Oviduct Tissue Research Articles

Identification of inorganic particles resulting from degradation of ESSURE® implants: Study of 10 cases

ObjectiveApproximately 750,000 women worldwide have undergone ESSURE hysteroscopic sterilization since 2002. In 2015, an increase in adverse effects was noted, with gynaecological and systemic symptoms reported. Scanning electron microscopy (SEM) analysis of fallopian tube and uterine horn tissues and implants, after hysterectomy or salpingectomy, revealed the presence of inorganic particles resulting from implant degradation. Study designTen patients (age 42–53 years) were included in this study. Of these, eight patients had undergone hysterectomy and two patients had undergone salpingectomy. Mean exposure time was 85.5 months (standard deviation 26.8 months, range 34–105 months). Mineralogical analyses were performed on 13 tissue biopsies and four implants by SEM coupled with energy dispersive x-ray spectrometry. ResultsIn five of the 10 patients, tin particles were observed in fallopian tube or uterine horn tissues with inflammatory cell reactions. In the other five cases, iron, chromium, nickel or platinum particles were observed. For implants, major deterioration of the weld zone was observed with either destroyed appearance or the presence of an organic coating containing numerous particles. Discussion and conclusionAnalysis of the preclinical studies performed by the manufacturer suggests that degradation of the tin weld plays a major role in these adverse events, with increasing leaching and corrosion between 3 and 6 months for an intratubal insert that si designed to remain in an woman’s body for her entire life. For patients with gynaecological symptoms (e.g. pain, metrorragies) needing explantation, these findings raise the question of a causal relationship between tin particles from implant degradation and the inflammatory tissue response. For patients with systemic symptoms (e.g. blurred vision, headache, asthenia, myalgia), the hypothesis that these symptoms may be related to the formation of organotin (chemical compounds based on tin with hydrocarbon substituents) in the body has yet to be proven. Tin levels in blood have to be measured before and after explantation. To the authors’ knowledge, this is the first study to report significant degradation of the ESSURE implant weld, evidenced by the detection of tin particles in the uterine tissue of patients and comparison of the welding zone between unused and used implants.

Localization and changes of tyrosine phosphorylated protein expressions in female reproductive tracts of PCOS rats

Polycystic ovary syndrome (PCOS) is a cause of female infertility and its mechanism is still unclear. Tyrosine phosphorylated (TyrPho) proteins have been shown to be important for male infertility. However, the biological functions of TyrPho proteins involved in PCOS condition have never been reported. This study aimed to localize and investigate the TyrPho protein expression in PCOS rats. Twenty female Sprague‐Dawley rats were divided into control and PCOS (induced by letrozole, 1 mg/KgBW, for 21 consecutive days) groups. The estrus cycle of animals was confirmed by vaginal smear daily. At the end of experiment, ovary, oviduct, and uterus tissues of both groups were localized for TyrPho proteins by immunohistochemistry. In addition, the expression of TyrPho proteins in reproductive tissues were investigated and quantified. The results showed that TyrPho proteins were localized in the developing ovarian follicles and epithelial cells of oviduct and uterus. Moreover, TyrPho proteins were significantly overexpressed in ovary (68, 52, 42 and 28 kDas), oviduct (56 and 42 kDa), and uterus (170, 53 and 42 kDas) of PCOS group as compared to control. We concluded that female reproductive tracts have tyrosine phosphorylation essential for female fertility.Support or Funding InformationKhon Kaen University

Local influence of the corpus luteum on the ipsilateral oviduct and early embryo development in the ewe

