Oviductal Epithelium Research Articles

Reply: Exposure of human fallopian tube epithelium to elevated testosterone results in alteration of cilia gene expression and beating

Study question How does exposure to a testosterone rich environment affect the function and gene expression of human fallopian tube epithelium (hFTE)? Summary answer Elevated testosterone level alters several gene transcripts that regulate cilia expression and negatively impacts the rate of cilia beating. What is known already The presence of estrogen in the follicular phase of the menstrual cycle increases the human fallopian tube ciliary beating frequency. The luteal phase, triggered by ovulation and increasing progesterone, is marked by a decrease in ciliary beating. Women with polycystic ovarian syndrome (PCOS) may have twice the serum level of testosterone than ovulatory women. To date, the effect of elevated androgens on the function of the human fallopian tube is not well-understood. We chose to examine the impact of elevated testosterone on hFTE. Study design, size, duration A prospective basic science study of human fallopian tube specimens from reproductive-aged women undergoing benign gynecologic surgery was performed. Fallopian tube removal at a large US academic center was collected and provided to us to continue with epithelium isolation and culturing. A total of 12 patients were analyzed in the study. Participants/materials, setting, methods Fallopian tube epithelium was isolated and exposed to two different conditions: normal with low testosterone concentration of 0.8 nM and PCOS-like, with high testosterone concentration of 2 nM. The study was conducted in both static and dynamic conditions in microfluidic devices for a total of 14 days, after which the tissue was collected for processing including RNA extraction, quantitative PCR and immunohistochemistry. After the first 7 days of each experiment, a sample of tissue from each condition was imaged to quantify cilia beating frequency. Main results and the role of chance hFTE exposed to the 2 nM testosterone displayed slower cilia beating, inhibited estrogen signaling and decreased expression of the ciliary marker FOXJ1 when compared to stimulation with 0.8 nM testosterone. Large scale data N/A. Limitations, reasons for caution The in vivo response to elevated testosterone may differ from in vitro studies. RNA amount was limited from tissue cultured in the microfluidic devices as compared to static culture. Wider implications of the findings Understanding elevated testosterone in tubal function may explain an additional contribution to subfertility in women with PCOS and other hyper-androgen disorders, aside from oligo-ovulation. Furthermore, this adds to the body of literature of fallopian tube function using a microfluidic device. Study funding/competing interest(s) NIH grants: UH3 ES029073 and R01 CA240301. There are no competing interests.

Endosalpingiosis Is Negative for GATA3.

Endosalpingiosis is a benign Müllerian inclusion that can mimic metastatic low-grade mammary carcinoma, particularly when encountered in axillary lymph nodes excised for breast cancer staging. Immunohistochemistry can be useful in histologically ambiguous cases, and a targeted immunopanel should include a marker of gynecologic tract origin and a marker of mammary origin. GATA3 is a sensitive immunomarker for breast carcinoma, but the immunoreactivity of GATA3 in endosalpingiosis has not been systematically evaluated. To evaluate whether GATA3 immunohistochemistry could be used to differentiate endosalpingiosis from metastatic mammary carcinoma. Whole slide sections of 15 cases of endosalpingiosis involving nonneoplastic tissues were subjected to GATA3 immunohistochemistry. Nuclear GATA3 labeling was scored as percentage and intensity labeling, with any labeling considered positive; GATA3 labeling was recorded in all cells present in the sections. Half (47%, n = 7 of 15) of the endosalpingiosis cases involved lymph nodes (2 axillary, 5 pelvic) and half (53%, n = 8 of 15) involved pelvic organs or soft tissue (3 myometrial, 2 paratubal, 2 periadnexal soft tissue, and 1 pelvic sidewall). GATA3 immunohistochemistry was negative in all cases of endosalpingiosis, with intact, positive control labeling in lymphocytes. The benign fallopian tube epithelium present on the sections of paratubal endosalpingiosis displayed focal (<5%), weak labeling for GATA3, specifically within the ciliated and secretory cells. These findings support the diagnostic utility of GATA3 immunohistochemistry and its use in a targeted immunopanel to resolve the differential diagnosis of metastatic low-grade mammary carcinoma (GATA3+) and nodal endosalpingiosis (GATA3-).

