Non-pregnant Endometrium Research Articles

NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium areprecursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

High Expression and Significance of Siglec10/CD24 in Unexplained Missed Abortion

ABSTRACT Objective To investigate the expression of Siglec10 and CD24 in normal early pregnancy and missed abortion, and their significance in the maternal-fetal interface. Methods For our research, we employed Q-PCR and WB techniques to evaluate the traits and expression of Siglec10 and CD24 in the nonpregnant endometrium, as well as in the villus and decidua of women in their 6–10 weeks of normal early pregnancy and those who experienced missed abortion. Additionally, we utilized ELISA to determine the levels of Siglec10 and CD24 in the peripheral blood of pregnancy, missed abortion, and non-pregnant individuals. T-test and ANOVA were used to compare groups. Results 1. Villous tissues in early pregnancy showed high expression of Siglec10 and CD24, with a significant increase in expression in the missed abortion group (P < 0.01). 2. Nonpregnant endometrial tissue showed low expression of Siglec10 and CD24, while early pregnancy decidua showed high expression, with even higher expression in missed abortion (all P < 0.05). 3. Serum levels of Siglec10 and CD24 in normal early pregnancy were significantly higher than non-pregnancy (P < 0.01). However, the missed abortion group showed significantly higher levels than normal pregnancy (P < 0.01). 4. CD24 expression in serum of missed abortion increases with Siglec10 expression, indicating a significant positive correlation (r = 0.500, P < 0.01). Conclusion Siglec10 and CD24 expression in villus, decidua, and peripheral blood are up-regulated in unexplained missed abortions than those of women with normal pregnancies. This suggests that the levels of serum Siglec10 and CD24 can be used as an effective predictor of missed abortion.

P-361 Is HIF-2α involved in pre-menstrual conditioning of the endometrium to ensure optimal repair and prevent heavy menstrual bleeding?

Abstract Study question Does HIF-2α play a key role in pre-menstrual conditioning of the non-pregnant endometrium to optimise menstrual breakdown/repair and prevent heavy menstrual bleeding (HMB)? Summary answer HIF-2 was higher in secretory endometrium of women with HMB versus controls, but manipulation of HIF-2α in the mouse model did not impact endometrial breakdown/repair. What is known already Hypoxia inducible factor (HIF) is the master regulator of the cellular response to hypoxia and has two common alpha isoforms (HIF-1α/HIF-2α) with overlapping but distinct target genes. We revealed that menstrual endometrial HIF-1α is necessary for normal menstrual endometrial repair. We detected HIF-2α in human endometrium exclusively during the secretory phase. The role of HIF-2α in the non-pregnant endometrium remains undetermined but was important for successful uterine implantation and lung vascular remodelling in mice. We hypothesised that endometrial HIF-2 is required for optimising the endometrial vasculature pre-menstrually to ensure appropriate vasoconstriction and endometrial repair during menstruation. Study design, size, duration HIF-2 was examined in human endometrial tissue and a mouse model of simulated menstruation. Hif2α +/- mice provided genetic reduction of HIF-2 during simulated menses versus wild-type littermates. HIF-2α was also pharmacologically inhibited during the secretory phase (PT2385 vs vehicle controls). To increase HIF-2, Hif1α +/- mice were treated with a HIF stabiliser (DMOG) during the secretory phase to increase HIF-2α followed by decreased HIF-1α at menses –mimicking human endometrial findings in women with HMB. Participants/materials, setting, methods Human secretory endometrium was collected (ethically approved and consented) from women with objectively measured HMB (n = 5) and controls (n = 4) and HIF-2 detected by Western blot. Mouse uterus was collected at the time of progesterone withdrawal (T0, decidualisation) and 8h (T8, endometrial breakdown) and 24h (T24, endometrial repair) after progesterone withdrawal. Histological endometrial repair and menstrual blood loss (MBL) were quantified. Decidualisation markers were examined by RT-qPCR. Endometrial vessel maturity and hypoxia were assessed using immunohistochemistry/immunofluorescence. Main results and the role of chance Human secretory endometrial HIF-2α protein levels were higher in those with HMB compared to controls (P &amp;lt; 0.05). Genetic reduction of HIF-2α in our mouse model of menses (Hif2α +/-) revealed no significant differences in endometrial breakdown (T8) or repair grade (T24) and no difference in MBL at T8 versus controls. There were no significant differences in expression of decidualisation markers (Prlr/Prl3c1) and vessel maturity at T0 (CD31/α-SMA immunofluorescence staining), or the presence of hypoxia at T8 (pimonidazole immunohistochemistry staining). Pharmacological inhibition of HIF-2 at the time of decidualisation also showed no significant difference in endometrial repair at T24 versus controls. Increasing pre-menstrual HIF-2 via pharmacological stabilisation of HIF-2α in Hif1α+/- mice similarly did not significantly affect endometrial breakdown (T8) or repair (T24) or MBL at T8 versus vehicle treated controls, in contrast to our findings in women with HMB. Limitations, reasons for caution Histological scoring of endometrial breakdown/repair in the menstruation mouse model may not be sensitive enough to detect subtle endometrial changes with increased HIF-2α. As HIF-1α delays endometrial repair, an additional timepoint 36h after progesterone withdrawal may better capture any delay in endometrial repair as a result of increased secretory HIF-2. Wider implications of the findings HIF-2α may play a greater role in implantation than pre-menstrual conditioning of the endometrium to optimise menstrual breakdown/repair and limit menstrual blood loss. Although endometrial breakdown was not significantly affected by HIF-2, the impact on repair warrants further investigation to fully delineate the role of HIF-2 in the non-pregnant endometrium. Trial registration number Not applicable

