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Intrauterine growth restriction (IUGR) is an obstetrical complication with an increased risk of perinatal mortality and morbidity. The uterus, once considered to be a sterile environment, has now been described in recent microbiome studies to harbor diverse commensal placenta microbiota, as well as potentially pathogenic flora known to cause infection. Therefore, in this pilot study, we tested whether IUGR was associated with changes to the reproductive microbiome. The reproductive microbiome was surveyed using 16S sequencing (20 IUGR, 20 controls). Alpha and beta diversity were compared, and differential taxa features associated with IUGR were identified. Microbial screening of the placenta demonstrated a diverse range of flora predominantly including Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. Neither alpha- nor beta-diversity was significantly different by IUGR status. However, at the taxa level, IUGR patients had significantly higher prevalence of Neisseriaceae, mucosal β-hemolytic bacteria known to uptake iron-bound host proteins including hemoglobin. Moreover, the increase in anaerobic bacteria such as Desulfovibrio reflects the emergence of a hypoxic environment in the IUGR placenta. Further analysis of the reproductive microbiome of IUGR samples showed lower levels of H202-producing Bifidobacterium and Lactobacillus that switch from respiration to fermentation, a less energetic metabolic process, when oxygen levels decrease. Source tracking analysis showed that the placental microbial contents were predominantly contributed from an oral source, as compared to a gut or vaginal source. Our results suggest that the reproductive microbiome profiles may, in the future, constitute potential biomarkers for fetal health during pregnancy, while Neisseriaceae may constitute promising therapeutic targets for IUGR treatment.

Perfluoroalkylated substances (PFAS) are man-made, persistent organic compounds with immune-modulating potentials. Given that pregnancy itself represents an altered state of immunity, PFAS exposure-related immunotoxicity is an important environmental factor to consider in SARS-CoV-2 infection during pregnancy as it may further affect humoral immune responses.To investigate the relationship between maternal plasma PFAS concentrations and SARS-CoV-2 antibody levels in a NYC-based pregnancy cohort.Maternal plasma was collected from 72 SARS-CoV-2 IgG + participants of the Generation C Study, a birth cohort established at the beginning of the COVID-19 pandemic in New York City. Maternal SARS-CoV-2 anti-spike IgG antibody levels were measured using ELISA. A panel of 16 PFAS congeners were measured in maternal plasma using a targeted UHPLC-MS/MS-based assay. Spearman correlations and linear regressions were employed to explore associations between maternal IgG antibody levels and plasma PFAS concentrations. Weighted quantile sum (WQS) regression was also used to evaluate mixture effects of PFAS. Models were adjusted for maternal age, gestational age at which SARS-CoV-2 IgG titer was measured, COVID-19 vaccination status prior to IgG titer measurement, maternal race/ethnicity, parity, type of insurance and pre-pregnancy BMI.Our study population is ethnically diverse with an average maternal age of 32 years. Of the 16 PFAS congeners measured, nine were detected in more than 60% samples. Importantly, all nine congeners were negatively correlated with SARS-CoV-2 anti-spike IgG antibody levels; n-PFOA and PFHxS, PFHpS, and PFHxA reached statistical significance (p 0.05) in multivariable analyses. When we examined the mixture effects using WQS, a quartile increase in the PFAS mixture-index was significantly associated with lower maternal IgG antibody titers (beta [95% CI] = -0.35 [-0.52, -0.17]). PFHxA was the top contributor to the overall mixture effect.Our study results support the notion that PFAS, including short-chain emerging PFAS, act as immunosuppressants during pregnancy. Whether such compromised immune activity leads to downstream health effects, such as the severity of COVID-19 symptoms, adverse obstetric outcomes or neonatal immune responses remains to be investigated.

