Uterine Tissue Research Articles

Loss of PRICKLE1 leads to abnormal endometrial epithelial architecture, decreased embryo implantation, and reduced fertility in mice.

Conservative cell division is essential to maintain apical-basal polarity and proper epithelial function in the uterus. Wnt/ Planar cell polarity signaling molecules are hypothesized to provide the spatial cues to organize unicellular, 2-dimensional sheet of epithelium in a plane orthogonal to the apical-basal polarity. Conditional ablation of Prickle1 , a crucial Wnt/ PCP gene, in mouse uterine epithelium results in aberrant expression of epithelial cadherin, altered plane of cell division, incomplete cytokinesis leading to binucleated/ multinucleated cells, epithelial - mesenchymal transition, and defective implantation. Role of Prickle1 in maintaining symmetric uterine epithelial cell division and tissue architecture is unique among Wnt/PCP genes, including previously described mouse models for Vangl2, Ror2, and Wnt5a . Classification: Biological Sciences (Major) Cell Biology (Minor), Physiology (Minor).

Apelin-13 alleviates intrauterine adhesion by inhibiting epithelial-mesenchymal transition of endometrial epithelial cells and promoting angiogenesis.

Intrauterine adhesion (IUA) is a common complication of surgical manipulation of the uterine cavity such as abortion. The pathology of IUA is characterized by fibrosis, but the pathogenesis is not fully understood. The function of Apelin-13 in IUA and related mechanisms were investigated in this study. The IUA rat model was established. The pathological changes and fibrosis degree of rat uterine tissues were detected by HE and Masson staining after intraperitoneal injection of Apelin-13. Epithelial-mesenchymal transition (EMT) of endometrial epithelial cells and endothelial-mesenchymal transition (EnMT) of vein endothelial cells were induced by TGF-β1. Tube-forming assay using HUVEC was implemented to detect the effect of Apelin-13 upon angiogenesis. IHC staining, immunofluorescence staining, and Western blot were conducted to detect the expression levels of EMT markers, angiogenesis, and key proteins of the TGF-β1/Smad signaling. Apelin-13 significantly alleviated IUA and fibrosis, and increased endometrial thickness and gland number in IUA rats. In addition, Apelin-13 significantly reversed EMT and EnMT induced by IUA modeling and TGF-β1, promoted the tube-forming ability of HUVEC, and up-regulated the expression of angiogenesis-related proteins. Mechanistically, Apelin-13 significantly suppressed smad2/3 phosphorylation and inhibited the TGF-β1/Smad signaling via its receptor APJ. Apelin-13 might alleviate IUA via repressing the TGF-β1/Smad pathway and is expected to be a potent therapeutic option for the clinical treatment of IUA.

Uterine Biosynthesis through Tissue Engineering: An Overview of Current Methods and Status.

In the last few decades, the rates of infertility among women have been on the rise, usually due to complications with the uterus and related tissue. A wide variety of reasons can cause uterine factor infertility and can be congenital or a result of disease. Uterine transplantation is currently used as a means to enable women with fertility issues to have a natural birth. However, multiple risk factors are involved in uterine transplantation that threaten the lives of the growing fetus and the mother, as a result of which the procedure is not prominently practiced. Uterine tissue engineering provides a potential solution to infertility through the regeneration of replacement of damaged tissue, thus allowing healing and restoration of reproductive capacity. It involves the use of stem cells from the patient incorporated within biocompatible scaffolds to regenerate the entire tissue. This manuscript discusses the need for uterine tissue engineering, giving an overview of the biological and organic material involved in the process. There are numerous existing animal models in which this procedure has been actualized, and the observations from them have been compiled here. These models are used to develop a further understanding of the integration of engineered tissues and the scope of tissue engineering as a treatment for uterine disorders. Additionally, this paper examines the scope and limitations of the procedure.

Immunohistochemical study of hormone receptors in endometrium with abnormal uterine bleeding

In the case of AUB, estrogen and progesterone exert their effects by acting on specific nuclei. Receptor proteins such as estrogen receptor (ER) and progesterone receptor (PR). And periodic changes in its expression have been shown in human uterine tissue. Immunohistochemistry (IHC). Immunohistochemistry using specific monoclonal antibodies estimate the content of the receptor at the cellular level and interpret their exact location and distribution.A two-year descriptive study was conducted. There were a total of 50 cases clinically diagnosed with AUB who underwent hysterectomy and endometrial curettage, and 20 non-AUB controls were included in this study. IHC was performed for ER and PR receptors in all cases and controls.The age range was 29 to 65 years and the average age of the patients was 43.21 years. Out of 50 patients, 26 were pre-menopausal and 24 were post-menopausal, and the mean hemoglobin, fertility and bleeding duration were 10.34 g/dL, 2.8 and 8.4 days per cycle. The average thickness of the endometrium was 10.5 mm in the cases and 6.25 mm in the control group. Increasing endometrial thickness was statistically significantly related to AUB and increased ER and PR HSCORE in stroma and glands (p<0.001). In terms of histomorphology, the highest number of cases was endometrial hyperplasia without atypia (27 cases), followed by early proliferative phase (7 cases). ER expression was more abundant in glands and PR expression was more in stroma. In endometrial hyperplasia with atypia, the expression of ER and PR was higher than in other endometrial stages.In addition to pelvic ultrasound and histology, immunohistochemistry. ER and PR have the advantage of allowing these hormone receptors to localize to the tissue.Endometrial aspiration samples and anxiliary tests in patient treatment allong with AUB. Endometrial hyperplasia is more common in patients with AUB. The concentration of estrogen and progesterone receptors is significantly higher. Therefore, the location and concentration of ER and PR receptors increases and this makes it possible for targeted therapies and avoid invasive surgical procedures using selective progesterone receptor modulators and progesterone antagonists in the treatment of patients with AUB and endometrial hyperplasia.