Immunohistochemical Expression Status of p53, CD44v9, and Ki-67 in a Series of Fallopian Tube Lesions of High-grade Serous Carcinoma.

Pelvic high-grade serous carcinoma (HGSC) has been postulated to arise via a stepwise accumulation of (epi)genetic alterations from normal epithelium to secretory cell outgrowth (SCOUT), p53 signature, and serous tubal intraepithelial carcinoma (STIC) to invasive HGSC. The aim of this study is to investigate alterations in p53 and CD44v9 expression and the status of Ki-67 labeling index in a series of fallopian tube lesions of HGSC patients. A total of 45 specimens were analyzed in 16 patients with HGSC, and their lesions were categorized as follows: morphologically normal fallopian tube epithelium (FTE, n=6 samples), SCOUT (n=5), p53 signature (n=4), dormant STIC (n=8), active STIC (n=6), and HGSC (n=16). Morphologic features and immunohistochemical expression patterns of the p53 protein, CD44v9 protein, and Ki-67 antigen were blindly evaluated by 2 pathologists. Increased nuclear p53 protein accumulation was observed in p53 signature, dormant STIC, active STIC and HGSC compared with normal FTE and SCOUT (P<0.001). Immunohistochemistry scores of CD44v9 protein expression were significantly higher in normal FTE, SCOUT, and p53 signature than in dormant STIC, active STIC, and HGSC (P<0.001). Both active STIC and HGSC had significantly higher Ki-67 labeling indices than normal FTE, SCOUT, p53 signature and dormant STIC (P<0.001). CD44v9 loss contributes to the stepwise progression of p53 signature to dormant STIC. In conclusion, p53 mutation followed by CD44v9 loss may be involved in the evolution of STIC, which may confer positive clonal selection with a growth and survival advantage.

Ovulatory Follicular Fluid Facilitates the Full Transformation Process for the Development of High-Grade Serous Carcinoma.

Simple SummaryOvulation is regarded as the culprit of ovarian high-grade serous carcinoma (HGSC). Previously, we discovered IGF2 in the ovulatory follicular fluid (FF), which bathe fallopian tube epithelium (FTE) during ovulation, promotes malignant transformation through the IGF-1R/AKT pathway. Transformed FTE cells then exfoliate and metastasize to the ovary and peritoneum to grow overt HGSC. In this study, we utilized immortalized FTE cells and HGSC cells carrying accumulating severity of driver mutations to explore FF’s role in the development of HGSC. We found FF promotes (by order of magnitude) migration, anchorage-independent growth (AIG), invasion, peritoneum attachment, anoikis resistance, and proliferation of the full panel of tested cells. The AIG activity largely depends on IGF-1R/AKT signaling, and both AKT- and non-AKT-mediated signals are responsible for other phenotypes. The results demonstrated an extensive transformation activity of FF in the full journey of HGSC development from FTE.Background: High-grade serous carcinoma (HGSC) is mainly derived from the stepwise accumulation of driver mutations in the fallopian tube epithelium (FTE), and it subsequently metastasizes to the ovary and peritoneum that develops into a clinically evident ovarian carcinoma. The developmental process involves cell proliferation/clonal expansion, cell migration, anoikis resistance, anchorage-independent growth (AIG), peritoneum attachment, and cell invasion. Previously, we discovered FTE could be transformed by follicular fluid (FF) released from ovulation, the most crucial risk factor of ovarian cancer, and IGF axis proteins in FF confers stemness activation and clonal expansion via IGF-1R/AKT pathway. However, whether other phenotypes in advanced cancer development are involved is unknown. Methods: A panel of FTE and ovarian HGSC cell lines with different severity of transformation were treated with FF with or without IGF-1R and AKT inhibitors and analyzed for the transformation phenotypes in vitro, ex vivo, and in vivo. Results: FF largely promotes (by order of magnitude) cell migration, AIG, cell invasion, peritoneum attachment, anoikis resistance, and cell proliferation. Most of these activities worked in the full panel of cell lines. The AIG activity largely depends on IGF-1R/AKT phosphorylation, and the proliferation activity depends on an AKT phosphorylation not mediated by IGF-1R. In contrast, both AKT- and non-AKT-mediated signals are responsible for the other transformation activities. Conclusions: Our data demonstrate an extensive transformation activity of FF in the full journey of carcinogenesis, and endorsed ovulation-inhibition for the prevention and AKT-inhibition for the treatment of ovarian HGSC.