Intrauterine growth restriction (IUGR) is an obstetrical complication with an increased risk of perinatal mortality and morbidity. The uterus, once considered to be a sterile environment, has now been described in recent microbiome studies to harbor diverse commensal placenta microbiota, as well as potentially pathogenic flora known to cause infection. Therefore, in this pilot study, we tested whether IUGR was associated with changes to the reproductive microbiome. The reproductive microbiome was surveyed using 16S sequencing (20 IUGR, 20 controls). Alpha and beta diversity were compared, and differential taxa features associated with IUGR were identified. Microbial screening of the placenta demonstrated a diverse range of flora predominantly including Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. Neither alpha- nor beta-diversity was significantly different by IUGR status. However, at the taxa level, IUGR patients had significantly higher prevalence of Neisseriaceae, mucosal β-hemolytic bacteria known to uptake iron-bound host proteins including hemoglobin. Moreover, the increase in anaerobic bacteria such as Desulfovibrio reflects the emergence of a hypoxic environment in the IUGR placenta. Further analysis of the reproductive microbiome of IUGR samples showed lower levels of H

Imprinted genes uniquely drive and support fetoplacental growth by controlling the allocation of maternal resources to the fetus and affecting the newborn’s growth. We previously showed that alterations of the placental imprinted gene expression are associated with suboptimal perinatal growth and respond to environmental stimuli including socio-economic determinants. At the same time, maternal psychosocial stress during pregnancy (MPSP) has been shown to affect fetal growth. Here, we set out to test the hypothesis that placental imprinted gene expression mediates the effects of MPSP on fetal growth in a well-characterized birth cohort, the Stress in Pregnancy (SIP) Study. We observed that mothers experiencing high MPSP deliver infants with lower birthweight (P=0.047). Among the 109 imprinted genes tested, we detected panels of placental imprinted gene expression of 23 imprinted genes associated with MPSP and 26 with birthweight. Among these genes, five imprinted genes (CPXM2, glucosidase alpha acid (GAA), GPR1, SH3 and multiple ankyrin repeat domains 2 (SHANK2) and THSD7A) were common to the two panels. In multivariate analyses, controlling for maternal age and education and gestational age at birth and infant gender, two genes, GAA and SHANK2, each showed a 22% mediation of MPSP on fetal growth. These data provide new insights into the role that imprinted genes play in translating the maternal stress message into a fetoplacental growth pattern.

While the developing fetus is largely shielded from the external environment through the protective barrier provided by the placenta, it is increasingly appreciated that environmental agents are able to cross and even accumulate in this vital organ for fetal development. To examine the potential influence of environmental pollutants on the placenta, we assessed the relationship between polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), 1,1-dichloro-2,2-bis(p- chlorophenyl) ethylene (DDE) and several epigenetic marks linked to fetoplacental development. We measured IGF2 / H19 imprint control region methylation, IGF2 and H19 expression, IGF2 loss of imprinting (LOI) and global DNA methylation levels in placenta ( n = 116) collected in a formative research project of the National Children’s Study to explore the relationship between these epigenetic marks and the selected organic environmental pollutants. A positive association was observed between global DNA methylation and total PBDE levels ( P

Experimental studies showed that genomic imprinting is fundamental in fetoplacental development by timely regulating the expression of the imprinted genes to overlook a set of events determining placenta implantation, growth and embryogenesis. We examined the expression profile of 22 imprinted genes which have been linked to pregnancy abnormalities that may ultimately influence childhood development. The study was conducted in a subset of 106 placenta samples, overrepresented with small and large for gestational age cases, from the Rhode Island Child Health Study. We investigated associations between imprinted gene expression and three fetal development parameters: newborn head circumference, birth weight, and size for gestational age. Results from our investigation show that the maternally imprinted/paternally expressed gene ZNF331 inversely associates with each parameter to drive smaller fetal size, while paternally imprinted/maternally expressed gene SLC22A18 directly associates with the newborn head circumference promoting growth. Multidimensional Scaling analysis revealed two clusters within the 22 imprinted genes which are independently associated with fetoplacental development. Our data suggest that cluster 1 genes work by assuring cell growth and tissue development, while cluster 2 genes act by coordinating these processes. Results from this epidemiologic study offer solid support for the key role of imprinting in fetoplacental development.

Objective The purpose of this study was to investigate imprinting patterns in first-trimester human placentas. Study Design Using samples of 17 first-trimester and 14 term placentas from uncomplicated pregnancies, we assessed loss of imprinting (LOI) at the RNA level in a panel of 14 genes that are known to be imprinted in the placenta with the use of a quantitative allele-specific reverse transcriptase polymerase chain reaction analysis of those genes that contained readout single nucleotide polymorphisms in their transcripts. Results There is significant LOI (ie, biallelic expression) in all 14 genes in first-trimester placentas. LOI was more variable and generally at lower levels at term. Although there is little difference in gene expression, the level of LOI is higher in the first-trimester placentas, compared with term placentas. Conclusion Genomic imprinting appears to be a dynamic maturational process across gestation in human placenta. In contrast with prevailing theories, epigenetic imprints may continue to evolve past 12 weeks of gestation.

