Placental Dysplasia Research Articles

Antiphospholipid antibody-activated NETs exacerbate trophoblast and endothelial cell injury in obstetric antiphospholipid syndrome.

Despite the widespread use of antiplatelets and anticoagulants, women with antiphospholipid syndrome (APS) may face pregnancy complications associated with placental dysplasia. Neutrophil extracellular traps (NETs) are involved in the pathogenesis of many autoimmune diseases, including vascular APS; however, their role in obstetric APS is unclear. Herein, we investigated the role of NETs by quantifying cell‐free DNA and NET marker levels. Live‐cell imaging was used to visualize NET formation, and MAPK signalling pathway proteins were analysed. Cell migration, invasion and tube formation assays were performed to observe the effects of NETs on trophoblasts and human umbilical vein endothelial cells (HUVECs). The concentrations of cell‐free DNA and NETs in sera of pregnant patients with APS were elevated compared with that of healthy controls (HCs) matched to gestational week. APS neutrophils were predisposed to spontaneous NET release and IgG purified from the patients (APS‐IgG) induced neutrophils from HCs to release NETs. Additionally, APS‐IgG NET induction was abolished with inhibitors of reactive oxygen species, AKT, p38 MAPK and ERK1/2. Moreover, NETs were detrimental to trophoblasts and HUVECs. In summary, APS‐IgG‐induced NET formation deserves further investigation as a potential novel therapeutic target in obstetrical APS.

Aberrant hydroxymethylation of ANGPTL4 is associated with selective intrauterine growth restriction in monochorionic twin pregnancies

ABSTRACT Selective intrauterine growth restriction (sIUGR) is a severe complication in monochorionic (MC) twin pregnancies, and it carries increased risks of poor prognosis. Current data suggest that vascular anastomoses and unequal placental sharing may be the key contributor to discordant foetal growth. While MC twins derive from a single zygote and have almost identical genetic information, the precise mechanisms remain unknown. DNA hydroxymethylation is a newly discovered epigenetic feature associated with gene regulation and modification. Here, we investigate discordant hydroxymethylation patterns between two placental shares of sIUGR and analyse the potential role of aberrant hydroxymethylation of angiopoietin-like 4 (ANGPTL4) in placental dysplasia. Hydroxymethylation DNA immunoprecipitation (hMeDIP)-chip and mRNA sequencing were performed to identify hydroxymethylation-associated genes. Real-time qPCR, western blotting, and immunohistochemistry were used to confirm ANGPTL4 expression. The mechanisms regulating ANGPTL4 were investigated by cell migration assay, invasion assay, viability assay, and apoptotic ratio assays, western blotting and hMeDIP-qPCR. Decreased ANGPTL4 was detected in the smaller placental shares of sIUGR. ANGPTL4 knockdown suppressed trophoblast invasiveness and migration, which possibly occurred through hypoxia inducible factor 1α (HIF-1α) and HIF-1 signalling pathway. Hypoxia leads to aberrant expression of ANGPTL4 and HIF-1α, positively correlated with their aberrant hydroxymethylation levels in promoter regions. Aberrant hydroxymethylation of ANGPTL4 may contribute to placental impairment by the HIF-1 signalling pathway in smaller placental shares of sIUGR.

MiRNA-210-3p regulates trophoblast proliferation and invasiveness through fibroblast growth factor 1 in selective intrauterine growth restriction.

Selective intrauterine growth restriction (sIUGR), which affects approximately 10%‐15% of monochorionic (MC) twin pregnancies, is highly associated with intrauterine foetal death and neurological impairment in both twins. Data suggest that unequal sharing of the single placenta is the main contributor to birth weight discordance. While MC twins and their placenta derive from a single zygote and harbour almost identical genetic material, the underlying mechanisms of phenotypic discrepancies in MC twins remain unclear. MicroRNAs are small non‐coding RNA molecules that regulate gene expression but do not change the DNA sequence. Our preliminary study showed that microRNA‐210‐3p (miR‐210‐3p) was significantly upregulated in the placental share of the smaller sIUGR twin. Here, we investigate the potential role of miR‐210‐3p in placental dysplasia, which generally results from dysfunction of trophoblast cells. Functional analysis revealed that miR‐210‐3p, induced by hypoxia‐inducible factor 1α (HIF1α) under hypoxic conditions, suppressed the proliferation and invasiveness of trophoblast cell lines. Further RNA sequencing analysis and luciferase reporter assays were performed, revealing that fibroblast growth factor 1 (FGF1) is an influential target gene of miR‐210‐3p. Moreover, correlations among miR‐210‐3p levels, HIF1α and FGF1 expression and the smaller placental share were validated in sIUGR specimens. These findings suggest that upregulation of miR‐210‐3p may contribute to impaired placentation of the smaller twin by decreasing FGF1 expression in sIUGR.

