Intrauterine Growth Restriction Placentas Research Articles

DNA methylation profiles of genes associated with angiogenesis in the samples of placenta in pregnancies complicated by intrauterine growth restriction

Background Impairment in placental angiogenesis is blamed for the etiopathogenesis of intrauterine growth restriction (IUGR). Aim To assess the genes related to angiogenesis in placental biopsies of pregnancies complicated by IUGR that could be aberrantly methylated and adversely affect placental angiogenesis. Methods The methylation profiles of soluble fms-like tyrosine kinase-1 (sFLT-1), vascular endothelial growth factor (VEGF), and the placental growth factor (PIGF) were evaluated using Illumina MiSeq™ System in placental biopsies from term IUGR pregnancies without preeclampsia (n = 18) and healthy controls (n = 17). DNA was isolated from samples of tissue collected from the fetal side of the placenta. In the targeted regions, we have identified 30, 24, and 29 CpG islands (CpGi) within sFLT-1, VEGF and PIGF genes, respectively. CpGi which are most methylated in the promoter regions of three genes were selected for the study from the database http://www.ensembl.org. Result(s) IUGR fetuses had significantly lower placental and fetal birth weight than controls. The promoter of sFLT-1 at three CpGi and VEGF at six CpGi were the regions with significant methylation differences between IUGR and control placentas. sFLT-1 was hypermethylated at 265 and 352 CpGi; however, hypermethylation was lower in IUGR group compared to control group at this position. sFLT-1 was hypomethylated at 456 CpGi in IUGR group and hypermethylated at the same region in control group. VEGF was hypomethylated at 668, 703, and 710 CpGi in control and IUGR groups; however, hypomethylation at these positions was significantly higher in control group compared to IUGR. 776, 845, and 863 CpGi of VEGF promoter were significantly hypermethylated in IUGR group whereas hypomethylated in control group. The methylation profile of PIGF did not differ between the groups. After adjustment for the factors known to affect fetal birth weight, DNA methylation of VEGF 668 CpGi had a significant negative association with fetal birth weight in the control and the IUGR group and a positive association with IUGR pregnancies. Conclusion(s) Our results do not support the hypothesis that altered DNA methylation in the placental angiogenic genes is a major mechanism generally involved in IUGR. Only a specific region (at 668 CpGi) corresponding to the promoter of VEGF may serve as an epigenetic marker of IUGR and may be involved in the mechanism of IUGR. Large sample-sized studies are needed to understand the effects of DNA methylation on placental gene function and how they might influence fetal growth.

LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

Abnormal placental development is one of the main etiological factors for intrauterine growth restriction (IUGR). Here, we show that LIN28A and LIN28B are significantly lower and lethal-7 (let-7) microRNAs (miRNAs) significantly higher in term human IUGR vs. normal placentas. We hypothesize that let-7 miRNAs regulate genes with known importance for human placental development [high-mobility group AT-hook 1 (HMGA1), transcriptional regulator Myc-like (c-myc), vascular endothelial growth factor A (VEGF-A), and Wnt family member 1 (WNT1)] by targeting the AT-rich interacting domain (ARID)-3B complex. ACH-3P cells with LIN28A and LIN28B knockout (DKOs) significantly increased let-7 miRNAs, leading to significantly decreased ARID3A, ARID3B, and lysine demethylase 4C (KDM4C). Similarly, Sw.71 cells overexpressing LIN28A and LIN28B (DKIs) significantly decreased let-7 miRNAs, leading to significantly increased ARID3A, ARID3B, and KDM4C. In ACH-3P cells, ARID3A, ARID3B, and KDM4C make a triprotein complex [triprotein complex comprising ARID3A, ARID3B, and KDM4C (ARID3B-complex)] that binds the promoter regions of HMGA1, c-MYC, VEGF-A, and WNT1. ARID3B knockout in ACH-3P cells disrupted the ARID3B-complex, leading to a significant decrease in HMGA1, c-MYC, VEGF-A, and WNT1. DKOs had a significant reduction, whereas DKIs had a significant increase in HMGA1, c-MYC, VEGF-A, and WNT1, potentially due to regulation by the ARID3B-complex. This is the first study showing regulation of let-7 targets in immortalized human trophoblast cells by the ARID3B-complex.-Ali, A., Anthony, R. V., Bouma, G. J., Winger, Q. A. LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