BACKGROUND: Endocrine disruptors (EDs) constitute a class of chemicals that can interfere with the endocrine system, possibly influencing breast cancer risk. EDs exposure has been associated with changes in the epigenome such as methylation; their ability to modify other epigenetic processes, such as microRNA, has not been thoroughly investigated, especially in the context of mammary development. METHODS: We investigated the influence on microRNA of three EDs widely used in personal care products: diethyl phthalate (DEP), methyl paraben (MPB) and triclosan (TCS). Sprague-Dawley rats were treated with these EDs at six windows of susceptibility (prenatal, neonatal, prepubertal and pubertal, parous, and nulliparous) from in utero to young adulthood. Oral treatment doses were selected to produce urinary metabolite levels comparable to those found in the US population. MicroRNAs were profiled using the NanoString platform. RESULTS: Of 420 microRNAs analyzed, 90 were stably detected in >90% samples across all windows, with let-7 family members showing the highest expression. In the meantime, 132 microRNA species were expressed below the detection level in >90% of the samples; other miRNAs were expressed at different levels in some but not all samples. Principal component analysis revealed that different developmental windows displayed markedly different microRNA profiles. Importantly, we found that ED exposure, TCS in particular, resulted in measurable difference in rat mammary microRNAs, and the changes were window-specific. More specifically, the TCS-related changes were mainly present in prenatal, neonatal and pubertal windows; among these the neonatal period appeared to be the most susceptible window to EDs with the largest number of differentially expressed microRNAs (7 microRNA species with adjusted p CONCLUSIONS: Low-dose ED exposure, even at levels comparable to human exposure, was able to modify microRNA expression in rat mammary tissues in a window-specific fashion. The study also demonstrates dynamic changes of microRNA profiles in developing mammary glands and highlights the importance of taking the normal developmental gene signal into account when searching for environment-induced gene signatures. Citation Format: Vasily N. Aushev, Maya Kappil, Kalpana Gopalakrishnan, Qian Li, Yula Ma, Luca Lambertini, Fabiana Manservisi, Laura Falcioni, Luciano Bua, Fiorella Belpoggi, Susan L. Teitelbaum, Jia Chen. Defining windows of susceptibility for low-dose exposure to endocrine disruptors in rat mammary development by microRNA profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-089. doi:10.1158/1538-7445.AM2015-LB-089

Breast cancer is hormonally driven. Endocrine disruptors (EDs) are exogenous substances that mimic or antagonize endogenous hormone action and disrupt endocrine system function. While EDs have been shown to modulate the risk for mammary tumors in animal models, their effects at biologically relevant doses and during critical developmental windows remain poorly understood. We sought to examine the individual and combinatorial effects of three common EDs, diethyl phthalate, methyl paraben and triclosan in rats exposed at five distinct developmental windows of susceptibility - prenatal, neonatal, prepubertal, pubertal and parous/nulliparous. Pathological features and whole genome expression of normal rat mammary tissues were profiled to provide both qualitative and quantitative biological effects of EDs. ED exposure at levels comparable to human exposure resulted in profound changes in both gross phenotypes (e.g. reproductive mortality and mammary gland morphology) as well as in molecular genome profiles. Specifically, ED exposure appeared to hamper normal breast development and resulted in increased mortality in the offspring, possibly due to reduced milk production. Whole genome expression profiling of mammary tissues revealed that in the course of development, the number of differentially expressed genes was lower in ED-exposed rats compared to controls, suggesting developmental delay or suppression by EDs. Pathway enrichment analysis indicated that several processes including inflammation, metabolism and development were disrupted by ED exposure. Taken together, our findings reveal novel mechanisms of ED action in mammary development and strongly suggest the importance of taking into account the timing of exposure when studying the relationship between environment and disease. Citation Format: Kalpana Gopalakrishnan, Qian Li, Yula Ma, Luca Lambertini, Jia Chen, Susan L. Teitelbaum, Fabiana Manservisi, Fiorella Belpoggi, Luciano Bua, Laura Falcioni. Windows of susceptibility to endocrine disruptors and gene expression in mammary tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3262. doi:10.1158/1538-7445.AM2014-3262

lipase, or MAGL) remodel tissues. Leptin activates a key ECB-degrading enzyme called fatty acid amide-hydrolase (FAAH) in immunologic and neuronal cells. Relationships between placental ECBs, placental structure, and maternal/fetal leptin have not been reported. This study aimed : 1) to evaluate expression of placental ECBs and their relationships to placental composition and to fetal and maternal leptin status in a baboon model; 2) to sequence baboon CB1 and CB2 receptors for the first phylogenetic comparison to humans. STUDY DESIGN: Placentas from eight baboons were collected and processed as described previously (Farley et al., 2009). Placental composition was estimated using computerized stereology on electron micrographs. Immunohistochemistry employed commercial antibodies. Slides were scanned and processed as previously described (Brocato et al., 2012). RT PCR was done with a kit according to manufacturer’s instructions. Twotailed correlation was applied for statistical analysis, P value of 0.05 was used to determine significance. RESULTS: The ratio of fetal endothelium to placental tissue correlated positively with MAGL and FAAH expressions (r 0.79, p 0.05 and r 0.70, p 0.05, respectively). Protein expression of FAAH in syncytiotrophoblast correlated positively with fetal leptin concentration (r 0.98, p 0.0001, Fig. 1A). CB1 receptors were expressed in fetal endothelial cells, cytotrophoblast, and fibroblasts of villous core (Fig.1B), displaying 100% homology on the protein level with human receptor. CONCLUSION: This is the first report regarding possible regulation of placental ECBs by fetal leptin and ECBs association with fetal endothelial remodeling. Placental FAAH may be a promising target for treatment of poor fetal growth.

gene NR3C1 and the association with birth weight and gestational age at delivery Jennifer Wong, Yula Ma, Luca Lambertini, Andrea Weintraub, Joanne Stone Mount Sinai School of Medicine, Maternal Fetal Medicine, New York, NY, Mount Sinai School of Medicine, Department of Preventive Medicine, New York, NY, Mount Sinai School of Medicine, Pediatrics, New York, NY OBJECTIVE: To determine the association of glucocorticoid receptor gene NR3C1 single nucleotide polymorphisms (SNPs) with birth weight and prematurity. STUDY DESIGN: This was a single center, pilot study of 80 umbilical cord blood samples collected prospectively from singleton pregnancies. DNA was isolated from each blood sample. SNPs were genotyped by Taqman assay (rs6193, rs6194, rs6195, rs6196, rs6198, rs258751) and sequencing (rs6189, rs6190). Linear regression models were calculated for birth weight (BW) and gestational age at delivery (GAd). RESULTS: The rs6195 SNP was not polymorphic in our sample, and was therefore excluded from further statistical analysis. The average BW was 3330g [1930-4660g]. The average GAd was 39.5 weeks [3241.6] with only 3 deliveries occurring prior to 37 weeks. The BW linear regression model showed a significant correlation with preterm labor, vaginal delivery, maternal Asian ethnicity, and the rs6196 variant (p 0.01; r2 0.270). The rs6196 GG genotype was associated with a 761g reduction in BW (95% CI 125-1398; p 0.02). The GAd linear regression model of gestational age at delivery also showed a significant correlation with preterm labor, betamethasone administration, neonatal intensive care unit admission, as well as the rs6196 GG genotype (p 0.01; r2 0.476). In this regression, the GG genotype was associated with an earlier GAd (95% CI 0.92-0.99; p 0.01). None of the other SNPs analyzed showed any significant correlation with either outcome. CONCLUSION: The GG genotype of the rs6196 SNP of the glucocorticoid receptor gene NR3C1 is associated with both lower birth weight and earlier gestational age at delivery. These data are consistent with existing scientific literature that correlates this and other SNP phenotypes to lower weight, BMI, waist/hip ratio, and decreased insulin resistance. This glucocorticoid-resistant phenotype may ultimately be protective postnatally among hypercortisolemic premature infants. Given the results from this interim analysis, our investigation will continue with a focus on umbilical cord blood collections from premature infants.