Evaluation of vascular effect of arsenic using in vivo assays.

Arsenic (As) is an abundant toxicant present in groundwater and soil in various parts of the world including eastern part of India. The epidemiological studies have shown that arsenic exposure is linked to developmental defects and miscarriage. Placenta is known to utilize vasculogenesis to develop its vasculature circulation. The effects of four different doses of sodium meta-arsenite (0, 10, 20, 75, and 150ppm) were assessed on the vascular structure using two different in vivo models, i.e., Matrigel and chorioallantoic membrane (CAM) assay. For the Matrigel assay, mice were exposed to different doses of arsenic through drinking water for 1month. Placenta and Matrigel plug (which was inserted on gestational day (GD 0.5)) were removed on GD 14. Similar arsenic concentration was used in CAM assay to observe the effect of vessel development in hen's eggs. The CAM assay outcome evaluated by Angiosys software showed that arsenic exposure reduced the total and mean tubule length in all the arsenic-treated groups. The percentage tubule inhibition was declined significantly in 20, 75, and 150ppm arsenic-treated groups as evaluated by ImageJ software. Analysis of the CAM outcome by both the image analysis software indicated the adverse effect of arsenic on the tubules. Further, a significant higher blood vessel density in 10ppm and lower vessel density in 20, 75, and 150ppm arsenic-exposed mice were also observed in Matrigel plug assay. The placental hypertrophy and dysplasia especially in the labyrinth zone (vasculature) were noted in placenta of arsenic-treated mice. The study indicated that higher arsenic exposures inhibited the angiogenesis which was dose-dependent in both CAM and Matrigel assay and altered structural morphology of placenta. However, no inhibition of blood vessels was noted at lower, i.e., 10ppm of arsenic-treated group.

Placental characteristics in monochorionic twins with selective intrauterine growth restriction assessed by gradient angiography and three-dimensional reconstruction

Objective: To investigate the placental characteristics in selective intrauterine growth restriction (sIUGR) using gradient angiography and three-dimensional (3D) reconstruction from computed tomography (CT) scan data.Methods: This study included 23 sIUGR cases and 16 monochorionic twin-pregnancies without sIUGR. We injected nonionic iodinated contrast agents into the umbilical arteries and veins. Placental characteristics were analyzed after CT scanning and 3D reconstruction.Results: 73.9% of smaller twins in sIUGR cases had marginal or velamentous cord insertions and less placental sharing. The terminal branch of the arterial tree was scored III–IV in smaller sIUGR twins, while it was scored V–VII in normal monochorionic twins and larger sIUGR twins. Arterio-arterial (A-A) anastomoses presented in all monochorionic placentas. Veno-venous (V-V) anastomoses present in 83.3% (5/6) of Type III sIUGR cases, which was higher than observed in Type I–II cases. The mean diameters of A-A and V-V anastomoses were larger in Type III sIUGR cases.Conclusions: Gradient angiography and 3D placental models displayed different placental angioarchitectures and voluminal placental sharing among three types of sIUGR cases. Placental dysplasia in the smaller twin may cause abnormal cord insertion and unequal placental sharing. The inter-twin anatomoses influence the umbilical cord artery (UA) Doppler and natural pathogenesis of sIUGR.

