Oviductal Epithelial Cells Research Articles

Scanning electron microscopic study of the human uterine tube epithelial lining: surgical biopsy samples and epithelial cell culture

This article summarizes the importance of the exact morphology of human uterine/fallopian tube epithelium at the scanning electron microscopy (SEM) level for the clinical outcome even nowadays. Visual referential micrographs from SEM reflect two ways to view human epithelial cell lining surfaces: the surface epithelial uterine tube from surgical tissue biopsy and human fallopian tube epithelial cells (HFTEC) culture monolayer surface. One colorized image visualizes ciliated cells, distinguishes them from non-ciliated cells, and provides an educational benefit. A detailed description of the ultrastructure in referential and pathologic human uterine tube epithelium is important in defining the morphological basis of high-grade carcinomas, in the mechanism of pathophysiology, and in discussing options for its prevention. Cell cultures of human fallopian tube epithelial cells offer new approaches in simulating the mechanisms of cancer genesis or may help to elucidate the genetic basis of several diagnoses. New technical approaches in SEM provide higher resolution and detailed surface images. The SEM modality is still one of the current options in diagnostics and may be useful for advancing human reproductive organ cancer research.

Distinct niche structures and intrinsic programs of fallopian tube and ovarian surface epithelial cells

Epithelial ovarian cancer (EOC) can originate from either fallopian tube epithelial (FTE) or ovarian surface epithelial (OSE) cells, but with different latencies and disease outcomes. To address the basis of these differences, we performed single cell RNA-sequencing of mouse cells isolated from the distal half of fallopian tube (FT) and surface layer of ovary. We find at the molecular level, FTE secretory stem/progenitor cells and OSE cells resemble mammary luminal progenitors and basal cells, respectively. An FT stromal subpopulation, enriched with Pdgfra + and Esr1 + cells, expresses multiple secreted factor (e.g., IGF1) and Hedgehog pathway genes and may serve as a niche for FTE cells. In contrast, Lgr5 + OSE cells express similar genes largely by themselves, raising a possibility that they serve as their own niche. The differences in intrinsic expression programs and niche organizations of FTE and OSE cells may contribute to their different courses toward the development of EOCs.

The conserved L1089 in the S2 subunit of avian infectious bronchitis virus determines viral kidney tropism by disrupting virus-cell fusion

The virulence of avian gamma-coronavirus infectious bronchitis viruses (IBV) for the kidney has led to high mortality in dominant-genotype isolations, but the key sites of viral protein that determine kidney tropism are still not fully clear. In this study, the amino acid sequences of the S2 subunit of IBVs with opposing adaptivity to chicken embryonic kidney cells (CEKs) were aligned to identify putative sites associated with differences in viral adaptability. The S2 gene and the putative sites of the non-adapted CN strain were introduced into the CEKs-adapted SczyC30 strain to rescue seven mutants. Analysis of growth characteristics showed that the replacement of the entire S2 subunit and the L1089I substitution in the S2 subunit entirely abolished the proliferation of recombinant IBV in CEKs as well as in primary chicken oviduct epithelial cells. Pathogenicity assays also support the decisive role of this L1089 for viral nephrotropism, and this non-nephrotropic L1089I substitution significantly attenuates pathogenicity. Analysis of the putative cause of proliferation inhibition in CEKs suggests that the L1089I substitution affects neither virus attachment nor endocytosis, but instead fails to form double-membrane vesicles to initiate the viral replication and translation. Position 1089 of the IBV S2 subunit is conservative and predicted to lie in heptad repeat 2 domains. It is therefore reasonable to conclude that the L1089I substitution alters the nephrotropism of parent strain by affecting virus-cell fusion. These findings provide crucial insights into the adaptive mechanisms of IBV and have applications in the development of vaccines and drugs against IB.

