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Background: The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the cause of the Coronavirus Diseases of 2019 (COVID-19). This virus has been responsible for the global pandemic of 2020 and 2021 with over 107 million confirmed cases and over 2.3 million deaths. An important population that deserves meticulous consideration during the COVID-19 pandemic is pregnant woman; however, in many places in the world there is inadequate screening for SARS-COV-2 during pregnancy. We aimed to determine the impact of maternal and neonatal cord blood SARS-COV-2 antibodies and placental transfer ratios in a region with a low screening plan. Methods: We performed a blind study in Babahoyo, Ecuador; one of the SARS-CoV-2epicenters in South America. Blood samples were collected from 100 serum samples collected from pregnant women at the time of delivery along with corresponding neonatal cord blood. SARS-CoV-2 IgG specific antibodies were determined using a enzyme linked immunosorbent assay (ELISA) for IgG and IgM SARS-CoV-2-specific nucleocapsid and spike antigens (DiaPro, Milano, Italy Cat number COV19G.CE). Findings: We observed 32% of pregnant women to be serological positive in areas lacking an appropriate screening program. We also show data that suggests an efficient passive immunization of the fetus to SARS-CoV-2 during pregnancy. Interpretation: There is a high incidence of SARS-CoV-2 infection during pregnancy. The best prevention for protecting the mother from infection is vaccination, which, will provide the fetus and neonate with a strong protection against SARS-CoV-2 infection through passive placental transfer of SARS-CoV-2 specific antibodies. This is especially important because newborns are highly susceptible to many viral infections. Funding: NIAID 1R01AI145829-01 Declaration of Interest: None to declare. Ethical Approval: This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines. This was retrospective study done with samples sent for tests evaluation that were originally not related to this study. A consent waiver was approved since the samples were not collected for the study and the samples used were defined as dischargeable material.

Introduction The limited cell number of primary trophoblasts and contamination of trophoblast cell lines promote us to develop a novel stable trophoblast cell line. Method of study Primary trophoblast cells were isolated from first-trimester placenta and telomerase-induced immortalization was used to immortalize these cells. Subsets of cells were then evaluated by flow cytometry using CK7, HLA-G, CD45 and CD14, specific markers for trophoblast cells, extra-villous trophoblast, pan leucocyte and monocyte/macrophage, respectively. Immunofluorescence staining and immunocytochemistry were used to detect CK7 expression in trophoblast cells. The level of secreted human Chorionic Gonadotropin (hCG) was measured by electrochemiluminescence (ECL). The Bio-Plex MAGPIX System was used to analyze the cytokines and chemokines produced by AL07 cell line. Results We were able to isolate primary trophoblast cells from several first-trimester placentas. One clone, AL07 trophoblast cells, isolated from a week 7 placenta, was morphologically stable and positive for the expression of CK7 by immunofluorescence and immunocytochemistry staining. Characterization of AL07 cells reveled that they are CD45 or CD14 negative and had constitutive secretion of hCG and low HLA-G expression. Furthermore, clone AL07 secret high levels of several cytokines and chemokines, including IL-6, IL-8 and VEGF, and moderately secreted MCP-1 IP-10 and RANTES. Discussion We report the successful isolation, immortalization and characterization of AL07 cells, a novel cell clone isolated from first trimester human placenta. The clone is free of contamination of immune cells, and exhibits similar cytokine profile as other trophoblast cell lines. This new cytotrophoblast-like AL07 cell, can be a valuable tool for in-vitro trophoblast studies in the future.