The objective of this study was to evaluate the local effect of the corpus luteum (CL) on ipsilateral oviduct-uterus functionality and early embryo development in ewes. A total of 499 embryos were transferred on Day 1 after in vitro fertilization into the ipsilateral (n = 250) and contralateral oviducts (n = 249) of 13 ewes on Day 1 after ovulation (18-20 embryos per oviduct). On Day 6, their reproductive tracts were collected and their uterine horns were flushed for embryo recovery. More recovered embryos, a higher proportion of blastocysts, and more viable embryos were collected when the embryos were transferred into the ipsilateral oviducts (P < 0.05). In addition, almost five times higher P4 concentrations and significantly lower E2 concentrations, with higher P4:E2 ratio, were found in the ipsilateral than contralateral oviductal tissue (P < 0.05). Furthermore, a higher concentration of adiponectin was found in the ipsilateral uterine tissue macerates than in the contralateral side to the CL. The ipsilateral oviductal tissue had a lower expression of PGR and IGFBP5, but the transcript expression of ADIPOR1 was higher in the ipsilateral oviductal tissue. In the uterus, the mRNA expression of ESR1, IGFBP3, IGFBP5, and LEPR was higher or tended to be higher in the ipsilateral than contralateral uterine tissue. Uterine flushing fluid collected from the ipsilateral uterine horn had lower insulin concentrations than the contralateral horn, while no differences were found in the P4 and E2 concentrations. In conclusion, on Day 6 post-ovulation, P4 was elevated in the ipsilateral oviductal tissue, embryo development was advanced, and differential gene expression of PGR, ESR1, IGFBP3, IGFBP5, LEPR, and ADIPOR1 in the oviductal or uterine tissue was found between the ipsilateral and contralateral side. This study demonstrates local regulation of the ovary on the ipsilateral oviduct/uterine horn in the ewe.

Glyco-scope into the Role of Protein Glycosylation in the Female Reproductive Tract

Aberrant glycosylation, in abundance or type, influences the overall physiology of tissues and the functions of proteins. This review is focused on the role of glycosylation in the reproductive process in the female reproductive tract from a glycoprotein perspec-tive. Many biological events are essential to allow for sperm-egg fertilisation in oviduct and subsequent blastocyst implantation in a receptive uterus during the window of implantation, including adhesion and proliferation. The process of blastocyst implantation is managed by a series of orchestrated events among several glycoproteins found on the surface of oviduct and uterine tissues. A change in the molecular surface of the oviduct and uterine tissues, such as the glycosylation of its membrane proteins, would affect the blas-tocyst journey through the female reproductive tract. In particular, the glycosylation presented by the mucins on the surface of the oviduct and uterus in diabetes, that is associated with reduced embryo implantation rates, is highlighted since these heavily glycosyl-ated proteins have been shown to play an important role in blastocyst implantation. An understanding of the glycosylation changes to the surface of the oviduct and uterus has potential for the development of a test for uterine receptivity and may provide the next leap forward for the treatment of infertility.

Factors in Oncogenesis: Viral Infections in Ovarian Cancer.

Ovarian cancer (OC) is one of the leading causes of cancer death in women, with high-grade serous ovarian cancer (HGSOC) being the most lethal gynecologic malignancy among women. This high fatality rate is the result of diagnosis of a high number of new cases when cancer implants have already spread. The poor prognosis is due to our inadequate understanding of the molecular mechanisms preceding ovarian malignancy. Knowledge about the site of origination has been improved recently by the discovery of tube intraepithelial cancer (TIC), but the potential risk factors are still obscure. Due to high tumoral heterogeneity in OC, the establishment of early stage biomarkers is still underway. Microbial infection may induce or result in chronic inflammatory infection and in the pathogenesis of cancers. Microbiome research has shed light on the relationships between the host and microbiota, as well as the direct roles of host pathogens in cancer development, progression, and drug efficacy. While controversial, the detection of viruses within ovarian malignancies and fallopian tube tissues suggests that these pathogens may play a role in the development of OC. Genomic and proteomic approaches have enhanced the methods for identifying candidates in early screening. This article summarizes the existing knowledge related to the molecular mechanisms that lead to tumorigenesis in the ovary, as well as the viruses detected in OC cases and how they may elevate this process.

Bona fide gene expression analysis of samples from the bovine reproductive system by microfluidic platform

Sample types such as those from reproductive systems often yield scarce material, which limits RT-qPCR analysis to only a few targets. Recently developed high-throughput systems can potentially change this scenario, however, the nanoliter scale of such platforms requires extra processing, e.g., preamplification, which needs to be defined through observation and experience. In order to establish best practices in high-throughput PCR approaches using samples from reproductive systems, we evaluated the Biomark™ HD performance using 11 different sample types from the bovine reproductive system: blastocyst (single/pool), oocyte (pool), cumulus, granulosa, and theca cells, oviduct tissue, fetal ovary, testicle (adult/fetal), and uterine horn. We observed that the preamplification step is not just reliable, but mandatory. Our results indicated that 14-preamplification cycles associated to 5- and 7-fold-dilution is the best approach for those samples. Additionally, the Biomark™ HD system has a high intra and inter reproducibility, therefore its performance in duplicate is unnecessary for the ΔCq analysis, taking in consideration the cutoff value 4 < Cq < 22. In summary, this high-throughput approach is a reliable and excellent tool for studying the bovine reproductive system, especially using quantitatively-limited samples, as a larger number of target genes can be assessed from a very low amount of starting material.