Therapeutic Targeting of Tumor Cells Rich in LGR Stem Cell Receptors.

LGR5 and LGR6 mark epithelial stem cells in many niches including the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. Human ovarian cancers express these receptors at high levels and express one of their ligands, RSPO1, at levels uniquely higher than all other tumor types except mesothelioma. Reasoning that these receptors are also important to tumor stem cells, arming the LGR binding domain of RSPO1 with a cytotoxin may permit depletion of the tumor stem cells. The Fu1-Fu2 receptor binding domain of RSPO1 (R1FF), containing a sortase recognition sequence at the C-terminal end, was produced in bacteria and a single molecule of MMAE was attached to each R1FF through a val-cit-PAB linker using the sortase reaction, thus producing a homogeneous population of armed molecules. R1FF-MMAE demonstrated (1) selective LGR-dependent binding, uptake, and cytotoxicity; (2) low nM cytotoxicity to multiple types of human tumor cell lines in vitro; (3) favorable plasma pharmacokinetic properties when administered iv with an elimination half-life of 27.8 h; (4) favorable absorption from the peritoneal cavity; and (5) therapeutic activity in aggressive xenograft models of ovarian cancer in the absence of any weight loss or other adverse events. These results demonstrate that the Fu1-Fu2 domain of RSPO1 can be exploited to deliver a potent cytotoxin to tumor cells that express the LGR4-6 family of stem cell receptors.

Patients with low nicotinamide N-methyltransferase expression benefit significantly from bevacizumab treatment in ovarian cancer

BackgroundThe role of nicotinamide N-methyltransferase (NNMT) in ovarian cancer is still elusive. Our aim is to explore the expression of NNMT in ovarian cancer and to assess its association with patient prognosis and treatment response.MethodsWe first analyzed the differential expression of NNMT among fallopian tube epithelium, primary ovarian cancers, metastatic ovarian cancers, and recurrent ovarian cancers using Gene Expression Ominus (GEO) database (GSE10971, GSE30587, GSE44104 and TCGA datasets). Then, we assessed the association of NNMT expression with clinical and molecular parameters using CSIOVDB database and GSE28739 dataset. Next, we evaluate the association of NNMT expression with the prognosis of ovarian cancer patients in both GSE9891 dataset and TCGA dataset. Finally, GSE140082 dataset was used to explore the association of NNMT expression with bevacizumab response.ResultsNNMT expression was significantly elevated in lymphovascular space invasion (LVSI)-positive ovarian cancers compared with that in LVSI-negative ovarian cancers (TCGA dataset, P < 0.05), Moreover, increased expression of NNMT was associated with increased tumor stage, grade, and mesenchymal molecular subtype (CSIOVDB database). Survival analysis indicated that increased expression of NNMT was associated with a reduced OS in both GSE9891 dataset (HR: 2.28, 95%CI: 1.51–3.43, Log-rank P < 0.001) and TCGA dataset (HR: 1.55, 95%CI: 1.02–2.36, Log-rank P = 0.039). Multivariate analysis further confirmed the negative impact of NNMT expression on OS in ovarian cancer patients in those two datasets. Furthermore, the NNMT-related nomogram showed that NNMT shared a larger contribution to OS, compared with debulking status. More interestingly, bevacizumab conferred significant improvements in OS for patients with low NNMT expression (HR: 0.56, 95%CI: 0.31–0.99, Log-rank P = 0.049). In contrast, patients with high NNMT expression didn’t benefit from bevacizumab treatment significantly (HR: 0.85, 95%CI: 0.48–1.49, Log-rank P = 0.561). NNMT expression was positively correlated with the expression of genes, LDHA and PGAM1, involved in Warburg effect.ConclusionsIn conclusion, NNMT expression is associated with the aggressive behavior of ovarian cancer, correlates with a poor prognosis, and is predictive of sensitivity to bevacizumab treatment.