Spermatozoa, acts as an external cue and alters the cargo and production of the extracellular vesicles derived from oviductal epithelial cells in vitro

The oviduct provides optimum physiological and biochemical milieu essential for successful fertilization, early embryo development and facilitates functional maturation of spermatozoa. A study has revealed that spermatozoa alters the gene expression in bovine oviductal epithelial cells (BOECs) remotely via bio-active particles, thus acting as a cue to the oviduct prior to their arrival. However, very little attention has been paid to the question of whether spermatozoa could alter the cargo of extracellular vesicles (EVs) derived from BOECs. Therefore, the aim of this study was to investigate the alterations in small non-coding RNAs in EVs cargo derived from BOECs when incubated with spermatozoa in contact and non-contact co-culture models. After 4 h of incubation the EVs were isolated from the conditioned media, followed by small non-coding sequencing of the BOEC derived EVs. Our results revealed that EVs from both co-culture models contained distinct cargo in form of miRNA, fragmented mRNA versus control. The pathway enrichment analysis revealed that EV miRNA from direct co-culture were involved in the biological processes associated with phagocytosis, macroautophagy, placenta development, cellular responses to TNF and FGF. The mRNA fragments also varied within the different groups and mapped to the exonic regions of the transcriptome providing vital insights regarding the changes in cellular transcriptome on the arrival of spermatozoa. The findings of this study suggest that spermatozoa, in contact as well as remotely, alter the EV cargo of female reproductive tract epithelial cells which might be playing an essential role in pre and post-fertilization events.Graphical abstract

102 Effect of conditioned media of oviductal epithelial cells on

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

77 Notch-inhibition stimulates secondary ciliation in re-differentiated equine oviduct epithelial cell monolayers

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Hypothalamic-Pituitary-Adrenal Hormones Impair Pig Fertilization and Preimplantation Embryo Development via Inducing Oviductal Epithelial Apoptosis: An In Vitro Study.

Previous studies show that stressful events after ovulation in sows significantly impaired the embryo cleavage with a significant elevation of blood cortisol. However, the effects of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol on fertilization and embryo development remain to be specified, and whether they damage pig embryos directly or indirectly is unclear. This study demonstrated that embryo development was unaffected when pig parthenotes were cultured with different concentrations of CRH/ACTH/cortisol. However, embryo development was significantly impaired when the embryos were cocultured with pig oviductal epithelial cells (OECs) in the presence of CRH/cortisol or cultured in medium that was conditioned with CRH/cortisol-pretreated OECs (CRH/cortisol-CM). Fertilization in CRH/cortisol-CM significantly increased the rates of polyspermy. CRH and cortisol induced apoptosis of OECs through FAS and TNFα signaling. The apoptotic OECs produced less growth factors but more FASL and TNFα, which induced apoptosis in embryos. Pig embryos were not sensitive to CRH because they expressed no CRH receptor but the CRH-binding protein, and they were tolerant to cortisol because they expressed more 11-beta hydroxysteroid dehydrogenase 2 (HSD11B2) than HSD11B1. When used at a stress-induced physiological concentration, while culture with either CRH or cortisol alone showed no effect, culture with both significantly increased apoptosis in OECs. In conclusion, CRH and cortisol impair pig fertilization and preimplantation embryo development indirectly by inducing OEC apoptosis via the activation of the FAS and TNFα systems. ACTH did not show any detrimental effect on pig embryos, nor OECs.

A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing.

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.

Spontaneous Transformation of a p53 and Rb-Defective Human Fallopian Tube Epithelial Cell Line after Long Passage with Features of High-Grade Serous Carcinoma.

