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We investigated whether excessive retroperitoneal adipose tissue (AT) expansion programmed by maternal obesity (MO) affects adipocyte size distribution and gene expression in relation to adipocyte proliferation and differentiation in male and female offspring (F1) from control (F1C) and obese (F1MO) mothers. Female Wistar rats (F0) ate a control or high-fat diet from weaning through pregnancy and lactation. F1 were weaned onto a control diet and euthanized at 110 postnatal days. Fat depots were weighed to estimate the total AT. Serum glucose, triglyceride, leptin, insulin, and the insulin resistance index (HOMA-IR) were determined. Adipocyte size and adipogenic gene expression were examined in retroperitoneal fat. Body weight, retroperitoneal AT and adipogenesis differed between male and female F1Cs. Retroperitoneal AT, glucose, triglyceride, insulin, HOMA-IR and leptin were higher in male and female F1MO vs. F1C. Small adipocytes were reduced in F1MO females and absent in F1MO males; large adipocytes were increased in F1MO males and females vs. F1C. Wnt, PI3K-Akt, and insulin signaling pathways in F1MO males and Egr2 in F1MO females were downregulated vs. F1C. MO induced metabolic dysfunction in F1 through different sex dimorphism mechanisms, including the decreased expression of pro-adipogenic genes and reduced insulin signaling in males and lipid mobilization-related genes in females.

Maternal metabolic status influences pregnancy and, consequently, the perinatal outcome. Resistin is a pro-inflammatory adipokine predominantly expressed and secreted by mononuclear cells, adipose tissue, and placental trophoblastic cells during pregnancy. Recently, we reported an inverse association between maternal resistin levels and fetal low-density lipoprotein cholesterol (LDL-C). Then, in this work, we used a human placental explant model and the trophoblast cell line JEG-3 to evaluate whether resistin affects placental LDL-C uptake. Resistin exposure induced the transcription factor SREBP-2, LDLR, and PCSK9 mRNA expression, and changes at the protein level were confirmed by immunohistochemistry and Western blot. However, for LDLR, the changes were not consistent between mRNA and protein levels. Using a labeled LDL-cholesterol (BODIPY FL LDL), uptake assay demonstrated that the LDL-C was significantly decreased in placental explants exposed to a high dose of resistin and a lesser extent in JEG-3 cells. In summary, resistin induces PCSK9 expression in placental explants and JEG-3 cells, which could be related to negative regulation of the LDLR by lysosomal degradation. These findings suggest that resistin may significantly regulate the LDL-C uptake and transport from the maternal circulation to the fetus, affecting its growth and lipid profile.

Evidence from studies in the general population suggests an association between vitamin D insufficiency/deficiency and COVID-19 susceptibility and disease severity. The present study was performed on 165 third-trimester pregnant women at the time of delivery. Seventy-nine women tested negative for SARS-CoV-2. From 86 women testing positive, 32 were asymptomatic, 44 presented a mild form of the disease, and 10 experienced severe symptoms. Serum 25-OH vitamin D levels were measured on blood samples collected on admission. Low vitamin D levels were detected in symptomatic but not asymptomatic COVID-19 patients compared to healthy women (p = 0.0227). In addition, 20 (45.4%) pregnant women in the mild COVID-19 group and 6 (60%) in the severe group were vitamin D deficient (p = 0.030). On the other hand, lasso regression analysis showed that 25-OH vitamin D deficiency is an independent predictor of severe COVID-19 with an odds ratio (OR) of 5.81 (95% CI: 1.108–30.541; p = 0.037). These results show the relationship between vitamin D deficiency in pregnant women and the severity of COVID-19 infection and support the recommendation to supplement with vitamin D to avoid worse COVID-19 outcomes during pregnancy.

An altered mitochondrial DNA copy number (mtDNAcn) at birth can be a marker of increased disease susceptibility later in life. Gestational exposure to acute stress, such as that derived from the earthquake experienced on 19 September 2017 in Mexico City, could be associated with changes in mtDNAcn at birth. Our study used data from the OBESO (Biochemical and Epigenetic Origins of Overweight and Obesity) perinatal cohort in Mexico City. We compared the mtDNAcn in the umbilical cord blood of 22 infants born before the earthquake, 24 infants whose mothers were pregnant at the time of the earthquake (exposed), and 37 who were conceived after the earthquake (post-earthquake). We quantified mtDNAcn by quantitative real-time polymerase chain reaction normalized with a nuclear gene. We used a linear model adjusted by maternal age, body mass index, socioeconomic status, perceived stress, and pregnancy comorbidities. Compared to non-exposed newborns (mean ± SD mtDNAcn: 0.740 ± 0.161), exposed and post-earthquake newborns (mtDNAcn: 0.899 ± 0.156 and 0.995 ± 0.169, respectively) had increased mtDNAcn, p = 0.001. The findings of this study point at mtDNAcn as a potential biological marker of acute stress and suggest that experiencing an earthquake during pregnancy or before gestation can have programing effects in the unborn child. Long-term follow-up of newborns to women who experience stress prenatally, particularly that derived from a natural disaster, is warranted.