Global gene expression in equine endometrium around the time of maternal recognition of pregnancy

The conceptus-maternal communication during the preimplantation period, also known as maternal recognition of pregnancy (MRP), is crucial for the establishment and maintenance of pregnancy. This communication affects the endometrial transcriptome and proteome with subsequent reduction of endometrial pulsatile prostaglandin F2α secretion and the consequent extension of the corpus luteum's lifespan during early pregnancy in the mare. Also, this communication results in the preparation of the uterine environment for implantation. Any alteration of these processes will result in early embryonic death. Therefore, a better understanding of the normal endometrial transcriptomic changes around the time of MRP might be the key to understanding and preventing early embryonic loss. This study aimed to evaluate the endometrial transcriptome from Day 14 (Day 0 = ovulation) of pregnant and non-pregnant (i.e. diestrus) mares. RNA was isolated from pregnant (n=5) and non-pregnant (n=4) endometrial tissue samples. RNA-Seq was performed using Illumina NovaSeq6000 and differentially expressed genes (DEGs) were evaluated using DESeq.2 based upon an FDR<0.05. Our study identified 1193 DEGs (570 upregulated DEGs and 623 downregulated DEGs) in the pregnant endometrium as compared to the non-pregnant endometrium. In the current study, the pregnant endometrium was associated with downregulation (9.6-fold change) of mRNA forprostaglandin-endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase-2 enzyme; COX2), which encodes for the rate-limiting enzyme in prostaglandin production. Gene ontology analysis of DEGs revealed that these genes were associated with relevant biological processes such as immune response (TNFAIP1, TNF, CXCR2, TNFSF15, IL15, TGFBR3, IL1B, TNFSF9, TLR8), regulation of trophoblast cell proliferation and migration (BOK, LIF, RBM46, TIMP1, CALR, GJA1), embryo growth and development (PTCH1, PTCH2R, DH10, GLI3, SHOX2, ZDBF2, EVX1, GREM2, KLF2, TWIST1), cell adhesion (COL28A1, EGFL6, ITGA3, FN1, NEDD9, LAMC2, RGMB, THBS4, RHOB, SELP, CHL1, CDHR1, MXRA8, FOLR3, NECTIN3), embryo implantation (MMP9 and LIF), placenta development (ADAM19, MAPK14, BIRC3), amino acids transportation (SLC1A1, SLC25A29, SLC36A2, SLC38A2, SLC7A5), steroid biosynthesis and metabolism (SRD5A1, HSD11B2, HSD17B7, CYP20A1), and progesterone receptors (PAQR5, and PGRMC1). Several of the DEGs identified in the current study are known to play a role in controlling endometrial receptivity to the conceptus in species other than the equine. These findings improve our understanding of the molecular changes taking place in equine endometrium around the time of MRP which might help us to understand and forestall early embryonic loss in the future.