Diffusion-weighted imaging of placenta in intrauterine growth restriction with worsening Doppler US findings

We aimed to compare the placental diffusion difference between intrauterine growth restriction (IUGR) patients with worsening Doppler ultrasonography (US) findings and control group with normal Doppler US findings by using diffusion-weighted imaging (DWI). We performed a prospective study to compare the placental diffusion difference in 63 patients (gestational week, 28-34 weeks), including 50 IUGR patients (mean gestational week, 30 weeks 3 days ±16.2 days) with worsening Doppler US findings and 13 patients with normal Doppler US findings (mean gestational week, 29 weeks 4 days ±12.3 days) by using DWI (b value, 0-1000 s/mm2). We classified IUGR patients into three groups according to the reference values of the umbilical artery pulsatility index (PI) chart. Placenta apparent diffusion coefficient (ADC) calculations were performed by freehand drawn regions-of-interest (ROIs) (min, 8.04 cm2; max, 200 cm2). Placental ADC values in IUGR patients (mean, 1.624±0.181 ×10-³ mm²/s; range, 1.35-1.96 ×10-3 mm2/s) were significantly reduced compared with the control group (mean, 1.827±0.191 ×10-³ mm²/s; range, 1.35-2.84 ×10-3 mm2/s) (P = 0.001). For adjusted ROI area calculation, ADC values were significantly lower in groups 3, 2 and 1, respectively, compared with the control group (P < 0.05); and there was no significant difference between groups 1 and 2 (P > 0.05). Preeclampsia significantly reduced the placental diffusion compared with patients without preeclampsia (P = 0.003). Gestational aging did not significantly affect ADC values in control patients (r=0.08, P = 0.561). The sensitivity, specificity, negative and positive predictive values of ADC to detect IUGR were 72%, 84.6%, 44%, and 94.7% with a cutoff value of 1.727 ×10-3 mm2/s, respectively. The diagnostic estimation of placental ADC values to predict the severity of IUGR is comparable to that of umbilical artery PI. Considering that at the very early onset of IUGR, placental diffusion diminishes, ADC as a marker for IUGR in lieu of umbilical artery PI has the potential to determine the threshold for decreased placental diffusion. Therefore, DWI should be added to routine fetal MRI to show diffusion changes in placenta.

Glucocorticoid receptors α and β are modulated sex specifically in human placentas of intrauterine growth restriction (IUGR).

The objective of this study was to analyze the expression of the glucocorticoid receptor (GR) subtypes GRα and GRβ in placentas affected by intrauterine growth restriction (IUGR). We analyzed the sex-specific placental expression of GRα and GRβ in 23 IUGR and 40 control placentas using immunohistochemistry and immunofluorescence. The GR gene, also known as nuclear receptor subfamily 3 group C member 1 (NR3C1), mRNA production in trophoblast-like cell line BeWo after stimulation with prednisolone was analyzed using quantitative polymerase chain reaction (qPCR) and on the protein level using western blot analysis. GR subtypes showed a sex-specific upregulation in placentas from IUGR compared to control placentas. An increased expression of GRα was detectable in female placental tissue, whereas GRβ was increased in males. Our data support previous findings suggesting that the glucocorticoid metabolism plays a role in the pathophysiology of IUGR. Furthermore, the data suggest that the underlying molecular mechanisms differ between male and female cases.

Placental ESRRG-CYP19A1 Expressions and Circulating 17-Beta Estradiol in IUGR Pregnancies.