Early and late onset severe preeclampsia: a clinicopathologic study of 178 placentas

To explore the pathologic features and prognosis of early and late onset severe preeclampsia. An observational study was conducted in 178 cases of severe preeclampsia collected during January 2010 to December 2014 from Haidian Maternal and Child Health Hospital.The cases were divided into two groups according to the onset of gestational age of the severe preeclampsia, with 54 cases of namely early onset (onset ≤ 34 weeks) and 124 cases of late onset (onset >34 weeks). Clinical characteristics of the patients, perinatal outcome and the pathologic characteristics of the placentas in each group were evaluated. Decidual vascular disease, placental infarction, abruptio placentae and placental villi dysplasia were seen in both groups. The incidence of placental villi dysplasia was the highest, followed by placental infarction. Incidence of severe decidual vascular disease of early and late onset severe decidual vascular disease were 16.7% (9/54) and 5.6% (7/124), respectively.Incidence of placental infarction of early and late onset severe preeclampsia were 48.1% (26/54) and 61.3% (76/124). Incidence of placental villi dysplasia of early and late onset severe preeclampsia were 79.6% (43/54) and 50.8% (63/124). Incidence of Severe decidual vascular disease, placental infarction and placental villi dysplasia were significantly different between early and late onset severe preeclampsia cases (P<0.05), while there was no difference in decidual vascular disease and placenta thrombi (P>0.05). Fetal survival rate of every group was 81.5% (44/54) and 93.5% (116/124). Incidence of fetal growth retardation was 55.6% (30/54) and 38.7% (48/124). The fetal survival rate and incidence of fetal growth retardation were different between two groups (P<0.05). The incidence of decidual vascular disease and placental villi dysplasia in early onset severe preeclampsia is higher than those in late onset severe preeclampsia. Neonatal outcome and prognosis in early onset severe preeclampsia are worse than those in late onset severe preeclampsia.

Maternal-foetal genomic conflict and speciation: no evidence for hybrid placental dysplasia in crosses between two house mouse subspecies.

Interspecific hybridization between closely related mammalian species, including various species of the genus Mus, is commonly associated with abnormal growth of the placenta and hybrid foetuses, a phenomenon known as hybrid placental dysplasia (HPD). The role of HPD in speciation is anticipated but still poorly understood. Here, we studied placental and foetal growth in F1 crosses between four inbred mouse strains derived from two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus. These subspecies are in the early stage of speciation and still hybridize in nature. In accordance with the maternal-foetal genomic conflict hypothesis, we found different parental influences on placental and foetal development, with placental weight most affected by the father's body weight and foetal weight by the mother's body weight. After removing the effects of parents' body weight, we did not find any significant differences in foetal or placental weights between intra-subspecific and inter-subspecific F1 crosses. Nevertheless, we found that the variability in placental weight in inter-subspecific crosses is linked to the X chromosome, similarly as for HPD in interspecific mouse crosses. Our results suggest that maternal-foetal genomic conflict occurs in the house mouse system, but has not yet diverged sufficiently to cause abnormalities in placental and foetal growth in inter-subspecific crosses. HPD is thus unlikely to contribute to speciation in the house mouse system. However, we cannot rule out that it might have contributed to other speciation events in the genus Mus, where differences in the levels of polyandry exist between the species.

Aberrant expression of Igf2/H19 in porcine parthenogenetic fetuses and placentas

The aberrant expression of imprinted genes induces parthenogenetic fetal and placental dysplasia, thus leading to failures in embryonic development. Igf2 and H19 are co-expressed in endoderm and mesoderm-derived tissues and play an important role in normal embryo and extraembryonic development. In this study, the expression and methylation of Igf2/H19 in porcine parthenogenetic fetuses and placentas which had grown 28 days was examined first time to further characterize mammalian parthenogenesis. Weight and morphological comparisons were conducted between parthenogenetic embryos on Day 28 and normal fertilized embryos (control). The results indicated that parthenogenetic fetuses and placentas had smaller weights and volumes than those of the control. In addition, quantitative RT-PCR (qRT-PCR) analysis was performed to determine Igf2/H19 expression levels, showing that the expression of H19 was up-regulated, while Igf2 expression was almost undetectable in both parthenogenetic fetuses and placentas. As a potential mechanism underlying this disrupted expression, the methylation of Igf2/H19 DMR3 was detected using bisulfite sequencing PCR analysis, which revealed the significant hypomethylation of DMR3 in parthenogenetic fetuses and placentas. These results suggest that disruption of Igf2/H19 expression in parthenogenetic fetuses and placentas contributes to implantation failure and/or abortion in swine parthenogenesis, which might be associated with differential methylation patterns in the imprinting control region of imprinted genes.

