Probes for INS

Androgens are steroid hormones crucial for sexual differentiation of the brain and reproductive function. In excess, however, androgens may decrease fertility as observed in polycystic ovary syndrome, a common endocrine disorder characterized by oligo/anovulation and/or polycystic ovaries. Hyperandrogenism may also disrupt energy homeostasis, inducing higher central adiposity, insulin resistance, and glucose intolerance, which may exacerbate reproductive dysfunction. Androgens bind to androgen receptors (AR), which are expressed in many reproductive and metabolic tissues, including brain sites that regulate the hypothalamo-pituitary-gonadal axis and energy homeostasis. The neuronal populations impacted by androgen excess, however, have not been defined. We and others have shown that, in mice, AR is highly expressed in leptin receptor (LepRb) neurons, particularly in the arcuate (ARH) and the ventral premammillary nuclei (PMv). Here, we assessed if LepRb neurons, which are critical in the central regulation of energy homeostasis and exert permissive actions on puberty and fertility, have a role in the pathogenesis of female hyperandrogenism. Prenatally androgenized (PNA) mice lacking AR in LepRb cells (LepRbΔAR) show no changes in body mass, body composition, glucose homeostasis, or sexual maturation. They show, however, a remarkable improvement of estrous cycles combined with normalization of ovary morphology compared to PNA controls. Our findings indicate that the prenatal androgenization effects on adult reproductive physiology (i.e., anestrus and anovulation) are mediated by a subpopulation of LepRb neurons directly sensitive to androgens. They also suggest that the effects of hyperandrogenism on sexual maturation and reproductive function in adult females are controlled by distinct neural circuits.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women. However, there is a lack of information regarding mechanisms and effects in humans. Our study aims to identify mechanisms of endocrine disruption using two EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ)1. Human ovarian cortical tissue obtained from Caesarean section patients was exposed to 10-9 M - 10-5 M KTZ and 10-10 M - 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied via histology analysis and liquid-chromatography-mass spectrometry-based steroid quantification. RNA-sequencing was performed on COV434, KGN, and primary ovarian cells that were exposed for 24 h. Significantly lower unilaminar follicle densities were observed in DES 10-10 M group, whereas low KTZ exposure reduced secondary follicle density. KTZ 10-5 M reduced levels of pregnenolone and progesterone. RNA-sequencing revealed that 445 and 233 differentially expressed genes (false discovery rate < 0.1) altogether in DES and KTZ exposed groups. Gene set variation analysis showed that both chemicals modulated pathways that are important for folliculogenesis and steroidogenesis. We selected stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) for further validation. Up-regulation of both genes in response to KTZ was confirmed by qPCR and in situ RNA hybridization. Further validation with immunofluorescence focused on the expression of SCD in growing follicles in exposed ovarian tissue. In conclusion, SCD may serve as a potential novel human-relevant biomarker of EDC exposure and effects on ovaries.

The estrogen receptor beta (ERβ) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERβ, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERβ (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERβ. Herein, we established an immunohistochemical detection protocol for ERβ protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERβ. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERβ. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERβ, is also suitable for reliable detection of murine ERβ. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERβ in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Introduction. During the COVID-19 pandemic, the question regarding an effect of related infection on the body of pregnant women and the fetoplacental complex has emerged, with many aspects of this issue still being unknown. At the moment, it has been proven that in some cases the course of COVID-19 can be accompanied by severe systemic inflammatory reaction leading to hypercoagulable state.Aim: to search for evidence of a direct and/or indirect effect of SARS-CoV-2 infection on human placenta structure.Materials and Methods. Taking into account the goal, this review was compiled according to the type of a narrative review of publications on a topic of interest. A search for English-language publications dated of 01.12.2019 till 01.12.2021 in PubMed/MEDLINE, Cochrane, Web of Science databases was made. The search queries included the following keywords: combinations of «coronavirus» and «infection during pregnancy», «placental structure» and «2019-nCoV», «COVID-19 and pregnancy», «SARSCoV-2 and pregnancy». In the process of writing the article, in order to improve the reader's understanding of the essence of debated issue, there was a need to discuss some of the results with literary sources published earlier 2019 that were not directly related to the topic of the new coronavirus infection (there are 6 such sources). We analyzed full-text publications, both reports on original research and meta-analyses on relevant topics. In total, 351 full-text publications met the query criteria, of which 54 were selected as meeting the objectives of the study. The select reports were discussed by the co-authors, duplicates were excluded and 34 of them were included in this review. In those that were excluded from the review, information about the clinical course of pregnancy and its outcome during novel coronavirus infection prevailed, or isolated cases of studying insignificant placental structural changes were discussed. Studies with a small number of observations were selected only in the case of the uniqueness of the published data, the absence of scientific papers where similar studies would have been conducted in larger sample.Results. Pregnancy complicated by COVID-19 may be accompanied by placental structural changes, which represent both a manifestation of compensatory-adaptive reactions and a consequence of the damaging effect to the placenta due to infectious process. In case of late (in the III trimester) disease in pregnant woman with mild COVID-19, placental disorders are predominantly of compensatory-adaptive nature, specific cytological signs of viral cell damage are uncharacteristic. During COVID-19 infection, chronic histiocytic intervillositis and syncytiotrophoblast necrosis occur more often than in average population, and adverse fetal outcomes are characterized by additional marked increase in intervillous fibrinoid deposition. Before COVID-19 pandemic, chronic histiocytic intervillositis was described in about 6 out of 10,000 placentas (0.6 %) in II and III trimesters.Conclusion. The high frequency of chronic histiocytic intervillositis, both in the placenta of paired women with live-born infants infected prenatally due to maternal virus transmission, and in the placentas of stillborn infected infants, allows us to cautiously assume that such placental structural changes are more characteristic for damage by SARS-CoV-2 rather than other infectious agents. It is necessary to study a relationship between placental structural changes occurred at different gestation ages, as well as clinical course and outcome of pregnancy during COVID-19.