Nayeli Martínez-Cruz,1 Agnese Maria Chiara Rapisarda,2 Karla Patricia Soriano-Ortega,3 Lidia Arce-Sánchez,1 Antonio Cianci,2 Carlos Ortega-Gonzalez,1 Ursula Torres-Herrera,1 Salvador Espino-Y-Sosa,3 Guadalupe Estrada-Gutierrez,4 Araceli Montoya-Estrada,5 José Romo-Yañez,5 Enrique Reyes-Muñoz5 1Department of Endocrinology, Instituto Nacional de Perinatología “Isidro Espinosa de los Reyes”, Mexico City, México; 2Department of General Surgery and Medical Surgical Specialties, University of Catania, Catania, Italy; 3Division of Clinical Research, Instituto Nacional de Perinatología “Isidro Espinosa de los Reyes”, Mexico City, México; 4Direction of Research, Instituto Nacional de Perinatología “Isidro Espinosa de los Reyes”, Mexico City, México; 5Department of Gynecological and Perinatal Endocrinology, Instituto Nacional de Perinatología “Isidro Espinosa de los Reyes”, Mexico City, MéxicoCorrespondence: Enrique Reyes-MuñozDepartment of Gynecological and Perinatal Endocrinology, Instituto Nacional de Perinatología Isidro Espinosa de los Reyes, Montes Urales 800 Lomas Virreyes, Miguel, Hidalgo 11000, Mexico CityMexicoTel +52 5555209900 Ext 307Email dr.enriquereyes@gmail.comPurpose: To compare the risk of adverse perinatal outcomes (APO) between pregnant women with mild gestational diabetes mellitus (GDM) diagnosed by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria, on no specific treatment, versus pregnant women without GDM.Patients and Methods: A retrospective cohort study of pregnant women referred to the Instituto Nacional de Perinatología, in Mexico City, for prenatal care and delivery. Eligibility criteria were singleton pregnancy, age >18 years, gestational age 20–28 weeks, and no history of pre-gestational diabetes. The study population was divided into two groups: Group 1, comprising women with mild GDM defined by one abnormal glucose value at the oral glucose tolerance test (OGTT) according to IADPSG criteria [fasting: 5.1–5.2 mmol/L (92–94 mg/dL) or 2h 8.5–8.56 mmol/L (153–154 mg/dL)], who did not receive specific treatment for GDM, and Group 2, comprising women without GDM, matched for maternal age and pre-gestational body mass index (BMI). Women with two or more abnormal OGTT values, pre-gestational diabetes, any chronic disease, or multiple pregnancies were excluded.Results: As many as 282 women were included in each group. There were no significant differences in basal characteristics between groups. APO analysis showed that newborn weight was significantly higher in Group 1 (3042.4±499g) vs Group 2 (2910±565g) p=0.003; conversely, the incidence of large for gestational age (LGA) and macrosomic neonates was similar in both groups (6 vs 5.7% and 2.1 vs 2.2%, respectively). There were no differences in rates of preeclampsia and gestational hypertension, cesarean and preterm delivery, or premature rupture of membranes. A sub-analysis by maternal pre-gestational BMI showed that LGA incidence was significantly higher among babies born to women with pre-gestational BMI ≥30 kg/m2 in both groups.Conclusion: The risk of APO was similar among Mexican women with mild untreated GDM diagnosed by IADPSG criteria, compared to pregnant women without GDM. Pre-gestational BMI was an independent risk factor for LGA.Keywords: gestational diabetes, large for gestational age, IADPSG, hyperglycemia, pregnancy &nbsp

Introduction: Lead (Pb) crosses the placenta and can cause oxidative stress, reduced fetal growth and neurological problems. The principal source of oxidative stress in human cells is mitochondria. Therefore, disruption of normal mitochondrial function during pregnancy may represent a primary mechanism behind the adverse effects of lead. We sought to assess the association of Pb exposure during pregnancy with mitochondrial DNA (mtDNA) content, a sensitive marker of mitochondrial function, in cord blood. Materials and methods: This study comprised mother-infant pairs from the Programming Research in Obesity, Growth, Environment and Social Stressors (PROGRESS) study, a prospective birth-cohort that enrolled 1050 pregnant women from Mexico City who were receiving prenatal care between December 2007 and July 2011. Quantitative PCR was used to calculate relative MtDNA content (mitochondrial-to-nuclear DNA ratio (mtDNA/nDNA)) in cord blood. Lead concentrations in both maternal blood (2nd and 3rd trimester and at delivery day) and in cord blood were measured by ICP-MS. Multivariable regression models adjusting for multiple confounders were fitted with 410 mother-infant pairs for whom complete data for mtDNA content, lead levels, and covariates were available. Results: Maternal blood Pb measured in the second (mean 3.79 μg/dL, SD 2.63; β = 0.059, 95% CI 0.008, 0.111) and third trimester (mean 3.90 μg/dL; SD 2.84; β = 0.054, 95% CI 0.002, 0.107) during pregnancy and PB in cord blood (mean 3.50 μg/dL, SD 2.59; β = 0.050, 95% CI 0.004; 0.096) were associated with increased cord blood mtDNA content (mean 1.46, SD 0.44). In two-way interaction analyses, cord blood Pb marginally interacted with gestational age leading to an increase in mtDNA content for pre-term births (Benjamini-Hochberg False Discovery Rate correction; BH-FDR = 0.08). Conclusion: This study shows that lead exposure in pregnancy alters mtDNA content in cord blood; therefore, alteration of mtDNA content might be a mechanism underlying the toxicity of lead. Keywords: mtDNA content, Lead exposure, Pregnancy, Mitochondrial dysfunction, Cord blood