Introduction: Sex steroids are regulating factors for intrauterine growth. 17-β Estradiol (E2) is particularly critical to a physiological pregnancy, as increased maternal E2 was correlated to lower fetal weight at delivery. The placenta itself is a primary source of estrogens, synthetized from cholesterol precursors. Cytochrome P450 aromatase (encoded by CYP19A1 gene) is a rate-limiting enzyme for E2 biosynthesis. CYP19A1 transcription is supported by Estrogen Related-Receptor Gamma (ERRγ- ESRRG gene), which thus has an indirect role in placental steroidogenesis. Here we investigated maternal E2 levels and placental CYP19A1 and ESRRG expressions in pregnancies with IntraUterine Growth Restriction (IUGR).Methods: Singleton pregnancies were studied. E2 was measured in maternal plasma by electrochemiluminescence in 16 term controls and 11 IUGR (classified by umbilical artery doppler pulsatility index) at elective cesarean section, and also in 13 controls during pregnancy at a gestational age comparable to IUGR. CYP19A1 and ESRRG expressions were analyzed in placental tissue. Maternal/fetal characteristics, placental and molecular data were compared among study groups and tested for correlations.Results: Maternal E2 plasma concentrations were significantly decreased in IUGR compared to controls at delivery. When analyzing normal pregnancies at a gestational age similar to IUGR, E2 levels were not different to pathological cases. However, E2 levels at delivery positively correlated with placental efficiency. Placental CYP19A1 levels were significantly higher in IUGR placental tissue vs. controls, and specifically increased in female IUGR placentas. ESRRG expression was not different among groups.Discussion: We report a positive correlation between 17-β Estradiol levels and placental efficiency, that might indicate a disrupted steroidogenesis in IUGR pregnancies. Moreover, we show alterations of CYP19A1 expression in IUGR placentas, possibly indicating a compensatory effect to the adverse IUGR intrauterine environment, also depending on fetal sex. Further studies are needed to deeper investigate IUGR alterations in the complex interaction among molecules involved in placental steroidogenesis.

Placental expression of angiogenesis-related genes and their receptors in IUGR pregnancies: correlation with fetoplacental and maternal parameters

Objectives: Aberrations in placental vascular development compromising fetal supply of oxygen and essential nutrients can be a significant contributor to intrauterine growth restriction (IUGR). The development of placental vascular tree is under the influence of two families of growth factors, namely the vascular endothelial growth factor (VEGF) family and angiopoietin/TEK family. In this study, we have examined the expression of angiogenesis-related growth factors, mainly VEGF family and angiopoietin-TEK (endothelial-specific receptor tyrosine kinase) family genes in placentae from IUGR pregnancies uncomplicated by preeclampsia (PE) compared to normal pregnancies.Methods: Placentae from normotensive IUGR (n = 42) and appropriate for gestational age (AGA) pregnancies (n = 47) were collected and examined histologically. Clinical parameters were obtained from the medical records. Real-time quantitative PCR was performed to assess placental transcript abundance of VEGF, PGF, FLT1, ANGPT1, ANGPT2, and TEK normalized to a panel of reference genes. Associations of placental transcript abundance of the genes with maternal, placental, and neonatal parameters were tested.Results: Placental transcript abundance for VEGF (relative expression 10.81 versus 12.98, p < .001), PGF (12.14 versus 13.8, p < .001) and ANGPT2 (3.67 versus 9.55, p = .002) were significantly lower in IUGR placentae compared to AGA. The transcript level of VEGF showed significant negative correlation with birth weight (r = −0.419, p = .006), placental weight (r = −0.318, p = .040), placental length (r = −0.389, p = .011) and breadth (r = −0.308, p = .047) only in the IUGR group. Presence of histopathological features of hypoxia correlated with significantly higher transcript levels of PGF in IUGR placentae (12.6 versus 10.9, p = .046).Conclusion: The low levels of VEGF transcripts may be responsible for the impaired angiogenesis in IUGR placentae. The significance of higher relative expression of PGF in the presence of chronic hypoxia needs to be explored.