Introduction Placental viral infections are associated with fetal inflammation and adverse pregnancy outcomes. However, there have been limited studies on how placental macrophages in the villous and adjacent fetal umbilical endothelial cells respond to a viral insult. This study aimed to evaluate the communication between Hofbauer cells (HBCs) and human umbilical vein endothelial cells (HUVECs) during a viral infection. Methods HBCs were either uninfected or infected with the γ-herpesvirus, MHV-68, and the conditioned medium (CM) collected. HUVECs were exposed to HBC CM and the levels of the pro-neutrophilic response markers: IL-8; E-selectin; intercellular adhesion molecule 1 (ICAM-1); and vascular adhesion molecule 1 (VCAM-1) measured by ELISA and qPCR. The role of HBC-derived IL-1β was investigated using an IL-1β blocking antibody (Ab) or IL-1 receptor antagonist (IL-1Ra). Results MHV-68 infection of HBCs induced a significant increase in IL-1β secretion. CM from infected HBCs induced HUVEC expression of IL-8, E-selectin, VCAM-1, ICAM-1 mRNA, and secretion of IL-8. The HUVEC response to the CM of MHV-infected HBCs was inhibited by a neutralizing IL-1β Ab and by IL-1Ra. Discussion Virally-induced HBC IL-1β activates HUVECs to generate a pro-neutrophilic response. This novel cell-cell communication pathway may play an important role in the genesis of fetal inflammation associated with placental viral infection.

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.

Objectives: To determine the combined efficacy of MIF, OPN, PROL and CA-125 in distinguishing ovarian cancer from healthy compared to CA-125 alone as a single biomarker. Methods: Serum samples from 153 ovarian cancer patients (37 Stage I/II and 116 Stage III/IV) and 279 healthy age-matched controls were utilized in this study. The ovarian cancer group (n=153, average age = 57.1 yr) was composed of women with newly diagnosed ovarian cancer following discovery of pelvic mass. Mean concentration of MIF, OPN, PROL and CA-125 was measured using a novel, fully automated, multi-analyte immunoassay platform, Ella. The serum samples were randomly divided into two different cohorts (n=153 and n=279) to be interchangeably used as training and testing sets for statistical analysis. Four statistical classification models, k-nearest neighbors, logistic regression, random forest, and support vector machines were used to calculate accuracy, sensitivity, specificity, positive-predictive value (PPV), and negative-predictive value (NPV) of the four protein biomarker panel and CA-125 alone. Download : Download high-res image (163KB) Download : Download full-size image Results: In distinguishing serum of ovarian cancer patients from healthy controls, the four-protein biomarker panel yielded an average accuracy of 91% compared to 85% when using CA-125 alone across the four classification models (p=3.224e-09). Further, the four-protein biomarker produced statistically significantly higher sensitivity (median of 76%), specificity (median of 98%), PPV (median of 91.5%), and NPV (median of 92%), compared to CA-125 alone in distinguishing ovarian cancer from healthy controls (Wilcoxon p-values: 2.22e-07, 0.02, 1.27e-2, and 3.629e-07, respectively; Figure 1). Importantly, the improved performance of the four-protein biomarker over CA-125 alone is maintained in the detection of early-stage ovarian cancer (Stage I/II) from health controls. Conclusions: Combining MIF, OPN, and PROL to CA-125 resulted in improved detection of ovarian cancer compared to CA-125 alone. This study is another step forward towards developing a useful serologic biomarker panel for early detection of ovarian cancer that is both highly accurate and specific. The multi-analyte platform, Ella, is fully automated, highly reproducible and a useful tool in future ovarian cancer biomarker discoveries.

