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Background: It is well established that obesity is associated with dysregulation of the ratio between the two major adipokines leptin and adiponectin. Furthermore, it was recently reported that maternal obesity has a significant impact on placental development. Leptin and adiponectin are present at the fetal-maternal interface and are involved in the development of a functional placenta. However, less is known about leptin and adiponectin's involvement in the placental alterations described in obese women. Hence, the objective of the present study was to characterize the placental expression and DNA methylation of these two adipokine systems (ligands and receptors) in obese women., Results: Biopsies were collected from the fetal and maternal sides of third-trimester placenta in obese and non-obese (control) women. In both groups, leptin levels were higher on the fetal side than the maternal side, suggesting that this cytokine has a pivotal role in fetal growth. Secondly, maternal obesity (in the absence of gestational diabetes) was associated with (i) elevated DNA methylation of the leptin promoter on fetal side only, (ii) hypomethylation of the adiponectin promoter on the maternal side only, (iii) significantly low levels of leptin receptor protein (albeit in the absence of differences in mRNA levels and promoter DNA methylation), (iv) significantly low levels of adiponectin receptor 1 mRNA expression on the maternal side only, and (v) elevated DNA methylation of the adiponectin receptor 2 promoter on the maternal side only., Conclusion: Our present results showed that maternal obesity is associated with the downregulation of both leptin/adiponectin systems in term placenta, and thus a loss of the beneficial effects of these two adipokines on placental development. Maternal obesity was also associated with epigenetic changes in leptin and adiponectin systems; this highlighted the molecular mechanisms involved in the placenta's adaptation to a harmful maternal environment.

We identified, through a genome-wide search for new imprinted genes in the human placenta, DSCAM (Down Syndrome Cellular Adhesion Molecule) as a paternally expressed imprinted gene. Our work revealed the presence of a Differentially Methylated Region (DMR), located within intron 1 that might regulate the imprinting in the region. This DMR showed a maternal allele methylation, compatible with its paternal expression. We showed that DSCAM is present in endothelial cells and the syncytiotrophoblast layer of the human placenta. In mouse, Dscam expression is biallelic in foetal brain and placenta excluding any possible imprinting in these tissues. This gene encodes a cellular adhesion molecule mainly known for its role in neurone development but its function in the placenta remains unclear. We report here the first imprinted gene located on human chromosome 21 with potential clinical implications.

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones., Competing Interests: The authors declare no conflict of interest.

Introduction: Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after somatic cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies., Methods: Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and somatic cloning pregnancies., Results: The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta., Discussion: In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes., Conclusions: These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Genomic imprinting characterizes genes with a monoallelic expression, which is dependent on the parental origin of each allele. Approximately 150 imprinted genes are known to date, in humans and mice but, though computational searches have tried to extract intrinsic characteristics of these genes to identify new ones, the existing list is probably far from being comprehensive. We used a high-throughput strategy by diverting the classical use of genotyping microarrays to compare the genotypes of mRNA/cDNA vs. genomic DNA to identify new genes presenting monoallelic expression, starting from human placental material. After filtering of data, we obtained a list of 1,082 putative candidate monoallelic SNPs located in more than one hundred candidate genes. Among these, we found known imprinted genes, such as IPW, GRB10, INPP5F and ZNF597, which contribute to validate the approach. We also explored some likely candidates of our list and identified seven new imprinted genes, including ZFAT, ZFAT-AS1, GLIS3, NTM, MAGI2, ZC3H12Cand LIN28B, four of which encode zinc finger transcription factors. They are, however, not imprinted in the mouse placenta, except for Magi2. We analyzed in more details the ZFAT gene, which is paternally expressed in the placenta (as ZFAT-AS1, a non-coding antisense RNA) but biallelic in other tissues. The ZFAT protein is expressed in endothelial cells, as well as in syncytiotrophoblasts. The expression of this gene is, moreover, downregulated in placentas from complicated pregnancies. With this work we increase by about 10% the number of known imprinted genes in humans.

