Human peritoneal fluid exerts ovulation- and nonovulation-sourced oncogenic activities on transforming fallopian tube epithelial cells.

Secretory cells in the fallopian tube fimbria epithelium (FTE) are regarded as the main cells of origin of ovarian high-grade serous carcinoma (HGSC). Ovulation is the main cause of FTE oncogenesis, which proceeds through a sequence of TP53 mutations, chromosomal instability due to Rb/cyclin E aberration, in situ carcinoma (STIC), and metastasis to the ovary and peritoneum (metastatic HGSC). Previously, we have identified multiple oncogenic activities of the ovulatory follicular fluid (FF), which exerts the full spectrum of transforming activity on FTE cells at different stages of transformation. After ovulation, the FF is transfused into the peritoneal fluid (PF), in which the FTE constantly bathes. We wondered whether PF exerts the same spectrum of oncogenic activities as done by FF and whether these activities are derived from FF. By using a panel of FTE cell lines with p53 mutation (FT282-V), p53/CCNE1 aberrations (FT282-CCNE1), and p53/Rb aberrations plus spontaneous transformation, and peritoneal metastasis (FEXT2), we analyzed the changes of different transformation phenotypes after treating with FF and PF collected before or after ovulation. Similar to effects exhibited by FF, we found that, to a lesser extent, PF promoted anchorage-independent growth (AIG), migration, anoikis resistance, and peritoneal attachment in transforming FTE cells. The more transformed cells were typically more affected. Among the transforming activities exhibited by PF treatment, AIG, Matrigel invasion, and peritoneal attachment growth were higher with luteal-phase PF treatment than with the proliferative-phase PF treatment, suggesting an ovulation source. In contrast, changes in anoikis resistance and migration activities were similar in response to treatment with PF collected before and after ovulation, suggesting an ovulation-independent source. The overall transforming activity of luteal-phase PF was verified in an i.p. co-injection xenograft mouse model. Co-injection of Luc-FEXT2 cells with either FF or luteal-phase PF supported early peritoneal implantation, whereas co-injection with follicular-phase PF did not. This study, for the first time, demonstrates that PF from ovulating women can promote different oncogenic phenotypes in FTE cells at different stages of malignant transformation. Most of these activities, other than anoikis resistance and cell migration, are sourced from ovulation.

Insulin-like growth factor (IGF) and hepatocyte growth factor (HGF) in follicular fluid cooperatively promote the oncogenesis of high-grade serous carcinoma from fallopian tube epithelial cells: Dissection of the molecular effects.

Incessant ovulation is believed to be a potential cause of epithelial ovarian cancer (EOC). Our previous investigations have shown that insulin-like growth factor (IGF2) and hepatocyte growth factor (HGF) in the ovulatory follicular fluid (FF) contributed to the malignant transformation initiated by p53 mutations. Here we examined the individual and synergistic impacts of IGF2 and HGF on enhancing the malignant properties of high-grade serous carcinoma (HGSC), the most aggressive type of EOC, and its precursor lesion, serous tubal intraepithelial carcinoma (STIC). In a mouse xenograft co-injection model, we observed that FF co-injection induced tumorigenesis of STIC-mimicking cells, FE25. Co-injection with IGF2 or HGF partially recapitulated the tumorigenic effects of FF, but co-injection with both resulted in a higher tumorigenic rate than FF. We analyzed the different transformation phenotypes influenced by these FF growth signals through receptor inhibition. The IGF signal was necessary for clonogenicity, while the HGF signal played a crucial role in the migration and invasion of STIC and HGSC cells. Both signals were necessary for the malignant phenotype of anchoring-independent growth but had little impact on cell proliferation. The downstream signals responsible for these HGF activities were identified as the tyrosine-protein kinase Met (cMET)/mitogen-activated protein kinase and cMET/AKT pathways. Together with the previous finding that the FF-IGF2 could mediate clonogenicity and stemness activities via the IGF-1R/AKT/mammalian target of rapamycin and IGF-1R/AKT/NANOG pathways, respectively, this study demonstrated the cooperation of the FF-sourced IGF and HGF growth signals in the malignant transformation and progression of HGSC through both common and distinct signaling pathways. These findings help develop targeted prevention of HGSC.

Effect of ovulation IGF and HGF signaling on the oncogenesis of murine epithelial ovarian cancer cell ID8.