Objectives: Yes-associated protein (YAP) is a transcriptional coactivator regulated via the Hippo pathway that has been shown to be tumorigenic in ovarian cancer (OC). YAP has also been identified to be uniquely expressed within induced regulatory T cells (Tregs). A conditional knockout of YAP in T cells decreased Tregs within the tumor microenvironment and increased antitumor immune response in a subcutaneous mouse melanoma model. Visudyne™ is a liposomal derivative of verteporfin (VP), a known YAP inhibitor that is FDA approved for the treatment of adult macular degeneration. The objective of our study is to: 1) evaluate the effect of Visudyne™ on YAP and its transcriptional targets, 2) determine cytotoxic effects of Visudyne™ in platinum sensitive and resistant OC cell lines, and 3) to evaluate its effect on immune regulatory cells. Methods: Western blot analysis and immunofluorescence were performed to determine YAP expression and localization in R182 and MR182 OC cell lines using specific antibodies for YAP, phosphorylated YAP (pYAP), and connective tissue growth factor (CTGF), a transcriptional target of YAP. Cell proliferation was measured using Incucyte real-time imaging following treatment with increasing doses of cisplatin, VP and Visudyne™ in A2780 wildtype and platinum resistant OC cells. IC50 was calculated using GraphPad PRISM. Three C57/B6 mice were injected intraperitoneally (IP) with triple knockout p53mut/PTENmut/mCherry OC cells. Tumor progression was monitored by mCherry signal with fluorescence spectroscopy. Visudyne™ was administered IP at 2 mg/kg and 3 days later at 4 mg/kg. Flow cytometry was performed using antibodies for CD4, CD8, FOXP3, and interferon gamma to identify Treg and cytolytic T cells in peritoneal lavage and spleen of the control and Visudyne™ treated mice. Download : Download high-res image (329KB) Download : Download full-size image Results: Treatment of OC cells with 1uM Visudyne™ is associated with a significant decrease expression of total YAP, pYAP, and CTGF (Figure 1A). Immunofluorescence demonstrated loss of nuclear YAP and increased pYAP in the cytoplasm of R182 and MR182 cells after VP treatment (Figure 1B). Increasing doses of Visudyne™ significantly decreased cell growth in platinum resistant A2780 OC cells when compared to cells treated with cisplatin alone. In vivo treatment of tumor bearing mice with Visudyne™ is associated with a significant decrease of Treg CD4+/FOXP3+ cells in peritoneal lavage of treated mice compared to the control (85.5% vs. 92.9%, Figure IC). No changes were observed in the spleen in treated mice vs. control. Conclusions: We propose that Visudyne™ should be further investigated as a novel therapeutic agent for the treatment of OC. Its effect on immune function, specifically Tregs, suggests a promising effect either as a single agent or in combination with other checkpoint inhibitors in platinum sensitive and resistant OC.

Objectives: The purpose of this study was to identify genes and pathways differentially expressed in platinum resistant high grade serous ovarian cancer (HGSOC) when compared to sensitive HGSOC. Methods: A total of 37 patients with HGSOC tissue samples underwent RNA sequencing performed by TEMPUS (N=37, 21 platinum sensitive, 16 resistant; 85% Stage III-IV; 58% received neoadjuvant chemotherapy). RNA gene expression data and significantly impacted pathways were analyzed using Advaita Bio's iPathwayGuide. Differentially expressed (DE) genes were identified using FDR of 0.05 and fold-change of 1.5. Genes from several impacted canonical metabolic pathways were validated by PCR against external data sets in a separate ovarian cancer sample group (n=15), platinum resistant ovarian cancer mouse tumor model, and wild-type sensitive and platinum resistant ovarian cancer cell lines. Relative gene expression was calculated using the comparative Ct method, also referred to as the “2 DDCT”, using L27 as internal control gene. Download : Download high-res image (333KB) Download : Download full-size image Results: We identified 177 differentially expressed (DE) genes out of a total of 16,607 genes (1.1%) with measured expression. 15 pathways were found to be significantly impacted. Of the 15 canonical pathways, all were up regulated in the resistant HGSOC and the majority of the most significantly altered (5/10) were related to metabolism (Retinol metabolism (p-value = 0.002); Tyrosine Metabolism (p-value = 0.005); Tryptophan Metabolism (p-value = 0.009); and Phenylalanine Metabolism (p-value = 0.012); CYP Drug Metabolism (p-value = 0.022)). A total of 3 separate genes from the CYP family and two from the Dopa Decarboxylase family of genes were validated against an external data set of human ovarian tissue samples, cell lines, mouse ovarian tumor model, and found to have similarly increased gene expression in the genes tested in the platinum resistant groups. Compilation of KEGG analysis and the common network genes revealed pathways associated with amino acid metabolism to be most significantly altered. Conclusions: We describe the identification of a unique transcriptomic profile associated with platinum resistance. Interestingly, the main pathways identified are related to metabolism, suggesting that the survival to chemotherapy demands a major metabolic adaptation. These findings also represent a first step towards the identification of biomarkers for the detection of chemo-resistant disease and metabolism-based drug targets specific for chemo-resistant tumors. Further validation of this model is required in order to determine its clinical value.