Retroelement Demethylation Associated with Abnormal Placentation in Mus musculus × Mus caroli Hybrids1

The proper functioning of the placenta requires specific patterns of methylation and the appropriate regulation of retroelements, some of which have been co-opted by the genome for placental-specific gene expression. Our inquiry was initiated to determine the causes of the placental defects observed in crosses between two species of mouse, Mus musculus and Mus caroli. M. musculus × M. caroli fetuses are rarely carried to term, possibly as a result of genomic incompatibility in the placenta. Taking into account that placental dysplasia is observed in Peromyscus and other Mus hybrids, and that endogenous retroviruses are expressed in the placental transcriptome, we hypothesized that these placental defects could result, in part, from failure of the genome defense mechanism, DNA methylation, to regulate the expression of retroelements. Hybrid M. musculus × M. caroli embryos were produced by artificial insemination, and dysplastic placentas were subjected to microarray and methylation screens. Aberrant overexpression of an X-linked Mus retroelement in these hybrid placentas is consistent with local demethylation of this retroelement, concomitant with genome instability, disruption of gene regulatory pathways, and dysgenesis. We propose that the placenta is a specific site of control that is disrupted by demethylation and retroelement activation in interspecific hybridization that occur as a result of species incompatibility of methylation machinery. To our knowledge, the present data provide the first report of retroelement activation linked to decreased methylation in a eutherian hybrid system.

36 CHANGES IN PLASMA CONCENTRATIONS OF PROGESTERONE AND ESTROGENS DURING GESTATION IN COWS WITH STILLBORN SOMATIC CELL CLONED CALVES

Cloning of mammals by nuclear transfer frequently results in gestational or neonatal failure with a variety of abnormalities that are likely caused by inappropriate epigenetic reprogramming. Early diagnosis of fetal abnormality is important for efficient production of cloned animals. Sex steroids are produced in the bovine placenta and their levels in the blood might be useful as a measure of fetal well being, as they are in humans. The objective of this study was to investigate whether changes in peripheral concentrations of progesterone and estrogens reflect fetal conditions. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were transferred into the uterine horn ipsilateral to the ovary bearing the CL of 29 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing oestrus (Day 0). Blood was collected at regular intervals from Day 40 until parturition. Plasma progesterone, estrone, oestradiol-17β, and estrone sulfate were measured by RIA in 4 recipient cows. 2 vaginally delivered healthy calves weighed 35 and 36 kg on days 278 and 280, respectively. The other 2 delivered stillborn calves weighed 42 and 31 kg by Caesarean section on days 267 and 287, respectively. Steroid profiles were compared with each other and with those in a cow made pregnant by embryo transfer. Statistical differences at stages of gestation were analysed with repeated-measures ANOVA. Stillborn calves were subjected to necropsy and histopathological analysis. Significant differences in steroid levels were observed individually and temporarily. In the cow bearing the former stillborn, progesterone and oestradiol-17β concentrations tended to be lower during mid and late gestation, respectively. Large offspring syndrome, ventricular septal defect, pulmonary oedema, hepatic fibrosis, and placental dysplasia were found in the calf by necropsy and histopathological analysis. In the cow bearing the second stillborn, plasma progesterone concentrations were temporarily increased from days 220 to 230 and reached a peak of 16 ng mL–1, which was approximately 3 times as high as before (5.0 ± 0.9; mean ± s.d.), then followed by a similar transient increase in estrogens during days 240 and 250 (170 pg mL–1, 16 pg mL–1, and 21 ng mL–1 at peak of estrone, oestradiol-17β, and estrone sulfate, respectively). Thereafter, oestrogen concentrations stayed low until parturition. As internal organs of the stillborn calf were severely autolysed with polyhydramnios at birth, the fetus might have been dead around Day 250. These results imply that changes in peripheral steroid concentrations in some cases reflect fetal condition.

