Assessing the risks for stillbirth in São Paulo, Brazil: protocol for a multidisciplinary case-control study - FetRisks.

Stillbirth is a fundamental component of childhood mortality, but its causes are still insufficiently understood. This study aims to explore stillbirth risk factors by using a multidisciplinary approach to stimulate public policies and protocols to prevent stillbirth, improve maternal care and support bereaved families. METHODS AND ANALYSIS: In this case-control study with stillbirths and live births in 14 public hospitals in São Paulo, mothers are interviewed at hospitals after delivery, and hospital records and prenatal care registries are reviewed. Maternal and umbilical cord blood samples and placentas are collected to analyse angiogenesis and infection biomarkers, and the placenta's anatomopathological exam. Air pollutant exposure is estimated through the participant's residence and work addresses. Traditional and non-invasive autopsies by image-guided histopathology are conducted in a subset of stillbirths. Subsample mothers of cases are interviewed at home 2 months after delivery on how they were dealing with grief. Information contained in the official prenatal care registries of cases and controls is being compiled. Hospital managers are interviewed about the care offered to stillbirth mothers. Data analysis will identify the main risk factors for stillbirth, investigate their interrelations, and evaluate health services care and support for bereaved families. We hope this project will contribute to the understanding of stillbirth's risk factors and related health services in Brazil, providing new knowledge about this central public health problem, contributing to the improvement of public policies and prenatal and puerperal care, helping to prevent stillbirths and improve the healthcare and support for bereaved families. ETHICS AND DISSEMINATION: This study protocol was approved by the Ethics Committee of the Municipal Health Secretary (process no 16509319.0.3012.5551) and of the Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (process no 16509319.0.0000.0068). Results will be communicated to the study participants, policy-makers and the scientific community.

Monitoring SARS-CoV-2 seroprevalence over time among pregnant women admitted to delivery units: Suitability for surveillance.

OBJECTIVES: To determine SARS-CoV-2 seroprevalence over time and risk factors among pregnant women at delivery in São Paulo, Brazil; and to evaluate the suitability of pregnant women as a sentinel population for SARS-CoV-2 serosurveillance. METHODS: Unselected consecutive pregnant women presenting at the labor ward of a single large hospital between July 20th 2020 to February 21st 2021 were enrolled and tested for SARS-CoV-2 serology using two assays: the rapid chromatic Wondfo One Step (for total IgA and IgG detection) and Roche Elecsys assay (detecting anti-nucleoprotein [N] IgG). SARS-CoV-2 seroprevalence was computed as smooth spline function over time with 95% confidence intervals (CI). Risk factors were evaluated for positivity by each assay. We compared timepoint seroprevalence by the two assays with four concomitant community household surveys (HHS), in which the Roche assay was used, to determine the sensitivity and relevance of the pregnant women population as sentinel population. RESULTS: Overall SARS-CoV-2 seroprevalence was 28.9% (221/763) by Roche and 17.9% (137/763) by Wondfo. Reported symptoms experienced during pregnancy were all significantly correlated with being SARS-CoV-2 seropositive at delivery with any assay (with odds-ratios ranging from 3.0 [95% CI: 2.1-4.3] for coryza to 22.8 [95% CI: 12.3-46.6] for ageusia). Seropositivity by either assay was high in women at delivery in the early period of the pandemic (June 2020), compared with seropositivity in women from the concomitant HHS: 44.1% (95% CI: 21.8-66.4) for Roche, 54.1% (30.9-78.5) for Wondfo, versus 11.4% (95% CI: 9.2-13.6) for HHS. For later periods (October 2020 and January 2021), the seropositivity in women at delivery measured by Roche corresponded well with the prevalence found among women in the HHS using the same assay, whilst prevalence measured by Wondfo dropped. CONCLUSIONS: Women at delivery represent a highly exposed and readily accessible population for sentinel surveillance of emerging infections such as SARS-CoV-2.

The complex search for the cause of gastroschisis.