Objectives: Emerging data suggests that key differences exist between BRCA1 and BRCA2 associated ovarian cancer, including response to therapy and survival outcomes. The purpose of this study was to identify gene expression profiles and interacting pathways unique to BRCA1- and BRCA2- associated high grade serous ovarian cancer (HGSOC) samples compared to one another as well as to BRCA wild type, homologous recombination proficient (HRP) tumors. Methods: Of 657 total HGSOC samples, 15 BRCA2 mutated (2.2%), 16 (2.4%) BRCA1 mutated, and 375 (57%) HRP samples were analyzed. BRCA mutated was defined as somatic variants that result in loss of function. The HRP control group was defined as samples negative for aberrations in BRCA1/2 and 28 HR genes. Gene expression data was collected from Tempus and unpaired t-tests were used to identify differentially expressed genes (DEG) with p-value Results: From 18,284 genes with measured expression, 843 (4.6%) DEG were found between BRCA2 vs BRCA1, 748 (4.1%) between BRCA2 vs HRP and 1,858 (10.2%) between BRCA1 and HRP. 8,296 mutated genes were similarly expressed in both BRCA2 and BRCA1 groups with FC Download : Download high-res image (139KB) Download : Download full-size image Conclusions: Our study identified genomic signatures for BRCA2 versus BRCA1 associated ovarian cancer, not shared by control HRP tumors in a sample representative of HGSOC tumor heterogeneity. Results suggest BRCA1/2 should not be considered a single entity, but rather separate phenotypes each with unique opportunities for targeted therapy.

Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE) is an endocrine disrupter and ultimate carcinogenic product of benzo(a)pyrene (BaP). Numerous studies have shown that BPDE causes trophoblast-related diseases, such as preeclampsia, growth restriction or miscarriages. However, the underlying mechanism, especially the mitochondria-related BPDE-induced trophoblast dysfunction remains unknown. In this study, we examined mitochondrial functions in BPDE-induced human trophoblast cell line Swan 71. BPDE decreased cell ability, attenuated cell invasion and HCG secretion, induced cell apoptosis, decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) and MDA, and decreased SOD activity in a dose-dependent manner. In the mechanism, BPDE significantly increased pro-apoptosis protein (P53 and Bak1) and decreased anti-apoptosis protein (Bcl-2). Furthermore, the protein expression levels of mitochondrial fusion genes (Mfn1, Mfn2, and OPA1) were decreased and those of fission genes (Fis1 and Drp1) were increased with increasing concentrations of BPDE and incubation time, resulting in the release of Cyt c and activation of Caspase 3, which irreversibly induced trophoblast cell apoptosis. This study reveals the mechanism of dysfunction of trophoblast cells through cell apoptosis due to the disorder of mitochondrial fission/fusion after exposure to BPDE, providing a further experimental understanding the adverse effects of BaP on trophoblast cells in early pregnancy.

Successful pregnancy relies on the accurate regulation of the maternal-fetal immune system. Without enough tolerance in the uterine microenvironment, the mother and the hemiallogeneic fetus could not peacefully coexist. T cell immunoglobulin and mucin domain (Tim)-3 is a molecule originally regarded as to be expressed on terminally differentiated IFN-γ expressing CD4+ T cells (Th1). The engagement of Tim-3 with its ligand, galectin-9 (Gal-9) could induce the exhaustion or apoptosis of effector T cells, and thus might regulate the tolerance. Tim-3 pathway also participates in regulating the activities of CD4+ regulatory T cells, monocyte-macrophages, dendritic cells and natural killer cells. Dysregulation of Tim-3 expression can elicit excessive or inhibited inflammatory responses and ultimately result in autoimmune diseases, viral or tumor evasion and pregnancy complications. In this review, we will mainly focus on the expression of Tim-3 on local immune cells and its function in pregnancy. In addition, meaningful questions that need further investigation and the potential roles of Tim-3 in fetal tolerance will be discussed. Deeper understanding of the immune checkpoint receptor Tim-3 will shed new light on exploring the pathogenesis of some pregnancy complications, including pre-eclampsia, intrauterine growth restriction, recurrent spontaneous abortion and preterm birth. Tim-3 pathway might be a new target of immune therapy for pregnancy complications in the future.