As renal development and maturation processes begin in utero and continue through early childhood, sensitive developmental periods arise during which metal exposures can program subclinical nephrotoxicity that manifests later in life. We used novel dentine biomarkers of established nephrotoxicants including arsenic (As), cadmium (Cd), lead (Pb), chromium (Cr), and lithium (Li), and their mixtures, to identify critical windows of exposure-associated kidney function alterations in preadolescents.Participants included 353 children in the Programming Research in Obesity Growth, Environment and Social Stressors (PROGRESS) longitudinal birth cohort study based in Mexico City. Estimated glomerular filtration rate (eGFR) was assessed in 8-12 year old children using serum cystatin C measures. Pre- and postnatal metal(loid) concentrations were assessed in weekly increments by analyzing deciduous teeth with laser ablation-inductively coupled plasma-mass spectrometry. We used reverse distributed lag models (rDLMs) and lagged Weighted Quantile Sum (L-WQS) regression to examine time-varying associations between weekly perinatal metal(loid) exposure or metal(loid) mixtures and preadolescent eGFR while adjusting for age, sex, BMI z-score, SES and prenatal tobacco smoke exposure.We identified a critical window of susceptibility to Pb exposure, in the late 3rd trimester (5 weeks prior to birth) during which higher Pb exposure was associated with children's increased eGFR. When all elements were assessed as a mixture, we identified late 2nd/early 3rd trimester (weeks 8-17 of gestation) as a window of vulnerability associated with decreased eGFR, with Li and Cr contributing the greatest weights to the association. When stratified by sex, we observed stronger effects among boys than girls.Using tooth-matrix biomarkers, we identified discrete developmental exposure windows wherein Pb and metal(loid) mixtures were associated with altered preadolescent kidney function.

A high-quality diet during pregnancy may have positive effects on fetal growth and nutritional status at birth, and it may modify the risk of developing chronic diseases later in life. The aim of this study was to evaluate the association between diet quality and newborn nutritional status in a group of pregnant Mexican women. As part of the ongoing Mexican prospective cohort study, OBESO, we studied 226 healthy pregnant women. We adapted the Alternated Healthy Eating Index-2010 for pregnancy (AHEI-10P). The association between maternal diet and newborn nutritional status was investigated by multiple linear regression and logistic regression models. We applied three 24-h recalls during the second half of gestation. As the AHEI-10P score improved by 5 units, the birth weight and length increased (β = 74.8 ± 35.0 g and β = 0.3 ± 0.4 cm, respectively, p &lt, 0.05). Similarly, the risk of low birth weight (LBW) and small for gestational age (SGA) decreased (OR: 0.47, 95%CI: 0.27–0.82 and OR: 0.55, 95%CI: 0.36–0.85, respectively). In women without preeclampsia and/or GDM, the risk of stunting decreased as the diet quality score increased (+5 units) (OR: 0.62, 95%IC: 0.40–0.96). A high-quality diet during pregnancy was associated with a higher newborn size and a reduced risk of LBW and SGA in this group of pregnant Mexican women.

Aims: The authors sought to examine associations between urinary exosomal miRNAs (exo-miRs), emerging biomarkers of renal health, and cardiorenal outcomes in early childhood. Materials & methods: The authors extracted exo-miRs in urine from 88 healthy Mexican children aged 4–6 years. The authors measured associations between 193 exo-miRs and cardiorenal outcomes: systolic/diastolic blood pressure, estimated glomerular filtration rate and urinary sodium and potassium levels. The authors adjusted for age, sex, BMI, socioeconomic status, indoor tobacco smoke exposure and urine specific gravity. Results: Multiple exo-miRs were identified meeting a false discovery rate threshold of q

Lead is a ubiquitous pollutant with deleterious effects on human health and remains a major current public health concern in developing countries. This heavy metal may interfere with nucleic acids via oxidative stress or epigenetic changes that affect biological markers of aging, e.g., telomere length and DNA methylation (DNAm). Telomere shortening associates with biological age in newborns, and DNA methylation at specific CpG sites can be used to calculate "epigenetic clocks".The aim of this study was to examine the associations of prenatal lead exposures with telomere length and DNA-methylation-based predictors of age in cord blood.The study included 507 mother-child pairs from the Programming Research in Obesity, Growth, Environment and Social Stressors (PROGRESS) study, a birth cohort in Mexico City. Maternal blood (second trimester, third trimester and at delivery) and bone lead levels (one month postpartum) were measured using inductively coupled plasma-mass spectrometry and X-ray fluorescence, respectively. Cord blood leukocyte telomere length was measured using quantitative PCR and apparent age by DNA methylation biomarkers, i.e., Horvath's DNA methylation age and the Knight's predictor of gestational age.Average maternal age was 28.5 ± 5.5 years, and 51.5% reported low socioeconomic status. Children's mean telomere length was 1.2 ± 1.3 relative units, and mean DNA methylation ages using the Horvath's and Knight's clocks were -2.6 ± 0.1 years and 37.9 ± 1.4 weeks (mean ± SD), respectively. No significant associations were found between maternal blood and bone lead concentrations with telomere length and DNAm age in newborns.We found no associations of prenatal lead exposure with telomere length and DNA methylation age biomarkers.