Mitochondrial and glycolysis-regulatory gene expression profiles are associated with intrauterine growth restriction

Introduction: Intrauterine growth restriction (IUGR) is a major pregnancy complication with significant postnatal implications. IUGR is characterized by high placental oxidative stress (OS) and increased mitochondrial DNA (mtDNA) abundance that altogether alter the placental metabolism. Such alterations may be captured by changes in the expression of mitochondrial-encoded oxidative phosphorylation genes and glycolysis-regulatory genes.Study design: We aimed here to determine the association between the placental expression of all 13 protein-coding mitochondrial-encoded genes and seven key nuclear glycolysis-regulatory genes, PDK1, PDK2, PDK3, PDK4, PKLR, PKM, OGT, with IUGR, within a case-control study including 50 IUGR and 100 control pregnancies. We additionally assessed placental mtDNA abundance and OS.Results: Three mitochondrial genes, MT-ND5, MT-ND6, and MT-ATP6 were found negatively associated with IUGR, while one glycolysis-regulatory gene, PDK1 was positively associated with IUGR. mtDNA abundance and OS were positively associated with IUGR. Our study confirmed the existing data on IUGR inducing increased placental OS and mtDNA abundance. Further, our data highlighted the significant involvement of mitochondria and glucose metabolism in the OS-challenged IUGR placentas, which might modulate the placental expression of genes affecting the OXPHOS and promoting glycolysis.Brief rationale: By using banked placenta samples available at Icahn School of Medicine at Mount Sinai, this study aims at laying the foundation for the characterization of the role of mitochondria epi/genetics in IUGR. IUGR is a highly prevalent pregnancy outcome with long-term effects on the progeny that, at present, has limited tools that can be used for its diagnosis and characterization, thus limiting the efficacy of both clinical and public health interventions. The alterations of mitochondrial copy number, OS and mitochondrial and glycolysis-regulatory gene expression that we detected, together, provide the first evidence that these phenomena are playing an important role in the pathophysiology of IUGR. These findings suggest possible new research paths for the full characterization of mitochondrial biomarkers of IUGR.

Antiphosphatidylserine/prothrombin Antibodies in Antiphospholipid Syndrome with Intrauterine Growth Restriction and Preeclampsia

Antibodies that recognize the phosphatidylserine/prothrombin complex (antiphosphatidylserine/prothrombin antibodies; aPS/PT) might reveal enhanced thrombotic risk in patients with systemic lupus erythematosus. Little is known about their association with pregnancy complications in the antiphospholipid syndrome (APS). We enrolled 55 patients with APS who were seeking pregnancy in 2 Italian hospitals. Antiphospholipid antibodies (aPL), including anticardiolipin antibodies, anti-β2-glycoprotein I antibodies, lupus-like anticoagulant, and aPS/PT antibodies were assessed, and the patients were prospectively followed for 24 months. There were 65% (36/55) of the APS patients who had aPS/PT antibodies. Forty-seven pregnancies were followed, including 33 of aPS/PT+ patients. Forty-one of the 47 patients (87%) who initiated a pregnancy eventually gave birth to a child. The pregnancy duration and the mean newborn weight at delivery were significantly lower in aPS/PT+ than in aPS/PT- patients (33.1 ± 4.7 vs 36.2 ± 3.4 wks of gestation, respectively, and 2058 ± 964 g vs 2784 ± 746 g, respectively, p < 0.05). Late pregnancy complications, including intrauterine fetal death, preterm delivery, preeclampsia, and intrauterine growth restriction (IUGR), were more frequent in aPS/PT+ patients, independent of the therapy. Titers of aPS/PT IgG were significantly inversely correlated with the neonatal weight at delivery. Vascular injury, as reflected by thrombosis, fibrinoid necrosis, ischemic and hemorrhagic areas, and presence of chorangiomas characterized the IUGR placentas in the presence of aPS/PT. The aPS/PT antibodies might represent markers of aPL-related pregnancy complications, IUGR/preeclampsia in particular, and could help identify beforehand patients who may require additional treatment.