Induction and characterization of a rat model of endometriosis

Endometriosis is a common condition that affects 5% to 10% of women during their reproductive years, although the aetiology and pathophysiology are still unknown. This study aimed to create an endometriosis model in rats to investigate the efficacy of natural and synthetic medications in treating endometriosis. An in vivo endometriotic model was established using a surgical induction method and the endocrine-disrupting drug diethylstilbestrol (DES). In brief, the experiment is categorised into three different groups. Each group contains five rats. The first group had no surgery, while in the in the second group of rats (n = 5), two small tissue grafts were fixed at the right and left walls of the abdomen. But in the in the third group of rats (n = 5), two small pieces of tissue have been grafted on the right and left abdomen walls by surgically along with DES treatments. Noninvasive photoacoustic imaging (PAI) was employed in the study to measure factors such as haemoglobin levels, oxygen saturation, and the size of endometriotic lesions. Histopathological analysis was carried out utilising staining techniques such as Hematoxylin and Eosin, Masson's Trichrome, and Periodic Acid Schiff, as well as immunohistochemistry with marker antibodies. Molecular markers in uterine tissue were examined using Western blots and real-time PCR. The developed endometriosis rat model showed a significant increase in the expression of anti-apoptotic Bcl-2, angiogenic marker VEGF and pro-inflammatory (COX-2 and IL-6) protein markers. In contrast to the control group, the treatment group had considerably lower Caspase-3 expression levels. Photoacoustic imaging (PAI) data demonstrated a constant increase in lesion size, as well as a decrease in oxygen saturation levels. The findings suggest that the in vivo endometriosis rat model may accurately assess the efficacy of natural or synthetic endometriosis treatments. This model may help in the improvement of disease understanding and the development of targeted therapeutic drugs.

Detection of sow pregnancy in day-20 urine samples using monoclonal antibody against synthesized porcine early pregnancy factor: Preliminary results

Early diagnosis of pregnancy is directly related to cost-effective livestock production. We produced a rat monoclonal antibody (mAb) against synthesized porcine early pregnancy factor (pEPF) using conventional hybridoma technology and used it as a tool for the detection of early pregnancy in Duroc sows. The rat pEPF-mAb showed reactivity to uterine tissues of pregnant sows 20 or 30 days post-mating (day 0 defined as the day of mating) and non-pregnant sows (confirmed signs of estrus) in western blotting. Immunohistochemical analysis confirmed that pEPF was located in the stromal and grand epithelial tissues of pregnant sows 20 or 30 days post-mating. In the enzyme-linked immunosorbent assay, pEPF expression in urine and blood showed similar results, with the highest expression observed in pregnant sows 20 days post-mating, whereas there was no significant difference in expression levels between non-pregnant sows and pregnant sows 30 days post-mating. The pEPF-mAb-based pregnancy diagnostic kit can be applied to pig urine samples non-invasively collected at 20 days post-mating with 70 % accuracy. Further improvements to the kit's diagnostic performance may lead to substantial benefits for the swine industry, facilitating more efficient and accurate reproductive management.

Surface-Conjugated Galactose on Electrospun Polycaprolactone Nanofibers: An Innovative Scaffold for Uterine Tissue Engineering.