The cytotrophoblast of human placenta transitions into an outer multinucleated syncytiotrophoblast (STB) layer that covers chorionic villi which are in contact with maternal blood in the intervillous space. During pregnancy, the Zika virus (ZIKV) poses a serious prenatal threat. STB cells are resistant to ZIKV infections, yet placental cells within the mesenchyme of chorionic villi are targets of ZIKV infection. We seek to determine whether ZIKV can open the paracellular pathway of STB cells. This route is regulated by tight junctions (TJs) which are present in the uppermost portion of the lateral membranes of STB cells. We analyzed the paracellular permeability and expression of E-cadherin, occludin, JAMs &ndash, B and &ndash, C, claudins -1, -3, -4, -5 and -7, and ZO-1, and ZO-2 in the STB of placentae from ZIKV-infected and non-infected women. In ZIKV-infected placentae, the pattern of expression of TJ proteins was preserved, but the amount of claudin-4 diminished. Placentae from ZIKV-infected women were permeable to ruthenium red, and had chorionic villi with a higher mean diameter and Hofbauer hyperplasia. Finally, ZIKV added to the basolateral surface of a trophoblast cell line reduced the transepithelial electrical resistance. These results suggest that ZIKV can open the paracellular pathway of STB cells.

Controversy remains surrounding vitamin D routine supplementation in healthy pregnancy, and the doses are unclear. The aim of this study was to describe maternal vitamin D status throughout pregnancy in a group of Mexican women and evaluate the effect of frequently prescribed doses of vitamin D3 on longitudinal 25-OH-D concentrations, adjusting for obesity, season, and other factors. We conducted a cohort study (Instituto Nacional de Perinatolog&iacute, a-INPer) (2017&ndash, 2020)) of healthy pregnant women without complications. Pregestational overweight/obesity (body mass index &ge, 25), vitamin D3 supplementation (prescribed by physician, 0&ndash, 250, 250&ndash, 400, and &gt, 400 IU/day), and serum 25-OH-D concentrations (ELISA) were evaluated in each trimester of pregnancy. Vitamin D deficiency or insufficiency was computed (&lt, 20 and &lt, 30 ng/mL, respectively). We studied 141 adult women, 58.5% had pregestational obesity or overweight. In the first trimester, 45.8% of the women were supplemented with vitamin D3, 51.4% had vitamin D insufficiency and 37.3%, deficiency. In the third trimester, 75.4% of the women were supplemented, and 20% of them still had deficiency. The final general mixed linear model showed that 25-OH-D significantly increased throughout pregnancy (p &lt, 0.001), the highest increase was observed in the third trimester in women with doses &gt, 400 IU/day of vitamin D3 (+4 ng/mL, 95% CI: 1.72&ndash, 8.11 ng/mL). In winter/autumn, 25-OH-D concentrations were also lower (p &le, 0.05). In this group of pregnant Mexican women, the prevalence of vitamin D deficiency and insufficiency was high. A higher increase in 25-OH-D concentrations during pregnancy was observed when the women were supplemented with &gt, 400 IU/day. Common supplementation doses of 250&ndash, 400 IU/day were insufficient for achieving an adequate maternal vitamin D status.

Preeclampsia (PE) and Intrauterine Growth Restriction (IUGR) are major contributors to perinatal morbidity and mortality. These pregnancy disorders are associated with placental dysfunction and share similar pathophysiological features. The aim of this study was to compare the placental gene expression profiles including mRNA and lncRNAs from pregnant women from four study groups: PE, IUGR, PE-IUGR, and normal pregnancy (NP). Gene expression microarray analysis was performed on placental tissue obtained at delivery and results were validated using RTq-PCR. Differential gene expression analysis revealed that the largest transcript variation was observed in the IUGR samples compared to NP (n = 461, 314 mRNAs: 252 up-regulated and 62 down-regulated, 133 lncRNAs: 36 up-regulated and 98 down-regulated). We also detected a group of differentially expressed transcripts shared between the PE and IUGR samples compared to NP (n = 39), including 9 lncRNAs with a high correlation degree (p &lt, 0.05). Functional enrichment of these shared transcripts showed that cytokine signaling pathways, protein modification, and regulation of JAK-STAT cascade are over-represented in both placental ischemic diseases. These findings contribute to the molecular characterization of placental ischemia showing common epigenetic regulation implicated in the pathophysiology of PE and IUGR.

