Human Cytomegalovirus Modifies Placental Small Extracellular Vesicle Composition to Enhance Infection of Fetal Neural Cells In Vitro.

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection.

Usutu Virus Infects Human Placental Explants and Induces Congenital Defects in Mice.

Usutu virus (USUV) is a neurotropic mosquito-borne flavivirus that has dispersed quickly in Europe these past years. This arbovirus mainly follows an enzootic cycle involving mosquitoes and birds, but can also infect other mammals, causing notably sporadic cases in humans. Although it is mainly asymptomatic or responsible for mild clinical symptoms, USUV has been associated with neurological disorders, such as encephalitis and meningoencephalitis, highlighting the potential health threat of this virus. Among the different transmission routes described for other flaviviruses, the capacity for some of them to be transmitted vertically has been demonstrated, notably for Zika virus or West Nile virus, which are closely related to USUV. To evaluate the ability of USUV to replicate in the placenta and gain access to the fetus, we combined the use of several trophoblast model cell lines, ex vivo human placental explant cultures from first and third trimester of pregnancy, and in vivo USUV-infected pregnant mice. Our data demonstrate that human placental cells and tissues are permissive to USUV replication, and suggest that viral transmission can occur in mice during gestation. Hence, our observations suggest that USUV could be efficiently transmitted by the vertical route.

Human Cytomegalovirus Infection Changes the Pattern of Surface Markers of Small Extracellular Vesicles Isolated From First Trimester Placental Long-Term Histocultures.

Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking.

Spontaneous pregnancy rate following surgery for deep infiltrating endometriosis in infertile women: The impact of the learning curve.

OBJECTIVES: To determine whether the surgical learning curve impact the spontaneous pregnancy rate in infertile patients undergoing removal of deep infiltrating endometriosis MATERIAL AND METHODS: Single center retrospective study including the first 50 consecutive infertile women suffering from deep infiltrating endometriosis and referred to a single surgeon. All patients underwent laparoscopic removal of deep endometriosis lesions. The study population was stratified in two subgroups, namely the early group (including the first 25 cases) and the late group (comprising the 25 subsequent cases). Pregnancy and live birth rates, surgical morbidity and clinical recurrence rate were compared between study groups. RESULTS: Overall, spontaneous pregnancy rate (40 % in the early group versus 56 % in the late group, p = 0.25), live birth rate (40 % versus 44 %, p = 0.77) and clinical recurrence rate (16 % versus 4%, p = 0.16) did not significantly differ between the study groups. Logistic regression analysis revealed that ASRM stage, EFI score, and body mass index were the only significant prognostic factors of postoperative spontaneous fertility. CONCLUSION: Surgical resection of deep infiltrating endometriosis in infertile women is associated with high spontaneous pregnancy and live birth rates. The surgeon's learning curve does not impact postoperative fertility outcomes.

Metabolic reprogramming by Zika virus provokes inflammation in human placenta.

The recent outbreak of Zika virus (ZIKV) was associated with birth defects and pregnancy loss when maternal infection occurs in early pregnancy, but specific mechanisms driving placental insufficiency and subsequent ZIKV-mediated pathogenesis remain unclear. Here we show, using large scale metabolomics, that ZIKV infection reprograms placental lipidome by impairing the lipogenesis pathways. ZIKV-induced metabolic alterations provide building blocks for lipid droplet biogenesis and intracellular membrane rearrangements to support viral replication. Furthermore, lipidome reprogramming by ZIKV is paralleled by the mitochondrial dysfunction and inflammatory immune imbalance, which contribute to placental damage. In addition, we demonstrate the efficacy of a commercially available inhibitor in limiting ZIKV infection, provides a proof-of-concept for blocking congenital infection by targeting metabolic pathways. Collectively, our study provides mechanistic insights on how ZIKV targets essential hubs of the lipid metabolism that may lead to placental dysfunction and loss of barrier function.

Genotype specific pathogenicity of hepatitis E virus at the human maternal-fetal interface.

Hepatitis E virus (HEV) infection, particularly HEV genotype 1 (HEV-1), can result in fulminant hepatic failure and severe placental diseases, but mechanisms underlying genotype-specific pathogenicity are unclear and appropriate models are lacking. Here, we model HEV-1 infection ex vivo at the maternal-fetal interface using the decidua basalis and fetal placenta, and compare its effects to the less-pathogenic genotype 3 (HEV-3). We demonstrate that HEV-1 replicates more efficiently than HEV-3 both in tissue explants and stromal cells, produces more infectious progeny virions and causes severe tissue alterations. HEV-1 infection dysregulates the secretion of several soluble factors. These alterations to the cytokine microenvironment correlate with viral load and contribute to the tissue damage. Collectively, this study characterizes an ex vivo model for HEV infection and provides insights into HEV-1 pathogenesis during pregnancy that are linked to high viral replication, alteration of the local secretome and induction of tissue injuries.