The uterus, a vital organ in the female reproductive system, nurtures and supports developing embryos until maturity. This study focuses on addressing uterine related problems by creating a nanofibrous scaffold to regenerate uterine myometrial tissue, closely resembling the native extracellular matrix (ECM) for enhanced efficacy. To achieve this, we utilized polycaprolactone (PCL) as a biomaterial and employed an electrospinning technique to generate PCL nanofibers in both random and aligned orientations. Due to the inherent hydrophobic nature of PCL nanofibers, a two-step wet chemistry surface modification technique is used, involving the conjugation of galactose onto them. Galactose, a lectin-binding sugar, was chosen to enhance the scaffold's hydrophilicity, thereby improving cell adhesion and fostering l-selectin-based interactions between the scaffold and uterine cells. These interactions, in turn, activated uterine fibroblasts, leading to ECM remodeling. The optimized electrospinning process successfully generated random and aligned nanofibers. Subsequent surface modification was carried out, and the modified scaffold was subjected to various physicochemical characterization, such as the ninhydrin assay, enzyme-linked lectin assay techniques that revealed successful galactose conjugation, and mechanical characterization to assess any changes in material bulk properties resulting from the modification. The tensile strength of random galactose-modified PCL fibers reached 0.041 ± 0.01 MPa, outperforming random unmodified PCL fibers (0.026 ± 0.01 MPa), aligned unmodified PCL fibers (0.011 ± 0.001 MPa), and aligned modified PCL fibers (0.016 ± 0.002 MPa). Cytocompatibility studies with human uterine fibroblast cells showed enhanced viability and proliferation on the modified scaffolds. Initial pilot studies were attempted in the current study involving subcutaneous implantation in the dorsal area of Wistar rats to assess biocompatibility and tissue response before proceeding to intrauterine implantation indicated that the modification did not induce adverse inflammation in vivo. In conclusion, our study introduces a surface-modified PCL nanofibrous material for myometrial tissue engineering, offering promise in addressing myometrial damage and advancing uterine health and reproductive well-being.

Reduced endometrial glycolysis concomitant with increased lesional fibrosis in patients with adenomyosis who complained of heavy menstrual bleeding

RESEARCH QUESTIONWhat role, if any, does the extent of lesional fibrosis play in adenomyosis-associated heavy menstrual bleeding (ADM-HMB)? DESIGNForty-eight patients with ADM-HMB were recruited, among them 25 reported moderate/heavy bleeding (MHB) and the remaining 23, excessive bleeding (EXB). The full-thickness uterine tissue columns were processed for Masson trichrome staining and immunohistochemistry analyses. The levels of HIF-1α, GLUT1, HK2, PFKFB3 and PKM2—proteins critically involved in glycolysis—in endometrial epithelial cells cultured on substrates of different stiffness, and the levels of glycolysis were quantitated. A mouse experiment with induced adenomyosis and simulated menstrual bleeding was conducted to assess the impact of adenomyosis on immunoexpression of proteins involved in glycolysis and inflammation as well as on endometrial repair and bleeding. RESULTSThe endometrial staining of HIF-1α, GLUT1, HK2, PFKFB3 and PKM2 was significantly lower in the EXB group as compared with MHB patients, concomitant with higher extent of fibrosis. The expression of HIF-1α, GLUT1, HK2, PFKFB3 and PKM2 was significantly reduced when endometrial epithelial cells were cultured in stiff substrate, concomitant with reduced glycolysis. Mice with induced adenomyosis exhibited reduced immunoexpression of Hif-1α, as well as those proteins each of which plays a vital, rate-limiting role in different steps of the glycolysis pathway, such as Glut1, Hk2, Pfkfb3 and Pkm2, and elevated fibrosis in endometrium, concomitant with disrupted endometrial repair and more bleeding. CONCLUSIONSLesional fibrosis results in reduced endometrial glycolysis in eutopic endometrium and subsequent imbalance in pro- and anti-inflammatory response, leading to ADM-HMB.

First identification of microplastics in human uterine fibroids and myometrium

Microplastics (MPs) pollution has received widespread attention in recent years as the use of plastics continues to increase. However, currently no studies have reported the finding of MPs in human uterine fibroids (UFs) and myometrium tissues. In this study, UFs tissues (n = 48) and myometrium tissues (n = 40) from 48 patients and myometrium tissues (n = 8) from healthy population were collected. Following digestion of the samples by 10% KOH and 30% H2O2, MPs were analyzed qualitatively and quantitatively using Raman spectroscopy. The 16 UFs and myometrium tissue samples contained an average of 1.5 ± 1.17 MP particles per gram of tissue. Notably, the abundance of MPs in the UFs tissues (2.13 ± 1.17 particles per gram) was higher than in the myometrium tissues (0.88 ± 0.78 particles per gram). In the same cohort of individuals with UFs, the quantities of MPs detected in the affected UFs tissue (2.63 ± 1.77 particles per gram) exceeded those detected in healthy tissue (1.08 ± 0.93 particles per gram), particularly in elderly patients. A correlation was observed between elevated MP levels and frequent consumption of takeout meals and bottled water among patients, indicating that MP ingestion through food sources might have contributed to the increased abundance and variety of MPs within UFs. Furthermore, UFs increased in size with higher concentrations of MPs, which may have been related to elevated levels of MPs-induced hormones. This study provides new insights into the assessment of the relationship between exposure to MPs and human disease risk.