PAX8, an Emerging Player in Ovarian Cancer.

Ovarian Cancer is one of the most lethal and widespread gynecological malignancies. It is the seventh leading cause of all cancer deaths worldwide. High-Grade Serous Cancer (HGSC), the most commonly occurring subtype, alone contributes to 70% of all ovarian cancer deaths. This is mainly attributed to the complete lack of symptoms during the early stages of the disease and absence of an early diagnostic marker.PAX8 is emerging as an important histological marker for most of the epithelial ovarian cancers, as it is expressed in about 90% of malignant ovarian cancers, specifically in HGSC. PAX8 is a member of the Paired-Box gene family (PAX1-9) of transcription factors whose expression is tightly controlled temporally and spatially. The PAX genes are well known for their role in embryonic development and their expression continues to persist in some adult tissues. PAX8 is required for the normal development of Müllerian duct that includes Fallopian tube, uterus, cervix, and upper part of vagina. In adults, it is expressed in the Fallopian tube and uterine epithelium and not in the ovarian epithelium. Considering the recent studies that predict the events preceding the tumorigenesis of HGSC from the Fallopian tube, PAX8 appears to have an important role in the development of ovarian cancer.In this chapter, we review some of the published findings to highlight the significance of PAX8 as an important marker and an emerging player in the pathogenesis of ovarian cancer. We also discuss regardingthe future perspectives of PAX8wherein itcould contribute to the betterment of ovarian cancer diagnosis and treatment.

Therapeutic effects of hysterosalpingography contrast media in infertile women: what do we know about the H2O in the H2Oil trial and why does it matter?

Recent interest in the use of oil-soluble contrast media (OSCM) for hysterosalpingography (HSG) and other tubal flushing procedures is largely the result of publication of the large, multicentre, randomized controlled H2Oil trial in the New England Journal of Medicine in 2017, addressing the long-held suspicion that pregnancy rates following HSG with OSCM are higher than if a water-soluble contrast media (WSCM) is used. However, the findings of this trial have been compromised by the WSCM selected for comparison with OSCM. The chosen WSCM belongs to a superceded class of ionic media, with an iodine concentration, osmolality and viscosity all lying at the extreme end of the range for water-soluble radiographic contrast. The requirement for safe, cheap, versatile WSCM for intravascular use during computerized tomography, angiography and interventional procedures has resulted in considerable refinement of WSCM, with current widespread use of non-ionic, low osmolality or iso-osmolar WSCM in radiology, including for HSG. However, the use of the earlier ionic, high osmolality agents such as that selected for the H2Oil trial persists for HSG in some centres, despite potential adverse effects on the endometrium and fallopian tubal epithelium compared with more modern, less toxic WSCM. Knowledge of the variety of physical and chemical characteristics of the available WSCM is essential for interpretation of the current literature and establishing the most effective and safe water-soluble agent to use for HSG. Design of future clinical trials to establish the potential superiority of OSCM over WSCM for fertility enhancement must include the use of the readily available, inexpensive modern WSCM. While the fertility rates following OSCM HSG have been shown to be high in women with idiopathic infertility, more robust trials are required before the widespread use of OSCM for HSG or other modalities such as ultrasound and laparoscopy should be adopted.

Female reproductive tract has low concentration of SARS-CoV2 receptors.

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.

Ultrastructural evidence for an unusual mode of ciliogenesis in mouse multiciliated epithelia.