Ovarian cancer is one of the most lethal gynecological cancers, and 80% are high-grade serous carcinomas (HGSOC). Despite advances in chemotherapy and the development of targeted therapies, the survival rate of HGSOC has only moderately improved. Therefore, a cell model that reflects the pathogenesis and clinical characteristics of this disease is urgently needed. We previously developed a human fallopian tube epithelial cell line (FE25) with p53 and Rb deficiencies. After long-term culture in vitro, cells at high-passage numbers showed spontaneous transformation (FE25L). This study aimed to compare FE25 cells cultured in vitro for low (passage 16-31) and high passages (passage 116-139) to determine whether these cells can serve as an ideal cell model of HGSOC. Compared to the cells at low passage, FE25L cells showed increased cell proliferation, clonogenicity, polyploidy, aneuploidy, cell migration, and invasion. They also showed more resistance to chemotherapy and the ability to grow tumors in xenografts. RNA-seq data also showed upregulation of hypoxia, epithelial-mesenchymal transition (EMT), and the NF-κB pathway in FE25L compared to FE25 cells. qRT-PCR confirmed the upregulation of EMT, cytokines, NF-κB, c-Myc, and the Wnt/β-catenin pathway. Cross-platform comparability found that FE25L cells could be grouped with the other most likely HGSOC lines, such as TYKNU and COV362. In conclusion, FE25L cells showed more aggressive malignant behavior than FE25 cells and hence might serve as a more suitable model for HGSOC research.

Fallopian tube secreted protein affects ovarian metabolites in high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC), the most lethal histotype of ovarian cancer, frequently arises from fallopian tube epithelial cells (FTE). Once transformed, tumorigenic FTE often migrate specifically to the ovary, completing the crucial primary metastatic step and allowing the formation of the ovarian tumors after which HGSOC was originally named. As only the fimbriated distal ends of the fallopian tube that reside in close proximity to the ovary develop precursor lesions such as serous tubal intraepithelial carcinomas, this suggests that the process of transformation and primary metastasis to the ovary is impacted by the local microenvironment. We hypothesize that chemical cues, including small molecules and proteins, may help stimulate the migration of tumorigenic FTE to the ovary. However, the specific mediators of this process are still poorly understood, despite a recent growth in interest in the tumor microenvironment. Our previous work utilized imaging mass spectrometry (IMS) to identify the release of norepinephrine (NE) from the ovary in co-cultures of tumorigenic FTE cells with an ovarian explant. We predicted that tumorigenic FTE cells secreted a biomolecule, not produced or produced with low expression by non-tumorigenic cells, that stimulated the ovary to release NE. As such, we utilized an IMS mass-guided bioassay, using NE release as our biological marker, and bottom-up proteomics to demonstrate that a secreted protein, SPARC, is a factor produced by tumorigenic FTE responsible for enhancing release of ovarian NE and influencing primary metastasis of HGSOC. This discovery highlights the bidirectional interplay between different types of biomolecules in the fallopian tube and ovarian microenvironment and their combined roles in primary metastasis and disease progression.

Oviduct Epithelial Cell-Derived Extracellular Vesicles Improve Porcine Trophoblast Outgrowth.

Porcine species have a great impact on studies on biomaterial production, organ transplantation and the development of biomedical models. The low efficiency of in vitro-produced embryos to derive embryonic stem cells has made achieving this goal a challenge. The fallopian tube plays an important role in the development of embryos. Extracellular vesicles (EVs) secreted by oviductal epithelial cells play an important role in the epigenetic regulation of embryo development. We used artificially isolated oviductal epithelial cells and EVs. In this study, oviductal epithelial cell (OEC) EVs were isolated and characterized through transmission electron microscopy, nanoparticles tracking analysis, western blotting and proteomics. We found that embryo development and blastocyst formation rate was significantly increased (14.3% ± 0.6% vs. 6.0% ± 0.6%) after OEC EVs treatment. According to our data, the inner cell mass (ICM)/trophectoderm (TE) ratio of the embryonic cell number increased significantly after OEC EVs treatment (43.7% ± 2.3% vs. 28.4% ± 2.1%). Meanwhile, the attachment ability of embryos treated with OEV EVs was significantly improved (43.5% ± 2.1% vs. 29.2% ± 2.5%, respectively). Using quantitative polymerase chain reaction (qPCR), we found that the expression of reprogramming genes (POU5F1, SOX2, NANOG, KLF4 and c-Myc) and implantation-related genes (VIM, KRT8, TEAD4 and CDX2) significantly increased in OEC EV-treated embryos. We report that OEC EV treatment can improve the development and implantation abilities of embryos.