The intestine represents the largest immune compartment in the human body, yet its development and organisation during human foetal development is largely unknown. Here we show the immune subset composition of this organ during development, by longitudinal spectral flow cytometry analysis of human foetal intestinal samples between 14 and 22 weeks of gestation. At 14 weeks, the foetal intestine is mainly populated by myeloid cells and three distinct CD3-CD7+ ILC, followed by rapid appearance of adaptive CD4+, CD8+ T and B cell subsets. Imaging mass cytometry identifies lymphoid follicles from week 16 onwards in a villus-like structure covered by epithelium and confirms the presence of Ki-67+ cells in situ within all CD3-CD7+ ILC, T, B and myeloid cell subsets. Foetal intestinal lymphoid subsets are capable of spontaneous proliferation in vitro. IL-7 mRNA is detected within both the lamina propria and the epithelium and IL-7 enhances proliferation of several subsets in vitro. Overall, these observations demonstrate the presence of immune subset-committed cells capable of local proliferation in the developing human foetal intestine, likely contributing to the development and growth of organized immune structures throughout most of the 2nd trimester, which might influence microbial colonization upon birth.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). While mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; Ift88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix (ECM), mesenchymal-epithelial transition, canonical Hh, and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

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 vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. 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 organoids3,4 and trophoblast stem cells5. 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.

Preeclampsia is a complicated syndrome with marked heterogeneity. The biomarker-based classification for this syndrome is more constructive to the targeted prevention and treatment of preeclampsia. It has been reported that preeclamptic patients had elevated miR-155 in placentas or circulation. Here, we investigated the characteristics of patients with high placental miR-155 (pl-miR-155).Based on the 95th percentile (P95) of pl-miR-155 in controls, preeclamptic patients were divided into high miR-155 group (≥P95) and normal miR-155 group (<P95). The changes of placental pathology, clinical manifestations, and placental transcriptome of preeclamptic patients were clustered by t-distributed stochastic neighbor embedding and hierarchical clustering analysis. The placental restricted miR-155 overexpression mouse model was constructed, and the phenotype, placental pathology, and transcriptome were evaluated. Furthermore, the therapeutic potential of antagonist of miR-155 was explored by administrating with antagomir-155.About one-third of preeclamptic patients had high pl-miR-155 expression, which was positively correlated with circulating miR-155 levels. These patients could be clustered as 1 group, according to clinical manifestation, placental pathology, or transcriptomes by t-distributed stochastic neighbor embedding and hierarchical clustering analysis. Further, the pregnant mice with placental restricted miR-155 overexpression could simulate the changes of clinical signs, pathology, and transcriptome of placentas in patients with high pl-miR-155. AntagomiR-155 treatment relieved the preeclampsia-like phenotype and improved the placental vascular development in mice.There is at least 1 type of preeclampsia with upregulated miR-155 presenting more severe clinical manifestations. MiR-155 may be a potential therapeutic target in patients with high pl-miR-155.

The pathogenesis of chronic intervillositis of unknown etiology (CIUE) may involve IFNγ overexpression. This study assesses the extent of IFNγ expression in CIUE by immunohistochemistry and compares it to spontaneous pregnancy losses. C4d deposition is also assessed to see whether IFNγ and C4d might represent separate diagnostic categories. Placenta from first to early second trimester with high grade CIUE (CHG; 17 cases) and low grade CIUE (CLG; 12 cases) is compared to euploid (SPLN; 18 cases), aneuploid spontaneous pregnancy losses (SPLA, 17 cases), normal placenta (NP, 13 cases). Protein level expression of IFNγ and C4d is assessed on whole tissue sections by immunohistochemistry. 35% of CHG and 42% of CLG show some level of IFNγ expression localized to the luminal surface of syncytiotrophoblast. 12% of SPLA and no SPLN or NP cases are IFNγ positive. C4d deposition is seen in 100% of CIUE, 88% of SPLA, 83% of SPLN, and 46% of NP samples. IFNγ overexpression occurs in approximately 40% of CIUE-related pregnancy losses. IFNγ expression restricted to a subgroup of CIUE implies that IFNγ may define a distinct disease process. The non-discriminatory pattern of C4d deposition suggests it is a non-specific phenomenon possibly related to placental damage.

As part of the Human Cell Atlas Initiative, our goal is to generate single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq], 86,708 cells) and regulatory (single-cell assay on transposase accessible chromatin sequencing [scATAC-seq], 59,830 cells) profiles of the normal postmenopausal ovary and fallopian tube (FT). The FT contains 11 major cell types, and the ovary contains 6. The dominating cell type in the FT and ovary is the stromal cell, which expresses aging-associated genes. FT epithelial cells express multiple ovarian cancer risk-associated genes (CCDC170, RND3, TACC2, STK33, and ADGB) and show active communication between fimbrial epithelial cells and ovarian stromal cells. Integrated single-cell transcriptomics and chromatin accessibility data show that the regulatory landscape of the fimbriae is different from other anatomic regions. Cell types with similar gene expression in the FT display transcriptional profiles. These findings allow us to disentangle the cellular makeup of the postmenopausal FT and ovary, advancing our knowledge of gynecologic diseases in menopause.