Multiciliogenesis is a cascading process for generating hundreds of motile cilia in single cells. In vertebrates, this process has been investigated in the ependyma of brain ventricles and the ciliated epithelia of the airway and oviduct. Although the early steps to amplify centrioles have been characterized in molecular detail, subsequent steps to establish multicilia have been relatively overlooked. Here, we focused on unusual cilia-related structures previously observed in wild-type mouse ependyma using transmission electron microscopy and analyzed their ultrastructural features and the frequency of their occurrence. In the ependyma, $\sim$5% of cilia existed as bundles; while the majority of the bundles were paired, bundles of more than three cilia were also found. Furthermore, apical protrusions harboring multiple sets of axonemes were occasionally observed (0-2 per section), suggesting an unusual mode of ciliogenesis. In trachea and oviduct epithelia, ciliary bundles were absent, but protrusions containing multiple axonemes were observed. At the base of such protrusions, certain axonemes were completely enwrapped by membranes, whereas others remained incompletely enwrapped. These data suggested that the late steps of multiciliogenesis might include a unique process underlying the development of cilia, which is distinct from the ciliogenesis of primary cilia.

DNA Methylation Profiling of Premalignant Lesions as a Path to Ovarian Cancer Early Detection

Genome-wide DNA methylation profiles of serous tubal intraepithelial carcinomas (STIC) resemble methylation profiles of high-grade serous ovarian carcinomas (HGSOC) more closely than normal fallopian tube epithelium. While STICs and HGSOCs share subsets of common hypermethylated regions, DNA methylation can distinguish STICs from HGSOCs to provide proof-of-principle that DNA methylation can identify HGSOC initiation.See related article by Pisanic et al., p. 6310.

Methylomic Landscapes of Ovarian Cancer Precursor Lesions.

The current paradigm in the development of high-grade serous ovarian carcinoma (HGSC) proposes that the majority of HGSCs arise from precursor serous tubal intraepithelial carcinoma (STIC) lesions of the fallopian tube. Here we survey genome-wide methylation in HGSC precursor lesions to identify genomic regions that exhibit high-specificity differential hypermethylation for potential use as biomarkers for detecting STIC and HGSC at stages when curative intervention likely remains feasible. We first identified quality control criteria for performing reliable methylomic analysis of DNA-limited tubal precursor lesions with the Illumina Infinium MethylationEPIC array. We then used this platform to compare genome-wide methylation among 12 STICs with paired adjacent-normal epithelia, one p53 signature lesion and two samples of concurrent HGSC. The resulting methylomic data were analyzed by unsupervised hierarchical clustering and multidimensional analysis. Regions of high-confidence STIC-specific differential hypermethylation were identified using selective bioinformatic criteria and compared with published MethylationEPIC data from 23 HGSC tumors and 11 healthy fallopian tube mucosae. Unsupervised analysis showed that STICs largely clustered with HGSCs, but were clearly distinct from adjacent-normal fallopian tube epithelia. Forty-two genomic regions exhibited high-confidence STIC-specific differential hypermethylation, of which 17 (40.5%) directly overlapped with HGSC-specific differentially methylated regions. Methylation at these shared loci was able to completely distinguish STIC and HGSC samples from normal and adjacent-normal specimens. Our results suggest that most STICs are epigenetically similar to HGSCs and share regions of differential hypermethylation that warrant further evaluation for potential use as biomarkers for early detection of ovarian HGSC.See related commentary by Ishak and De Carvalho, p. 6083.

411 Sperm movement, storage, and release from the oviduct

Abstract After semen deposition, a fraction of sperm is transported through the female reproductive tract to the lower oviduct, the isthmus, where sperm are retained to form a reservoir. Some of these sperm are released to move to the upper oviduct, the site of fertilization. For sperm to make this journey, they must overcome challenges including possible phagocytosis, high fluid viscosity, and peristaltic contractions of the tract. The study of sperm transport is complex because so few sperm reach the site of fertilization. We have focused our studies on how sperm are retained in the reservoir, how storage prolongs sperm lifespan, and how sperm are released to fertilize oocytes. Sperm storage is particularly important in situations in which ovulation is not tightly synchronized with semen deposition. This occurs in domestic animals, but is especially notable in some species of bats, birds, and insects. Using porcine sperm and an array of 400 common glycans, we identified two specific glycan motifs found in all structures that bound sperm, a Lewis X trisaccharide and a branched 6-sialylated oligosaccharide. Tandem MS profiling indicated that both motifs were abundant in the asparagine-linked glycans of the oviduct epithelium and several larger oligosaccharides were identified that contained both motifs. When these motifs were immobilized, each could retain sperm, suppress Ca2+ influx and lengthen sperm lifespan. Secretions from the cumulus-oocyte complex (COC) and progesterone released sperm from immobilized oviduct glycans, suggesting that COCs can themselves signal sperm release. Progesterone-induced release required CatSper channels, sperm hyperactivation and was dependent on sperm protein degradation. These studies support a model in which sperm are retained in the isthmus by specific glycans on the epithelium, which extends sperm lifespan until COCs produce releasing factors that promote sperm liberation from the isthmus and movement to the ampulla to fertilize the COCs.