Regulation of Porcine Oviduct Epithelium Functions via Progesterone and Estradiol Is Influenced by Cortisol.

Preimplantation maternal stress, characterized by elevated glucocorticoids (GCs), has been linked to reproductive failures caused by impaired oviduct functionality, which is known to be predominantly regulated by the sex steroids, progesterone (P4) and (17)estradiol (E2). Although steroid receptors share analogous structures and binding preferences, the interaction between GCs and E2/P4 in the oviduct has attracted little attention. Using an air-liquid interface culture model, porcine oviduct epithelial cells were stimulated with single (cortisol, E2, P4) or hormone mixtures (cortisol/E2, cortisol/P4) for 12 hours and 72 hours. Cultures were subsequently assessed for epithelial morphometry, bioelectrical properties, and gene expression responses (steroid hormone signaling, oviductal function, immune response, and apoptosis). Results confirmed the suppressive role of P4 in regulating oviduct epithelium characteristics, which was partially opposed by E2. Besides increasing the ratio of ciliated cells, cortisol antagonized the effect of P4 on epithelial polarity and modified sex steroid-induced changes in transepithelial electrical properties. Both sex steroids affected the glucocorticoid receptor expression, while cortisol downregulated the expression of progesterone receptor. The overall gene expression pattern suggests that sex steroid dominates the cotreatment, but cortisol contributes by altering the gene responses to sex steroids. We conclude that besides its individual action, maternal cortisol interplays with sex steroids at phenotypic and molecular levels in the oviduct epithelium, thereby influencing the microenvironment of gametes and early embryos.

Integrative analysis identifies potential ferroptosis-related genes of hypoxia adaptation in yak.

There are studies on the hypoxia adaptation in yak, but there are few studies on the regulation of ferroptosis by hypoxia. This study was the first time to explore ferroptosis-related genes about hypoxia in yak. In this study, the oviduct epithelial cells between yak and bovine are performed by integrative analysis for functions, regulating network and hub genes. The results showed 29 up-regulated ferroptosis genes and 67 down-regulated ferroptosis genes, and GO-KEGG analysis showed that up-regulated differentially expressed genes (DEGs) were significantly enriched in ribosome pathway and oxidative phosphorylation pathway. Down-regulated DEGs were significantly enriched in longevity regulating pathway-mammal pathway. Mitophagy-Animal Pathway was a significant enrichment pathway for the up-regulated differentially expressed ferroptosis genes (DE-FRGs). HIF-1 signaling pathway is a significant pathway for the down-regulated DE-FRGs. By constructing DE-FRGs protein-protein interaction (PPI) network, 10 hub DE-FRGs (Jun, STAT3, SP1, HIF1A, Mapk1, Mapk3, Rela, Ulk1, CDKN1A, EPAS1) were obtained. The bta-mir-21-5p, bta-mir-10a and bta-mir-17-5p related to STAT3 were predicted. The results of this study indicated the important genes and pathways of the hypoxia in yak, and it was the first time to study ferroptosis genes and pathways related to the hypoxia adaptation by bulk-seq in yak. This study provided sufficient transcriptome datas for hypoxia adaptation.

Effects of an obesogenic diet on the oviduct depend on the duration of feeding.