Cyclopamine‐induced synophthalmia in sheep: defining a critical window and toxicokinetic evaluation

Cyclopamine, a steroidal alkaloid, from the plant Veratrum californicum is teratogenic, causing a range of different birth defects. The critical window for cyclopamine-induced synophthalmia formation has been reported to be gestational day (GD) 14. The objectives of this study were to better describe cyclopamine-induced craniofacial deformities, to better define the window of susceptibility to synophthalmia formation, and to characterize cyclopamine toxicokinetics in sheep. Ewes were dosed i.v. with purified cyclopamine for toxicokinetic analysis. Another four groups of ewes were dosed orally twice daily with 0.88 g/kg of V. californicum on GD 13, 14 or 15 or consecutively on GD days 13-15. Pregnancy and pre-partum fetal malformations were determined by ultrasound imaging on GD 60. At parturition lambs were assessed for gross malformations. The elimination half-life of cyclopamine in ewes was determined to be 1.1 +/- 0.1 h. The rapid clearance of cyclopamine indicates that ingestion of V. californicum must occur during a very narrow window for synophthalmia formation to occur. Ewes dosed with V. californicum on GD 13 or 14 had lambs with various craniofacial malformations including cyclopia, maxillary dysplasia and mandibular micrognathia. Ewes dosed on GD 15 delivered normal lambs. Ewes dosed consecutively on GD 13-15 were not pregnant at GD 60 and Veratrum-induced embryonic death was assumed to be the cause. Interestingly, lambs with cyclopia were smaller, under-developed and appeared premature even though their twin appeared fully developed. Initial evaluations suggest this was due to placental dysplasia.

The Role of Parental-Specific Methylation Imprint on Mammalian Development.

Germ cells are unique totipotent cells from which individuals are generated by the fusion of male and female gametes. However, in mammals bi-parental reproduction is necessary for the production of descendants because parent-specific epigenetic modifications occur in the genome during gametogenesis. Therefore mouse parthenogenetic embryos die by E10. However, ng/fg bi-maternal embryos that contain a non-growing (ng) oocyte genome and a fully grown (fg) oocyte genome develop to E14. Imprinted genes have prominent effects on foetal and placental development. The Igf2-H19 genes, which are controlled by paternally methylated regions on chromosomes 7, contribute to the further extended development of the bi-maternal embryos. It was found that the bi-maternal embryos harboring a 13-kb deletion (Δchr7) that spanned the differentially methylated region and the transcription unit of the H19 gene developed to term. Extended development, however, was accompanied by foetal severe growth retardation and placental dysplasia. Because of these abnormalities the foetuses died after they were recovered from the uterus. It is also known that the genes, Dlk1-Gtl2, controlled by the paternally methylated region of chromosome 12, contribute to foetal development. To obtain an insight into the association between Dlk1-Gtl2 and development of bi-maternal embryos, we reconstructed bi-maternal embryos using ng oocytes harboring an IG-DMR deletion at chromosome 12 (Δchr12). Assessment of developmental ability showed that these bi-maternal embryos also developed to term. However, the phenotype is apparently differed between the ngΔchr7/fg and ngΔchr12/fg bi-maternal fetuses/placentae. In the ngΔchr12/fg bi-maternal foetuses. Extremely severe growth retardation was observed in the latter foetuses. The ngΔchr7/fg placenta is exhibited severe dysplasia, including an expanded spongiotrophoblast layer and a malformed labyrinthine zone. The placental dysplasia was restored in the ngΔchr12/fg placentae, although the mass was severely reduced. Gene expression analysis revealed that the expressions of imprinted genes regulated by IG-DMR was corrected in the bi-maternal embryos. In order to understand more the features of bi-maternal embryos, we characterized the gene expression profiles of the ngΔchr7/fg and the ngΔchr12/fg bi-maternal foetuses and placentae by using the Affymetrix Mouse Genome 430 2.0 Array. After describing a Venn diagram using the GeneSpring software, we constructed lists including genes in the ngΔchr7/fg and ngΔchr12/fg bi-maternal embryos and placentae. Based on the obtained extensive gene expression data and developmental features, we discuss complementary role of the 2 sets of imprinted genes (Igf2-H19 and Dlk1-Gtl2) on the development of bi-maternal mouse embryos.