In the reproductive phase, women experience cyclic changes in the ovaries and uterus, and hormones regulate these changes. Menopause is the permanent loss of menstruation after 12 months of amenorrhea. Menopause is also linked to a decrease in estrogen production, causing an imbalance in oxidative stress. We aimed to compare the three stages of lipid peroxidation, protein oxidative damage, and total antioxidant capacity (TAC) between reproductive-aged women (RAW) and postmenopausal women (PMW) in Mexico. We carried out a cross-sectional study with 84 women from Mexico City, including 40 RAW and 44 PMW. To determine the oxidative stress of the participants, several markers of lipid damage were measured: dienes conjugates (DC), lipohydroperoxides (LHP), and malondialdehyde (MDA), exposure to protein carbonyl is indicative of oxidative modified proteins, and TAC is indicative of the antioxidant defense system. Biomarkers of oxidative stress were significantly lower in RAW vs. PMW. DC were 1.31 &plusmn, 0.65 vs. 1.7 &plusmn, 0.51 pmol DC/mg dry weight (p = 0.0032), LHP were 4.95 &plusmn, 2.20 vs. 11.30 &plusmn, 4.24 pmol LHP/mg dry weight (p &lt, 0.0001), malondialdehyde was 20.37 &plusmn, 8.20 vs. 26.10 &plusmn, 8.71 pmol MDA/mg dry weight (p = 0.0030), exposure of protein carbonyl was 3954 &plusmn, 884 vs. 4552 &plusmn, 1445 pmol PC/mg protein (p = 0.042), and TAC was 7244 &plusmn, 1512 vs. 8099 &plusmn, 1931 pmol Trolox equivalent/mg protein (p = 0.027). PMW display significantly higher oxidative stress markers compared to RAW, likewise, PMW show a higher TAC.

Obesity is associated with inflammatory changes and accumulation and phenotype polarization of adipose tissue macrophages (ATMs). Obese pregnant women have alterations in adipose tissue composition, but a detailed description of macrophage population is not available. In this study, we characterized macrophage populations in visceral adipose tissue (VAT) from pregnant women with normal, overweight, and obese pregestational weight. Immunophenotyping of macrophages from VAT biopsies was performed by flow cytometry using CD45 and CD14 as markers of hematopoietic and monocyte linage, respectively, while HLA-DR, CD11c, CD163, and CD206 were used as pro- and anti-inflammatory markers. Adipocyte number and size were evaluated by light microscopy. The results show that pregnant women that were overweight and obese during the pregestational period had adipocyte hypertrophy. Two different macrophage populations in VAT were identified: recruited macrophages (CD45+CD14+), and a novel population lacking CD45, which was considered to be a resident macrophages subset (CD45−CD14+). The number of resident HLA−DRlow/− macrophages showed a negative correlation with body mass index (BMI). Both resident and recruited macrophages from obese women expressed higher CD206 levels. CD11c expression was higher in resident HLA-DR+ macrophages from obese women. A strong correlation between CD206 and CD11c markers and BMI was observed. Our findings show that being overweight and obese in the pregestational period is associated with adipocyte hypertrophy and specific ATMs populations in VAT.

Pregnancy is a complex process where several physiological pathways interact. The down-regulated inflammatory response and the abundance of anti-inflammatory molecules during gestation may explain the acceptance of the fetus and the lack of immune response against it, even though it is a foreign tissue for the mother. NF-κB is a key regulator of the transcription of inflammatory genes, such as IL-8, IL-1β, TNF-α, or IL-6. Increased NF-κB activity that leads to the production of proinflammatory cytokines may induce obstetric disorders, such as preterm birth or abortion. Low activity of this transcription factor is associated with the beneficial anti-inflammatory environment during fetus development until delivery. Galectin-1 (Gal-1) is a lectin-type glycan-binding protein that is able to down-regulate inflammation. It has been shown that Gal-1 is abundantly expressed at the feto-maternal interface in humans, where it promotes maternal immune tolerance to the fetal semi-allograft. Gal-1 tolerance-promoting mechanisms have been established for adaptive immune cells, such as T cells and dendritic cells. However, the role of this lectin has not been established in non-immune cells at the feto-maternal interface. Here, we determined that Gal-1 is able to block the stimulating effect of LPS on IL-6 in human decidua cells. Our results show that Gal-1 acts by inhibiting the stimulation of the LPS-induced IκBζ expression, an NF-κB regulator involved in IL-6 gene transcription.

Problem Infection of human fetal membranes elicits secretion of pro-inflammatory modulators through its innate immune capacities. We investigated the effect of lipopolysacharide (LPS) and progesterone (P4) upon expression of TLR-4/MyD88, TNFα, IL-6, IL-8, IL-10, and HBD2 on the human amniotic epithelium. Method of study Explants of the human amniotic epithelium were pre-treated with 0.01, 0.1, and 1.0 μm of P4; then cotreated with 1000 ng/mL LPS. TLR-4 was immuno-detected, and concentrations of MyD88, TNFα, IL-6, IL-8, IL-10, and HBD2 were quantified by ELISA. Results P4 significantly reduced the expression of LPS-induced TLR-4/MyD88. LPS increased the concentrations of TNFα, IL-6, IL-8, IL-10, and HBD2 by factors of 30-, eight, three, three, and fivefold, respectively. P4 at 1.0 μm was the most effective dose to blunt the secretion of TNFα, IL-6, and HBD-2. RU-486 blocks the effect of P4. Conclusion P4 inhibited LPS-induced TLR-4/MyD88 and pro-inflammatory factors in the human amniotic epithelium. These results could explain partially how P4 can protect the amniotic region of fetal membranes and generate a compensatory mechanism that limits the secretion of pro-inflammatory modulators, which could jeopardize the immune privilege during pregnancy.