Prosaposin in the rat oviductal epithelial cells.

Prosaposin (PSAP) has two forms: a precursor and a secreted form. The secreted form has neurotrophic, myelinotrophic, and myotrophic properties. The precursor form is a precursor protein of saposins A-D. Although the distribution of PSAP in male reproductive organs is well known, its distribution in female reproductive organs, especially in the oviduct, is unclear. Immunoblots and immunohistochemistry of oviducts showed that oviductal tissues contain PSAP proteins, and a significant increase in PSAP was observed in the estrus-metestrus phase compared to the diestrus-proestrus phase in the ampulla. To identify PSAP trafficking in cells, double-immunostaining was performed with antibodies against PSAP in combination with sortilin, mannose 6 phosphate receptor (M6PR), or low-density lipoprotein receptor-related protein 1 (LRP1). PSAP and sortilin double-positive reactions were observed near the nuclei, as well as in the apical portion of microvillous epithelial cells, whereas these reactions were only observed near the nuclei of ciliated epithelial cells. PSAP and M6PR double-positive reactions were observed near the nuclei of microvillous and ciliated epithelial cells. PSAP and M6PR double-positive reactions were also observed in the apical portion of microvillous epithelial cells. PSAP and LRP1 double-positive reactions were observed in the plasma membrane and apical portion of both microvillous and ciliated epithelial cells. Immunoelectron staining revealed PSAP immunoreactive small vesicles with exocytotic features at the apical portion of microvillous epithelial cells. These findings suggest that PSAP is present in the oviductal epithelium and has a pivotal role during pregnancy in providing an optimal environment for gametes and/or sperm in the ampulla.

Association between vitamin D and ovarian cancer development in BRCA1 mutation carriers.

Objective: Women with inherited mutations in BRCA1 gene have a high (40–70%) genetic risk of developing ovarian cancer. Epidemiological studies suggest an inverse correlation between serum vitamin D (VD) levels and the risk of ovarian cancer, but there is a lack of data from BRCA1 mutation (BRCA1mut) carriers. Therefore, we investigated VD levels and actions in cancer free women with BRCA1 mutations.Materials and Methods: Blood, ovary and fallopian tube samples were collected from healthy pre-menopausal women with BRCA1mut and without BRCA1 mutations (BRCAwt). Serum calcifediol (major circulating form of VD) concentrations were measured by electrochemiluminescence immunoassay. Immunohistochemistry was performed on paraffin-embedded ovarian and fallopian tube sections to determine vitamin D receptor (VDR) expression. Ovarian surface epithelial cells (OSEs) from BRCA1mut carriers were cultured with or without calcitriol supplementation for 72 hrs. VDR protein levels, cell proliferation and cell viability were analyzed.Results: BRCA1mut women had lower serum calcifediol levels compared to BRCAwt women (p = 0.003). VDR protein expression was evident in ovarian and the fallopian tube epithelium of BRCAwt patients, but was reduced in BRCA1mut women. Calcitriol (biologically active VD) supplementation elevated VDR expression in cultured BRCA1mut OSEs (p = 0.005) and decreased cell proliferation rates in a dose-dependent manner without inducing apoptosis.Conclusions: VD biosynthesis and signaling via VDR in the ovarian and fallopian tube epithelium are impaired in BRCA1mut women. VD treatment may limit BRCA1mut epithelial cell proliferation without affecting cell viability, providing a rationale for exploring the potential for VD in ovarian cancer prevention in BRCA1mut carriers.