Expression patterns of inflammatory and oxidative stress-related genes in the uterine and ovarian tissues of dogs diagnosed with pyometra based on cervical patency status

This study aimed to investigate the expression patterns of genes associated with inflammation and oxidative stress in ovarian and uterine tissues of dogs with pyometra, categorized by cervical status (open cervix or closed cervix), which influences disease severity. The control group comprised healthy animals undergoing elective ovariohysterectomy. Tissue inflammatory gene expression and Malondialdehyde (MDA) levels were determined while microbial and histopathological examinations were conducted, along with immunohistochemical evaluations. In the closed-cervix group, uterine TNF and IL6 were upregulated approximately 10-fold while IL10 was upregulated nearly 5-fold. TNF expression differed remarkably between the pyometra groups. In the closed-cervix group, PTGS2 and HMOX1 were upregulated approximately 5-fold whereas NFE2L2 expression was downregulated. The closed-cervix group also had the highest uterine MDA levels. Regarding ovarian tissue, MDA levels were higher in the closed-cervix group than in the open-cervix group while IL10 expression was lower in the closed-cervix group than the open-cervix group. In the closed-cervix group, NFE2L2 was downregulated whereas HMOX1 was upregulated. Uterine TNF levels were positively correlated with IL6, IL10, PTGS2, and HMOX1, but negatively correlated with NFE2L2. IL6 was positively correlated with IL10, PTGS2, and HMOX1. NFE2L2 was negatively correlated with IL6 and HMOX1. IL10 was positively correlated with PTGS2 and HMOX1. MDA was positively correlated with TNF, IL6, IL10, PTGS2, NFE2L2, and HMOX1. TNF levels were positively correlated with ovarian PTGS2, and with IL6 and NFE2L2. MDA was positively correlated with PTGS2 and HMOX1. MDA could be an important biomarker for understanding the severity of pyometra. Moreover, TNF expression and its relationships with various studied parameters such as IL10 may contribute to treatment and prognostic biomarker studies in closed-cervix pyometra pathology.

Effect of electroacupuncture pre-conditioning on the expression rhythm of core clock gene Bmal1 in uterine tissue of controlled hyperstimulation rats.

To observe the effect of electroacupuncture (EA) pre-conditioning on the expression rhythm of clock gene Bmal1 in the uterine tissue of rats with controlled ovarian hyperstimulation(COH), so as to explore its mechanisms underlying improvement of the endometrial receptivity of ovarian superovulation during implantation. Seventy-two female SD rats with typical estrous cycles were randomly divided into normal control, model and EA pre-conditioning (pre-EA) groups, with 24 rats in each group. The COH model was established by giving the rats with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) by intraperitoneal injection. The rats of the pre-EA group received EA stimulation (1 Hz/5 Hz, a tolerable strength) of "Guanyuan"(CV4) and "Sanyinjiao"(SP6) for 15 min each time, once daily (at 21：00 every day). After successive EA intervention during the first two estrous cycles, the modeling began in the third estrus cycle and the EA intervention was continued till the end of modeling, followed by raising the rats with superovulation induction and male rats undergoing vasoligation in one cage (1∶1). The rats during the estrum in the normal control group or those of the model group at the end of modeling were raised together with the male rats undergoing vasoligation in one cage. On the 5th day (04：00 AM) after raising in one cage, the rats in the three groups were sacrificed in six batches every 4 hours, with 4 rats in each group in each batch. The H.E. staining was used for revealing alterations of the endometrial thickness, number of glands and blood vessels and tissue histology, and ELISA employed to ascertain the contents of estradiol (E2) and progesterone (Pg) in serum. The expression rhythm of core clock gene Bmal1 ［In the present study, Zeitgeber time (ZT) is an artificially set laboratory time, i.e., ZT7 (07：00) is light on and ZT19 (19：00) is light off.］ and the expression of endometrial HoxA10 and leukemia inhibitory factor (LIF) mRNAs were detected by quantitative real-time PCR. The Western blot was employed to detect the expression levels of HoxA10 and LIF proteins. Findings of the clock gene Bmal1 level showed that the expression peak was at ZT12 and the valley value at ZT20 in the normal control group, and that of the peak value was at ZT20 and valley value at ZT12 in the model group, while in the pre-EA group, the peak value was at ZT8, and the valley value at ZT4. The difference of Bmal1 levels among the three groups was most significant at ZT12 (12：00), therefore, the tissue samples were taken at ZT12 in this study for comparison of the levels of different indexes among the 3 groups. Compared with the control group, the endometrial thickness, number of glands and blood vessels, HoxA10 and LIF mRNAs and proteins were significantly down-regulated (P<0.01, P<0.05), and contents of serum E2 and Pg were considerably up-regulated in the model group (P<0.01, P<0.05). Relevant to the model group, the pre-EA group had an apparent increase in the endometrial thickness, number of glands and blood vessels, and expression levels of HoxA10 and LIF mRNAs and proteins (P<0.05, P<0.01), and a marked decrease in the serum Pg (P<0.05). At the ZT12 (12：00 noon), compared with the normal control group, the mRNA level of Bmal1 was significantly decreased in the model group (P<0.01)；and compared with the model group, the level of Bmal1 mRNA was significantly increased in the pre-EA group (P<0.05). In addition, at the node of ZT16, the mRNA level of Bmal1 was significantly decreased in the model group in comparison with the normal control group (P<0.01). EA preconditioning can improve the endometrial receptivity during the implantation window period in rats with COH, which may be related to its functions in regulating the expression of clock gene Bmal1 in the uterine tissue and in correcting the disturbance of clock gene rhythm.

