathway enrichment analysis of upregulated DEGs in the aging mid-secretory endometrium. Statistical analysis was performed by two-sided unpaired Student’s t-test or Mann–Whitney U rank-sum test. Data are presented as mean ± s.d. All replicates were biological replicates. FC, fold change; mid-aged, middle-aged; ns, not significant; P.adj, adjusted P value; PC, principal component; PCA, principal component analysis.

Source data

a, Consistent gene expression changes between A485/CON and Aging/Young. b, Consistent gene expression changes between RU486/CON and Aging/Young. c, Heatmap showing gene expression of DEGs between the young and aging murine uterus. d, Heatmap showing gene expression of DEGs between the CON and A485 murine uterus. e, Heatmap showing gene expression of DEGs between the CON and RU486 murine uterus. f, Venn diagram illustrating the overlap among different groups of DEGs. g, KEGG enrichment analysis of different groups of DEGs. h, GSEA of gene expression changes in the murine uterus (A485 or RU486 versus CON) against the cell cycle and vasculogenesis gene sets. CON, control; FC, fold change; P.adj, adjusted P value.

Supplementary information

Supplementary Fig. 1 and Tables 1–6.

Reporting Summary

Supplementary Data 1

DEGs between the mid-aged and young human mid-secretory endometrium.

Supplementary Data 2

DEGs between mid-aged and young human mid-secretory endometrial stromal cells.

Supplementary Data 3

Differentially binding peaks of H3K27ac between mid-aged and young human mid-secretory endometrial epithelial cells.

Supplementary Data 4

Differentially binding peaks of H3K27ac between mid-aged and young human mid-secretory endometrial stromal cells.

Supplementary Data 5

Differentially binding peaks of PGR between mid-aged and young human mid-secretory endometrial stromal cells.

Supplementary Data 6

DEGs between the uterus of aging and young mice.

Supplementary Data 7

DEGs between the uterus of A485 and control (CON) mice.

Supplementary Data 8

DEGs between the uterus of RU486 and control (CON) mice.

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Wang, Y., Zhou, P., Shan, H. et al. Endometrial aging is accompanied by H3K27ac and PGR loss. Nat Aging 5, 816–830 (2025). https://doi.org/10.1038/s43587-025-00859-5

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Received: 11 May 2024
Accepted: 31 March 2025
Published: 20 May 2025
Issue Date: May 2025
DOI: https://doi.org/10.1038/s43587-025-00859-5