Cloning in mammals suffers from high rates of pregnancy losses associated with abnormal placentation, mainly placentomegaly, leading to fetal death. Placental growth is dependent on the regulated expression of many genes of which imprinted genes play a fundamental role. Among them, the Phlda2 gene is expressed from the maternal allele and acts to limit placental growth in mouse and human. Here we used Northern blots, quantitative RT-PCR and in situ hybridization to analyze the expression patterns of bovine PHLDA2 and to compare its expression levels in normal and somatic cell nuclear transfer (SCNT) placentas over a range of gestational stages. PHLDA2 is not expressed in extra-embryonic tissues before d32 of gestation but the level of expression increases throughout pregnancy until term in the placental villi collected from pregnancy obtained by artificial insemination (AI). At all stages of pregnancy, PHLDA2 mRNA are specifically localized in the trophoblast mononucleated cells contrasting with lack of expression in the binucleated cells and uterine tissues. In SCNT placentas, a similar pattern of expression was observed during early pregnancy. In contrast the level of expression is significantly reduced around d200 of gestation in the placental villi from pathological clones. The reduced expression of PHLDA2 was obvious particularly in the placental villi anchored within the uterine crypts with expression confined to the trophoblast of the chorionic plate. Altogether, these results highlight a similarity in expression patterns for PHLDA2 bovine and human where expression is localized to the trophoblast throughout pregnancy and parallels the continuous growth of the placenta. Moreover, the lack of expression in the fetal villi from oversized bovine cloned placenta is consistent with the function of PHLDA2 in restraining placental growth and underlines an aberrant expression of this gene after somatic cloning.

Genomic imprinting regulates the expression of a group of genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting genes from the recently described imprinted gene network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated network of imprinted genes results in a compensatory process capable of correcting potential dysfunction of placenta.

Background: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice., Methodology/principal Findings: Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations., Conclusions: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.

Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Zn finger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents., (Copyright 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors (SERPINs). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5, and B7 presented significant differences in expression in >or=1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5, and A8 presented complex methylation profiles. For SERPINA3, the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.

Background: As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes., Results: Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of Intra-Uterine Growth Retardation (IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals., Conclusion: We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5 x 10(-5)), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the "hypoxic" condition (2% O2), whereas apoptotic genes are overrepresented in the "normoxic" condition (20% O2). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O2 tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.

Growth hormone releasing factor (GHRH) has been described in the rat, mouse and human placentae. This study reports the presence of an immunoreactive GHRH activity (IR-GHRH) in the ovine placenta. This activity was detected by radioimmunoassay from day 50 (D50) until the end of pregnancy. Higher IR-GHRH concentration in placental tissue was observed on days 100 (543 +/- 123 pg/g) and 140 (550 +/- 62 pg/g) and, when compared with the GHRH content of the ovine hypothalamus (1.2 ng/hypothalamus), represents a considerable amount of GHRH per placenta (a mean of 200 ng). Perifused placenta explants released IR-GHRH in vitro at a mean rate of 200 pg/g/h. Depolarization by 55 mM KCl increased the IR-GHRH concentration of the perifusion media 1.7 times over basal values. The elution position of GHRH immunoreactivity in the gel filtration chromatography profiles was the same for placenta and hypothalamus extracts and lay very near to the molecular weight of bovine GHRH. Northern blot hybridization analysis revealed the existence of a placental transcript whose size (0.75 kb) was comparable to the size of the ovine hypothalamus and rat placenta GHRH transcripts. Hybridization signal was observed at each stage studied from D50 until D120 of pregnancy. This study demonstrated the existence of a IR-GHRH peptide in the ovine placenta.

Abstract: Since the first success in cloning sheep, the production of viable animals by somatic cell nuclear transfer (SCNT) has developed significantly. Cattle are by far the most successfully cloned species but, despite this, the technique is still associated with a high incidence of pregnancy failure and accompanying placental and fetal pathologies. Pre- and early post-implantation losses can affect up to 70% of the pregnancies. In the surviving pregnancies, placentomegaly and fetal overgrowth are commonly observed, but the incidence varies widely, depending on the genotype of the nuclear donor cell and differences in SCNT procedures. In all cases, the placenta is central to the onset of the pathologies. Although cellular organisation of the SCNT placenta appears normal, placental vascularisation is modified and fetal-to-maternal tissue ratios are slightly increased in the SCNT placentomes. In terms of functionality, steroidogenesis is perturbed and abnormal estrogen production and metabolism probably play an important part in the increased gestation length and lack of preparation for parturition observed in SCNT recipients. Maternal plasma concentrations of pregnancy-associated glycoproteins are increased, mostly due to a reduction in turnover rate rather than increased placental production. Placental glucose transport and fructose synthesis appear to be modified and hyperfructosemia has been observed in neonatal SCNT calves. Gene expression analyses of the bovine SCNT placenta show that multiple pathways and functions are affected. Abnormal epigenetic re-programming appears to be a key component of the observed pathologies, as shown by studies on the expression of imprinted genes in SCNT placenta. [Copyright &y& Elsevier]