Targeted therapy or molecular targeted therapy has been defined as a type of treatment that blocks the growth of cancer cells by interfering with specific cell molecules required for carcinogenesis and tumor growth, rather than by simply interfering with all rapidly dividing cells as with traditional chemotherapy. There is a growing number of FDA approved monoclonal antibodies and small molecules targeting specific types of cancer suggestive of the growing relevance of this therapeutic approach. Targeted cancer therapies, also referred to as "Personalized Medicine", are being studied for use alone, in combination with other targeted therapies, and in combination with chemotherapy. The objective of personalized medicine is the identification of patients that would benefit from a specific treatment based on the expression of molecular markers. Examples of this approach include bevacizumab and olaparib, which have been designated as promising targeted therapies for ovarian cancer. Combinations of trastuzumab with pertuzumab, or T-DM1 and mTOR inhibitors added to an aromatase inhibitor are new therapeutic strategies for breast cancer. Although this approach has been seen as a major step in the expansion of personalized medicine, it has substantial limitations including its high cost and the presence of serious adverse effects. The Cancer Genome Atlas is a useful resource to identify novel and more effective targets, which may help to overcome the present limitations. In this review we will discuss the clinical outcome of some of these new therapies with a focus on ovarian and breast cancer. We will also discuss novel concepts in targeted therapy, the target of cancer stem cells.

The aim of this review is to discuss the role of Toll-like receptors at the maternal-fetal interface and the capacity of trophoblast cells to initiate innate immune responses towards infection. The maternal-fetal interface represents an immunologically unique site that must promote tolerance to the allogenic fetus, whilst maintaining host defense against a diverse array of possible pathogens. Clinical studies have shown a strong association between certain complications of pregnancy and intrauterine infections. Therefore, innate immune responses against microorganisms at the maternal-fetal interface may have a significant impact on the success of a pregnancy. There is growing evidence that trophoblast cells are able to recognize and respond to pathogens through the expression of Toll-like receptors, an important part of innate immunity. This review will discuss the role of Toll-like receptors at the maternal-fetal interface, the potential for trophoblast cells to function as components of the innate immune system and the impact TLR-mediated trophoblast responses may have on pregnancy outcome.

Objective The goal of this work was to determine whether exposure to estrogen following treatment of low-grade endometrial stromal sarcomas affects clinical outcome. Methods Twenty-two patients with low-grade endometrial stromal sarcomas were reviewed to determine whether they were exposed to exogenous or endogenous estrogen and/or progestins following their diagnosis and whether exposure to these hormones might have influenced their prognosis. Estrogen receptor (ER) α and β and progestin receptor (PR) status were analyzed from paraffin-embedded tissue by immunohistochemistry and ER mRNA was measured in fresh tissue by reverse transcription polymerase chain reaction (RT-PCR). Results Ten of the twenty-two patients with low-grade endometrial stromal sarcomas developed recurrent disease. Four of five patients (80%) who received estrogen replacement therapy (ERT) recurred. Four of eight patients (50%) with retained ovaries recurred. Eight of the ten specimens available for analysis were positive for ERα, none were positive for ERβ, and 9 of 10 were positive for PR. Four of thirteen patients who received progestins as adjuvant therapy recurred, compared with 6 of 9 patients who did not receive progestins (31% vs 67%). Eight recurrences were treated with progestin therapy and 7 (88%) of them had either stable disease (3/8, 38%) or complete response (4/8, 50%). Conclusions Our results suggest that ERT may be detrimental in patients with low-grade endometrial stromal sarcoma. Retention of normally functioning ovaries, on the other hand, may not significantly affect the recurrence rate following hysterectomy alone in Stage I patients. The lack of ERβ expression in endometrial stromal sarcomas compared with normal endometrial stromal cells suggests that loss of ERβ may be a marker for malignancy. Progestin therapy should be routinely considered for adjuvant therapy and for the treatment of recurrent endometrial stromal sarcomas.