Objective To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa. Design Case-control, prospective study. Setting Academic centers. Patient(s) Fertile and subfertile subjects. Intervention(s) Semen samples from subfertile and fertile men, 11 per group, were analyzed for basic semen parameters and early (annexin-V binding) and late (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with M540 to assess the presence of M540 apoptotic bodies. Main Outcome Measure(s) Presence of M540 apoptotic bodies. Result(s) Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups. Conclusion(s) This study confirmed the occurrence of M540 bodies in semen and that male factor infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that the presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.

Objective To study the association of obesity and overweight with the prevalence of insulin resistance (IR), pre-diabetes and clinical–biochemical characteristics among infertile Mexican women with polycystic ovary syndrome (PCOS). Design Retrospective cross-sectional study. Setting Level-three medical institution, an infertility clinic in Mexico City. Participants We included infertile Mexican women with diagnosis of PCOS according to the Rotterdam criteria: group 1 (n=83), normal weight (body mass index (BMI) 18.5–24.9 kg/m2); group 2 (n=217), overweight (BMI 25–29.9 kg/m2); and group 3 (n=238), obese (BMI≥30 kg/m2). Primary and secondary outcome measures IR was determined by homeostatic model assessment (HOMA) >2.5 and pre-diabetes by fasting glucose between 5.6 and 6.9 mmol/L and/or glucose value between 7.8 and 11 mmol/L at 2 hours during an oral glucose tolerance test. We compared clinical–biochemical characteristics among groups. Results Prevalence of IR for groups 1, 2 and 3 was 19.3%, 56.2% and 78.2%; overweight and obesity increase the IR OR (CI 95%) to 5.3 (2.9 to 9.8) and 14.9 (8.0 to 28), respectively. Prevalence of pre-diabetes for groups 1, 2 and 3 was 7.2%, 17.5% and 31.5%; overweight and obesity increase the pre-diabetes OR (CI 95%) to 2.7 (1.1 to 6.7) and 5.9 (2.4 to 14), respectively. Acanthosis nigricans was more frequent in group 3 than group 1. Free Androgen Index (FAI) and thyroid-stimulating hormone (TSH) levels were lower in group 1 than in groups 2 and 3. Progesterone and sex hormone-binding globulin (SHBG) levels were higher in group 1 than in groups 2 and 3. Dehydroepiandrosterone sulfate (DHEA-S) was higher in group 1 than group 3. Conclusions Obese and overweight infertile Mexican women with PCOS, attending to an infertility clinic, have a higher prevalence of IR and pre-diabetes compared with normal-weight women with PCOS. Therapeutic interventions should include those that improved metabolic functioning prior to attempting pregnancy in these groups of women.

Rupture of the fetal membranes during human labor is associated with an inflammatory process localized to the maternal-fetal interface. There is evidence that specific leukocytes subsets are attracted to the choriodecidua, and that after homing they condition a local inflammatory microenvironment, possibly being directly involved in rupture of the membranes. In this study our aim was to compare the phenotypes and function of leukocytes located in the placental intervillous blood with peripheral leukocytes obtained before or after labor, including expression of modulators of inflammation in these cells. Flow cytometry revealed that the proportion of CD14(+) cells is increased in intervillous blood, suggesting the participation of monocytes/macrophages during labor. Real time qRT-PCR showed that at term gestation and particularly during labor, placental blood leukocytes adopt a different expression pattern of pro-inflammatory cytokines than leukocytes in peripheral blood, including IL-1beta and IL-1RA. During labor, both placental and peripheral leukocytes increase their secretion of matrix metalloproteinase-9. Moreover, we showed that placental leukocytes respond differently than peripheral leukocytes to bacterial lipopolysaccharide, secreting differential amounts of TNF-alpha, IL-1beta and IL-6. Finally, a preliminary proteomic characterization of placental leukocytes revealed a significantly higher number of individual proteins than in peripheral leukocytes. Our results support the existence of selective subsets of leukocytes recruited to the maternal-fetal interface that may participate in the triggering of parturition.

Ascendant colonization of pathogenic microorganisms from the vagina to the uterus is strongly associated to preterm labour and premature rupture of membranes. This study evaluated the secretion of interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, prostaglandin E(2) (PGE(2)), and metalloproteinases 9 and 2 by the human chorioamnion stimulated with Candida albicans. Chorioamniotic membranes were obtained after delivery by elective Cesarean section from women at 37-40 weeks of gestation without evidence of active labour. The membranes were mounted in Transwell devices that form two independent compartments, which allow testing the individual responses and contributions of the amnion and choriodecidua. One million CFU ml(-1) of C. albicans was added to either the amniotic or choriodecidual surface and secretions of the markers were measured in both compartments using specific enzyme-linked immunosorbent assay and zymography. Fetal membranes followed different secretion patterns of proinflammatory cytokines depending on the side to which the stimulus was applied. IL-1beta was produced in higher amounts in the presence of C. albicans when applied to the choriodecidual side; TNFalpha and IL-6 secretion did not change in either the amnion or choriodecidual region. PGE(2) synthesis depicted a different pattern, the amniotic tissue was more responsive than the choriodecidual tissue, and this response tended to be higher even when only the amniotic side was stimulated. Matrix metalloproteinases (MMP)-9 increased after stimulation, being the choriodecidua its main source. Selective stimulation with C. albicans induced a differential secretion of IL-1beta, PGE(2), and MMP-9, resulting from a cooperative and bidirectional communication between the amnion and the choriodecidua.

Extracellular matrix degradation in fetal membranes leading to its rupture is coupled to myometrial activity and cervical ripening during human normal labor. Mechanisms which modulate collagen degradation in amniochorion during labor have not been elucidated. Initial characterization of the effect of different blood compartments on connective tissue degradation in amniochorion during human labor was explored. Amniochorion explants were stimulated with plasma of maternal venous blood, umbilical cord blood or placental blood, obtained from women with pregnancies at term, with or without labor. MMP-2 and MMP-9 activities were quantified in conditioned media by gelatin-zymography as an index of connective tissue degradation. Collagen content was measured in tissue explants and collagen fibrils distribution was examined by electron microscopy. Placental plasma from term pregnancies, with or without labor, is enriched with soluble signals that enhance the in vitro MMP-9 production by amniochorion. Accompanying ultrastructural distortion of collagen fibers and demonstration of collagen degradation fragments confirmed induction of extracellular matrix degradation. Control experiments in which MMP-9 activity was blocked with TIMP-1 resulted in inhibition of all the above mentioned changes. These results suggest that placental intervillous space is a functional compartment in which mediators capable to induce collagen degradation in amniochorion are selectively expressed during human labor.

Extracellular matrix homeostasis is a key process in the maintenance of the tensile strength of the amniochorion. This tensile strength guarantees the role of the membranes as a physical and functional boundary for the fetus during human pregnancy. Pathological rupture of these structures before 37 completed weeks of gestation is known as preterm prelabour rupture of the membranes (PPROM) and it is a major cause of spontaneous preterm labour and preterm birth. A mechanism involving the activation of matrix metalloproteinases (MMP)-9, a 92-kDa type IV collagenase, as an essential mediator of tissue damage is under investigation. The proposed mechanism involves the abnormal expression and activity of MMP-9 with subsequent connective tissue degradation taking place at a time that does not synchronise with other events of labour. The local physiological signal by amniochorion cells to induce MMP-9 expression is not known, but bacterial products and/or the proinflammatory cytokines, IL-1h and TNF-a, as paracrine or autocrine signals may trigger these processes in pregnancies complicated with intra-amniotic infection. These signalling pathways indicate complex cooperative and bidirectional communications between amnion and choriodecidua in response to bacterial products, which include intermembranous cytokine traffic and signalling between tissues. Products secreted in culture by amniochorion and choriodecidual leucocytes, obtained from women who delivered following normal labour in the absence of infection, condition a specific microenvironment that induces collagen degradation in fetal membranes. Further characterisation of the role of choriodecidual leucocytes in the control of extracellular matrix degradation in amniochorion is currently under way.

Objective To compare the inflammatory response preserved ex vivo by decidual cells isolated from women who experienced preterm labor with and without subclinical intrauterine infection. Methods Fetal membranes were obtained after cesarean section from 35 women who delivered before 37 weeks of gestation following spontaneous preterm labor, with no clinical evidence of intrauterine infection. Decidua was microbiologically tested and cultured. Concentrations of anti-inflammatory cytokines (IL-2, IL-4, IL-10), pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α), and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) were measured in the supernatants using Bio-Plex, and prostaglandin E(2) (PGE(2)) was measured by enzyme immunoassay. Results Subclinical infection was confirmed in 10 women (28.5%). Microorganisms isolated were Ureaplasma urealyticum (4), group B streptococci (3), Gardnerella vaginalis (1), and Escherichia coli (2). We found a significant increase of pro-inflammatory cytokines and a significant decrease of anti-inflammatory cytokines in supernatants from decidual cells obtained from women with preterm labor and subclinical intrauterine infection compared to women without infection. Secretion of MMP-1, MMP-8, MMP-9 and PGE(2) was significantly higher in infected women. Secretion of IL-8 by decidual cells from infected women persisted upon repeated in vitro culture passages. Conclusions Almost 30% of idiopathic preterm labor cases were associated with subclinical intrauterine infection, and decidual cells isolated from these cases preserved an ex vivo inflammatory status after in vivo bacterial exposure.