How long does it take for an obesogenic (high-fat/high-sugar, HF/HS) diet to influence the oviductal microenvironment? What are the affected cellular pathways and are they dependent on the genetic background of the mouse model? Female Swiss (outbred) and C57BL/6N (B6, inbred) mice were fed either a control (10% fat) or HF/HS (60% fat, 20% fructose) diet. Body weight was measured weekly. Mice were sacrificed at 3 days (3d), 1 week (1w), 4w, 8w, 12w and 16w on the diet (n = 5 per treatment per time point). Total cholesterol concentrations and inflammatory cytokines were measured in serum. Oviductal epithelial cells (OECs) were used to study the expression of genes involved in (mitochondrial) oxidative stress (OS), endoplasmic reticulum (ER) stress and inflammation using qPCR. Body weight and blood cholesterol increased significantly in the HF/HS mice in both strains compared to controls. In Swiss mice, HF/HS diet acutely increased ER-stress and OS-related genes in the OECs already after 3d. Subsequently, mitochondrial and cytoplasmic antioxidants were upregulated and ER-stress was alleviated at 1w. After 4-8w (mid-phase), the expression of ER-stress and OS-related genes was increased again and persisted throughout the late-phase (12-16w). Serum inflammatory cytokines and inflammatory marker-gene expression in the OECs were increased only in the late-phase. Some of the OEC stress responses were stronger or earlier in the B6. OECs are sensitive to an obesogenic diet and may exhibit acute stress responses already after a few days of feeding. This may impact the oviductal microenvironment and contribute to diet-induced subfertility.

Chlamydia transmitting from the genital to gastrointestinal tract and inducing tubal disease: Double attack pattern.

Chlamydia trachomatis ( CT ) genital tract infection is insidious, and patients often have no conscious symptoms.Delayed treatment after infection can lead to serious complications. Chlamydia muridarum ( CM ) genital tract infection in female mice can simulate CT genital tract infection in women, which is an ideal model to investigate the pathogenesis of CT . CM plasmid protein pGP3, chromosomal protein TC0237/TC0668, CM -specific CD8 + T cells, TNF-α, and IL-13 can induce genital tract inflammation, CD4 + T cells are responsible for CM clearance. However, tubal inflammation persists after genital tract CM is removed. Genital tract CM can spread spontaneously in vivo and colonize the gastrointestinal (GI) tract, but the GI tract CM cannot reverse spread to the genital tract. The survival time and number of CM transmitted from genital tract to GI tract are positively correlated with the long-term lesion of oviduct, while the CM inoculated directly into the GI tract has no pathogenicity in both the genital and GI tract. The double attack pattern of Chlamydia -induced genital tract inflammatory lesions is as follows: CM infection of oviduct epithelial cells initiates the process of oviduct repair as the first attack. After genital CM spreads to the GI tract, activated chlamydia-specific CD8 + T cells are recruited to the genital tract and secreted pro-fibrotic cytokines such as TNF-α and IL-13. This process is called the second attack which transform tubal repair initiated by the first attack into long-term tubal fibrosis/hydrosalpinx. Elucidating the pathogenic mechanism of Chlamydia infection can provide new ideas for the development of Chlamydia vaccine, which is expected to solve the problems of infertility caused by repeated CT infection in women.

Morphogenesis of the female reproductive tract along antero-posterior and dorso-ventral axes is dependent on Amhr2+ mesenchyme in mice†.

Morphogenesis of the female reproductive tract is regulated by the mesenchyme. However, the identity of the mesenchymal lineage that directs the morphogenesis of the female reproductive tract has not been determined. Using in vivo genetic cell ablation, we identified Amhr2+ mesenchyme as an essential mesenchymal population in patterning the female reproductive tract. After partial ablation of Amhr2+ mesenchymal cells, the oviduct failed to develop its characteristic coiling due to decreased epithelial proliferation and tubule elongation during development. The uterus displayed a reduction in size and showed decreased cellular proliferation in both epithelial and mesenchymal compartments. More importantly, in the uterus, partial ablation of Amhr2+ mesenchyme caused abnormal lumen shape and altered the direction of its long axis from the dorsal-ventral axis to the left-right axis (i.e., perpendicular to the dorsal-ventral axis). Despite these morphological defects, epithelia underwent normal differentiation into secretory and ciliated cells in the oviduct and glandular epithelial cells in the uterus. These results demonstrated that Amhr2+ mesenchyme can direct female reproductive tract morphogenesis by regulating epithelial proliferation and lumen shape without affecting the differentiation of epithelial cell types.

