Human Placental-Specific Epipolymorphism and its Association with Adverse Pregnancy Outcomes

Interindividual variation in DNA-methylation level is widespread in the human genome, despite its critical role in regulating gene expression. The nature of this variation, including its tissue-specific nature, and the role it may play in human phenotypic variation and disease is still poorly characterized. The placenta plays a critical role in regulating fetal growth and development in ways that have lifelong effects on health. To identify genes with a high degree of interindividual DNA methylation variation in the human placenta, we surveyed the human genome using the Illumina GoldenGate Methylation Cancer panel targeting 1505 CpG sites of 807 genes. While many sites show a continuous pattern of methylation levels, WNT2, TUSC3 and EPHB4 were identified to have a polymorphic “on-or-off” pattern of DNA methylation variation at their promoter region which was confirmed by pyrosequencing. Methylation of these genes can be found in 7%–25% of over 100 placentas tested. The methylation state at the promoter of these genes is concordant with mRNA allelic expression. In three informative cases TUSC3 was observed to be methylated on the maternal allele, and it is thus possible this represents a polymorphically imprinted gene. Furthermore, TUSC3 promoter methylation showed evidence for association with preeclampsia. A biological significance of these methylation allelic polymorphisms (MAPs) to human placental diversity is further implied by their placental specificity and absence in mouse. An extended study of blood suggests that MAPs may also be found in other tissues, implicating their utility for tissue-specific association with complex disorders. The identification of such “epipolymorphism” in other tissues and their use in association studies, should improve our understanding of interindividual phenotypic variability and complex disease susceptibility

WNT5A, β‑catenin and SUFU expression patterns, and the significance of microRNA deregulation in placentas with intrauterine growth restriction

Placental insufficiency is a common cause of intrauterine growth restriction (IUGR). It affects ~10% of pregnancies and increases fetal and neonatal morbidity and mortality. Although Wnt and Hh pathways are crucial for embryonic development and placentation, their role in the pathology of IUGR is still not sufficiently explored. The present study analyzed the expression of positive regulators of the Wnt pathway, WNT5A and β‑catenin, and the expression of the Hh pathway negative regulator suppressor of fused (SUFU). Immunohistochemical and reverse transcription‑quantitative PCR (RT‑qPCR) assays were performed on 34 IUGR and 18 placental tissue samples from physiologic singleton‑term pregnancies. Epigenetic mechanisms of SUFU gene regulation were also investigated by methylation‑specific PCR analysis of its promoter and RT‑qPCR analysis of miR‑214‑3p and miR‑378a‑5p expression. WNT5A protein expression was higher in endothelial cells of placental villi from IUGR compared with control tissues. That was also the case for β‑catenin protein expression in trophoblasts and endothelial cells and SUFU protein expression in trophoblasts from IUGR placentas. The SUFU gene promoter remained unmethylated in all tissue samples, while miR‑214‑3p and miR‑378a‑5p were downregulated in IUGR. The present results suggested altered Wnt and Hh signaling in IUGR. DNA methylation did not appear to be a mechanism of SUFU regulation in the pathogenesis of IUGR, but its expression could be regulated by miRNA targeting

Epigenetic analysis of cell-free DNA as a tool to study pathogenesis and maternal syndrome of preeclampsia

El ADN libre circulante (ADNlc) ha surgido como una valiosa fuente de información en el campo de la biomedicina. En concreto, la existencia de diferencias epigenéticas en el ADNlc entre individuos sanos y enfermos evidencia la utilidad del estudio de estas marcas y su potencial aplicación en enfermedades que no conllevan mutaciones del ADN. La preeclampsia es una complicación del embarazo considerada una de las principales causas de muerte materna alrededor del mundo. A pesar de su gran importancia, su fisiopatología todavía no se conoce con exactitud. Este síndrome representa un modelo de patología sistémica en la que, además de verse comprometida la salud del feto, los efectos sobre la madre implican una respuesta inflamatoria generalizada, hipertensión y daños hepático y renal. Por esta razón, consideramos que la preeclampsia podría servir como modelo para evaluar el potencial de la secuenciación del ADNlc metilado con el fin de descubrir de cambios epigenéticos específicos de enfermedad. Basándonos en esto, nuestra hipótesis es que existen diferencias de metilación en el ADNlc en mujeres embarazadas control y con preeclampsia grave (sPE, del inglés), reflejando las respuestas sistémicas propias de la patología. Por tanto, el objetivo de este estudio es comparar la metilación del ADNlc entre ambos grupos, para ello utilizamos la secuenciación del ADN previamente capturado utilizando una proteína de unión a metilcitosina (del inglés, MBD-seq). Nuestros resultados revelaron la existencia de regiones diferencialmente metiladas (DMRs, del inglés) entre los grupos de estudio. La anotación mostró un enriquecimiento en promotores e islas CpG entre las regiones hipermetiladas en sPE. Los análisis funcionales revelaron la presencia de cambios de metilación en genes involucrados en rutas previamente relacionadas con la preeclampsia. Además, el análisis de asociación gen-enfermedad mostró cambios en genes relacionados con los síntomas de preeclampsia, así como con algunas de sus secuelas a largo plazo. Entre las DMRs cabe resaltar la presencia de cambios gen-específicos que podrían ser utilizados en el futuro para el desarrollo de biomarcadores, como los detectados en el gen eNOS. Por otro lado, llevamos a cabo un análisis de los tamaños de las moléculas de ADNlc metilado. Encontramos un enriquecimiento en fragmentos pequeños asociado a sPE. El hecho de que este cambio correlacione negativamente con la fracción fetal podría indicar que es debido a un aumento en la contribución materna al ADNlc como respuesta al estado patológico, apoyando estudios publicados anteriormente. En conclusión, nuestros resultados resaltan el potencial del análisis de la metilación del ADNlc en el estudio de la preeclampsia, así como el de la contribución de los distintos tejidos al ADNlc en dicha patología. Además, el descubrimiento de tales diferencias abre la puerta a su estudio en otras patologías sistémicas