BACKGROUND: To reveal the complex etiology of gastroschisis through two independent cases. CASES: Case 1 involves gastroschisis recurrence in a consanguineous marriage, and Case 2 concerns a fetus with gastroschisis whose mother had undergone gastroplasty. Methylation array was carried out in both cases (two fetuses with gastroschisis, their two mothers, one father from the consanguineous marriage), and in 16 controls (fetuses and their respective mothers). CONCLUSION: The two cases presented different noninherited methylation profiles.

Vertical transmission of SARS-CoV2 during pregnancy: A high-risk cohort.

OBJECTIVE: Identify the potential for and risk factors of SARS-CoV-2 vertical transmission. METHODS: Symptomatic pregnant women with COVID-19 diagnosis in whom PCR for SARS-CoV-2 was performed at delivery using maternal serum and at least one of the biological samples: cord blood (CB), amniotic fluid (AF), colostrum and/or oropharyngeal swab (OPS) of the neonate. The association of parameters with maternal, AF and/or CB positivity and the influence of SARS-CoV-2 positivity in AF and/or CB on neonatal outcomes were investigated. RESULTS: Overall 73.4% (80/109) were admitted in hospital due to COVID-19, 22.9% needed intensive care and there were four maternal deaths. Positive RT-PCR for SARS-CoV-2 was observed in 14.7% of maternal blood, 13.9% of AF, 6.7% of CB, 2.1% of colostrum and 3.7% of OPS samples. The interval between COVID-19 symptoms and delivery was inversely associated with SARS-CoV-2 positivity in the maternal blood (p = 0.002) and in the AF and/or CB (p = 0.049). Maternal viremia was associated with positivity for SARS-CoV-2 in AF and/or CB (p = 0.001). SARS-CoV-2 positivity in the compartments was not associated with neonatal outcomes. CONCLUSION: Vertical transmission is possible in pregnant women with COVID-19 and a shorter interval between maternal symptoms and delivery is an influencing factor.

Individual exposure to urban air pollution and its correlation with placental angiogenic markers in the first trimester of pregnancy, in São Paulo, Brazil.

Pollution of the atmosphere is known that may lead to adverse obstetric outcomes, including fetal growth restriction, gestational hypertension, and preeclampsia. Such disorders are correlated with imbalances in angiogenic factors, which may also be involved in the pathological mechanism as the pollutants impact placental and maternal physiology. In the first trimester of gestation, this study assessed the outcomes of personal maternal short period exposure to air pollution on soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PLGF) of pregnant women blood concentrations. This was a cross-sectional study, held in the city of São Paulo, Brazil, and conducted with low-risk pregnant women, who carried personal passive nitrogen dioxide (NO2) and ozone (O3) monitors for about a few days preceding the ultrasound evaluation, and on this day, the venous blood sample was collected to measure the angiogenic factors sFlt1 and PLGF and their ratio (sFlt1/PLGF) by enzyme-linked immunosorbent assay (ELISA). By means of multiple regression models, the effect of the studied pollutants on the log-transformed concentrations of the angiogenic factors was evaluated. One hundred thirty-one patients were included. The log of the sFlt1/PLGF ratio increased with rising NO2 levels (p = 0.021 and beta = 0.206), and the log of the PLGF concentration showed a negative correlation with NO2 (p = 0.008 and beta = - 0.234). NO2, an indicator of the levels of primary air pollutants, presented significant positive correlation with an increased sFlt1/PLGF ratio and diminished PLGF levels, which may reflect an antiangiogenic state generated by air pollution exposure.

COL1A1, COL4A3, TIMP2 and TGFB1 polymorphisms in cervical insufficiency.