ZIKA virus reveals broad tissue and cell tropism during the first trimester of pregnancy.

The outbreak of the Zika Virus (ZIKV) and its association with fetal abnormalities have raised worldwide concern. However, the cellular tropism and the mechanisms of ZIKV transmission to the fetus during early pregnancy are still largely unknown. Therefore, we ex vivo modeled the ZIKV transmission at the maternal-fetal interface using organ culture from first trimester pregnancy samples. Here, we provide evidence that ZIKV strain circulating in Brazil infects and damages tissue architecture of the maternal decidua basalis, the fetal placenta and umbilical cord. We also show that ZIKV replicates differentially in a wide range of maternal and fetal cells, including decidual fibroblasts and macrophages, trophoblasts, Hofbauer cells as well as umbilical cord mesenchymal stem cells. The striking cellular tropism of ZIKV and its cytopathic-induced tissue injury during the first trimester of pregnancy could provide an explanation for the irreversible congenital damages.

Natural cytotoxicity receptor splice variants orchestrate the distinct functions of human natural killer cell subtypes.

The natural cytotoxicity receptors NKp46/NCR1, NKp44/NCR2 and NKp30/NCR3 are critical for natural killer (NK) cell functions. Their genes are transcribed into several splice variants whose physiological relevance is not yet fully understood. Here we report that decidua basalis NK (dNK) cells of the pregnant uterine mucosa and peripheral blood NK (pNK) cells, two functionally distinct subsets of the physiological NK cell pool, display differential expression of NKp30/NCR3 and NKp44/NCR2 splice variants. The presence of cytokines that are enriched within the decidual microenvironment is sufficient to convert the splice variant profile of pNK cells into one similar to that of dNK cells. This switch is associated with decreased cytotoxic function and major adaptations to the secretome, hallmarks of the decidual phenotype. Thus, NKp30/NCR3 and NKp44/NCR2 splice variants delineate functionally distinct NK cell subsets. To our knowledge, this is the first conclusive evidence underlining the physiological importance of NCR splice variants.

Human cytomegalovirus infection elicits new decidual natural killer cell effector functions.

During the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus (HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we demonstrate that CD56(pos) dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through which these cells may operate to limit the spreading of viral infection to fetal tissues.

Audit of preoperative and early complications of laparoscopic lymph node dissection in 1000 gynecologic cancer patients.

OBJECTIVE: Establish the reliability and safety of minimal invasive surgery in gynecologic oncology in a large-scale study. Estimate the complication rate on a large sample size. STUDY DESIGN: From December 1998 to November 2004, 1000 gynecologic cancer patients underwent pelvic and/or aortic lymphadenectomies by laparoscopy. A total of 1192 pelvic and aortic lymphadenectomies have been performed: 777 pelvic (757 transperitoneal, 20 extraperitoneal) and 415 aortic lymphadenectomies (155 transperitoneal, 260 extraperitoneal). Main indications for laparoscopic lymph node dissection were: early cervical carcinoma (n = 456), advanced cervical carcinoma (n = 219), vaginal carcinoma (n = 4), endometrial carcinoma (n = 182), and ovarian carcinoma (n = 139). Surgical laparoscopic management via laparoscopy was achieved during the same operative session in 372 patients. RESULTS: No lethality occurred. Thirteen open surgeries (1.3%) were required as a result of failure to complete a satisfactory laparoscopic procedure. Intraoperative, early postoperative complication rate, and lymphocyst formation rate were 2.0%, 2.9%, and 7.1%, respectively. A laparotomy was required for complication in seven patients (7 per 1000), including five returns to operating room. Eleven significant intraoperative vascular injuries occurred, but none required a laparotomy. The most frequently encountered visceral complications were bowel complications (n = 7), urinary tract complications (n = 5), and nerve injuries (n = 5). CONCLUSION: Evidence is given on a large series that laparoscopic lymph node dissection is safe. Laparoscopic surgery may be considered as the gold standard of assessment of the status of regional lymph nodes in gynecologic malignancies.