Successful treatment of endometrial hyperplasia with progestins is commonly accompanied by the finding of an inactive or suppressed endometrium after therapy. However, approximately 30% of the endometrial hyperplasia cases do not respond to progestins and hyperplastic glands persist. The Fas/FasL system is known to play a role in tissue remodeling as a result of changes in menstrual hormone levels. The aims of this study are to examine Fas/FasL expression in endometrial hyperplasia of pre- and postprogestin treatment samples and to study the Fas/FasL regulation in vitro with Ishikawa cells after progestin stimulation.Pre- and posttreatment paraffin-embedded endometrial hyperplasia tissue samples from 26 women were examined by immunohistochemistry for changes in Fas/FasL expression related to the administration of progestins. Among 26 patients, 18 were successfully treated with progestins and 8 failed treatment. Fas/ FasL positivity was defined by the presence of 10% or more immunoreactive epithelial cells in each specimen. In positive cases, a percentage or an immunoscore of immunoreactive cells was given by counting 500 cells. Cell viability was evaluated by the MTT assay. The in vitro effects of progesterone on Fas/FasL expression and apoptosis in Ishikawa cells were examined by using Western blot and TUNEL assays, respectively.Fas immunoreactivity was present in 4/26 (15%) preprogestin cases with an average of 16% of the epithelial cells expressing Fas. FasL was expressed in 21/26 (80%) pretreatment cases with an average of 42% of the hyperplastic glandular cells being positive. In postprogestin cases, an increase of Fas expression (14/18, 77%) with an average of 47% stained cells was seen in responders (P0.001), while FasL was found in 16/18 (89%) responders with an average of 65% of cells positive (P = 0.587). In nonresponders, no significant changes in Fas/FasL expression were detected compared to pretreatment samples. With in vitro Ishikawa cells, a slight increase (10-20%) of Fas and FasL protein expression was detected after 24 h of progesterone treatment, but a more significant increase (220-343%) of both Fas and FasL expression was found after 48 h of withdrawing progesterone, which parallels apoptotic activity.The Fas/FasL system may be involved in the development of endometrial hyperplasia. Part of the molecular mechanisms of progestin therapy for endometrial hyperplasia is through upregulation of Fas/FasL expression. Dysregulation of Fas/FasL expression in hyperplastic endometrium may be part of the molecular mechanisms for nonresponders to progestin treatment. Intermittent, rather than continuous, progestin treatment may be more effective clinically for the treatment of endometrial hyperplasia.

The brain has an intrinsic capacity to remove infiltrating T cells by inducing apoptosis. However, the pathways and cellular components driving this process are still under debate. Astrocytes seem to play an important role because they colocalize with apoptotic lymphocytes in vivo and induce apoptosis of transformed T cells in vitro. Since we previously demonstrated the expression of the death ligand CD95L (APO-1L/FasL) on astrocytes in the brain, we wanted to know whether nontransformed astrocytes induce cell death in nontransformed T cells, reflecting the in vivo situation and, if so, whether CD95/CD95 ligand interaction is important. T cell apoptosis measured by Annexin V binding and DNA fragmentation was significantly lower using CD95 ligand-deficient (gld) astrocytes compared to nondeficient controls. Moreover, neutralizing anti-CD95 ligand antibody reduced astrocyte-induced T cell apoptosis. Thus, adult astrocytes are capable of inducing the apoptotic death of T cells by involving the CD95/CD95 ligand pathway without undergoing cell death in vitro. Since astrocytic end-feet contribute to the formation of the blood-brain barrier, this depletion mechanism may play an important role as the first line of defense in the brain.

For centuries, the question of "whether there is life after death" has intrigued the mind of philosophers and the same question fascinates researchers in the field of apoptosis today. The death of a cell is by no means the end of the story. On the contrary, growing evidence suggests that the clearance of apoptotic bodies by macrophages is an important regulatory component in tissue renewal. Without death by apoptosis, the life of reproductive tissues and their function would not be possible. The survival signals that counteract cell death also prepare the cells for apoptosis, and dead cells are important stimuli for tissue survival. The Fas/Fas ligand system is an important mediator of apoptosis and is an excellent example of this apparently contradictory phenomenon.