The induction of the expression of matrix metalloproteinases (MMPs) and their extracellular activation are key processes in connective tissue degradation in the chorioallantoid membrane during rat labour. However, the regulatory mechanisms remain largely unknown. Here, we report the identification of a calcium-dependent high molecular weight complex composed of MMP-9, MMP-3, MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, identified by zymography and western blotting. Molecular sieve chromatography confirmed the presence of a complex of MMPs and TIMPs with an exclusion volume670 kDa. Differential scanning calorimetry of the complex confirmed the existence of a macromolecular complex that unfolds with a broad transition; it is denatured over a wide range of temperatures and has a T(m) of 72 degrees C in the presence of Ca(2+). When denatured in the absence of Ca(2+), there were at least eight transitions with T(m)s that corresponded to pro-MMP-9, MMP-9, pro-MMP-3, MMP-3, pro-MMP-2, MMP-2, TIMP-1 and TIMP-2. Co-localization of the same molecular components was demonstrated by confocal microscopy using cell-depleted chorioallantoid membranes. The assembly and disassembly of the complex can be reproduced at physiological concentrations of Ca(2+). This complex provides a potential mechanism for the enzymatic regulation of MMPs, which may participate in connective tissue degradation leading to the rupture of the fetal membranes during labour.

Background During intrauterine infection, amniochorionic membranes represent a mechanical and immunological barrier against dissemination of infection. Human beta defensins (HBD)-1, HBD-2, and HBD-3 are key elements of innate immunity that represent the first line of defense against different pathogen microorganisms associated with preterm labor. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of HBD-1, HBD-2 and HBD-3, after stimulation with Candida albicans. Methods Full-thickness human amniochorionic membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation with no evidence of active labor. The membranes were cultured in a two-compartment experimental model in which the upper compartment is delimited by the amnion and the lower chamber by the choriodecidual membrane. One million of Candida albicans were added to either the AMN or the CHD face or to both and compartmentalized secretion profiles of HBD-1, HBD-2, and HBD-3 were quantified by ELISA. Tissue immunolocalization was performed to detect the presence of HBD-1, -2, -3 in tissue sections stimulated with Candida albicans. Results HBD-1 secretion level by the CHD compartment increased 2.6 times (27.30 [20.9-38.25] pg/micrograms protein) when the stimulus with Candida albicans was applied only on this side of the membrane and 2.4 times (26.55 [19.4-42.5] pg/micrograms protein) when applied to both compartments simultaneously. HBD-1 in the amniotic compartment remained without significant changes. HBD-2 secretion level increased significantly in the CHD when the stimulus was applied only to this region (2.49 [1.49-2.95] pg/micrograms protein) and simultaneously to both compartments (2.14 [1.67- 2.91] pg/micrograms protein). When the stimulus was done in the amniotic compartment HBD-2 remained without significant changes in both compartments. HBD-3 remained without significant changes in both compartments regardless of the stimulation modality. Localization of immune-reactive forms of HBD-1, HBD-2, and HBD-3 was carried out by immunohistochemistry confirming the cellular origin of these peptides. Conclusion Selective stimulation of amniochorionic membranes with Candida albicans resulted in tissue-specific secretion of HBD-1 and HBD-2, mainly in the CHD, which is the first region to become infected during an ascending infection.

Objective: Leukocytes arriving to choriodecidua during labor are capable to secrete cytokines and collagenases that may play a role in extracellular matrix degradation leading to the rupture of amniochorion. The aim of this work was to study the role of amniochorion in the active recruitment of leukocytes through production of specific chemokines during labor. Methods: Amniochorion explants were obtained from women at term with spontaneous labor (n = 4) and subjected to cesarean section without labor (n = 4). Explant cultures were carried out during 24 hr and a homogenate including the culture media was made after this period. Cell free extracts were tested in Boyden chambers for chemotaxis using leukocytes from maternal blood obtained from women with (n = 2) and without labor (n = 2). Attracted cells were analyzed by flow cytometry for count and immunophenotype. Chemokines were identified in the extracts using a commercial chemiarray. Results: All tested amniochorion extracts induced chemotaxis for leukocytes; however, those from labor tissues induced a higher chemotactic activity than the correspondent non-in-labor tissues (P = 0.003). Chemotactic effect was higher over leukocytes from labor women (P = 0.001). More than 80% of the attracted cells were polymorphonuclear cells in all cases. IL-8 was the main chemokine found in all extracts; however its concentration was increased in extracts from labor. Conclusions: Fetal membranes induced chemotaxis for leukocytes and this condition is enhanced by the presence of labor. Amniochorion under active labor secretes IL-8 which induces a preferential chemotaxis for polymorphonuclears. Infiltration of these cells in choriodecidua during labor may play a role in the amniochorion degradation associated to rupture.

Background During human pregnancy, infection/inflammation represents an important factor that increases the risk of developing preterm labor. The purpose of this study was to determine if pre-treatment with progesterone has an immunomodulatory effect on human placenta production of endotoxin-induced inflammation and degradation of extracellular matrix markers. Methods Placentas were obtained under sterile conditions from pregnancies delivered at term before the onset of labor by cesarean section. Explants from central cotyledons of 10 human placentas were pre-treated with different concentrations of progesterone (0.01, 01, 1.0 μM) and then stimulated with 1000 ng/mL of LPS of Escherichia coli. Cytokines TNFα, IL-1β, IL-6, IL-8, MIP-1α, IL-10 concentrations in the culture medium were then measured by specific ELISA. Secretion profile of MMP-9 was evaluated by ELISA and zymogram. Statistical differences were determined by one-way ANOVA followed by the appropriate ad hoc test; P