Construction and Investigation of an LINC00284-Associated Regulatory Network in Serous Ovarian Carcinoma.

The low survival rate associated with serous ovarian carcinoma (SOC) is largely due to the lack of relevant molecular markers for early detection and therapy. Increasing experimental evidence has demonstrated that long noncoding RNAs (lncRNAs) are involved in cancer initiation and development, and a competitive endogenous RNA (ceRNA) hypothesis has been formulated. Therefore, the characterization of new lncRNA and lncRNA-related networks is crucial for early diagnosis and targeted therapy of SOC. Data on lncRNAs, mRNAs, and miRNAs with differential expression in SOC, compared to normal ovarian tissue, were obtained from the Gene Expression Omnibus (GEO) database. Data on lncRNA expression and clinical data in SOC were obtained from The Cancer Genome Atlas (TCGA). lncRNA-miRNA interactions were predicted by the miRBase database. Different online tools, i.e., TargetScan, RNA22, miRmap, microT, miRanda, StarBase, and PicTar, were cooperatively utilized to predict the mRNAs targeted by miRNAs. The plugin of BiNGO in Cytoscape and KOBAS 3.0 were used to conduct the functional and pathway enrichment analyses. The lncRNA, miRNAs, and mRNAs identified to be expressed at statistically significant and different levels between SOC and healthy fallopian tube tissues were further validated using qRT-PCR. A total of 4 lncRNAs (LINC00284, HAGLR, HCAT158, and BLACAT1) and 111 mRNAs were found to be upregulated in SOC tissues compared to normal tissues, based on the GEO database. LINC00284 was found to be highly expressed in SOC, in association with the upregulation of the transcription factor SOX9. The high LINC00284 expression was associated with poor prognosis and proved to be an independent risk factor in patients with SOC, based on TCGA database. The qRT-PCR validation results closely recapitulated the expression profiles and prognostic scores of the aforementioned bioinformatic analyses. The LINC00284-related ceRNA network was found to be associated with SOC carcinogenesis by biofunctional analysis. In conclusion, the LINC00284-related ceRNA network may provide valuable information on the mechanisms of SOC initiation and progression. Importantly, LINC00284 proved to be a new potential prognostic biomarker for SOC.

Relative mRNA expression of CRBP IV gene and its association with layer economic traits in Rhode Island Red chicken

Cellular retinol-binding protein (CRBP) IV gene plays an important role in absorption, transport, metabolism and homeostasis of retinol (Vitamin A) and its derivatives. Vitamin A plays vital role in ovarian follicular growth, oviduct and uterine environments, embryo and its development, and oocyte maturation. Present investigation was carried out in Rhode Island Red (RIR) chicken to assess relative mRNA expression of CRBP IV gene in kidney, liver and oviduct tissues, collected from 12 birds at 40 weeks of age from four different egg production-body weight groups, viz. high egg production-high body weight (HEP-HBW), high egg production-low body weight (HEP-LBW), low egg production-high body weight (LEP-HBW) and low egg production-low body weight (LEPLBW), using qRT-PCR method. Data on body weight at 20 weeks of age (BW20) and layer traits, viz. age at sexual maturity (ASM), egg weight at 28 (EW28) and 40 (EW40) weeks and egg production up to 40 weeks of age (EP40) were analyzed by LS ANOVA taking sire as random and hatch as fixed effects. LS means of ASM, BW20, EW28, EW40 and EP40 were 135.19±1.15 days, 1347.13±15.28 g, 42.49±0.27 g, 48.19±0.42 g and 124.55±1.94 eggs, respectively. Expression data was analyzed by LS ANOVA using SAS. The basal mRNA expression of CRBP IV gene differed significantly among different egg production-body weight groups in kidney only. The 40-ΔCt values of mRNA expression levels of CRBP IV gene in the four groups were 35.81±1.60, 29.49±1.60, 32.16±1.60 and 39.02±1.60, respectively. Significantly lower expression was observed in HEP-LBW group. Similarly, tissues differed significantly for the CRBP IV gene expression. The 40-ΔCt values of mRNA expression levels of CRBP IV gene in kidney, liver and oviduct were 34.12±0.87, 31.67±0.87 and 37.97±0.87, respectively. Highest expression was observed in oviduct. The study generated significant information on basal mRNA expression of CRBP IV gene in poultry which may have great potential for use in selection for higher reproductive efficiencies.