Important reproductive events including implantation, trophoblast invasion, placental development, and immune protection are regulated by the immune system at the maternal-fetal interface. This maternal-fetal immune interaction is complex, and it is difficult to perceive the whole process based on one mechanism of action. Clearly, there are multiple mechanisms for the induction of peripheral and local tolerance during pregnancy that prevent fetal rejection while maintaining a strong and active immune surveillance against viral or bacterial infections, which may endanger the successful outcome and the survival of the species.

The phenotype and frequency of cells in normal human peripheral blood spontaneously secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-alpha ex vivo was determined using ELIspot assays. CD4+ T cells were the dominant source of IL-2 and IL-4 while multiple cell types (primarily CD8+ lymphocytes) produced IFN. Fewer than 0.05% of mononuclear cells were spontaneously secreting these T cell derived factors. By comparison, IL-6, IL-10 and TNF-alpha were produced by 0.7-20% of PBMC. The primary sources of the latter cytokines were CD14+ macrophages/monocytes. A significant positive correlation was found in the frequency of cells secreting IL-6, IL-10 and TNF-alpha ex vivo, suggesting that the release of such factors was coordinately regulated. No such correlation was found among IL-2, IL-4 and IFN secreting cells, indicating that the production of predominantly T cell derived cytokines was regulated independently.

The objective of the study was to evaluate the safety of semen washing with intrauterine insemination (SW-IUI) for achieving pregnancy when the man is human immunodeficiency virus (HIV) infected and the woman is HIV negative.We conducted a retrospective analysis of 635 HIV-discordant couples enrolled in a SW-IUI program and followed up 367 Italian women. We computed pregnancy, live birth, and multiple delivery rates and assessed the women's postinsemination HIV status.The retrospective analysis included 635 couples (2113 SW-IUI cycles): 41% of the women (95% confidence interval [CI], 37-45%) had a live birth (per-cycle live birth rate 13%; 95% CI, 11-14%). HIV status after SW-IUI was negative when available but unknown for 26% of the women: missing HIV status was not associated with correlates of HIV risk. The follow-up study included 367 couples (1365 cycles): 47% of the women (95% CI, 42-52%) had a live birth (per-cycle rate 14%; 95% CI, 12-16%). Ascertainment of postinsemination HIV status was complete and confirmed no HIV transmission attributable to SW-IUI. The upper 95% confidence limit of the HIV transmission rate was 1.8 per 1000 cycles in the retrospective analysis and 2.7 per 1000 cycles in the follow-up study.SW-IUI appears to be a safe and effective method for achieving pregnancy in HIV-discordant couples in which the man is HIV infected.

Objective We evaluated the hypothesis that ovarian cancer patients have significantly higher levels of serum macrophage migration inhibitory factor (MIF). Study Design MIF levels were determined by enzyme-linked immunosorbent assay (ELISA) in epithelial ovarian cancer cell lines and immortalized normal ovarian surface epithelial cells and in serum of ovarian cancer patients (n = 54) and age-matched healthy women (n = 60). To determine the impact of Toll-like receptor-4 ligation on MIF levels, cells were treated for 48 hours with lipopolysaccharide. Results Cancer cells, but not normal cells, secrete significant amounts of MIF. This correlates in vivo , where serum MIF levels are significantly higher in ovarian cancer patients. Treatment of cancer cells with lipopolysaccharide induced a significant increase in MIF secretion. Conclusion MIF may be relevant in the process of ovarian cancer formation and progression. The events leading to the induction of MIF expression and its contribution to ovarian cancer progression may open new venues for targeted therapy.