The incidence and mortality of epithelial ovarian cancer (EOC) are increasing in Taiwan and worldwide. The prognosis of this disease has improved little in the last few decades due to insufficient knowledge of the etiology. Previous studies on the role of ovulation in the development of EOC have unveiled IGF2, HGF, and other carcinogens in ovulatory follicular fluid (FF) that exert transformation activities on the exposed fallopian tube fimbria epithelium. However, an orthotopic proof in an animal model is lacking. By using the murine ID8 EOC cells and the syngenic transplantation model, this study explored the effect of FF on the oncogenesis of mouse ovarian cancer. We found FF promoted clonogenicity and anchorage-independent growth of ID8 cells, largely through the IGF-1R and cMET signaling. In contrast, FF modestly promoted cell proliferation independent of the two signals and did not affect cell migration and invasion. Transplantation of ID8 cells into the ovarian bursa of C57BL6/J mice orthotopically grew ovarian tumors and metastasized to the peritoneum with ascites formation. The tumorigenic rate and severity of the disease were positively correlated with the level of IGF-1R and cMET receptors on the cell surface. Our data demonstrated that ovulation, through the signaling of IGF/IGF-1R and HGF/cMET, promotes oncogenic phenotypes in a murine EOC model. The results provide further proof of the carcinogenic effect of ovulation in the development of EOC.

Successful Live Twin Birth through IVF/ICSI from a Couple with an Infertile Father with Pericentric Inversion of Chromosome 9 (p12q13): A Case with a High Aneuploidy Rate.

Evidence suggests that the pericentric inversion of chromosome 9 (inv(9)) does not affect the aneuploidy rate (38.5%) after IVF. Herein, we report a successful live female twin birth through IVF/ICSI with a high aneuploidy rate from a couple within which the infertile father has inv(9)(p12q13). A couple (a 34-year-old male and a 35-year-old female) was referred to our clinic due to infertility. The wife has a child with her previous husband. Results from the infertility workup of both parents were normal. Karyotyping revealed that the inv(9)(p12q13) of the father was the only cytogenetic abnormality. Preimplantation genetic testing for aneuploidies (PGT-A) after IVF/ICSI revealed a high aneuploidy rate (77%; 10/13). Two euploid blastocysts were transferred, resulting in a successful live female twin birth. The presented case highlights the possibility that inv(9)(p12q13) in males may impact the fertility and euploidy rate. PGT-A facilitates the selection of qualified blastocysts for the optimization of live-birth outcomes.

Haemoglobin in pelvic fluid rescues Fallopian tube epithelial cells from reactive oxygen species stress and apoptosis.

Fallopian tube fimbrial epithelium is considered to be the major site of origin of ovarian high-grade serous carcinoma, with p53 loss being the earliest and universal change. We previously reported that reactive oxygen species (ROS) in the ovulatory follicular fluids (FFs) are mutagenic and cytotoxic to fimbrial epithelial cells, which are bathed in the peritoneal fluid mixed with FFs. Here, we observed that ferryl haemoglobin (Hb), which was abundantly present in ovulatory FFs and pelvic peritoneal fluids, could rescue p53-deficient immortalized fimbrial epithelial (FE25) cells and oviduct epithelial cells from Trp53-null mice from lethal ovulatory ROS stress. Ferryl Hb and FF containing high Hb levels protected FE25 cells from apoptosis, mainly by consuming extracellular ROS and reducing NADPH oxidase-mediated cell death. The remaining extracellular ROS could still induce DNA double-strand breaks in the fimbrial epithelial cells. Our study revealed that ferryl Hb in peritoneal fluid rescued ROS-stressed, DNA-damaged fimbrial epithelial cells from death, and suggested that peritoneal blood from various sources may contribute to the ovulation-induced transformation of Fallopian tube epithelium. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mutagenic, surviving and tumorigenic effects of follicular fluid in the context of p53 loss: initiation of fimbria carcinogenesis.

Ovulation is the strongest risk factor for ovarian high-grade serous carcinoma (HGSC) that largely originates from the fallopian tube fimbriae and always carries loss-of-function mutations of TP53 in both early and late lesions. Mature ovarian follicle contains high level of reactive oxygen species (ROS). When released from ovulation, follicular fluid (FF) bathes the fimbriae and may lead to DNA double-strand break (DSB) and neoplastic transformation. In this study, we examined the mutagenic and tumorigenic activities of human pre-ovulatory FFs. A subset (6/11) of FFs was found with high levels of ROS whereas the antioxidant capacities were indifferent. These ROS(high) FFs induced intracellular ROS and DSBs in the secretory cell population of fimbriae epithelium. When p53 and Rb were turned down, the FF-exposed secretory cells overcame apoptosis and expanded the population carrying ROS and DSB. The cancer initiation and promotion effects of FF were further recapitulated in Trp53 (-/-) mice. When introduced into the mammary fat pad, ROS(high) but not ROS(low) FFs induced early-onset B-cell lymphoma. Cotreatment with physiological concentration of melatonin, a potent antioxidant, ameliorated the mutagenic and tumorigenic effect of ROS(high) FF in vitro and in vivo. The study revealed ROS and mitogens in mature ovarian follicles could initiate the transformation of fimbria epithelium in the context of p53 loss and melatonin is a potent preventive agent.

Ovulation provides excessive coagulation and hepatocyte growth factor signals to cause postoperative intraabdominal adhesions.

Postoperative adhesions show a higher occurrence in females aged 16-60, especially after pelvic surgeries. This study explores the role of ovulation in adhesion formation in mice. Ovarian surgery in mice with normal- or super-ovulation led to pronounced adhesions, whereas ovulation-defective Pgr-KO mice showed minimal adhesions. Specifically, exposure to ovulatory follicular fluid (FF) markedly increased the adhesion. The hazardous exposure time window was one day before to 2.5 days after the surgery. Mechanistically, early FF exposure triggered adhesions via the blood coagulation cascade, while later exposure relied on the HGF/cMET signaling pathway. Prophylactic administration of a thrombin inhibitor pre-operatively or a cMET inhibitor postoperatively effectively mitigated FF-induced adhesions, while COX inhibitor treatment exhibited no discernible effect. These findings underscore ovulation as a pivotal factor in the development of pelvic wound adhesions and advocate for targeted preventive strategies such as c-MET inhibition, scheduling surgeries outside the ovulatory period, or employing oral contraceptive measures.

Internal pudendal artery injury during prolapse surgery using nonanchored mesh.

Herein is presented the case of a patient with stage 2 uterine prolapse treated surgically using nonanchored mesh. Complications were internal pudendal artery injury and a massive presacral hematoma that formed after surgery. Transcatheter arterial embolization was performed immediately, and the bleeding stopped. The patient subsequently experienced difficulty micturating and defecating because of presacral hematoma compression. Self-micturation and defecation capabilities were regained gradually at approximately 1 week after surgery. The hematoma resolved completely by 71 days postoperatively. Comprehensive knowledge of pelvic anatomy is important when performing surgery to treat prolapse using mesh kits. Removing the mesh and prophylactic antibiotic therapy is a means of conservatively managing a pelvic hematoma caused by prolapse surgery.

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

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.

Insuline-Like Growth Factor-2 (IGF2) and Hepatocyte Growth Factor (HGF) Promote Lymphomagenesis in p53-null Mice in Tissue-specific and Estrogen-signaling Dependent Manners.

Background: Trp53-/- mice are prone to develop lymphomas at old ages. Factors promoting this tumorigenesis are unknown. Here, we showed human ovulatory follicular fluid (FF) largely promotes lymphomagenesis in Trp53-/- mice at earlier ages. Meanwhile, we clarified that IGF2 and HGF are important cell transforming factors within FF. Methods: To induce tumor formation, 5% FFs, 100 ng/ml IGF2, 20 ng/ml HGF, or both IGF2 and HGF in a volume of 200 µl PBS, was injected into 8-wk-old female Trp53 -/- mice at the mammary fat pad. The injection was repeated weekly for up to 7 weeks or extending to 13 weeks to observe the accumulative incidence of lymphomagenesis. Immunohistochemistry staining and gene rearrangement analysis were used to identify the tumor type. Results: By injecting FF into the mammary fat pad weekly, lymphomas developed in 8/16 (50%) of mice by seven weeks. We identified IGF2 and HGF in FF is largely responsible for this activity. The same weekly injection of IGF2, HGF, and their combination induced lymphomas in 4/11 (36%), 3/8 (38%), and 6/9 (67%) mice, respectively. Interestingly, tumorigenesis was induced only when those were injected into the adipose tissues in the mammary gland, but not when injected into non-adipose sites. We also found this tumor-promoting activity is estradiol (E2)-dependent and relies on estrogen receptor (ER) α expression in the adipose stroma. No tumor or only tiny tumor was yielded when the ovaries were resected or when ER is antagonized. Finally, an extension of the weekly FF-injection to 13 weeks did not further increase the lymphomagenesis rate, suggesting an effect on pre-initiated cancer cells. Conclusions: Taken together, the study disclosed a robust tumor-promoting effect of IGF2 and HGF in the p53 loss-initiated lymphomagenesis depending on an adipose microenvironment in the presence of E2. In light of the clarity of this spontaneous tumor promotion model, we provide a new tool for studying p53-mediated lymphomagenesis and suggest that, as a chemoprevention test, this is a practical model to perform.

Ovulation sources coagulation protease cascade and hepatocyte growth factor to support physiological growth and malignant transformation.

The fallopian tube fimbrial epithelium, which is exposed to the follicular fluid (FF) contents of ovulation, is regarded as the main origin of ovarian high-grade serous carcinoma. Previously, we found that growth factors in FF, such as IGF2, are responsible for the malignant transformation of fallopian tube epithelium. However, ovulation is a monthly transient event, whereas carcinogenesis requires continuous, long-term exposure. Here, we found the transformation activity of FF sustained for more than 30 days after drainage into the peritoneal fluid (PF). Hepatocyte growth factor (HGF), activated through the ovulation injury-tissue factor-thrombin-HGF activator (HGFA)-HGF cleavage cascade confers a sustained transformation activity to fallopian tube epithelium, high-grade serous carcinoma. Physiologically, the high reserve of the coagulation-HGF cascade sources a sustained level of HGF in PF, then to the blood circulation. This HGF axis promotes the growth of the corpus luteum and repair of tissue injury after ovulation.

IGF-axis confers transformation and regeneration of fallopian tube fimbria epithelium upon ovulation.

BACKGROUND: The fallopian tube fimbria is regarded as the main tissue of origin and incessant ovulation as the main risk factor of ovarian high-grade serous carcinoma. Previously, we discovered the tumorigenesis activity of human ovulatory follicular fluid (FF) upon injection to the mammary fat pad of Trp53-null mice. We also found a mutagenesis activity of FF-ROS and a apoptosis-rescuing activity of Hb from retrograde menstruation. However, neither of them can explain the tumorigenesis activities of FF. METHODS: From two cohorts of ovulatory FF retrieved from IVF patients, the main growth factor responsible for the transformation of human fimbrial epithelial cells was identified. Mechanism of activation, ways of signal transduction of the growth factor, as well as the cellular and genetic phenotypes of the malignant transformation was characterized. FINDINGS: In this study, we showed that insulin-like growth factor (IGF)-axis proteins, including IGFBP-bound IGF2 as well as the IGFBP-lytic enzyme PAPP-A, are abundantly present in FF. Upon engaging with glycosaminoglycans on the membrane of fimbrial epithelial cells, PAPP-A cleaves IGFBPs and releases IGF2 to bind with IGF-1R. Through the IGF-1R/AKT/mTOR and IGF-1R/AKT/NANOG pathways, FF-IGF leads to stemness and survival, and in the case of TP53/Rb or TP53/CCNE1 loss, to clonal expansion and malignant transformation of fimbrial epithelial cells. By depleting each IGF axis component from FF, we proved that IGF2, IGFBP2/6, and PAPP-A are all essential and confer the majority of the transformation and regeneration activities. INTERPRETATION: This study revealed that the FF-IGF axis functions to regenerate tissue damage after ovulation and promote the transformation of fimbrial epithelial cells that have been initiated by p53- and Rb-pathway disruptions. FUND: The study was supported by grants of the Ministry of Science and Technology, Taiwan (MOST 106-2314-B-303-001-MY2; MOST 105-2314-B-303-017-MY2; MOST 107-2314-B-303-013-MY3), and Buddhist Tzu Chi General Hospital, Taiwan (TCMMP104-04-01).

Laparoscopic management of tubal ectopic pregnancy.

OBJECTIVES: To study the surgical morbidity associated with laparoscopic management of tubal ectopic pregnancy compared with that of open laparotomy. METHODS: A retrospective study in an academic tertiary obstetrics and gynecology referral center was conducted from 2005 through 2007. Forty-nine patients who had pathology-confirmed tubal ectopic pregnancies were divided into 2 groups, laparoscopy (n=38) and laparotomy (n=11). The main outcome measures included operative time, blood loss, and complications. RESULTS: No significant differences existed in gestational age, beta-hCG level, history of previous surgeries, pelvic inflammatory disease, or endometriosis. The laparotomy group included more patients with a history of previous ectopic pregnancy. The length of hospital stay following laparoscopic management was significantly less than that in the laparotomy group. CONCLUSION: Laparoscopic management of ectopic pregnancy can be the most beneficial procedure with maximum safety and efficacy.

Trends of Opportunistic Salpingectomy.

BACKGROUND AND OBJECTIVES: This study analyzed the trends of opportunistic salpingectomy (OS) accompanied by hysterectomy in a 9-year follow-up period at a single institute. METHODS: This retrospective cohort study included 1184 women at Hualien Tzu Chi Hospital from 2007 to 2015 who underwent hysterectomy performed with or without OS. Parameters including patient age, operating time, surgical approach, length of hospital stay, and perioperative complications were evaluated. RESULTS: There was an increase in the number of hysterectomies with OS (from 8% to 80%; P < .001) over the study period. Minimal additional operating time was necessary for hysterectomy with OS (3.7 and 3.6 minutes in open and laparoscopic surgery, respectively). No significant differences were observed in the risks of hospital readmission or blood transfusions between women who underwent hysterectomy with OS performed with the open approach and those who underwent the procedure using the laparoscopic approach. From 2007 to 2015, the proportion of open hysterectomies decreased from 56% to 6%. CONCLUSION: The results of this 9-year follow-up study revealed that, as a cancer prevention method, OS seems to be feasible and safe, requires minimal extra time, and does not increase the morbidity or long-term sequelae.

Platelet-Derived Growth Factor in the Ovarian Follicle Attracts the Stromal Cells of the Fallopian Tube Fimbriae.

During human ovulation, the fallopian tube fimbriae must move to the ovulation site to catch the oocyte. As the tissue-of-origin of the majority of ovarian high-grade serous carcinoma (HGSC), the fallopian tube fimbriae carrying a precursor cancer lesion may also approach the ovulatory site for metastasis. We hypothesize that platelet-derived growth factor (PDGF) in mature follicle fluid (FF) attracts the migration of PDGFR-expressing fimbriae toward the ovulating follicle. We observed that more PDGFR-ß was expressed in the distal part than in the proximal parts of the fallopian tube, particularly in stromal cells in the lamina propria. The stromal cells, but not the epithelial cells, from normal fimbriae and fallopian tube HGSC were highly chemotactic to mature FF. The chemotactic activities were positively correlated with PDGF-BB and estradiol levels in FF and were abolished by a blocking antibody of PDGFR-ß and by tyrosine kinase inhibitor imatinib. When PDGF-BB/AB was depleted from the FF, more than 80% of chemotaxis activities were diminished. This study suggests an ovarian follicle-directed and PDGF-dependent attraction of fallopian tube fimbriae before ovulation. The same mechanism may also be crucial for the ovarian homing of HGSC, which largely originates in the fimbriae.

Prenatal diagnosis of the maternal derivative chromosome der(15)t(Y;15)(q12;p13) in a dizygotic twin pregnancy.

Sex chromosome translocations are unique and must be considered separately from translocations between autosomes. Here, we describe the first prenatal case of one twin fetus with an unbalanced translocation between chromosome Y and chromosome 15, presenting a 46,XY,der(15)t(Y;15) karyotype. The other twin had a normal 46,XY karyotype. Cytogenetic analysis of the parental chromosomes revealed that the father had a normal 46,XY karyotype, whereas the mother exhibited a 46,XX,der(15) t(Y;15) karyotype. Thus, the proband inherited this translocation from the mother. Fluorescence in situ hybridization analyses demonstrated that the breakpoint on chromosome Y involved a heterochromatin region (Yq12), while that on chromosome 15 involved a p-arm region (15p13). At 37 gestational weeks, healthy twins were delivered vaginally. We conclude that accurate identification of der(15) chromosomal content can facilitate not only prenatal diagnosis of a chromosomal aberration in one twin, but also prediction of the fetal phenotype.