The evaluation of the oxidative stress index of reproductive tissues and serum thiol/disulphide homeostasis during estrous cycle in bitches

The aim of this study was firstly to determine the relationship between estrous cycle and oxidative stress in reproductive tissues in bitches. This research was performed in twenty-nine healthy bitches from different breeds and of varied ages (the range was 2-5 years) that were brought to the clinic for routine ovariohysterectomy. The stages of estrous cycle were detected using by vaginal cytology, blood progesterone level and histological findings. Ovarian, oviduct and uterine tissues were taken into Eppendorf tubes for oxidative stress index and stored at -80°C until analyses. Also, another part of these tissues were fixed in 10% formalin solution. There were no significant differences (P &amp;gt; 0.05) among total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) values in the reproductive tissues in concerning estrous cycle stages. However, there were remarkable correlations between oxidative stress parameters and reproductive tissues in different estrus stages in bitches. In conclusion, the physiological values of TAS and TOS concentration, and OSI in the ovarian, oviduct and uterine tissues during estrous cycle were firstly defined in this article. The serum thiol/disulphide homeostasis was also determined in the estrous cycle of bitches. We have found that there are remarkable variations of oxidative stress balance in the ovary, oviduct and uterus concurrently, during estrous cycle in the bitches. Besides, significant correlations between oxidative stress parameters and estrous cycle stages in the reproductive tissues were observed in the present study.

Alpine and lowland grazing differentially alter the reproductive tract redox milieu and amino acid composition in cattle

An alpine environment is unique due to pasture biodiversity, with an abundant content of natural antioxidant polyphenols. The present study investigated the effects of lowland and alpine grazing on the oviduct and uterine tissue redox status and amino acid concentrations in plasma and reproductive fluids. In the first experiment, heifers grazed on lowland (H-LOW: n = 13) and on alpine (H-ALP: n = 15) pastures. In the second experiment, heifers grazed on the same lowland (HS-LOW: n = 6) and on a different alpine (HS-ALP: n = 6) pasture. The abundance of mRNA transcripts for antioxidant enzymes in the oviduct (glutathione S-transferase alpha 2, glutathione synthetase (GSS)) and the endometrium (catalase, glutathione-disulfide reductase, GSS) was less (P < 0.05), and for glutathione peroxidase 4 in the endometrium greater (P = 0.006) in the H-LOW than in the H-ALP group. The abundance of mRNA transcript for catalase was less in the endometrium in the H-LOW than in the H-ALP (P = 0.001) group. Catalase and NAD(P)H quinone dehydrogenase 1 concentrations in the oviduct were greater in the HS-LOW than in the HS-ALP group (P < 0.05). Of 32 amino acids analysed, there were differences in concentrations in the H-LOW and H-ALP group of 13, seven and 15 in plasma, oviduct and uterine fluids, respectively (P < 0.05). Comparing the HS-LOW to the HS-ALP groups, there were 13, one and three amino acids in the plasma, oviduct and uterine fluids, respectively, that were differentially abundant (P < 0.05). The grazing systems had some effect on the redox status and amino acid patterns in reproductive tissues.

190 Effect of TCM-199 and synthetic oviductal fluid medium supplemented with varying hormone concentrations on invitro maturation of canine oocytes