Arsenic Exposure in Pregnant Mice Disrupts Placental Vasculogenesis and Causes Spontaneous Abortion

Arsenic is an abundant toxicant in ground water and soil around areas with extractive industries. Human epidemiological studies have shown that arsenic exposure is linked to developmental defects and miscarriage. The placenta is known to utilize vasculogenesis to develop its circulation. The hypothesis tested here states the following: arsenic exposure causes placental dysmorphogenesis and defective placental vasculogenesis resulting in placental insufficiency and subsequent spontaneous abortion. To test this hypothesis, pregnant mice were exposed to sodium arsenite (AsIII) through drinking water from conception through weanling stages. Neonatal assessment of birth rates, pup weights, and litter sizes in arsenic exposed and control mothers revealed that AsIII-exposed mothers had only 40% the fecundity of controls. Preterm analysis at E12.5 revealed a loss of fecundity at E12.5 from either 20 ppm or greater exposures to AsIII. There was no loss of fecundity at E7.5 suggesting that spontaneous abortion occurs during placentation. Histomorphometry on E12.5 placentae from arsenic-exposed mice revealed placental dysplasia especially in the vasculature. These results suggest that arsenic toxicity is causative for mammalian spontaneous abortion by virtue of aberrant placental vasculogenesis and placental insufficiency.

Carboxypeptidase E in the mouse placenta

Carboxypeptidase E (CPE) has important functions in processing of endocrine pro-peptides, such as pro-insulin, pro-opiomelanocortin, or pro-gonadotropin-releasing hormone, as evidenced by the hyper-pro-insulinemia, obesity, and sterility of Cpe mutant mice. Down-regulation of Cpe in enlarged placentas of interspecific hybrid (interspecies hybrid placental dysplasia (IHPD)) and cloned mice suggested that reduced CPE enzyme and receptor activity could underlie abnormal placental phenotypes. In this study, we have explored the role of Cpe in murine placentation by determining its expression at various stages of gestation, and by phenotypic analysis of Cpe mutant placentas. Our results show that Cpe and Carboxypeptidase D ( Cpd), another carboxypeptidase with a very similar function, are strictly co-localized in the mouse placenta from late mid-gestation to term. We also show that absence of CPE causes a sporadic but striking placental phenotype characterized by an increase in giant and glycogen cell numbers and giant cell hypertrophy. Microarray-based transcriptional profiling of Cpe mutant placentas identified only a very small number of genes with altered expression, including Dtprp, which belongs to the prolactin gene family. Concordant deregulation of Cpe and Cpd in abnormal placentas of interspecies hybrids before the onset of IHPD phenotype and recapitulation of some phenotypes of IHPD hyperplastic placentas in Cpe mutant placentas suggests that these two genes are causally involved in IHPD and may function as speciation genes in the genus Mus.

Expression and Functional Analysis of Fibulin-1 ( Fbln1) During Normal and Abnormal Placental Development of the Mouse

The extracellular matrix protein fibulin-1 (FBLN1) is an important component of blood vessel walls, as shown by the lethality of mice with homozygous targeted deletion of the Fbln1 gene. Here, we show that a murine placental overgrowth phenotype is associated with elevated Fbln1 transcript levels, suggesting that the gene and its product have a functional role in placentation. Fbln1 exhibits a specific expression pattern in the mouse placenta. Transcripts could not be detected prior to day 12. In subsequent stages, Fbln1 was expressed strongly in the spongiotrophoblast. Other sites of expression were endothelia of large fetal blood vessels, a tissue type reported to not express this gene. In addition, a subset of giant cells expressed the gene. This giant cell specific expression was strongly increased in hyperplastic placentas. Analysis of the placentation in fibulin null mice did not show any abnormality. Attempts to rescue the placental phenotypes of a congenic model of interspecies hybrid placental dysplasia (IHPD) by normalizing expression of Fbln1 proved that Fbln1 alone is not the key cause of phenotypes in these models of placental hyperplasia.