The Correlation Between Serum Pregnancy Associated Plasma Protein A (PAPP-A) with the Endometrial Thickness and Resistance Index and their Effect on Pregnancy Outcome for Infertile Women Undergo ICSI Cycle

Pregnancy-associated plasma protein-A (PAPP-A) is approved to be not pregnancy specific; it could be localized in nonpregnant women endometrium. Thus, it was of interest to see if PAPP-A can be capable of preparing the uterine endometrium for implantation. However, the relationship between serum PAPP-A and endometrium receptivity remains unclear. To evaluate if there is any correlation between the level of serum pregnancy associated plasma protein-A (PAPP-A) with the endometrial thickness and resistance index and evaluate their effects of pregnancy outcome for patient's candidate for intracytoplasmic sperm injection (ICSI). This is a prospective study that was submitted at the infertility center of High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain University, Baghdad, Iraq, from November 2020 till March 2021. Forty-five infertile females participated in the study, younger than 40 years old submitted to antagonist protocol. For assessment of endometrium receptivity, transvaginal ultrasound was done at days of oocyte pickup and embryo transfer to measure the endometrial thickness and the resistance index. Serum PAPP-A was measured at the day of oocyte pickup and at day of embryo transfer. Two weeks later, a pregnancy test was done for all patients and according to the result the patients divided into two groups pregnant (n=20) and non-pregnant (n=25). There was insignificant positive correlation between serum PAPP-A level with endometrial thickness (ET) and insignificant negative correlation with (RI) at day of oocyte pick up and day embryo transfer. High serum Pregnancy associated plasma protein-A (PAPP-A) may be associated with an increase in endometrial thickness and low level of resistance index that both may promote the possibility of implantation.

Revisiting the Transcriptome Landscape of Pig Embryo Implantation Site at Single-Cell Resolution.

Litter size is one of the most economically important traits in commercial pig farming. It has been estimated that approximately 30% of porcine embryos are lost during the peri-implantation period. Despite rapid advances over recent years, the molecular mechanism underlying embryo implantation in pigs remains poorly understood. In this study, the conceptus together with a small amount of its surrounding endometrial tissues at the implantation site was collected and subjected to single-cell RNA-seq using the 10x platform. Because embryo and maternal endometrium were genetically different, we successfully dissected embryonic cells from maternal endometrial cells in the data according to single nucleotide polymorphism information captured by single-cell RNA-seq. Undoubtedly, the interaction between trophoblast cells and uterine epithelial cells represents the key mechanism of embryo implantation. Using the CellChat tool, we revealed cell-cell communications between these 2 cell types in terms of secreted signaling, ECM-receptor interaction and cell-cell contact. Additionally, by analyzing the non-pregnant endometrium as control, we were able to identify global gene expression changes associated with embryo implantation in each cell type. Our data provide a valuable resource for deciphering the molecular mechanism of embryo implantation in pigs.

Cell and Tissue Nanomechanics: From Early Development to Carcinogenesis.

Cell and tissue nanomechanics, being inspired by progress in high-resolution physical mapping, has recently burst into biomedical research, discovering not only new characteristics of normal and diseased tissues, but also unveiling previously unknown mechanisms of pathological processes. Some parallels can be drawn between early development and carcinogenesis. Early embryogenesis, up to the blastocyst stage, requires a soft microenvironment and internal mechanical signals induced by the contractility of the cortical actomyosin cytoskeleton, stimulating quick cell divisions. During further development from the blastocyst implantation to placenta formation, decidua stiffness is increased ten-fold when compared to non-pregnant endometrium. Organogenesis is mediated by mechanosignaling inspired by intercellular junction formation with the involvement of mechanotransduction from the extracellular matrix (ECM). Carcinogenesis dramatically changes the mechanical properties of cells and their microenvironment, generally reproducing the structural properties and molecular organization of embryonic tissues, but with a higher stiffness of the ECM and higher cellular softness and fluidity. These changes are associated with the complete rearrangement of the entire tissue skeleton involving the ECM, cytoskeleton, and the nuclear scaffold, all integrated with each other in a joint network. The important changes occur in the cancer stem-cell niche responsible for tumor promotion and metastatic growth. We expect that the promising concept based on the natural selection of cancer cells fixing the most invasive phenotypes and genotypes by reciprocal regulation through ECM-mediated nanomechanical feedback loop can be exploited to create new therapeutic strategies for cancer treatment.