OBJECTIVES: To investigate the association between selected single nucleotide polymorphisms (SNPs) with cervical insufficiency and its relationship with obstetric history. METHODS: Twenty-eight women with cervical insufficiency (case group) and 29 non-pregnant women (control group) were included. The SNPs sequenced included rs2586490 in collagen type I alpha 1 chain (COL1A1), rs1882435 in collagen type IV alpha 3 chain (COL4A3), rs2277698 in metallopeptidase inhibitor 2 (TIMP2), and rs1800468 in transforming growth factor beta 1 (TGFB1). RESULTS: We found a higher frequency of the normal allele in the control group (65.5%) and the homozygous mutated genotype in the case group (64.3%) for rs2586490 in COL1A1 (p=0.023). An unplanned finding in the cervical insufficiency group was a higher gestational age of delivery (median≥38 weeks) in the mutated allele than in the wild-type genotype (median of 28.2 weeks) for rs2857396, which is also in the COL1A1 gene (p=0.011). CONCLUSIONS: The findings of the present study corroborate the hypothesis that cervical insufficiency has a genetic component and probably involves genes encoding proteins in the extracellular matrix, in addition to inflammatory processes.

Expression of matrix metalloproteinases and their inhibitors at the implantation site in ampullary ectopic pregnancies.

OBJECTIVE: This study investigated the qualitative and semi-quantitative expression of metalloproteinases (MMP) and their tissue inhibitors (TIMP) in trophoblastic tissue during ampullary ectopic pregnancies and correlated that expression with the degree of tubal invasion. STUDY DESIGN: It is a prospective study that included 34 patients diagnosed with ampullary tubal pregnancy who underwent salpingectomy. A histological evaluation of the depth of trophoblastic invasion in the tubes obtained was performed. Subsequently, the expression of the MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2 and TIMP-3 markers was qualitatively and semi-quantitatively evaluated by indirect immunohistochemistry. In addition, the degree of trophoblastic invasion was correlated with the expression of each marker and with the metalloproteinase/inhibitor ratios. RESULTS: MMP-2 (11.2 %; 3.6-17.9) was the marker with greater expression at the implantation site, both in the qualitative and semi-quantitative assessment, while MMP-9 (2.23 %; 0.2-5.4) and TIMP-3 (2.53 %; 0.1-15.3) were only weakly expressed. CONCLUSION: There was wide variation in expression among the markers and metalloproteinase/inhibitor ratios studied compared to the degrees of invasion.

Preditores moleculares de evolução da mola hidatiforme no tecido trofoblástico / Molecular prognostic predictors in trophoblastic tissue of hydatidiform mole

A mola hidatiforme (MH) é a forma mais comum de doença trofoblástica gestacional e representa uma condição benigna que em alguns casos pode sofrer malignização. Todas as pacientes diagnosticadas com doenças molares são acompanhadas por pelo menos seis meses para detecção precoce da neoplasia trofoblástica gestacional. No momento, existem poucas ferramentas para avaliação prognóstica da mola hidatiforme. Foi descrita a expressão diferencial de diversos fatores em tecido molar em comparação ao trofoblasto não neoplásico. Essas moléculas podem estar relacionadas com o comportamento agressivo da MH e consequentemente poderiam servir para melhor entendimento do processo de malignização e como preditoras da evolução da doença trofoblástica gestacional.

The hydatidiform mole (HM) is the most common form of gestational trophoblastic disease and a benign condition that in some cases may undergo malignant transformation. All patients diagnosed with molar diseases are monitored for at least six months for early detection of gestational trophoblastic neoplasia. Currently, there are few prognostic tools for the prediction of hydatidiform mole evolution. Differential expression on molar tissue of different molecular factors have been described when compared to non-neoplastic trophoblast. These markers may be associated with aggressive behavior of HM and therefore could serve as predictors of the development of gestational trophoblastic disease and to better understand molar malignant transformation. This review article will summarize and evaluate prognostic molecular markers of HM.

NADPH oxidase as an important source of reactive oxygen species at the mouse maternal-fetal interface: putative biological roles.