Synthesis of Cyclodextrin-Based Multifunctional Biocompatible Hydrogels and Their Use in the Prevention of Intrauterine Adhesions (Asherman's Syndrome) after Surgical Injury.

Asherman's syndrome, which can occur during the regeneration of damaged uterine tissue after surgical interventions, is a significant health problem in women. This study aimed to acquire and characterize cyclodextrin-based hydrogels, which can be used to prevent Asherman's syndrome, and investigate their effectiveness with biomedical applications. A series of hydrogels were synthesized from the cross-linking of β-cyclodextrin and different polyphenols with epoxy-functional PEG. Their chemical, physical, and biological properties were subsequently determined. The results demonstrated that the cyclodextrin-based hydrogels had a porous structure, high swelling ratio, good injectability, drug release ability, and antioxidant activity. Cell culture results illustrated that the hydrogels had no significant cytotoxicity toward L929 fibroblast cells. Considering all properties, the β-CD-PEG-600-Ec hydrogel showed the most satisfactory properties rather than other ones. The potential of this hydrogel in preventing Asherman's syndrome was evaluated in a rat model. The results revealed that the β-estradiol- and melatonin-loaded cyclodextrin-based multifunctional hydrogel group both structurally and mechanically showed an antiadhesion effect in the uterus and a therapeutic effect on the damage with the β-estradiol and melatonin that it contains compared to the Asherman (ASH) group. This double drug-loaded hydrogel can be a promising candidate for preventing Asherman's syndrome due to its versatile properties.

Investigation of the Biosafety of Commercially Available Natural Rubber Latex and Synthetic Polyurethane Condom Materials

To investigate the biological safety of commercially available natural rubber latex and synthetic polyurethane condoms. Natural rubber latex condom brands of A1 and A2 and polyurethane condom brands of B1 and B2 were purchased from large chain pharmacies in Chengdu, with three packages randomly selected for each brand. The study assessed the toxic effects of condom extracts on L-929 mouse fibroblasts according to GB/T standards. Gross observation and histopathological evaluation were conducted to assess the irritation reactions of condoms on the vagina and penis of rabbits (3 rabbits were used for each brand), as well as their sensitization effects on guinea pig skin. Additionally, the impact of continuous perfusion of condom extracts of the vaginas of SD rats for 30 days on their reproductive systems was evaluated, following GB/T standards (5 rats were used for each brand). Extracts from natural rubber latex condom brands A1 and A2, at concentrations of 100% and 50%, exhibited significant cytotoxicity, with optical density (OD) values being significantly lower than those of the blank control group and the polyurethane condom brands B1 and B2 (P<0.01). There was no significant difference in cell morphology and OD values between the extracts of B1 and B2 and the blank control group (P>0.05). Vaginal congestion was found in 3 rabbits from A1 group and 1 rabbit from the A2 group, while no obvious congestion was noted in rabbits from the B1 and the B2 groups. Histopathological examination showed scattered inflammatory cell infiltration in the vaginal tissue of 3 rabbits from the A1 group and 2 rabbits from the A2 group, and slight congestion in the blood vessels of the lamina propria. No obvious pathological changes were observed in the vaginal tissue of polyurethane brand rabbits. Two rabbits from the A1 group and 1 rabbit from the A2 group showed transient and mild erythema on the penis during the experiment. Histopathological examination showed that 1 rabbit from A1 group had small foci of pericapillary lymphocytes in the dermis of the penis, while no significant pathological changes were observed in the penile tissue of A2, B1, and B2 groups. After 30 days of continuous vaginal perfusion with condom extract, 3 rats in A1 group and 2 rats in the A2 group had uterine congestion, with the degree of congestion being lower in the A2 group. No significant congestion or pathological changes were observed in the vaginal and penile tissues of rabbits, or in the uterine tissues of rats from the polyurethane groups. None of the 4 groups of guinea pigs showed significant skin allergic reactions to the condom extracts. Significant differences in biosafety exist among condoms of various materials and brands. To ensure product safety, it is crucial to strengthen quality control and regulatory oversight after condoms become commercially available.

Transcriptomic and metabolomic data of goat ovarian and uterine tissues during sexual maturation

The ovaries and uterus are crucial reproductive organs in mammals, and their coordinated development ensures the normal development of sexual maturity and reproductive capacity. This study aimed to comprehensively capture the different physiological stages of the goat’s sexual maturation by selecting four specific time points. We collected samples of ovarian and uterine tissues from five female Jining Gray goats at each time point: after birth (D1), 2-month-old (M2), 4-month-old (M4), and 6-month-old (M6). By combining transcriptomic sequencing of 40 samples (including rRNA-depleted RNA-seq libraries with 3607.8 million reads and miRNA-seq libraries with 444.0 million reads) and metabolomics analysis, we investigated the transcriptomic mechanisms involved in reproductive regulation in the ovary and uterus during sexual maturation, as well as the changes in metabolites and their functional potential. Additionally, we analyzed blood hormone indices and uterine tissue sections to examine temporal changes. These datasets will provide a valuable reference for the reproductive regulation of the ovary and uterus, as well as the regulation of metabolites during sexual maturation in goats.

Cardioprotective and antispasmodic effects of Humulus lupulus (Hops) in an oral subacute treatment in rats

Hops (Humulus lupulus) was used to treat menopausal symptoms because it contains phytoestrogens. Since these compounds prevent cardiac ischemic diseases, it was evaluated whether oral administration of an ethanol extract (Hops-T) to rats during a week could reduce cardiac post-ischemic dysfunction. Moreover, its effects on visceral peristaltism and the mechanisms of action were studied. Rat isolated hearts were perfused inside a calorimeter, left intraventricular pressure and calorimetrical signals were recorded during ischemia/reperfusion. The NO-synthases and mKATP channels roles were evaluated by respectively blocking them with L-NAME and 5-HD before ischemia. After the experiment, western-blots for cellular pathways (p-AKT, p-PKCϵ, GSK3b and BCL-2) were done. In isolated intestinal, bladder and uterine rat tissues, contractile carbachol concentration-response curves (CCh-CRCs) were performed. The HPLC-DAD mainly showed prenylated flavonoids, beta acids and isoflavones. Hops-T increased contractile and energetic recoveries more significantly in the male rat hearts compared to females, due to beneficial NO production and mitochondrial mKATP channels activation. However, the p-AKT, p-PKCϵ, GSK3b or BCL-2 expressions were not affected after reperfusion. Hops-T inhibited CCh-CRCs in intestinal, bladder and uterine tissues through a non-competitive mechanism. Additionally, it showed a non-competitive antagonism of the Ca2+-CRCs in bladder tissues, similar to the effects observed with verapamil. In conclusion, oral Hops-T prevented cardiac mitochondrial and contractile ischemic dysfunction, and it was a good visceral antispasmodic medicine by interfering with Ca2+ influx. This is a significant finding since it pre-clinically supports the use of hops extract for prevention of visceral spasms and cardiac diseases associated with coronary insufficiency.

P-788 Analysis of tissue regeneration after transplantation of bioengineered uterine grafts in a sheep model of uterine injury

Abstract Study question Can we regenerate a damaged uterus using decellularized (further repopulated with stem cells or not) uterine scaffolds in a sheep model? Summary answer Bioengineered uterine grafts can promote the regeneration of a damaged uterus, but this process is highly dependent on the basal immune status of the recipient. What is known already Uterus bioengineering aims to develop scaffolds to repair defective uterine tissue that may cause infertility. The remaining extracellular matrix after uterus decellularization has frequently been used as a scaffold material for preclinical treatment applications. So far, multiple in vivo studies using rodents have shown successful restoration of uterine tissue and even fertility when small grafts were used. Furthermore, this pro-regenerative effect is enhanced when repopulating the scaffolds with mesenchymal stem cells (MSCs). However, studies using larger animal models and larger scaffolds are still missing. Therefore, we investigated the therapeutic potential of transplanting these scaffolds in the sheep model. Study design, size, duration All experiments were performed in university facilities, including the sheep animal work. Uterine scaffold donor sheep (from a local abattoir) and recipient sheep (n = 10) were of the same mixed sheep breed (Swedish fin-ull and Dutch texel mixed breed) and age (8-12 months old). Recipient sheep were transplanted with uterine decellularized (either recellularized or not) grafts that were maintained for 6 weeks. Participants/materials, setting, methods We implemented our previously established decellularization protocol (2% sodium deoxycholate) for sheep uterine scaffold production, generating 3x2cm grafts; half of these were recellularized with sheep MSCs. Decellularized (DC; n = 7) and recellularized (RC; n = 8) grafts were transplanted as replacements for native uterine tissue. After six weeks, histological, immunohistochemistry (CD31, CD45), and gene expression (progesterone/estrogen receptors, HOXA10, VEGF, vWF, TNFA, IFNG) analysis were conducted. Simultaneously, we used cell sorting to monitor the systemic immune response over time. Main results and the role of chance Angiogenesis was increased in the RC group as more new blood vessels (CD31+ cells) were evidenced (P=0.0120). VEGF and vWF gene expression also indicated the presence of blood vessels in the grafts but without differences among the groups. Regarding the immune response, CD45 staining revealed leukocyte recruitment into both transplanted grafts, with an increased significance in the DC group (P=0.0071) compared to the RC (P=0.0181). Furthermore, the pro-inflammatory genes TNFA and IFNG expression remained invariable compared to the native tissue. Finally, hormone receptor gene levels were comparable to those in the native tissue, with HOXA10 presenting higher values in the RC group compared to the DC (P=0.0548). So, the RC group showed signs of better uterine regeneration, probably due to the MSC’s anti-inflammatory action. Still, differences were subtle between groups. So, we blindly analyzed the histology of all grafts and detected substantially but also poorly regenerated grafts in both DC and RC groups. The analysis of the systemic immune response likely answers this, as when segregating individuals in substantially and poorly regenerated grafts, there was a significant difference in the percentage of activated CD4+ T-cells, being higher (P&amp;lt;0.0001) in those with poorly regenerated grafts already at day 0. Limitations, reasons for caution Due to the lack of available sheep antibodies, we did not study specific subtypes of leukocytes by immunohistochemistry as we did for the cell sorting analysis. Also, despite experienced group members performing the blinded histological analysis of the grafts to detect substantially/poorly regenerated grafts, this method requires refinement. Wider implications of the findings Basal immune system status should be considered before transplanting uterine grafts, as inflammation seems crucial in tissue regeneration. Trial registration number Not applicable

O-268 Re-considering the endometrial microbiome: a comparison of DNA- and RNA-based sequencing techniques within the same cohort

Abstract Study question Does the analysis of the endometrial microbiome provide the same information when using DNA or RNA sequencing-based techniques? Summary answer DNA vs. RNA-based microbiome analysis techniques demonstrate significant microbial compositional differences, meaning that the previous endometrial 16S rRNA gene-based microbiome analysis results might be overestimated. What is known already In recent years, high-throughput sequencing technologies have revolutionised the field of reproductive microbiome research. Our understanding of the composition of endometrial microbiome predominantly relies on DNA-based 16S rRNA gene profiling method. Regardless of its effectiveness and low cost, its underestimation of microbial diversity, abundance, or functionality and lack of detection precision on species level have been highlighted. The meta-transcriptome analysis, RNA-based method, overcomes these limitations and identifies functional genes that are actively expressed by the alive microbes. This study aims to elucidate for the first time the endometrial microbiome using a dual approach of 16S rRNA gene-method and meta-transcriptomic analyses. Study design, size, duration In total forty-seven women with infertility at ages 27-42 years old were enrolled in the present study. Paired endometrial samples, endometrial brushing and Pipelle biopsy were collected from each participant at the mid-secretory menstrual phase (LH + 7/9). Participants/materials, setting, methods Endometrial samples were collected using Tao Brush for 16S rRNA analysis (DNA analysis) and Pipelle endometrial curette for meta-transcriptomic analysis (total RNA analysis). QIAamp UCP Pathogen Mini Kit was used for DNA extraction and 16S rRNA gene V3-V4 regions were sequenced on MiSeq. RNA was extracted using miRNeasy Micro kit, with rRNA removal by RiboZero kit and Libraries were generated using Stranded Total RNA Prep. Taxonomy was assigned by using Kraken2 (v2.2.1), Bracken (v2.7). Main results and the role of chance To the best of our knowledge, this is the first study to compare endometrial microbial identification using both metagenomics (16S rRNA sequencing) and meta-transcriptomics (meta-RNA sequencing) within the same cohort. Analysis of the composition of the microorganisms by bacterial 16S rRNA gene sequencing revealed that the most abundant bacterial genus in the endometrium was Lactobacillus. In the meta-transcriptome analysis, no microorganisms belonging to the Lactobacillus genus were detected in high abundance. This indicates that the relative abundance of this genus at the DNA level does not necessarily imply microbial activity or the presence of viable Lactobacillus in the endometrium. The meta-transcriptomics analysis detected microbial taxa such as Staphylococcus, Bacillus, Streptococcus, and Burkholderia as the most dominant. The results suggest that DNA-based detection of microorganisms in the endometrium may be overestimated due to the presence of naked DNA sequences or microorganisms from the vagina and cervix. Furthermore, RNA-based analysis demonstrates a population of active microorganisms different from the image shown by DNA analysis. Altogether, our study results indicate that the uterine microenvironment, as previously identified through DNA sequencing, may partly reflect the vaginocervical microenvironment, with the uterine tissue being a low biomass site not dominated by lactobacilli. Limitations, reasons for caution The endometrial samples were paired; however, DNA was analysed in the endometrial brushing sample while RNA in the tissue biopsy, which might result in some differences in microbial composition. Wider implications of the findings Contrary to the general belief of the Lactobacillus dominance in the human endometrium, our study suggests that the endometrial microenvironment may be harbouring DNA fragments and/or cells of lactobacilli originating from the lower reproductive tract. Our study results call out to re-consider/re-analyse the endometrial microbiome in health and disease. Trial registration number not applicable

P-209 Development and characterization of human uterine myometrium in prenatal stage: insights from a single-cell atlas and three-dimensional imaging

Abstract Study question What’s the timepoint of human uterine myometrial formation and how dose it develop in the prenatal stage. Summary answer This study identified that the myometrium layer initially forms at postconceptional week (PCW) 11, ESR1+, IGFBP5, POSTIN may play pivotal role in myometrial development. What is known already Using immunohistochemistry staining, previous studies observed a faint expression of α-SMA in the outer mesenchyme at approximately 8-9 weeks of gestation and showed the early development of muscular layer in the uterine wall, and the expression of α-SMA becomes more prominent in the uterine corpus and cervix by 18 weeks, suggesting the maturation of smooth muscle cells may commence from 18 weeks of gestation. ESR-1 gene encodes estrogen receptor alpha, which plays a pivotal role in menstruation, pregnancy, and parturition. In adult female myometrium, estrogen performs functions in proliferation before term and contractile at term. Study design, size, duration This is an observational study. To investigate when exactly the smooth-muscle tissue appears during uterine development, we have collected 14 uterine tissue samples from 14 aborted fetuses across seven gestational time points from PCW 8 to PCW 15. Participants/materials, setting, methods Uterus from aborted fetuses from PCW8- PCW15 cover the fusion of Mullerian tube and development of Myometrium. The 10X genomics scRNA-seq and immune labeling were performed. In addition, we have integrated scRNA-seq data from adult uterine tissue for comparison. Main results and the role of chance We provide a single cell atlas of the human developing uterus, and we observed that smooth-muscle cells (SMCs), identified by ACTA2, TAGLN, MYH11, exhibited a significant increase in abundance starting from PCW 11, but weaker signals of SMCs were also detected at earlier stages. Using the iDISCO technique with continuous α-SMA labeling to examine the localization of α-SMA in the uterus between PCW10- PCW12, we observed the entire process of myometrial development in 3-D level. We utilized an oblique section of 3-D reconstruction uterus and extended it to 150 μm. Subsequently, apparent signals became visible in the middle of the uterine wall and the uterine fundus. Collectively, these findings suggest that the anatomical myometrium layer initially forms at PCW 11. We used the CytoTRACE to assess the capacity of differentiation of adult and fetal SMCs, SMCs come from fetus appeal a significant grander potential. Specifically, fetal uterine SMCs showed higher expression of ESR1, IGFBP5, and POSTN, while adult uterine SMCs exhibited higher expression of PDGFRB and IGFBP7. These results suggested ESR1+ SMCs could be the precursor of SMCs and SMCs in fetal uterus could be in a primary state. Limitations, reasons for caution While our study did not capture later stages of development or postnatal changes. Future studies are needed to understand the full spectrum of myometrial development. It is also important to note that the findings in this study are specific to human and may not directly translate to other species. Wider implications of the findings Our study provides a basis for studying abnormal myometrial development and its association with various reproductive disorders, such as the abnormal uterine development in female fetus with Turner syndrome. Potential biomarkers or therapeutic targets for uterine malformations and abnormal uterine contractility may also of research interest. Trial registration number not applicable

Puerarin inhibits Staphylococcus aureus-induced endometritis through attenuating inflammation and ferroptosis via regulating the P2X7/NLRP3 signalling pathway.

Endometritis is one of the important causes of infertility. Puerarin (PU) can inhibit oxidative stress and reduce inflammation; however, it is unclear whether PU has a protective effect on the endometritis. In our study, we used Staphylococcus aureus to induce mouse endometritis. The PU group (100 mg/kg PU) and the S. aureus + PU group received daily intraperitoneal injection of PU (25, 50 or 100 mg/kg PU). The results showed that S. aureus significantly increased the levels of MPO, TNF-α, IL-1β and IL-6 in uterine tissue, and increased the expression of p-p65 and p-IκBα proteins in uterine tissue to induce endometritis in mice (p < 0.05). Furthermore, it has been found that S. aureus promotes the occurrence of ferroptosis by reducing GSH and ATP content, increasing MDA and iron content and reducing GPX4 and SLC7A11 protein expression levels (p < 0.05). S. aureus significantly increase the expression of NLRP3, ASC, caspase-1 and P2X7 proteins in uterine tissue (p < 0.05). However, PU obviously reduced the inflammatory response and reversed the changes of ferroptosis and the expression of P2X7 receptor/NLRP3 pathway associated proteins of the uterus induced by S. aureus (p < 0.05). Taken together, these findings emphasize the protective effect of PU on endometritis by regulating the P2X7 receptor/NLRP3 signalling pathway and inhibiting ferroptosis.

Analysis of Expression of the ANG1, CaSR and FAK Proteins in Uterine Fibroids.

Understanding the molecular factors involved in the development of uterine myomas may result in the use of pharmacological drugs instead of aggressive surgical treatment. ANG1, CaSR, and FAK were examined in myoma and peripheral tissue samples taken from women after myoma surgery and in normal uterine muscle tissue samples taken from the control group. Tests were performed using tissue microarray immunohistochemistry. No statistically significant differences in ANG1 expression between the tissue of the myoma, the periphery, and the normal uterine muscle tissue of the control group were recorded. The CaSR value was reduced in the myoma and peripheral tissue and normal in the group of women without myomas. FAK expression was also lower in the myoma and periphery compared to the healthy uterine myometrium. Calcium supplementation could have an effect on stopping the growth of myomas.