235 Uterine Function During Maternal Recognition of Pregnancy Differs Due to Size of the Ovarian Reserve in Beef Heifers

Abstract The relationship of ovarian reserve parameters to fertility in mammalian females continues to be debated. We demonstrated improved uterine function, creating an environment that is more supportive of early embryonic development in beef heifers with increased numbers of follicles. In the present study we hypothesized that timing of luteolysis differs between heifers with increased compared to heifers with diminished numbers of follicles. Angus heifers (n = 20/ group) were classified as low (14.7 ± 1.1) or high (32 ± 1.1) antral follicle number based on ovarian ultrasonography, and artificially inseminated (d0) following a Select Synch protocol. At d16 after insemination, heifers were slaughtered, and reproductive tracts collected. Tracts were flushed with 20 mL of physiological saline to determine pregnancy status by presence of a conceptus and endometrial samples from the horn ipsilateral to the CL were frozen for determination of transcript abundance. Real-time RT-PCR was performed to determine relative transcript abundance of oxytocin receptor (OXTR) and interferon-stimulated gene-15 (ISG15) with glyceraldehyde-3-phospate dehydrogenase as the endogenous reference gene. Transcript abundance was analyzed using the MIXED procedure of SAS with follicle group, pregnancy status and the interaction as fixed effects. Transcript abundance of ISG15 was greater in pregnant endometrium than non-pregnant endometrium (2.75 ± 0.25 vs 0.16 ± 0.22-fold; P &amp;lt; 0.0001) with no influence of follicle group or the interaction. The interaction of follicle group and pregnancy status influenced OXTR transcript abundance (P = 0.06). Luteolytic mechanisms were activated in non-pregnant heifers with diminished numbers of follicles (1.46 ± 0.25-fold) and were not activated in pregnant heifers (0.06 ± 0.22-fold) or heifers with increased numbers of follicles that were not pregnant (0.32 ± 0.30-fold). These data indicate a wider window of recognition of pregnancy in beef heifers with increased numbers of ovarian follicles. USDA is an equal opportunity provider and employer.

Sex Hormones and Aging Modulate Interferon Lambda 1 Production and Signaling by Human Uterine Epithelial Cells and Fibroblasts.

Estradiol (E2) and progesterone (P) have potent effects on immune function in the human uterine endometrium which is essential for creating an environment conducive for successful reproduction. Type III/lambda (λ) interferons (IFN) are implicated in immune defense of the placenta against viral pathogens, which occurs against the backdrop of high E2 and P levels. However, the effect of E2 and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown. We generated purified in vitro cultures of human uterine epithelial cells and stromal fibroblast cells recovered from hysterectomy specimens. Poly (I:C), a viral dsRNA mimic, potently increased secretion of IFNλ1 by both epithelial cells and fibroblasts. The secretion of IFNλ1 by epithelial cells significantly increased with increasing age following poly (I:C) stimulation. Stimulation of either cell type with E2 (5x10-8M) or P (1x10-7M) had no effect on expression or secretion of IFNλ1 either alone or in the presence of poly (I:C). E2 suppressed the IFNλ1-induced upregulation of the antiviral IFN-stimulated genes (ISGs) MxA, OAS2 and ISG15 in epithelial cells, but not fibroblasts. Estrogen receptor alpha (ERα) blockade using Raloxifene indicated that E2 mediated its inhibitory effects on ISG expression via ERα. In contrast to E2, P potentiated the upregulation of ISG15 in response to IFNλ1 but had no effect on MxA and OAS2 in epithelial cells. Our results demonstrate that the effects of E2 and P on IFNλ1-induced ISGs are cell-type specific. E2-mediated suppression, and selective P-mediated stimulation, of IFNλ1-induced ISG expression in uterine epithelial cells suggest that the effects of IFNλ1 varies with menstrual cycle stage, pregnancy, and menopausal status. The suppressive effect of E2 could be a potential mechanism by which ascending pathogens from the lower reproductive tract can infect the pregnant and non-pregnant endometrium.

Differential Expression Pattern of Goat Uterine Fluids Extracellular Vesicles miRNAs during Peri-Implantation.

Early pregnancy failure occurs when a mature embryo attaches to an unreceptive endometrium. During the formation of a receptive endometrium, extracellular vesicles (EVs) of the uterine fluids (UFs) deliver regulatory molecules such as small RNAs to mediate intrauterine communication between the embryo and the endometrium. However, profiling of small RNAs in goat UFs’ EVs during pregnancy recognition (day 16) has not been carried out. In this study, EVs were isolated from UFs on day 16 of the estrous cycle or gestation. They were isolated by Optiprep™ Density G radient (ODG) and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Immunostaining demonstrated that CD63 was present both in the endometrial epithelium and glandular epithelium, and stain intensity was greater in the pregnant endometrium compared to the non-pregnant endometrium. Small RNA sequencing revealed that UFs’ EVs contained numerous sRNA families and a total of 106 differentially expressed miRNAs (DEMs). Additionally, 1867 target genes of the DEMs were obtained, and miRNA–mRNA interaction networks were constructed. GO and KEGG analysis showed that miRNAs were significantly associated with the formation of a receptive endometrium and embryo implantation. In addition, the fluorescence in situ hybridization assay (FISH) showed that chi-miR-451-5p was mainly expressed in stromal cells of the endometrium and a higher level was detected in the endometrial luminal epithelium in pregnant states. Moreover, the dual-luciferase reporter assay showed that chi-miR-451-5p directly binds to PSMB8 and may play an important role in the formation of a receptive endometrium and embryo implantation. In conclusion, these results reveal that UFs’ EVs contain various small RNAs that may be vital in the formation of a receptive endometrium and embryo implantation.