Fetal-Maternal Exposure to Endocrine Disruptors: Correlation with Diet Intake and Pregnancy Outcomes

Endocrine-disrupting chemicals (EDCs) are exogenous substances able to mimic or to interfere with the endocrine system, thus altering key biological processes such as organ development, reproduction, immunity, metabolism and behavior. High concentrations of EDCs are found in several everyday products including plastic bottles and food containers and they could be easily absorbed by dietary intake. In recent years, considerable interest has been raised regarding the biological effects of EDCs, particularly Bisphenol A (BPA) and phthalates, on human pregnancy and fetal development. Several evidence obtained on in vitro and animal models as well as by epidemiologic and population studies strongly indicated that endocrine disruptors could negatively impact fetal and placental health by interfering with the embryonic developing epigenome, thus establishing disease paths into adulthood. Moreover, EDCs could cause and/or contribute to the onset of severe gestational conditions as Preeclampsia (PE), Fetal Growth Restriction (FGR) and gestational diabetes in pregnancy, as well as obesity, diabetes and cardiovascular complications in reproductive age. Therefore, despite contrasting data being present in the literature, endocrine disruptors must be considered as a therapeutic target. Future actions aimed at reducing or eliminating EDC exposure during the perinatal period are mandatory to guarantee pregnancy success and preserve fetal and adult health