Abstract 5160: Identifying TK1 localization in immortalized placental cell lines and in conditioned placental tissue

Abstract Many parallels in growth pattern can be observed between placental and cancer development. In fact, placental trophoblast and cancer cells share similarities in migration and invasion patterns, immune escape strategies, and angiogenesis induction. A common feature of pregnancy complication is aberrant trophoblast behavior. Understanding trophoblast cell behavior can help us identify common patterns in cancer gene and protein regulation. Thymidine kinase 1 (TK1) has been extensively studied as a serum and tissue cancer biomarker. Recently, TK1 has been shown to be upregulated in some cancer tissues and localized on the surface of certain cancer cell lines, and has been suggested as a possible immunotherapeutic target in some cancers. Our goal for this study was to investigate TK1 correlation to placental invasive potential, and therefore establish a function for TK1 localization in these tissues. To do this, we wanted to investigate TK1 placental tissue expression/ localization during normal and obstetric complications and its expression in current placental cell lines. We obtained human placental tissue from various pregnancy conditions including preeclampsia (PE), diabetes (GDM; treated with diet (D) or Insulin (I)) and intrauterine growth restriction (IUGR). For our cell study, immortalized placental cell lines, SW71, JEG-3 and BeWo were used. Immunofluorescence was used for TK1 cell expression in human placental sections. Flow cytometry was performed for TK1 presence in cultured cells. We observed high staining for nuclear and cytosolic expression of TK1 in the control placenta tissues. Compared to controls, there was a marked reduction in TK1 staining in the IUGR and GDM-I placentas which lack nuclear expression of TK1. A decreased for both nuclear and cytosolic TK1 staining, was present in the PE and GDM-D placenta. Flow cytometry showed that SW71 cells had high expression of TK1 on their surface (56%), JEG-3 had low expression of TK1 on their surface (12%), and BeWo cells did not express TK1 on their surface. These findings are interesting because they suggest TK1 surface expression directly correlates with the invasive potential. TK1 protein expression was also measured using standard western blotting techniques to support the data obtained by immunohistochemistry. Furthermore, we established the invasion potential of these placental cell lines using the xCELLigence RTCA DP system between the TK1 positive and negative placenta cell lines. Further confirmation is required; however, these results could assist in understanding the placental/trophoblast invasive mechanism and its correlation in cancer development. Citation Format: Eliza E. Lawrence, Evita G. Weagel, Juan F. Mejia, Juan Arroyo, Shalee Killpack, Kim L. O'Neill, Richard Robison. Identifying TK1 localization in immortalized placental cell lines and in conditioned placental tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5160.

Stillbirth at Patan Hospital, Nepal

Introductions: Stillbirth (SB) is one of the most common adverse outcomes of pregnancy. The aim of this study was to determine the SB rate and to identify the likely causes contributing to SB. &#x0D; Methods: This cross-sectional study was conducted at Patan Hospital from 15th June 2014 to 14th June 2017 for all the cases of SBs, at or after 22 weeks, birth weight of 500 gm or more. The perinatal outcome, demographic profile, fetal characteristics, causes and contributing factors were analyzed. &#x0D; Results: There were 262 SB out of total 23069 deliveries, (11.24 per 1000) and 119 (46.12%) had antenatal check-up (ANC) at Patan Hospital. The 214 (82.95%) SB were among 20-34 years mothers, 133 (51.55%) being multigravida. Antepartum SB were 234 (89.31%), macerated 213 (81.30%), birth weight &lt;1000gm 86 (32.82%) and male 156 (59.54%). The intrauterine growth restriction (IUGR) was present in 60 (22.90%), unexplained casue in 43 (16.41%), prematurity 28 (10.69%), congenital anomalies 26 (9.92%), pre-eclampsia 19 (7.25%), gestational diabetes, and abruptio placenta each 13 (4.96%). Delay in seeking care in 202 (78.30%) was a potential contributing factor. &#x0D; Conclusions: The SB was 11.24/1000 births. The causes in descending order were IUGR, unexplained, prematurity, congenital anomalies, pre-eclampsia, gestational diabetes and abruptio placenta. Delay in seeking care was found as a potential contributing factor.

Alterations in placental long chain polyunsaturated fatty acid metabolism in human intrauterine growth restriction.