Invitro oocyte maturation (IVM) in the domestic canine is yet to be optimized, with low rates of cumulus-oocyte complexes (COCs) reaching MII. This limits the progression of assisted reproductive technologies, which could benefit breeding programs for assistance dogs and endangered Canidae. Canine oocyte maturation differs from that in other mammals, with the ovulation of a COC in the germinal vesicle stage and nuclear maturation occurring in the oviduct. Because of this, the environment in which a canine COC matures is unlike that of other mammals, meaning that IVM protocols cannot be readily adapted. The aim of the current work was to determine (1) the effects of varying concentrations of FSH, human chorionic gonadotrophin (hCG), and oestradiol (E2) during IVM on meiotic resumption and nuclear maturation of canine COCs; and (2) the optimal medium base, either synthetic oviductal fluid (SOF) or tissue culture medium-199 (TCM). Reproductive tracts of bitches (6 months to 7 years of age) were collected from veterinary clinics within 2h of routine spaying. Ovaries were sliced using a scalpel blade, releasing the COCs into aspiration medium. The COCs were randomly allocated to a maturation medium consisting of one of the hormones at two concentrations (FSH: 5 or 10µgmL−1; hCG: 5 or 10IUmL−1; E2: 1 or 5µgmL−1) and for both SOF and TCM base. Each hormone was tested individually for a replicate of eight animals per hormone (total of 12 experimental groups; 24 animals). The COCs were cultured for 72h in their allocated medium and then denuded and stained with Hoechst 33258. Fluorescence microscopy was used to determine nuclear maturation stage. Nuclear maturation rates to MII were analysed using a general linear model with pairwise comparison (SPSS version 25; SPSS Inc./IBM Corp.) with each individual animal acting as a replicate. Canine COCs matured in a SOF-based media had higher rates of meiotic resumption (MI and MII) (SOF: 38.68%, n=515; TCM: 25.78%, n=542; P&amp;lt;0.05) and number reaching MII (SOF: 7.54%; TCM: 4.39%; P&amp;lt;0.05) compared with TCM-based medium. Resumption of meiosis and nuclear maturation to MII did not differ between media with differing E2 or hCG concentrations. The use of FSH at 10µgmL−1 in SOF medium decreased resumption of meiosis (8.57%) and MII rates (0%) compared with 5µgmL−1 FSH in SOF (29.41% and 3.92%, respectively; P&amp;lt;0.05). In summary, our data indicated that higher concentrations of FSH during IVM have a negative effect on meiotic resumption and maturation to MII, whereas canine COCs resume meiosis and mature to MII in higher rates in a SOF-based medium compared with a TCM base. An IVM medium that replicates the invivo environment in which canine COCs mature is beneficial. However, rates of IVM canine oocytes reaching maturity are low, signifying that future research must investigate a greater range of hormone concentrations and combinations to better mimic invivo conditions to assess the possible benefits for canine IVM.

Kallistatin inhibits tumour progression and platinum resistance in high-grade serous ovarian cancer

Ovarian cancer is the most lethal gynaecologic malignancy. Although there are various subtypes of ovarian cancer, high-grade serous ovarian cancer (HGSOC) accounts for 70% of ovarian cancer deaths. Chemoresistance is the primary reason for the unfavourable prognosis of HGSOC. Kallistatin (KAL), also known as SERPINA4, is part of the serpin family. Kallistatin has been discovered to exert multiple effects on angiogenesis, inflammation and tumour progression. However, the roles and clinical significance of kallistatin in HGSOC remain unclear. Here, we showed that kallistatin was significantly downregulated in HGSOC compared to normal fallopian tube (FT) tissues. Low expression of kallistatin was associated with unfavourable prognosis and platinum resistance in HGSOC. Overexpression of kallistatin significantly inhibited proliferation and metastasis, and enhanced platinum sensitivity and apoptosis in ovarian cancer cells. Collectively, these findings demonstrate that kallistatin serves as a prognostic predictor and provide a potential therapeutic target for HGSOC.

Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients

Viral and bacterial infections are detected in epithelial ovarian cancer (EOC) tissues. Since the fallopian tubes are often affected by pelvic inflammatory disease (PID) and the majority of serous EOCs appear to originate from dysplastic lesions in the distal tube, it is relevant to consider the potential role that infectious agents may play in ovarian carcinogenesis. We sought to analyze the prevalence of human papillomavirus (HPV) and cytomegalovirus (CMV) in EOC tissue and fallopian tube specimens obtained at tumor resection. Ovarian cancer and fallopian tube tissue samples obtained from patients with EOC were analyzed by both qualitative and quantitative PCR to detect and quantify viral DNA. The presence of CMV and HPV DNA was detected in 70% and 74% cancerous ovarian tissues, respectively, and was significantly higher in EOC than in benign tumor cases (P ≤ 0.01). CMV or HPV infection was observed also in the fallopian tube samples. Infection with HPV16 was determined in 70% of EOC cases. Almost two thirds of EOC patients demonstrated coinfection with CMV and HPV in the pathological samples. The results revealed that the presence of CMV and HPV in EOC samples is common. CMV and HPV infections can be potential risks for EOC development.

Expression analysis of BMPR1B, BMP15, and GDF9 in prolific and non-prolific sheep breeds during the follicular phase

To elucidate how expression characteristics of BMPR1B, BMP15, and GDF9 are associated with sheep reproduction, tissue-specific mRNA expression of these genes in Small Tail Han (STH) ewes (a polytocous breed) and Sunite (SNT) ewes (a monotocous breed) in the follicular phase were investigated using transcription profiling and quantitative real-time PCR (qPCR). Expression levels of the three genes were all highest in ovaries of the two sheep breeds, and BMPR1B and GDF9 expression in the ovarian tissue was significantly higher in STH sheep compared with SNT sheep (P &amp;lt; 0.01), whereas BMP15 expression in pituitary, ovarian, oviduct, and uterine tissues was significantly lower in STH sheep compared with SNT sheep (P &amp;lt; 0.01). This study revealed that BMPR1B, BMP15, and GDF9 may play important roles in the hypothalamic-pituitary-gonadal axis, especially in the ovary, which may affect sheep prolificacy. BMPR1B and GDF9 expression might increase litter size, whereas BMP15 expression may decrease litter size. These results provide preliminary information regarding molecular regulatory mechanisms underlying sheep prolificacy.

Differential DNA methylation in high-grade serous ovarian cancer (HGSOC) is associated with tumor behavior

The epigenome offers an additional facet of cancer that can help categorize patients into those at risk of disease, recurrence, or treatment failure. We conducted a retrospective, nested, case-control study of advanced and recurrent high-grade serous ovarian cancer (HGSOC) patients in which we assessed epigenome-wide association using Illumina methylationEPIC arrays to characterize DNA methylation status and RNAseq to evaluate gene expression. Comparing HGSOC tumors with normal fallopian tube tissues we observe global hypomethylation but with skewing towards hypermethylation when interrogating gene promoters. In total, 5,852 gene interrogating probes revealed significantly different methylation. Within HGSOC, 57 probes highlighting 17 genes displayed significant differential DNA methylation between primary and recurrent disease. Between optimal vs suboptimal surgical outcomes 99 probes displayed significantly different methylation but only 29 genes showed an inverse correlation between methylation status and gene expression. Overall, differentially methylated genes point to several pathways including RAS as well as hippo signaling in normal vs primary HGSOC; valine, leucine, and isoleucine degradation and endocytosis in primary vs recurrent HGSOC; and pathways containing immune driver genes in optimal vs suboptimal surgical outcomes. Thus, differential DNA methylation identified numerous genes that could serve as potential biomarkers and/or therapeutic targets in HGSOC.

High-grade Pelvic Serous Carcinoma Within the Fallopian Tube Lumen: Real or Artifact?

Tumor cells are occasionally observed in the lumen in histologic sections of the fallopian tube from women with gynecologic cancer. There is some evidence that this finding may be important in endometrial cancer, but its significance is unknown in women with extrauterine pelvic serous carcinomas (tubo-ovarian high-grade serous carcinoma). Fallopian tube sections from 213 women with extrauterine pelvic serous carcinoma were reviewed, and luminal tumor cells were correlated with clinical and pathologic features. Intraluminal tumor cells were found in 84 patients (39%). The presence or absence of luminal tumor cells correlated significantly with serous tubal intraepithelial carcinoma (52% and 33%, respectively, P=0.004), tubal lymphatic invasion (32% and 12%, respectively, P=0.0002), and number of tube sections reviewed (6.6 and 4.9 for lumen-positive and lumen-negative cases, respectively, P=0.0056). There was no correlation with the presence of ascites, peritoneal cytopathologic findings, lymph node metastases, or FIGO stage. In the setting of pelvic serous carcinoma, a substantial portion of fallopian tube tissue is often distorted, fibrotic, and difficult to identify. Since the identification of luminal tumor cells, serous tubal intraepithelial carcinoma and tubal lymphatic invasion all depend on identification of fallopian tube tissue, these correlates with luminal tumor cells could be a result of a higher likelihood of their observation when tubal tissue can be more readily identified and may not necessarily reflect a biologically important phenomenon. It remains unclear whether and in what proportion this finding reflects an artifact of specimen handling.

Abstract GMM-030: MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS

Abstract INTRODUCTION: Ovarian cancer is the 5th deadliest cancer found in women and is the deadliest involving the gynecological tract. Most epithelial ovarian cancers have extra-ovarian origins and can be stratified into various histotypes: high and low-grade serous (HGS and LGS), endometrioid (ENOC), clear cell (CCOC), and mucinous – each of which are proposed to have distinct precursor lesions. We present organoids as a useful model to study precursor lesions and the process of tumorigenesis in epithelial ovarian carcinomas. Organoids recapitulate the in vivo growth microenvironment and are useful to study the mechanisms of tumorigenesis from healthy cells. We have previously proposed that ENOC arise from the secretory cell lineage, while CCOC originate from the ciliated cell lineage, and organoids are an ideal model to examine in greater depth the impact of mutation on specific cell populations, such as ciliated cells. METHODS: Surgical fallopian tube and endometrial tissues, removed for non-cancer reasons, were cultured in 2D followed by plating into Matrigel. Matrigel cultures were supplemented with media containing stem/progenitor differentiation factors promoting organoid growth. To study the effect of mutations often found in ovarian cancers on organoid growth and development, gene knockouts were produced using CRISPR lentiviruses on cells prior to Matrigel culture. Lentiviral transductions were optimized for organoid formation and for minimizing invasiveness accrued on cells. CRISPR gRNA constructs were validated by Western Blot and qPCR. Organoids containing knockouts of p53, BRCA1 and BRCA2 were used to model precursor lesions of HGS, whereas ARID1A knockouts and an inducible PIK3CA activating mutations were used to model CCOC. To gain further insight into ciliated cells of the endometrium, organoids were treated with the notch inhibitor-DBZ to drive differentiation of cells towards a ciliated cell lineage. We analyzed organoids by single-cell RNA sequencing (scRNA-seq), immunohistochemistry (IHC), and immunofluorescence staining (IF). Single cells were derived by purifying the organoids from Matrigel followed by a chemical and physical digestion. scRNA-seq was performed utilizing the 10X Genomics Platform and analyzed by in-house bioinformaticians. Bioinformatic analyses included stringent QC to remove low-quality and dead cells, before applying unsupervised learning algorithms like PCA and Gaussian mixture modeling as well as differential expression analysis to understand both how samples relate to each other and cell types discovered within each sample. RESULTS: We successfully recapitulated the histology observed in tissues by growing endometrial and fallopian tube organoids. The notch inhibitor, DBZ forced ciliated cell differentiation, as observed by IHC, IF and scRNA-seq. scRNA-seq clustering of DBZ-treated organoid cultures revealed a possible intermediary state between progenitor and ciliated cells. Initial IHC and IF analyses of CRISPR-mediated organoids reveal successful gene manipulation. CONCLUSIONS: Organoid cultures present as a powerful method for modelling precursor lesions; they can be readily manipulated genetically and with rapid turnaround compared to conventional mouse models. Organoids are also amenable to sequencing at single-cell resolution. The ability to model ovarian cancers with permanent knockouts in human tissue serves as a necessary link between animal models and human therapy. Citation Format: Germain C. Ho, Dawn R. Cochrane, Evan W. Gibbard, Kieran Campbell, Basile Tessier-Cloutier, Kendall Greening, Forouh Kalantari, Genny Trigo-Gonzalez, Yemin Wang, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-030.

Sperm Gatekeeping: 3D Imaging Reveals a Constricted Entrance to Zebra Finch Sperm Storage Tubules

Females across many internally fertilizing taxa store sperm, often in specialized storage organs in their reproductive tracts. In birds, several hundred sperm storage tubules exist in the utero-vaginal junction of the oviduct, and there is growing evidence that sperm storage in these tubules is selective. The mechanisms underlying female sperm storage in birds remain unknown because of our limited ability to make three-dimensional, live observations inside the large, muscular avian oviduct. Here, we describe a new application of fluorescence selective plane illumination microscopy to optically section oviduct tissue from zebra finch Taeniopygia guttata females label free by harnessing tissue autofluorescence. Our data provide the first description of the three-dimensional structure of sperm storage organs in any vertebrate to the best of our knowledge and reveal the presence of gate-like constricted openings that may play a role in sperm selection.