How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.

CircRNA landscape of non-pregnant endometrium during the estrus cycle in dairy goats

Endometrial development is a complicated process involving numerous regulatory factors. Circular RNAs (circRNAs) have been known as a member of the naturally occurring non-coding RNA family, and are reportedly crucial for a variety of physiological processes. This study investigated the circRNA landscape of non-pregnant endometrium of dairy goats during estrus. Non-pregnant endometrial samples of goats at estrus day 5 (Ed5) and estrus day 15 (Ed15) were used to methodically analyze the circRNA landscape using strand-specific Ribo-Zero RNA-Seq. A total of 2 331 differentially expressed (P<0.05) circRNAs (DEciRs) between Ed5 and Ed15 were discovered in the goat endometrium. It was found that Nipped-B-like (NIPBL) and calcium responsive transcription factor (CARF) may participate in the development of the endometrium by decreasing (P<0.05) the levels of their circRNA-transcript forms. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEciR host genes (hgDEciRs) revealed that tight junctions and GTPases may be involved in endometrial development during the estrus cycle. A total of 2 331 DEciRs were discovered in the endometrium at Ed5 and Ed15. Based on GO and KEGG enrichment analyses, it could be inferred that tight junctions and GTPases are likely to play an important role in the development of goat endometrium during the estrus cycle. This circRNA study greatly enhances our knowledge of global trends in the development of non-pregnant endometrium during the estrus cycle in goats; these results help us to better understand the molecular regulation of endometrial development in dairy goats.

Investigation of infertility using endometrial organoids.

Infertility is a common problem in modern societies with significant socio-psychological implications for women. Therapeutic interventions are often needed which, depending on the cause, can either be medical treatment, surgical procedures or assisted reproductive technology (ART). However, the treatment of infertility is not always successful due to our limited understanding of the preparation of the lining of the uterus, the endometrium, for pregnancy. The endometrium is of central importance for successful reproduction as it is the site of placental implantation providing the interface between the mother and her baby. Due to the dynamic, structural and functional changes the endometrium undergoes throughout the menstrual cycle, it is challenging to study. A major advancement is the establishment of 3D organoid models of the human endometrium to study this dynamic tissue in health and disease. In this review, we describe the changes that the human endometrium undergoes through the different phases of the menstrual cycle in preparation for pregnancy. We discuss defects in the processes of endometrial repair, decidualization and acquisition of receptivity that are associated with infertility. Organoids could be utilized to investigate the underlying cellular and molecular mechanisms occurring in non-pregnant endometrium and early pregnancy. These studies may lead to therapeutic applications that could transform the treatment of reproductive failure.

Analysis and Screening of Reproductive Long Non-coding RNAs Through Genome-Wide Analyses of Goat Endometrium During the Pre-attachment Phase.

Reproduction in goat is highly impeded by implantation failure. Of concern, the underlying mechanism leading to embryo implantation remains unclear. In this study, deep sequencing was employed through strand-specific Ribo-Zero RNA-Seq to characterize transcriptome changes in the endometrium during the maternal recognition of pregnancy. A total of 996 differential transcripts (115 lncRNAs and 881 mRNAs) existing between the pregnant and non-pregnant endometrium were revealed through bioinformatics analysis. The screening was performed on lncRNAs (XR_001918173.1, LNC_002760, and LNC_000599) and LNC_009053, to determine their potential role in regulating the synthesis of retinol and endometrium remolding through the proteasome pathway, respectively. The hypothesis of whether certain lncRNAs, namely, LNC_007223, LNC_005256, and LNC_010092 could play important roles in embryo implantation was tested. These novel findings are of paramount relevance to further elucidate the molecular mechanisms of embryo implantation and uncover new targets to improve goat reproduction.

Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy losses.

Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). RPL is associated with a compromised decidual vascular phenotype. Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. N/A.

Immune cells in normal pregnancy and gestational trophoblastic diseases

A healthy pregnancy requires the development of maternal–fetal immune tolerance against the semi-allogeneic fetus. The interactions between the trophoblastic cells and the maternal immune cells (p.e., natural killer cells, T cells, macrophages, dendritic cells and B-cells) are important for the development of the maternal–fetal immune tolerance and the placental growth and function. These interactions are mediated by cell to cell contact and secreted molecules such as cytokines, chemokines, angiogenic factors and growth factors. The maternal immune cells are present in normal non-pregnant and pregnant endometrium and there are several lines of evidence based on immunohistochemical and RNA sequencing data that the decidual immune cells and immune-related pathways display alterations in GTD, which may have pathogenetic and clinical significance.The present review focuses on the usefulness of the immunohistochemical analysis which provides multiparametric in situ information regarding the numbers, the immunophenotypes and the immunotopographical distributions of the decidual immune cells in tissue sections from normal pregnancy and GTD. We also discuss the significance of the immunohistochemical information in order to gain insight in the putative mechanisms explaining the alterations of the decidual immune cells in GTD and the potential implications of these alterations in the pathogenesis and the clinical behavior of GTD.

HYPOXIA AND REPRODUCTIVE HEALTH: The presence and role of hypoxia in the endometrium.

The endometrium is a multicellular tissue that is exquisitely responsive to the ovarian hormones. The local mechanisms of endometrial regulation to ensure optimal function are less well characterised. Transient physiological hypoxia has been proposed as a critical regulator of endometrial function. Herein, we review the literature on hypoxia in the non-pregnant endometrium. We discuss the pros and cons of animal models, human laboratory studies and novel in vivo imaging for the study of endometrial hypoxia. These research tools provide mounting evidence of a transient hypoxic episode in the menstrual endometrium and suggest that endometrial hypoxia may be present at the time of implantation. This local hypoxia may modify the inflammatory environment, influence vascular remodelling and modulate endometrial proliferation to optimise endometrial function. Finally, we review current knowledge of the impact of this hypoxia on endometrial pathologies, with a focus on abnormal uterine bleeding. Throughout the manuscript areas for future research are highlighted with the aim of concentrating research efforts to maximise future benefits for women and society.

Endometrial and decidual stromal precursors show a different decidualization capacity.

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context - that is, non-pregnancy vs pregnancy - of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.

Tissue stiffness at the human maternal-fetal interface.

STUDY QUESTIONWhat is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta?SUMMARY ANSWERThe stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta.WHAT IS KNOWN ALREADYMechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal–fetal interface.STUDY DESIGN, SIZE, DURATIONAn observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5).PARTICIPANTS/MATERIALS, SETTING, METHODSAtomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis.MAIN RESULTS AND THE ROLE OF CHANCEWe observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONTissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women.WIDER IMPLICATIONS OF THE FINDINGSThese results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z).

Effects of l-arginine on endometrial estrogen receptor α/β and progesterone receptor expression in nutrient-restricted sheep

This study aimed to determine the effects of l-arginine (L-Arg) supplementation on steroid hormone receptors in non-pregnant ovine endometrium. All experimental ewes were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on estrogen receptor α/β (ERα/β) and progesterone receptor (PGR) expression in the ovine endometrium were assessed. Our results showed that levels of ERβ and PGR expression were significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on ERβ and PGR expression (p < 0.05). Also, expression of endometrial ERα was substantially increased (p < 0.05) by L-Arg supplementation. Furthermore, ERα/β and PGR were mainly detected in the endometrial luminal epithelium and glandular epithelium. Therefore, we isolated and identified endometrial epithelial cells (EECs) from sheep. Different concentrations of L-Arg were added to investigate the effects on ERα/β and PGR in EECs. The expression levels of endothelial nitric oxide synthase, ERβ, and PGR were significantly increased in response to low-concentration (200 μmol) L-Arg supplementation, which subsequently decreased with a high concentration (800 μmol) (p < 0.05). Otherwise, ERα expression was remarkably increased at both L-Arg concentrations in EECs (p < 0.05). Overall, the results indicated that L-Arg performed crucial roles in the regulation of ovine steroid hormone receptor expression in the endometrium. The results of this study provide a theoretical basis and technical means for the normal function of endometrium in response to low nutrient levels.