Fatty acids (FA) are critical for fetal brain development and are transferred across the placenta by membrane-bound FA transport proteins (FATP), translocases (FAT/CD36), and cytosolic binding proteins (FABP). The cytosolic protein perilipin-2 aids in neutral lipid storage within lipid droplets. Decreased placental nutrient transport is believed to contribute to intrauterine growth restriction (IUGR); however, IUGR placental lipid transport and metabolism are poorly understood. We hypothesized that protein expression of FATPs, FABPs, and perilipin-2 in human placenta is decreased and placental lipid content and incorporation into lipid classes are reduced in IUGR. Placental tissue of idiopathic IUGR (n=25) and gestational age-matched, appropriately grown for gestational age (AGA) fetuses (n=19) was collected. We determined protein expression of FABP4 and perilipin-2 in placental homogenate and FATPs (2, 4, 6, CD36) in syncytiotrophoblast microvillous plasma membrane (MVM) by Western blot. Lipid droplet area (Oil Red O stain) and cellular FA content (GC/MS) were measured in chorionic villous tissue. MVM expression of FATP6 and CD36 was significantly increased in IUGR. The concentrations of seven n-6 and n-3 species long chain polyunsaturated FAs (LCPUFA) were significantly increased in the triglyceride fraction in IUGR vs AGA placenta. In summary, MVM FATP6 and CD36 protein expression is increased and LCPUFA are preferentially routed toward cellular storage in TG in the IUGR placenta, possibly to protect against oxidative stress associated with cellular FA accumulation. We speculate that these changes may be caused by impaired efflux of FA across the fetal-facing syncytiotrophoblast basal plasma membrane in IUGR placenta.

CD15 as a marker of fetoplacental endothelial immaturity in IUGR placentas

Background: Structural or functional defects in the placenta, are the primary cause of growth restriction of the fetus. Morphological examination of such placentas from intrauterine growth restricted (IUGR) fetuses often appears deceptively normal. Evaluation of angiogenesis and fetoplacental vasculature is critical to understand the underlying pathogenesis of fetal growth restriction in both idiopathic as well as cases where it is thought to be secondary to complications like preeclampsia (PE). We analyzed the immaturity of fetoplacental vasculature using CD15, which is a stage specific embryonic antigen known to be expressed in immature endothelium.Material and methods: One hundred and twelve placentas (81 from IUGR and 31 from gestationally appropriate samples (appropriate for gestational age (AGA)) were collected based on stringent inclusion criteria, and subjected to detailed examination of morphology and microscopy along with immunostaining for CD15. IUGR placentas known to have villous immaturity such as those associated with gestational diabetes, Rh negative pregnancies and anemia were excluded. The time of clinical onset of IUGR, associated complications like PE and oligohydramnios along with clinical variables were recorded. CD15 expression was scored in both distal and proximal vasculature and the values in IUGR and AGA pregnancies were compared and correlated with clinical variables.Results: The mean CD 15 scores in both proximal vasculature (PV) as well as distal (DV) vasculature were significantly higher in the IUGR group compared to AGA (17.7 versus 5.16 in PV and 50.8 versus 23.7 in distal vasculature (DV)). Gestational age had no influence on CD15 staining in PV or DV in IUGR group, whereas preterm AGAs expressed higher CD15 only in the distal vessels. PE, oligohydramnios and the time of onset of IUGR did not influence the fetal vascular immaturity, as measured by CD15 scores. Although none of the clinical or obstetric factors influenced CD15 staining in AGA, fetal vessel immaturity in the IUGR group remained high even after adjusting for confounding variables like maternal age, gestational age and birth weight. Histological features suggestive of chronic hypoxia were significantly higher in IUGR placentas, compared to AGA and correlated positively with CD15 expression.Conclusion: Fetoplacental endothelium in both PV and DV is immature in IUGR irrespective of the gestational age or any other associated factors and CD15 immunodetection is a valuable marker for assessment of immaturity.