DNA Methylation in Placentas of Interspecies Mouse Hybrids

Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids.

Proliferation and growth factor expression in abnormally enlarged placentas of mouse interspecific hybrids.

It has been shown previously that abnormal placental growth occurs in crosses and backcrosses between different mouse (Mus) species. In such crosses, late gestation placentas may weigh between 13 and 848 mg compared with a mean placental weight of approximately 100 mg in late gestation M. musculus intraspecific crosses. A locus on the X-chromosome was shown to segregate with placental dysplasia. Thus in the (M. musculus x M. spretus)F1 x M. musculus backcross, placental hyperplasia cosegregates with a M. spretus derived X-chromosome. Here we have investigated whether increased cell proliferation and aberrant expression of two genes that are involved in placental growth control, Igf2 and Esx1, may cause, or contribute to placental hyperplasia. Increased bromodeoxyuridine labeling of nuclei, reflecting enhanced proliferation, was indeed observed in hyperplastic placentas when compared with normal littermate placentas. Also, increased expression of Igf2 was seen in giant cells and spongiotrophoblast. However, when M. musculus x M. spretus F1 females were backcrossed with males that were heterozygous for a targeted mutation of the Igf2 gene, placentas that carried a M. spretus derived X-chromosome and were negative for a functional Igf2 allele exhibited an intermediate placental phenotype. Furthermore, in early developmental stages of placental hyperplasia, we observed a decreased expression of the X-chromosomal Esx1 gene. This finding suggests that abnormal expression of both Igf2 and Esx1 contributes to abnormal placental development in mouse interspecific hybrids. However, Esx1 is not regulated by IGF2.

Genetic and developmental analysis of X-inactivation in interspecific hybrid mice suggests a role for the Y chromosome in placental dysplasia.

It has been shown previously that abnormal placental growth, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. A locus that contributes to this abnormal development has been mapped to the X chromosome. Unexpectedly, an influence of fetal sex on placental development has been observed, in that placentas attached to male fetuses tended to exhibit a more pronounced phenotype than placentas attached to females. Here, we have analyzed this sex dependence in more detail. Our results show that differences between male and female placental weights are characteristic of interspecific matings and are not observed in intraspecific Mus musculus matings. The effect is retained in congenic lines that contain differing lengths of M. spretus-derived X chromosome. Expression of the X-linked gene Pgk1 from the maternal allele only and lack of overall activity of two paternally inherited X-linked transgenes indicate that reactivation or lack of inactivation of the paternal X chromosome in trophoblasts of interspecific hybrids is not a frequent occurrence. Thus, the difference between male and female placentas seems not to be caused by faulty preferential X-inactivation. Therefore, these data suggest that the sex difference of placental weights in interspecific hybrids is caused by interactions with the Y chromosome.

Genetic dissection of X-linked interspecific hybrid placental dysplasia in congenic mouse strains.

Interspecific hybridization in the genus Mus results in male sterility and X-linked placental dysplasia. We have generated several congenic laboratory mouse lines (Mus musculus) in which different parts of the maternal X chromosome were derived from M. spretus. A strict positive correlation between placental weight and length of the M. spretus-derived part of the X chromosome was shown. Detailed analysis was carried out with one congenic strain that retained a M. spretus interval between 12.0 and 30.74 cM. This strain consistently produced hyperplastic placentas that exhibited an average weight increase of 180% over the weight of control placentas. In derived subcongenic strains, however, increased placental weight could no longer be observed. Morphometric analysis of these placentas revealed persistence of abnormal morphology. Fully developed placental hyperplasia could be reconstituted by recombination of proximal and central M. spretus intervals with an intervening M. musculus region. These results may suggest that placental dysplasia of interspecific mouse hybrids is caused by multiple loci clustered on the X chromosome that act synergistically. Alternatively, it is possible that changes in chromatin structure in interspecific hybrids that influence gene expression are dependent on the length of the alien chromosome.

Analysis of differentially expressed genes in mouse interspecific hybrid placental dysplasia

Analysis of differentially expressed genes in mouse interspecific hybrid placental dysplasia