Objective Toll-like receptors (TLRs) recognize microbial ligands and host products that are released during tissue damage, the so-called “danger signals.” This study was conducted to determine whether changes in TLR-4 and TLR-2 expressions can be detected in the trophoblasts at the placental bed of women with and without preeclampsia. Study design Placental bed biopsy specimens were obtained from women with: (1) normal term pregnancies with and without labor (each n = 20); (2) preeclampsia who delivered preterm (n = 15); and (3) preterm labor and intact membranes with and without chorioamnionitis (each n = 15). The expression pattern of TLR-4 and TLR-2 in the trophoblasts was analyzed by double immunohistochemistry. Results (1) The median percentage of TLR-4 positive interstitial trophoblasts was significantly higher in patients with preeclampsia than in patients with preterm labor without or with histologic chorioamnionitis (P = .0002 and P = .02, respectively). (2) The median percentage of TLR-2 positive interstitial trophoblasts was not different among the study groups (P > .05). (3) TLR-4 positive trophoblasts were also frequently immunoreactive to activated nuclear factor–κB, tumor necrosis factor–α, and M30 (a specific apoptosis antigen for trophoblast). (4) Lipopolysaccharide treatment inhibited the migration of trophoblast cell lines in vitro. Conclusion TLR-4 protein expression is increased in interstitial trophoblasts of patients with preeclampsia. We propose that “danger signals” at the feto-maternal interface, which are recognized by trophoblasts through TLR-4, may play a key role in the creation of a local abnormal cytokine milieu. This suggests a novel mechanism that links the activation of the innate immune system through TLR-4 and preeclampsia.

IMMUNE SYSTEM, TROPHOBLAST APOPTOSIS AND PREECLAMPSIA? YEON MEE KIM, SEO YOUNG OH, JYH KAE NIEN, RICARDO GOMEZ, CHONG JAI KIM, MOSHE MAZOR, VIKKI ABRAHAMS, GIL MOR, SHIGERU SAITO, ROBERTO ROMERO, Wayne State University School of Medicine, Department of Pathology, Detroit, Michigan, Perinatology Research Branch, NICHD, NIH, DHHS, Bethesda, Maryland, CEDIP, Sotero del Rio Hospital, Puente Alto, Chile, Chile, Seoul National University, Seoul, Korea, South Korea, Soroka University Medical Center, Beer Sheva, Israel, Israel, Yale University, Department of Obstetrics and Gynecology, New Haven, Connecticut, Toyama Medical and Pharmaceutical University, Department of Obstetrics and Gynecology, Toyama, Japan, Japan OBJECTIVE: Toll-like receptors (TLRs), discovered to regulate patterning during embryonic development, were subsequently found to play a role in innate immunity. TLRs recognize microbial ligands as well as host products released during tissue damage or ‘‘danger signals’’ (Science 2002;296:301). Engagement of TLR-4 can induce trophoblast cells to produce pro-inflammatory cytokines. Such cytokines may promote trophoblast cell apoptosis, which is increased in preeclampsia. This study was conducted to determine TLR-4 expression patterns in the extravillous trophoblasts (EVT) in the placental bed of women with and without preeclampsia. STUDY DESIGN: Placental bed biopsies were obtained from patients with: (1) normal pregnancy at term (n = 40); (2) severe preeclampsia (n = 15); and (3) preterm delivery and intact membranes (PTD) with and without histologic chorioamnionitis (n = 15 for each group). The expression pattern of TLR-4 in the EVT was examined by double immunohistochemistry. Image analysis was conducted and non-parametric statistics were employed for analysis. RESULTS: (1) The median percentage of TLR-4 positive EVT was significantly higher in patients with preeclampsia than in patients with PTD or normal patients at term (PTD: P = .0001; women at term: P ! .0001). (2) The median percentage of TLR-4 positive EVT was significantly higher in patients with preeclampsia than in those with PTD with histologic chorioamnionitis (P = .0057). (3) The median percentage of TLR-4 positive EVT in the placental bed was significantly higher in patients with PTL and histologic chorioamnionitis than in those without these conditions (P = .037). CONCLUSION: TLR-4 expression is increased in the extravillous trophoblasts in women with preeclampsia. We propose that ‘‘danger signals’’ may increase the expression of TLR-4 by EVT in the placental bed, which might lead to trophoblast apoptosis and defective hemochorional placentation in preeclampsia.