Long-term culture of feline oviduct epithelial cells on permeable filter supports

Basic knowledge about cellular and molecular mechanisms underlying feline reproduction is required to improve reproductive biotechnologies in endangered felids. Commonly, the domestic cat (Felis catus) is used as a model species, but many of the fine-tuned, dynamic reproductive processes can hardly be observed in vivo. This necessitates the development of in vitro models. The oviduct is a central reproductive organ hosting fertilization in the ampulla and early embryonic development in the isthmus part, which also functions as a sperm reservoir before fertilization. In other species, culturing oviduct epithelial cells in compartmentalized culture systems has proven useful to maintain oviduct epithelium polarization and functionality. Therefore, we made the first attempt to establish a compartmentalized long-term culture system of feline oviduct epithelial cells from both ampulla and isthmus. Cells were isolated from tissue samples (n = 33 animals) after routine gonadectomy, seeded on permeable filter supports and cultured at the liquid–liquid or air–liquid interface. Cultures were harvested after 21 days and microscopically evaluated for epithelial differentiation (monolayer formation with basal–apical polarization) and protein expression of marker genes (oviduct-specific glycoprotein, acetylated tubulin). Due to the heterogeneous and undefined native tissue material available for this study, the applied cell culture approach was only successful in a limited number of cases (five differentiated cultures). Even though the protocol needs optimization, our study showed that the compartmentalized culture approach is suitable for maintaining differentiated epithelial cells from both isthmus and ampulla of the feline oviduct.

Oviduct-mimicking microfluidic chips decreased the ROS concentration in the in vitro fertilized embryos of CD-1 mice

BackgroundThe process of the assisted reproductive technology (ART) cycle is extremely complicated, and various factors in each step may influence the final clinical outcomes; thus, optimizing culture conditions for embryos is crucial in the ART cycle, particularly when the traditional petri-dish method remains unchanged for decades. In the current study, we intend to culture embryos in a dynamic environment on chips to optimize the embryo culture conditions. MethodsMultilayer soft lithography technology was utilized to establish a microfluidics-based oviduct. Mouse primary oviduct epithelial cells were identified by immunofluorescence staining and then loaded into the chip to coculture with the embryos. The development potential parameters of embryos on chips with cells, on chips without cells, and in drops were compared, as well as reactive oxygen species (ROS) in embryos. ResultsThere were no obvious differences regarding the fertilization rate, 4-Cell embryo rate, cleavage rate, high-quality embryo rate, or blastocyst formation rate. However, the intracellular ROS levels in 4-Cell stage embryos on chips with cells were statistically significantly lower than those in drops (P < 0.001). This organ-on-chip device allowed the probability of mammalian embryo culture in a microfluidic-based manner. ConclusionsOur findings demonstrated that this novel oviduct-on-chip model may optimize embryo culture conditions by reducing intracellular ROS levels, which may be a competent alternative to the existing stable embryo culture system.

Evaluation of sperm integrin α5β1 as a potential marker of fertility in humans.

Sperm selection for assisted reproduction techniques is generally based on basic parameters, while key aspects of sperm competence and its journey from the deposition site to the fertilization site are overlooked. Consequently, identifying molecular markers in spermatozoa that can efficiently predict the fertility of a semen sample could be of great interest, particularly in cases of idiopathic male infertility. When spermatozoa reach the female reproductive tract, it provides to them the cellular and molecular microenvironment needed to acquire fertilizing ability. In this sense, considering the role that integrin α5β1 of spermatozoa plays in reproduction-related events, we investigated the correlation between the subcellular localization of sperm integrin α5β1 and early embryo development outcome after in vitro fertilization (IVF) procedures in human. Twenty-four semen samples from normozoospermic men and metaphase II (MII) oocytes from healthy women aged under 38 years, from couples who underwent IVF cycles, were used in this work. Sperm α5β1 localization was evaluated by immunofluorescence assay using an antibody against integrin α5 subunit. Integrin α5β1 was mainly localized in the sperm acrosomal region (45.33±7.89%) or the equatorial segment (30.12±7.43%). The early embryo development rate (data obtained from the Fertility Center) correlated positively with the localization of α5β1 in the acrosomal region (number of usable embryos / inseminated oocytes: ρ = 0.75; p<0.01 and number of usable embryos/total number of two pronuclear zygotes: ρ = 0.80; p<0.01). However, this correlation was not significant when the equatorial segment mark was evaluated. In addition, human sperm released from co-culture with bovine oviductal epithelial cells (BOEC) showed a significant enrichment in the acrosomal localization pattern of α5β1 compared to those sperm that were not co-cultured with BOEC (85.20±5.35% vs 35.00±17.09%, respectively, p<0.05). In conclusion, the evaluation of sperm integrin α5β1 immunolocalization could be a useful tool to select sperm with fertilizing ability from human semen samples before IVF procedures.