Optical Resolution Photoacoustic Microscopy of Ovary and Fallopian Tube

Ovarian cancer is the leading cause of death among gynecological cancers, but is poorly amenable to preoperative diagnosis. In this study, we investigate the feasibility of “optical biopsy,” using high-optical-resolution photoacoustic microscopy (OR-PAM) to quantify the microvasculature of ovarian and fallopian tube tissue. The technique is demonstrated using excised human ovary and fallopian tube specimens imaged immediately after surgery. Quantitative parameters are derived using Amira software. The parameters include three-dimensional vascular segment count, total volume and length, which are associated with tumor angiogenesis. Qualitative results of OR-PAM demonstrate that malignant ovarian tissue has larger and more tortuous blood vessels as well as smaller vessels of different sizes, while benign and normal ovarian tissue has smaller vessels of uniform size. Quantitative analysis shows that malignant ovaries have greater tumor vessel volume, length and number of segments, as compared with benign and normal ovaries. The vascular pattern of benign fallopian tube is different than that of benign ovarian tissue. Our initial results demonstrate the potential of OR-PAM as an imaging tool for fast assessment of ovarian tissue and fallopian tube and could avoid unnecessary surgery if the risk of the examined ovary is extremely low.

Clinicopathological study of SET subtype of ovarian high-grade serous carcinoma

Objective: To investigate the clinicopathological characteristics and significance of solid, endometrioid and transitional (SET) ovarian high-grade serous carcinoma (HGSC). Methods: A total of 408 cases of ovarian HGSC admitted to Peking University People's Hospital from January 2011 to September 2016 were collected. (1) According to the proportion of tumors with SET form in all tumors, they were divided into three groups: HGSC-classic group (<25%), HGSC-SET Ⅰ (25%-50%) and HGSC-SET Ⅱ (>50%) group. The clinical and pathological characteristics of three groups of ovarian HGSC patients were compared respectively. (2) According to the growth pattern, that was, the proportion of pushing/expanding invasive tumors in the whole pelvic disseminated tumors of pelvic disseminated tumors, the three groups were divided into four subgroups: group A (0-25%), group B (26%-50%), group C (51%-75%) and group D (>75%). Differences in progression-free survival (PFS) among the four subgroups in each group were compared respectively. Results: The median age of 408 cases with ovarian HGSC was 63.3 years (47-78 years), including 152 cases premenopausal and 256 cases postmenopausal. Among 408 cases of ovarian HGSC, 290 cases were in HGSC-classic group, 91 cases in HGSC-SET Ⅰ and 27 cases in HGSC-SET Ⅱ group. (1) There were significant differences in age, proportion of menopausal patients, tumor necrosis (including map necrosis or acne necrosis), response rate to primary chemotherapy, 5-year mortality rate and PFS between HGSC-SET Ⅰ and HGSC-SET Ⅱ (P<0.05). There was no significant difference among the above indexes between HGSC-SET Ⅰ and HGSC-SET Ⅱ (P>0.05). In HGSC-classic group, HGSC-SET Ⅰ and HGSC-SET Ⅱ, the proportion of family members or patients with history of epithelial ovarian cancer or breast cancer increased in turn, and the detection rate of serous tutal intraepithelial carcinoma (STIC) in fallopian tube tissue decreased in turn. There were significant differences between the two groups (P<0.05). (2) In HGSC-classic group, there were 147 cases in group A, 124 cases in group B and 19 cases in group C (0 case in group D), with median PFS of 17.4, 17.7 and 16.5 months respectively (P<0.05); 10, 6, 29 and 46 cases in group A, B, C and D in HGSC-SET Ⅰ, with median PFS of 9.6, 12.7, 30.1 months and 39.0 months respectively, which there were significant difference among group A and C and D (all P<0.05); among group B, C and D group in HGSC-SET Ⅱ, there were respectively 3, 12 and 12 cases (0 case in group A), and the median PFS was 13.5, 34.2 and 47.8 months (P<0.05). PFS was positively correlated with the increase of push/expansive infiltration ratio. Conclusions: The detection rate of STIC in ovarian HGSC patients with SET is higher, the effect of primary chemotherapy is better, and PFS is prolonged. PFS was significantly prolonged in patients with pelvic disseminated tumors of HGSC-SET, the infiltration of which were predominated by pushing or expanding boarder.