MicroRNA regulation of Transthyretin in trophoblast differentiation and Intra-Uterine Growth Restriction

Placental trophoblast cells produce various cytokines, transporters vital to normal embryogenesis. Transthyretin (TTR) aids trans-placental passage of maternal thyroxin (TH) to fetal circulation. Inadequate TH delivery leads to developmental abnormality. Regulation of TTR biosynthesis in placenta is critical for normal embryo development. We showed here that TTR transcripts were expressed more in fetal placenta. Using bioinformatic analysis and confirmation with dual-luciferase reporter assays, we found that miR-200a-3p and miR-141-3p inhibited TTR expression by directly binding to the 3′UTR of TTR, which is reversed by mutation in the microRNA binding site. Differentiation of human trophoblast BeWo cells was associated with decreased TTR transcript and protein levels with concomitant increase in the levels of both microRNAs. Interestingly, ectopic overexpression of the microRNA mimics abrogated thyroxin uptake by BeWo cells, which was reversed by the corresponding inhibitors. Furthermore, in a rat model of intra-uterine growth restriction (IUGR), TTR expression decreased significantly in placenta with reciprocal rise in miR-141-3p but not 200a-3p. In human IUGR placenta, TTR transcript and protein levels were significantly lower associated with high expression of miR-141-3p but not 200a-3p. These data provides new insight into physiological role of miR-141-3p in regulating TTR during trophoblast differentiation and IUGR.

Genetic and epigenetic changes in the placenta in intrauterine growth restriction (IUGR)

Genetic and epigenetic changes in the placenta in intrauterine growth restriction (IUGR)

Fatty acid profile of maternal and fetal erythrocytes and placental expression of fatty acid transport proteins in normal and intrauterine growth restriction pregnancies

Long-chain polyunsaturated fatty acids (LC-PUFA), mainly docosahexaenoic (DHA) and arachidonic acids (AA), are critical for adequate fetal growth and development. We investigated mRNA expression of proteins involved in hydrolysis, uptake and/or transport of fatty acids in placenta of fifteen full term normal pregnancies and eleven pregnancies complicated by intrauterine growth restriction (IUGR) with normal umbilical blood flows. The mRNA expression of LPL, FATPs (−1, −2 and −4) and FABPs (−1 and −3) was increased in IUGR placentas, however, tissue profile of LC-PUFA was not different between groups. Erythrocytes from both mothers and fetuses of the IUGR group showed lower concentrations of AA and DHA and inferior DHA/ALA ratio compared to normal pregnancies (P < 0.05). We hypothesize that reduced circulating levels of AA and DHA could up-regulate mRNA expression of placental fatty acids transporters, as a compensatory mechanism, however this failed to sustain normal LC-PUFA supply to the fetus in IUGR.

Dexamethasone-Induced Intrauterine Growth Restriction Is Associated With Altered Expressions of Metastasis Tumor Antigens and Cell Cycle Control Proteins in Rat Placentas.

Molecular mechanisms affecting placental formation in intrauterine growth-restricted (IUGR) pregnancies are not clearly understood. Since metastasis tumor antigens (MTAs) MTA1 and MTA2 promote cell proliferation and MTA3 suppresses it, we hypothesized that IUGR alters cell survival/cell death programs driven by placental MTAs. To induce IUGR, pregnant Sprague Dawley rats were given daily intraperitoneal injections of either saline or dexamethasone (0.4 mg/kg) starting from 14 days of gestation (dg) to either 19 dg or 21 dg. Gene and protein expressions of MTA1-3 in the placental basal and labyrinth zones were investigated by real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We also explored the expressions of proliferating cell nuclear antigen (PCNA), caspase-3, p53, p21, and β-catenin. Dexamethasone-induced IUGR resulted in decreased expression of MTA1 in the nuclei of cells in the basal zone. The expression of p21 was increased and that of PCNA was reduced in both placental zones of IUGR rats. Cytoplasmic expression of MTA1 and p53 increased in the labyrinth zone of IUGR placentas in association with an increase in cell death as indicated by an increased caspase-3 expression. The labyrinth zone of IUGR placentas showed a significant reduction in MTA2-MTA3 gene expression and an increase in p53 protein levels. Total MTA3 level increased and β-catenin level decreased in the labyrinth zone of IUGR placentas associated with a reduction in cell proliferation. Taken together, these results strongly suggest that dexamethasone-induced IUGR is associated with changes in MTA expression, decreased cell proliferation, and increased cell death in placentas.

Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency.

Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2-/- ) were generated. Emp2-/- females are fertile but have reduced litter sizes when carrying Emp2-/- but not Emp2+/- fetuses. Placentas of Emp2-/- fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2-/- fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans, and suggest that it may be a new biomarker for placental insufficiency. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Differential microRNA expression in human placentas of term intra-uterine growth restriction that regulates target genes mediating angiogenesis and amino acid transport.

Placental insufficiency leading to intrauterine growth restriction (IUGR) demonstrates perturbed gene expression affecting placental angiogenesis and nutrient transfer from mother to fetus. To understand the post-transcriptional mechanisms underlying such placental gene expression changes, our objective was to identify key non-coding microRNAs that express biological function. To this end, we initially undertook microarrays targeting microRNAs in a small sub-set of placentas of appropriate (AGA) versus small for gestational age (SGA) weight infants, and observed up-regulation of 97 miRs and down-regulation of 44 miRs in SGA versus AGA. In a larger cohort of samples (AGA, n = 21; SGA, n = 11; IUGR subset, n = 5), we validated by qRT-PCR differential expression of three specific microRNAs (miR-10b, -363 and -149) that target genes mediating angiogenesis and nutrient transfer. Validation yielded an increase in miR-10b and -363 expression of ~2.5-fold (p<0.02 each) in SGA versus AGA, and of ~3-fold (p<0.005) in IUGR versus AGA, with no significant change despite a trending increase in miR-149. To further establish a cause-and-effect paradigm, employing human HTR8 trophoblast cells, we assessed the effect of nutrient deprivation on miR expression and inhibition of endogenous miRs on target gene expression. In-vitro nutrient deprivation (~50%) increased the expression of miR-10b and miR-149 by 1.5-fold (p<0.02) while decreasing miR-363 (p<0.0001). Inhibition of endogenous miRs employing antisense sequences against miR-10b, -363 and -149 revealed an increase respectively in the expression of the target genes KLF-4 (transcription factor which regulates angiogenesis), SNAT1 and 2 (sodium coupled neutral amino acid transporters) and LAT2 (leucine amino acid transporter), which translated into a similar change in the corresponding proteins. Finally to establish functional significance we performed dual-luciferase reporter assays with 3’-insertion of miR-10b alone and observed a ~10% reduction in the 5’-luciferase activity versus the control. Lastly, we further validated by microarray and employing MirWalk software that the pathways and target genes identified by differentially expressed miRs in SGA/IUGR compared to AGA are consistent in a larger cohort. We have established the biological significance of various miRs that target common transcripts mediating pathways of importance, which are perturbed in the human IUGR placenta.

Assessment of the Placenta in Intrauterine Growth Restriction by Diffusion-Weighted Imaging and Proton Magnetic Resonance Spectroscopy.

Placental insufficiency is a major cause of intrauterine growth restriction (IUGR) and seriously affects fetal development. When placental insufficiency happened, the diffusion of water molecule was restricted and the metabolic balance was destroyed in the placenta. In this prospective study, we aimed to evaluate the diagnostic value of diffusion-weighted imaging (DWI) in combination with proton magnetic resonance spectroscopy (1H MRS) for placental insufficiency in IUGR. The apparent diffusion coefficient (ADC) values were calculated using DWI, and the metabolism of N-acetylaspartate (NAA), choline, and lipid and their ratios in the placenta were calculated using 1H MRS. Data were statistically analyzed by receiver operating characteristic (ROC) curves and logistic regression analysis. The NAA and choline peaks were decreased in IUGR placentas compared with normal placentas, while lipid peaks showed an opposite trend. The average ADC value and the NAA/lipid and choline/lipid ratios were lower in the IUGR group than in the normal group ( P < .01). Logistic regression with multiple parameters showed that combination of the ADC value and choline/lipid ratio was more favorable than either parameter alone in analyzing placental insufficiency of IUGR ( P < .05). The area under the ROC curve for the combination was 0.939, indicating reasonably good discrimination. We concluded that reduced ADC and NAA/lipid and choline/lipid ratios could serve as potential markers for placental insufficiency of IUGR. Furthermore, the combination of ADC value and choline/lipid ratio greatly improved the diagnostic value.

P45 NF-E2 regulates syncytiotrophoblast differentiation by post-translational GCM1 modifications in human intrauterine growth restriction

Placental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.