Extracellular Vesicle Mediated Crosstalk Between the Gametes, Conceptus, and Female Reproductive Tract.

Extracellular vesicles (EVs) mediated intracellular communication plays an imperative role in the proper completion of different physiological events. Most of the bio-fluids are enriched with several subpopulations of EVs including exosomes and microvesicles (MVs), with the capacity of transferring different functional molecules (lipids, proteins, and nucleic acids) to target cells. Recipient cells upon receiving the signal molecules undergo different changes that positively affect the structural and functional integrity of the cells. This article was aimed to highlight the role of EVs secreted by gametes, the female reproductive tract, and the growing conceptus in the successful completion of different reproductive events related to gestation. EVs associated with the reproductive system are actively involved in the regulation of different physiological events including gamete maturation, fertilization, and embryo and fetal development. In the reproductive system, EVs mediated intracellular communication is not unidirectional but is rather regulated through crosstalk between the reproductive tract and the growing conceptus. These vesicles are secreted from the ovary, oviductal epithelium, endometrium, developing embryo, and the placenta. The cargo inside these vesicles exerts pleiotropic effects on both maternal and embryonic environments. A better understanding of the EVs-mediated crosstalk will be helpful in the development of useful tools serving both the diagnostic as well as therapeutic needs related to female fertility.

Oviductal regulation of fertilization and early embryonic development

During the period of late follicular development and the first four days of the oestrous cycle, the oviduct occupies a central role in the establishment of pregnancy. Oviductal function is regarded as being either 'passive' or biologically active, providing an environment that sustains and enhances fertilization and early cleavage-stage embryonic development. Recent reports have focused on this microenvironment and shown that ovarian steroids induce marked morphological, physiological and biochemical changes. Alterations include changes in the biosynthetic activity and release of macromolecules by the oviductal epithelium which become part of the luminal microenvironment. Furthermore, both regional and temporal differences in activity and protein production occur through hormonal changes during the oestrous cycle and early pregnancy. Studies on identification, characterization and regulation of several proteins synthesized de novo have indicated oocyte-oviduct and embryo-oviduct interactions. However, the identification of oviduct-derived proteins, their regulation and their potential function in vivo needs to be examined. Studies in other species also suggest roles for growth factors in early embryonic development, but little information is available for the pig. We propose that ovarian hormones control changes in synthetic activity, synthesis of some oviduct-derived proteins and the presence of specific factors in the luminal microenvironment which sustain and enhance fertilization and early cleavage-stage embryonic development.

Mating induces early transcriptional response in the rat endosalpinx: the role of TNF and RA.

During mating, males provide not only the spermatozoa to fertilize the oocyte but also other stimuli that are essential for initiating and maintaining the reproductive programme in females. In the mammalian oviduct, mating regulates sperm storage, egg transport, fertilization, early embryonic development, and oestradiol metabolism. However, the main molecules underlying these processes are poorly understood. Using microarray analyses, we identified 58 genes that were either induced or repressed by mating in the endosalpinx at 3 h post-stimulus. RT-qPCR confirmed that mating downregulated the expression of the Oas1h and Prim1 genes and upregulated the expression of the Ceacam1, Chad, Chst10, Slc5a3 and Slc26a4 genes. The functional category 'cell-to-cell signalling and interaction' was over-represented in this gene list. Network modelling identified TNF and all-trans retinoic acid (RA) as upstream regulators of the mating-induced transcriptional response, which was confirmed by intraoviductal injection of TNF or RA in unmated rats. It partially mimicked the transcriptional effect of mating in the rat endosalpinx. Furthermore, mating decreased RA levels in oviductal fluid, and RA-receptor-gamma (RARG) exhibited a nuclear location in oviductal epithelium in both unmated and mated rats, indicating RA-RARG transcriptional activity. In conclusion, the early transcriptional response regulated by mating in the rat endosalpinx is mediated by TNF and RA. These signalling molecules regulate a cohort of genes involved in 'cell-to-cell signalling and interactions' and merit further studies to understand the specific processes activated in the endosalpinx to sustain the events that occur in the mammalian oviduct early after mating.