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment

Platsenta geeniekspressioon normaalses ja komplitseeritud raseduses

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Platsenta on organ, mille kaudu on loode ühendatud ema organismiga. Raseduse jooksul osaleb platsenta loote ja ema ainevahetuses, sekreteerib rasedushormoone ning kaitseb arenevat beebit ema immuunsüsteemi ning välistegurite eest. Platsenta toimimiseks on vajalik täpne ajaline ja ruumiline platsenta geenide avaldumine. Seni on platsenta geeniekspressiooni dünaamika ning rasedustüsistustega seotud molekulaarsed mehhanismid põhjalikult kaardistamata. Käesolev doktoriväitekiri käsitleb platsenta geeniekspressiooni kahest vaatenurgast. Esiteks uuritakse embrüo implantatsiooniks ning raseduse varajaseks säilitamiseks olulise koorionkonadotropiini hormooni (hCG) -alaühikut kodeerivate CGB geenide avaldumist platsentas. Töös kirjeldatakse CGB geenide ekspressioon läbi raseduse ning näidatakse, et piisava hCG taseme tagamiseks on vajalik mõlemalt vanemalt päritud geenialleelide avaldumine. Korduva spontaanabordi juhte iseloomustab oluliselt langenud CGB geenide ekspressioon. Lisaks leiti kolmel patsiendil isalt päritud CGB alleelide vaigistamine DNA metülatsiooni vahendusel. Neil juhtudel avaldusid platsentas vaid emalt päritud alleelid, mis võis viia hCG taseme languseni ema veres ning põhjustada raseduse katkemise. Töö teises osas uuriti mikrokiipide abil globaalset platsenta geeniekspressiooni dünaamikat varajasest rasedusest raseduse keskpaigani. Leiti mitmed geenid, mille ekspressioon platsentas on kõrgeim raseduse keskpaigas. Lisaks tuvastati häired nende geenide avaldumises ema preeklampsia, gestatsiooni diabeedi ning loote kasvuanomaaliatega seotud platsentades. Sanniokaltsiin 1 (STC1) on uus potentsiaalne biomarker, mille taseme tõus ema veres on mõõdetav preeklampsia ja loote kasvupeetuse patsientidel. Töös kirjeldatud raseduse keskpaigas platsentas avalduvad geenid on lisaks seotud täiskasvanuea krooniliste haigustega, mis viitab üsasisese arengu rollile sünnijärgse tervise kujundamisel. Kuna rahvastiku kasv ja vananev elanikkond mõjutavad ühiskonda järjest enam, on tööea pikendamine haiguste ennetamise teel üks tuleviku teaduse ja meditsiini väljakutsetest. Platsenta mõjutab ema ja loote heaolu nii raseduse ajal kui ka nende edasises elus. Seega omab platsenta geeniregulatsiooni mõistmine normaalses ja komplitseeritud raseduses suurt kliinilist ja sotsiaalset tähtsust.Placenta is a unique temporary feto-maternal endocrine organ that mediates interactions between the mother and the fetus during gestation. Successful pregnancy requires spatial and temporal regulation of molecular, histological and functional changes in placenta to guarantee fetal growth and development as well as maternal adaptation to pregnancy. Despite its leading role in shaping in utero environment, the temporal dynamics of placental gene expression remains under-investigated. In this doctoral thesis, transcriptional and epigenetic regulation of placental gene expression was compared in normal and complicated pregnancies, and genes with altered transcription in pregnancy complications were identified. The first part of the thesis involved in-depth expression analysis of the CGB genes encoding the placental human chorionic gonadotropin (HCG). The HCG hormone is essential for embryo implantation, placentation, and maintenance of early pregnancy. The study described CGB transcription during normal pregnancy, and demonstrated that both parental CGB alleles are required for sufficient production of HCG. In contrast, cases of recurrent miscarriage showed significantly decreased CGB transcription in first trimester placentas compared to normal controls. Monoallelic maternal expression and promoter hemimethylation was detected in one normal and two complicated samples. The second part of the thesis involved transcriptional profiling of human placentas of early and mid-term pregnancies, and revealed genes with specific up-regulation during mid-gestation. Aberrant expression of mid-gestation placental genes was associated to pregnancy complications such as restricted fetal growth, maternal preeclampsia and gestational diabetes mellitus. The study highlighted the stanniocalcin-1 (STC1) protein as a novel prognostic biomarker for non-invasive determination of preeclampsia and restricted fetal growth from maternal serum. All complication-associated mid-gestation placental genes have been previously linked to adult complex diseases, emphasizing the role of placenta in developmental programming and in utero origin of adult disease. In summary, in utero development has a central role in determining the health of the child and the mother during pregnancy and also in their later lives. As the growth and aging of the global human population increasingly impact the society, disease prevention and extension of working age are important challenges of future science and medicine. Thus, understanding the role of placenta in healthy and complicated pregnancies is of great clinical and societal importance

From animal models to patients : the role of placental microRNAs, miR-210, miR-126, and miR-148a/152 in preeclampsia

Placental microRNAs (miRNAs) regulate the placental transcriptome and play a pathological role in preeclampsia (PE), a hypertensive disorder of pregnancy. Three PE rodent model studies explored the role of placental miRNAs, miR-210, miR-126, and miR-148/152 respectively, by examining expression of the miRNAs, their inducers, and potential gene targets. This review evaluates the role of miR-210, miR-126, and miR-148/152 in PE by comparing findings from the three rodent model studies with in vitro studies, other animal models, and preeclamptic patients to provide comprehensive insight into genetic components and pathological processes in the placenta contributing to PE. The majority of studies demonstrate miR-210 is upregulated in PE in part driven by HIF-1a and NF-?Bp50, stimulated by hypoxia and/or immune-mediated processes. Elevated miR-210 may contribute to PE via inhibiting anti-inflammatory Th2-cytokines. Studies report an up- and downregulation of miR-126, arguably reflecting differences in expression between cell types and its multifunctional capacity.MiR-126 may play a pro-angiogenic role bymediating the PI3K-Akt pathway. Most studies report miR-148/152 family members are upregulated in PE. Evidence suggests they may inhibit DNA methylation of genes involved in metabolic and inflammatory pathways. Given the genetic heterogeneity of PE, it is unlikely that a single placental miRNA is a suitable therapeutic target for all patients. Investigating miRNAs in PE subtypes in patients and animal models may represent a more appropriate approach going forward. Developing methods for targeting placental miRNAs and specific placental cell types remains crucial for research seeking to target placental miRNAs as a novel treatment for PE

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment