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The relationship between the human placenta—the extraembryonic organ made by the fetus, and the decidua—the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels 1 . Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia 2 . Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal–fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids 3 , 4 and trophoblast stem cells 5 . We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell–cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy. A multiomics single-cell atlas of the human maternal–fetal interface including the myometrium, combining spatial transcriptomics data with chromatin accessibility, provides a comprehensive analysis of cell states as placental cells infiltrate the uterus during early pregnancy.

Abstract Uterine fibroids are benign monoclonal neoplasms of the myometrium, representing the most common tumors in women worldwide. To date, no long-term or noninvasive treatment option exists for hormone-dependent uterine fibroids, due to the limited knowledge about the molecular mechanisms underlying the initiation and development of uterine fibroids. This paper comprehensively summarizes the recent research advances on uterine fibroids, focusing on risk factors, development origin, pathogenetic mechanisms, and treatment options. Additionally, we describe the current treatment interventions for uterine fibroids. Finally, future perspectives on uterine fibroids studies are summarized. Deeper mechanistic insights into tumor etiology and the complexity of uterine fibroids can contribute to the progress of newer targeted therapies. Graphical Abstract Graphical Abstract

Background Cesarean scar defects can lead to long-term complications, such as cesarean scar disorders, cesarean scar pregnancy, and the risk of uterine scar dehiscence and rupture in subsequent pregnancy. However, the optimal closure technique to prevent the development of cesarean scar defects (CSD) remains unclear. Therefore, this study aimed to explore whether two-layer interrupted versus two-layer continuous sutures could prevent the formation of CSD. Methods A randomized controlled trial was conducted in a single university hospital in Japan. We recruited pregnant women with ≥ 20 primary or previous cesarean sections. Participants were randomly assigned to either a two-layer interrupted or a two-layer continuous suture group. Residual myometrial thickness (RMT) and the depth of CSD were measured using sonohysterography, 6–8 months post-cesarean section. In addition, the rate of severe CSD, defined as a loss of over 50% of the myometrium, was examined. Results Of the 220 study participants, 43 dropped out; 89 in the interrupted group and 88 in the continuous group underwent sonohysterography. No significant difference in RMT was observed in the interrupted and continuous groups (median 8.1 [interquartile range, 6.2–9.9] mm and 7.9 [4.6–10.3] mm, respectively). However, the incidence of severe CSD in the interrupted group was significantly lower than that in the continuous group (2% versus 22%, p  < 0.0001). Multivariate logistic regression analysis revealed that the factors contributing to developing severe CSD were interrupted suture (odds ratio [OR]: 0.04, 95% confidence interval [95%CI]: 0.006–0.281, p  = 0.0011), the difference in myometrial thickness between the fundal and cervical sides at the center of the uterine wound before suturing (OR: 1.65, 95%CI: 1.144–2.367, p  = 0.0072), and retroversion of the uterus at 6–8 months after cesarean section (OR: 3.42, 95%CI: 1.074–10.946, p  = 0.0374). Conclusion This study suggested that two-layer interrupted sutures are superior to two-layer continuous sutures in preventing the development of severe CSD. Trial registration Clinical trial identification number: University Hospital Medical Information Network registration code, UMIN000040601. URL of the registration site: https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000046334 .

Uterine transplantation is currently the only solution that enables women with absolute uterine factor infertility to become pregnant and give birth to a child. In the preparatory phase of a human uterus transplantation, the sheep is the most recommended species. Cold ischaemia, i.e., a period of reduced or absent blood flow at cold conditions, can significantly impair the function of the transplanted organ. Cold ischaemia impairs smooth muscle function in general and reduces smooth muscle contractile activity. However, it seems to provide some protection against cold storage. Our main goal was to investigate the molecular mechanisms leading to reversible changes in myometrial myofilaments and to distinguish these from permanent changes, which was supported by histological imaging of uterine samples. Using fluorescence spectroscopy, we investigated important interactions between major components of smooth muscle such as actin and tissue-specific actin-binding proteins. We characterized functional changes by denaturation sensitivity and protein-protein interactions under low and high salt conditions by intrinsic tryptophan, Alexa488-phalloidin and eosin fluorescence emission spectroscopy assays. Our results suggest that short-term cold ischaemia causes minor disruption of muscle cells. The protein extracts of myometrium contained large amounts of actin, which was present in soluble complexes with actin-binding proteins after ischaemic stress. The results indicate that the contractile filament system underwent molecular stabilization and reassembly due to ischaemic stress and that the actin monomers were unable to form polymers due to increased heterologous protein-protein interactions. The content of necrotic proteins cannot be detected after brief ischaemia, but eosin selectively binds to large proteins (caldesmon, myosin chains, tropomyosin) and protein complexes. Based on these results, we can assume that short-term preservation of cold ischaemia in uterine transplantation reduces the risk of using it in clinical trials for complete myometrial recovery after reperfusion.

Uterine leiomyomas (fibroids) are a major source of gynecologic morbidity in reproductive age women and are characterized by the excessive deposition of a disorganized extracellular matrix, resulting in rigid benign tumors. Although down regulation of the transcription factor AP-1 is highly prevalent in leiomyomas, the functional consequence of AP-1 loss on gene transcription in uterine fibroids remains poorly understood. Using high-resolution ChIP-sequencing, promoter capture Hi-C, and RNA-sequencing of matched normal and leiomyoma tissues, here we show that modified enhancer architecture is a major driver of transcriptional dysregulation in MED12 mutant uterine leiomyomas. Furthermore, modifications in enhancer architecture are driven by the depletion of AP-1 occupancy on chromatin. Silencing of AP-1 subunits in primary myometrium cells leads to transcriptional dysregulation of extracellular matrix associated genes and partly recapitulates transcriptional and epigenetic changes observed in leiomyomas. These findings establish AP-1 driven aberrant enhancer regulation as an important mechanism of leiomyoma disease pathogenesis. Somatic mutations in MED12 have been implicated as the causal genetic lesion in the majority of uterine leiomyomas. Here, the authors profile the chromatin landscape of matched normal and leiomyoma tissues and find that changes in enhancer acetylation, enhancer-promoter interaction strength, differential enhancer usage and transcription factor AP-1 occupancy are significant drivers of transcriptional dysregulation in MED12 mutant leiomyomas.

Background Preterm birth, a leading cause of perinatal mortality and morbidity, is often associated with inflammation and aberrant myometrial contractions. This study investigates the role of Piezo1, a mechanosensitive ion channel, in myometrium contraction and inflammation-associated preterm birth. Methods We employed Western blotting, Immunofluorescence, and Quantitative real-time PCR techniques to examine Piezo1 expression in uterine tissues. Functional assays, including myometrial contractility studies and cell contraction assays, were conducted to elucidate the effects of Piezo1 on myometrial contractions. Piezo1 inhibitors and gene knockdown techniques were used to investigate the impact of Piezo1 on inflammation-associated preterm birth, complemented by inflammatory cytokine profiling and calcium imaging to investigate the mechanism. Results Our findings reveal that Piezo1 is the predominant mechanosensitive channel in mouse myometrium tissue and mouse primary uterine smooth muscle (pUSMCs), with increased expression during mouse and human pregnancy. Following lipopolysaccharide (LPS) intrauterine injection, Piezo1 mRNA and protein levels were elevated in the mouse uterine smooth muscle layer. Direct pharmacologic activation of Piezo1 by Yoda1 increased the contraction of pUSMCs and shortened the pregnancy duration. In contrast, inhibition with Gsmtx4 or siRNA knockdown of Piezo1 attenuated LPS-induced pUSMCs contraction and spontaneous uterine myometrium contraction. Additionally, blocking or knocking down Piezo1 prolonged the pregnancy in an LPS-induced preterm birth model. Yoda1 stimulation increased intracellular Ca 2+ levels in pUSMCs, while Gsmtx4 reduced these levels. Gsmtx4 decreased cox-2 expression and inflammation factors in LPS-stimulated pUSMCs. Conclusions These results suggest that Piezo1 acts as a critical regulator of uterine function, and its overexpression may predispose to preterm labor through heightened myometrial activity and inflammation. The study underscores the potential of targeting Piezo1 as a therapeutic strategy to mitigate preterm birth associated with uterine inflammation.

Labor onset is a complex process influenced by multiple factors. Although the mechanisms initiating labor remain incompletely understood, accumulating evidence suggests that maternal immune responses serve as critical drivers of this process. Ferroptosis, an iron-dependent form of programmed cell death, has emerged as a potential modulator of the immune microenvironment. But its role in labor onset remains largely unexplored. This study examined ferroptosis occurrence and how it affects cell functions during labor onset. We analyzed multi-cohort transcriptome datasets to identify immune cell infiltration using CIBERSORT and ssGSEA. Meanwhile, with via LASSO regression, we identified four core immune cells including M2 macrophages in labor onset. Differential analysis revealed 49 ferroptosis-related differential genes (FRDGs) in this process. Ferroptosis and iron ion homeostatic pathways were enriched by KEGG and GO analysis. Our ROC model using the key FRDGs for diagnosing labor onset performed excellently. We further identified a significant negative correlation between M2 macrophages and the FRDGs HMOX1 by Spearman. Finally, we performed in vitro and in vivo experiments to confirm the ferroptosis involvement in myometrium and to affect the specific functions of human uterine smooth cells (HUSMCs) and THP1. Several key FRDGs are significantly differentially expressed at both mRNA and protein levels in the labor myometrium. In addition, elevated Fe²⁺, decreased T-GSH, and increased MDA reflected higher levels of ferroptosis during labor. In vitro, we detected a shift towards a pro-inflammatory phenotype induced by ferroptosis in M2 macrophages using flow cytometry and PCR arrays, which enhanced cytokine secretion and promoted uterine smooth muscle contraction. Conversely, inducing ferroptosis in HUSMCs suppressed cytoskeletal remodeling and gap junction protein expression, impairing contraction. The study aims to provide a new theoretical basis for the mechanism of labor onset and a potential therapeutic target for abnormal timing of delivery.

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation,myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNAdependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation. Summary Sentence Progesterone independently regulates the maternal immune system and the onset of parturition in the mouse.

Adenomyosis is a benign gynaecological disorder in which endometrial glands and stroma enter the uterine myometrium with varying degrees of spreading. To analyse the presence of developmentally displaced endometrial glands and stroma in the foetal myometrium, a retrospective cohort of 420 foetal uteri, including one monozygotic twin pair, was histopathologically evaluated. The gestational age ranged between 18 and 37 weeks; the clinical characteristics included various foetal malformations with a predominantly normal karyotype, except in one case with trisomy 18. Ectopic endometrial tissue enclosed in the myometrium was discovered in twelve individual foetuses from the cohort (12/420). The investigation of the histogenetic attributes of the misplaced endometrial tissue in both monozygotic twins’ (MZ) foetal uteri revealed isolated glands and thin channels containing cords of endometrial-type glands penetrating the myometrium. Through immunohistochemistry, low levels of oestrogen receptors (ERs) were detected, whereas a moderate level of progesterone receptor (PR) expression was observed in the ectopic glandular and stromal cell nuclei in all cases. Additionally, the surrounding periglandular component consistently expressed the vimentin and CD10 stromal cell markers, while the myometrial smooth muscle cells revealed the strong expression of both alpha-Smooth Muscle Actin (α-SMA) and desmin marker proteins.

Deregulation in uterine contractility can cause common pathological disorders of the female reproductive system, including preterm labor, infertility, inappropriate implantation, and irregular menstrual cycle. A better understanding of human myometrium contractility is essential to designing and testing interventions for these important clinical problems. Robust studies on the physiology of human uterine contractions require in vitro models, utilizing a human source. Importantly, uterine contractility is a three-dimensionally (3D)-coordinated phenomenon and should be studied in a 3D environment. Here, we propose and assess for the first time a 3D in vitro model for the evaluation of human uterine contractility. Magnetic 3D bioprinting is applied to pattern human myometrium cells into rings, which are then monitored for contractility over time and as a function of various clinically relevant agents. Commercially available and patient-derived myometrium cells were magnetically bioprinted into rings in 384-well formats for throughput uterine contractility analysis. The bioprinted uterine rings from various cell origins and patients show different patterns of contractility and respond differently to clinically relevant uterine contractility inhibitors, indomethacin and nifedipine. We believe that the novel system will serve as a useful tool to evaluate the physiology of human parturition while enabling high-throughput testing of multiple agents and conditions.