Chemopreventive effect of progestin and vitamin D in the mogp-TAG ovarian cancer mouse model

Objective: Growing evidence suggests the fallopian tube epithelium harbors the precursor for high-grade serous ovarian cancer, creating opportunities for targeting the fallopian tube for effective chemoprevention. Animal and human evidence has shown that progestins confer potent cancer preventive effects in the gynecologic tract by activating molecular pathways that clear genetically damaged cells. in addition, it has been shown in vitro that the potency of progestins can be enhanced with vitamin D.

Fallopian tube epithelial cells express androgen receptor and have a distinct hormonal responsiveness when compared with endometrial epithelium.

How does steroid receptor expression, proliferative activity and hormone responsiveness of the fallopian tube (FT) epithelium compare to that of the endometrial epithelium? Proliferative indices, hormone receptor expression-scores and in vitro response to oestrogen and androgens of the human FT demonstrate a distinct pattern from the matched endometrium. The FT epithelium exists as a continuum of the endometrium, and both express steroid hormone receptors. The ovarian steroid hormones regulate cyclical proliferation and regeneration of the endometrium, but their effects on steroid hormone receptor expression and proliferation in the FT have not yet been fully elucidated. We included women with proven fertility, undergoing hysterectomy and bilateral salpingo-oophorectomy for benign, gynaecological conditions at Liverpool Women's NHS Foundation Trust. They had no known endometrial or tubal pathology and were not on hormonal treatments for at least 3 months preceding sample collection in this prospective observational study (conducted between 2010 and 2018). A full-thickness sample of the endometrium and a sample from the FT were collected from each woman. The differential protein and mRNA levels of steroid hormone receptors, oestrogen receptors α and β, androgen receptor (AR) and progesterone receptor (PR), and the proliferative marker (Ki67) of the endometrium and the FT tissue samples from 47 healthy women undergoing surgery (37 premenopausal and 10 postmenopausal) were investigated using immunohistochemistry and quantitative real-time PCR. The comparative responsiveness to oestrogen and androgen of the endometrium and the fimbrial end of the FT was analysed using an in vitro short-term explant culture model. The endpoints assessed in the explants were the changes in mRNA and protein levels for AR, PR and the epithelial proliferative index after 24 h treatment with oestradiol (E2) or dihydrotestosterone (DHT). The premenopausal endometrial functionalis glands (FG) displayed the well-known cyclic variation in cellular proliferation and steroid receptor scores. Compared with the endometrial FG, the matched FT epithelium (both fimbrial or isthmic ends) displayed a significantly lower proportion of cells expressing Ki67 (2.8% ± 2.2%, n = 18 vs 30.0% ± 26.3%, n = 16, P = 0.0018, respectively) accompanied with a significantly higher AR immunoscores (6.7 ± 2.7, n = 16 vs 0.3 ± 1.0, n = 10, P = 0.0136). The proportion of cells expressing Ki67 and the AR immunoscores of the FT epithelium correlated positively with endometrial luminal epithelium (r = 0.62, P = 0.005, and r = 0.68, P = 0.003, respectively). In vitro experiments suggested the tubal explants to be apparently less responsive to E2 yet more sensitive to DHT compared with the matched endometrium explants. The short-term in vitro nature of the tissue explant cultures used in the study may not be representative of how different anatomical regions of the endometrium and FT behave in vivo. Our study included a high proportion of older premenopausal women with a regular menstrual cycle, which may therefore affect extrapolation of findings to a younger group. Advancing our understanding of tubal and endometrial epithelial cell function has important implications for the diagnosis and treatment of diseases such as infertility, ectopic pregnancy, endometriosis and cancer. The work included in this article was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.) and Wellbeing of Women Entry-Level Scholarship ELS706 (A.M). A.M. was also supported by an NIHR ACF fellowship grant. Further support received from Liverpool Women's Hospital NHS Trust (S.M.), University of Liverpool (E.B. and A.W.). All authors declare there are no conflicts of interest. N/A.