Oxygen derivatives that comprise the large family of reactive oxygen species (ROS) are actively involved in placental biology. They are generated at the maternal-fetal interface at the level of decidual, trophoblast and mesenchymal components. In normal conditions, ROS produced in low concentrations participate in different functions as signalling molecules, regulating activation of redox-sensitive transcription factors and protein kinases involved in cell survival, proliferation and apoptosis, hence much of cell functioning. Physiological ROS generation is also associated with such defence mechanisms as phagocytosis and microbiocidal activities. In mice, particularly but not exclusively, trophoblast cells phagocytose intensively during implantation and post-implantation periods and express enzymic machinery to address a ROS-producing response to changes in the environment. The cells directly associated with ROS production are trophoblast giant cells, which mediate each and every relationship with the maternal organism. In this review, the production of ROS by the implanting mouse trophoblast is discussed, focusing on NADPH oxidase expression, regulatory mechanisms and similarities with NOX2 from phagocytes. Some of the current controversies are assessed by attempting to integrate data from studies in human trophoblast and mouse models.

Bothrops jararaca peptide with anti-hypertensive action normalizes endothelium dysfunction involved in physiopathology of preeclampsia.

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, is a major cause of fetal and maternal morbidity and mortality especially in developing countries. Bj-PRO-10c, a proline-rich peptide isolated from Bothrops jararaca venom, has been attributed with potent anti-hypertensive effects. Recently, we have shown that Bj-PRO-10c-induced anti-hypertensive actions involved NO production in spontaneous hypertensive rats. Using in vitro studies we now show that Bj-PRO-10c was able to increase NO production in human umbilical vein endothelial cells from hypertensive pregnant women (HUVEC-PE) to levels observed in HUVEC of normotensive women. Moreover, in the presence of the peptide, eNOS expression as well as argininosuccinate synthase activity, the key rate-limiting enzyme of the citrulline-NO cycle, were enhanced. In addition, excessive superoxide production due to NO deficiency, one of the major deleterious effects of the disease, was inhibited by Bj-PRO-10c. Bj-PRO-10c induced intracellular calcium fluxes in both, HUVEC-PE and HUVEC, which, however, led to activation of eNOS expression only in HUVEC-PE. Since Bj-PRO-10c promoted biological effects in HUVEC from patients suffering from the disorder and not in normotensive pregnant women, we hypothesize that Bj-PRO-10c induces its anti-hypertensive effect in mothers with preeclampsia. Such properties may initiate the development of novel therapeutics for treating preeclampsia.

Bothrops jararaca Peptide with Anti-Hypertensive Action Normalizes Endothelium Dysfunction Involved in Physiopathology of Preeclampsia

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, is a major cause of fetal and maternal morbidity and mortality especially in developing countries. Bj-PRO-10c, a proline-rich peptide isolated from Bothrops jararaca venom, has been attributed with potent anti-hypertensive effects. Recently, we have shown that Bj-PRO-10c-induced anti-hypertensive actions involved NO production in spontaneous hypertensive rats. Using in vitro studies we now show that Bj-PRO-10c was able to increase NO production in human umbilical vein endothelial cells from hypertensive pregnant women (HUVEC-PE) to levels observed in HUVEC of normotensive women. Moreover, in the presence of the peptide, eNOS expression as well as argininosuccinate synthase activity, the key rate-limiting enzyme of the citrulline-NO cycle, were enhanced. In addition, excessive superoxide production due to NO deficiency, one of the major deleterious effects of the disease, was inhibited by Bj-PRO-10c. Bj-PRO-10c induced intracellular calcium fluxes in both, HUVEC-PE and HUVEC, which, however, led to activation of eNOS expression only in HUVEC-PE. Since Bj-PRO-10c promoted biological effects in HUVEC from patients suffering from the disorder and not in normotensive pregnant women, we hypothesize that Bj-PRO-10c induces its anti-hypertensive effect in mothers with preeclampsia. Such properties may initiate the development of novel therapeutics for treating preeclampsia.

Trophoblast phagocytic program: roles in different placental systems.