The role of mesenchymal stem cells in the ovarian cancer precarcinoma microenvironment (091)

<b>Objectives:</b> Research on ovarian cancer (OC) initiation has primarily focused on the transformation from normal epithelium to invasive serous carcinoma; however, the role of the stromal microenvironment in cancer initiation is largely unexplored. We previously demonstrated that mesenchymal stem cells (MSCs), stromal progenitor cells in the OC microenvironment, are reprogrammed by tumor cells into cancer-associated MSCs (CA-MSCs). These reprogrammed CA-MSCs promote cancer growth, chemoresistance, and metastases. We sought to determine if changes in the stromal microenvironment occur prior to OC initiation. We identified a subset of MSCs in the fallopian tubes of women with germline <i>BRCA</i> mutations without cancer that have acquired a CA-MSC phenotype, which we term high-risk MSCs (HR-MSCs). We hypothesized that HR-MSCs acquire a tumor supportive phenotype prior to OC initiation that enhances the transformation of the normal epithelium into carcinoma. <b>Methods:</b> MSCs and fallopian tube epithelial cells (FTE) were isolated from fallopian tubes of women with germline <i>BRCA</i> mutations undergoing risk-reducing salpingo-oophorectomies without findings of cancer or pre-cancerous lesions upon standard pathologic examination. MSCs were characterized as low-risk (LR-MSCs) or high-risk (HR-MSCs) using a previously described MSC classifier score. Co-cultures were performed using normal FTE, <i>BRCA</i>-mutated (<i>BRCA</i>mut) FTE, and <i>BRCA</i>/p53-mutated FTE with LR-MSCs or HR-MSCs. FTE and MSC growth and proliferation were assessed using <i>in vitro</i> assays. We investigated the impact of HR-MSCs on FTE DNA damage after co-culture using FTE alone as control with yH2AX foci quantification. Long-term organoid co-cultures of MSCs and FTE were implanted into NSG mice to assess tumor initiation capacity. <b>Results:</b> HR-MSCs significantly enhance <i>BRCA</i>mut FTE growth compared to LR-MSCs. HR-MSCs also induced DNA damage in FTE. Strikingly, DNA damage induced by HR-MSCs was similar to that seen with hydrogen peroxide (H2O2) treatment of FTE. HR-MSCs also increased the survival of <i>BRCA</i>mut FTE after the induction of DNA damage by H2O2 treatment, indicating that HR-MSCs induce FTE DNA damage and promote survival of DNA-damaged FTE. HR-MSCs were also able to induce full malignant transformation as injecting HR-MSCs + <i>BRCA</i>mut FTE organoids-initiated tumors in one of three mice, and HR-MSCs + <i>BRCA</i>/p53-mut FTE organoids-initiated tumors in two of three mice (one of which developed metastasis). No mice injected with LR-MSCs or FTE alone developed tumors. <b>Conclusions:</b> A subset of women with <i>BRCA</i> mutations at high risk for OC contains fallopian tube-derived MSCs, which functionally support ovarian cancer initiation. These HR-MSCs increase FTE proliferation, induce FTE DNA damage, enhance survival of DNA-damaged FTE, and ultimately have the capacity to induce full malignant transformation with tumor initiation and metastasis <i>in vivo</i>. This highlights a critical role for the stromal microenvironment in OC initiation.Fig. 1