Although not belonging to the class of professional phagocytes, in many species trophoblast cells exhibit intense phagocytic activity. The complete range of physiological functions of trophoblast phagocytosis has not yet been fully characterized. Close association between the trophoblast and nutrition was determined many years ago. Hubrecht (1889) when proposing for the first time the name trophoblast to the external layer of the blastocyst, directly established the nutritive significance of this embryonic layer. Indeed, histotrophic phagocytosis, i.e. the internalization of maternal cells and secreted materials, is considered an important function of the trophoblast before the completion of the placenta. Recently, however, unexpected characteristics of the trophoblast have significantly enhanced our understanding of this process. Roles in acquisition of space for embryo development, in tissue remodeling during implantation and placentation and in defense mechanisms are highlighting how this cellular activity may be relevant for the maternal-fetal relationship beyond its nutritional function.

Signaling molecules involved in IFN-gamma-inducible nitric oxide synthase expression in the mouse trophoblast.

PROBLEM: We have previously shown that trophoblast can generate nitric oxide (NO) and express inducible isoform of nitric oxide synthase (iNOS). Moreover, this production was changed by the presence of interferon-gamma (IFN-gamma) establishing a relationship between trophoblast inductive response and this proinflammatory cytokine. METHOD OF STUDY: As the intracellular signal transduction pathway used by IFN-gamma in target cells is the Janus kinase (JAK)-signal transducer and transcription activator (STAT), here we analyzed in the mouse trophoblast the effect of IFN-gamma and staurosporine on mRNA and protein expressions of IFN-gamma signaling molecules correlating them with iNOS expression. RESULTS: Interferon-gamma induced iNOS expression and upregulated Jaks and Stat1, but not Stat2 transcriptions. The protein distribution matched the mRNA expression pattern. These effects were abrogated when IFN-gamma receptor was blocked by staurosporine. CONCLUSION: Due to the biological effects of NO-iNOS generated on induction of apoptosis and inflammatory responses, interaction between iNOS expression and IFN-gamma-mediated signaling is very important for understanding the physiology of trophoblast at the maternal-fetal interface. Our data indicate IFN-gamma acts specifically on trophoblast, regulating the expression of signaling molecules and is fundamental for iNOS expression.

Modulation of the induction of lung and airway allergy in the offspring of IFN-gamma-treated mother mice.

Recent studies have highlighted the influence of fetal/maternal interactions on the development of asthma. Because IFN-gamma reduces Th2-mediated allergic responses, we assessed its capacity to modulate asthma in the offspring when injected into mothers during pregnancy. IFN-gamma was injected in CD1 female mice on day 6.5 of gestation. Immediately after birth, male newborns were housed in cages with interchanged mothers: the offspring from IFN-gamma-treated mothers were breastfed by normal mothers (IFN/nor), and those from normal mothers were breastfed by IFN-gamma-treated (Nor/IFN) or normal mothers (Nor/nor). Immediately after weaning, the spleen cells from IFN/nor and Nor/IFN mice produced less IL-4 and more IFN-gamma than Nor/nor mice when stimulated with Con A. At the age of 6-7 wk, mice were immunized with OVA on days 0 and 7. From day 14 to 16, they were exposed to aerosolized OVA. The bronchoalveolar lavage fluid from Nor/nor mice showed eosinophilia, a large number of these cells being present in perivascular and peribronchial regions of lung tissues. IFN/nor or Nor/IFN mice showed greatly reduced eosinophil numbers in bronchoalveolar lavage fluid. In addition, lung sections from IFN/nor, but not Nor/IFN mice showed almost normal histology. In OVA-sensitized IFN/nor and Nor/IFN mice, the production of IFN-gamma, IL-4, and IL-5 by spleen cells was significantly reduced as compared with cells from the OVA-sensitized Nor/nor group. IgE and anaphylactic IgG1 were also reduced in plasma of IFN/nor mice. In conclusion, the presence of IFN-gamma during pregnancy confers to the fetus a protection against allergenic provocations in the adult life.