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Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth restriction in mice

Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth... Background In utero exposure to obesity is consistently associated with increased risk of metabolic disease, obesity and cardiovascular dysfunction in later life despite the divergence of birth weight outcomes. The placenta plays a critical role in offspring development and long-term health, as it mediates the crosstalk between the maternal and fetal environments. However, its phenotypic and molecular modifications in the context of maternal obesity associated with fetal growth restriction (FGR) remain poorly understood. Methods Using a mouse model of maternal diet-induced obesity, we investigated changes in the placental transcriptome through RNA sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA) at embryonic day (E) 19. The most differ- entially expressed genes (FDR < 0.05) were validated by Quantitative real-time PCR (qPCR) in male and female placentae at E19. The expression of these targets and related genes was also determined by qPCR at E13 to examine whether the observed alterations had an earlier onset at mid-gestation. Structural analyses were performed using immunofluorescent staining against Ki67 and CD31 to investigate phenotypic outcomes at both timepoints. Results RNA-seq and IPA analyses revealed differential expression of transcripts and pathway interactions related to placental vascular development and tissue morphology in obese placentae at term, including downregulation of Muc15, Cnn1, and Acta2. Pdgfb, which is implicated in labyrinthine layer development, was downregulated in obese placentae at E13. This was consistent with the morphological evidence of reduced labyrinth zone (LZ) size, as well as lower fetal weight at both timepoints irrespective of offspring sex. Conclusions Maternal obesity results in abnormal placental LZ development and impaired vascularization, which may mediate the observed FGR through reduced transfer of nutrients across the placenta. Introduction The prevalence of obesity has nearly tripled since 1975 [1]. As a consequence, the number of women who are classified These authors contributed equally: Daniela de Barros Mucci, Laura C. Kusinski as overweight or obese during pregnancy has risen sub- stantially, estimated at 38.9 million in 2014 [2]. This is Supplementary information The online version of this article (https:// especially concerning as offspring born to obese mothers doi.org/10.1038/s41366-020-0561-3) contains supplementary material, which is available to authorized users. are more likely to have poor neonatal outcomes [3] and to * Daniela de Barros Mucci Nutritional Biochemistry Laboratory, Institute of Nutrition Josué danimucci@gmail.com de Castro, Federal University of Rio de Janeiro, Rio de Janeiro, * Laura C. Kusinski Brazil lck34@medschl.cam.ac.uk Nutritional Epidemiology Observatory, Institute of Nutrition Josué de Castro, Federal University of Rio de Janeiro, Rio de Janeiro, Metabolic Research Laboratories and MRC Metabolic Diseases Brazil Unit, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, UK 1234567890();,: 1234567890();,: 1088 D. de Barros Mucci et al. develop obesity, insulin resistance, hypertension and dys- and were carried under the Home Office Animals (Scientific lipidemia later in life [4]. Interestingly, maternal obesity Procedures) Act 1986. The model has been described in leads to divergent birth weight outcomes; while it is often detail previously [20, 22]. Briefly, female C57BL/6J mice, shown to increase the risk of macrosomia [5, 6], a higher proven breeders, were randomly assigned either a standard incidence of low birth weight is also documented [6–8]. chow RM1 diet [7% simple sugars, 3% fat (wt/wt), Although both are similarly associated with metabolic dis- 10.74 kJ/g] or an energy-rich highly palatable obesogenic ease in later life, distinct placental alterations seem to diet [10% simple sugars, 20% animal lard (wt/wt), 28.43 kJ/g] mediate these contrasting offspring phenotypes [9]. supplemented with sweetened condensed milk [55% simple Changes in placental function are thought to be pivotal in sugar, 8% fat (wt/wt); Nestle, Croydon, UK], and fortified the development of pregnancy complications [10, 11] and with mineral and vitamin mix AIN93G. Both diets were fed could also be a key link between the maternal and intrau- ad libitum and purchased from Special Dietary Services terine milieu and long-term health of the offspring [12, 13]. (Witham, UK). Body composition was monitored (TD- Alterations in placental function and structure in response NMR, Bruker Minispec) and females were set up to breed if to obesity and their underlying molecular mechanisms body fat was between the thresholds of 10–12% or 35–40% have been explored both in humans and in animal models for Control and Obese dams, respectively. After mating for [9, 14–17]. Yet, even though fetal growth restriction (FGR) the second time with RM1 fed males, dams were killed at is recognized as a placenta-related disorder [18], the impact either E13 or E19 by rising CO concentration. Fetal and of maternal obesity on the placental transcriptome in this placental weights were recorded. Placentae for molecular context remains largely unknown. analysis were immediately snap frozen on dry ice and stored It has been shown that placentae from high fat diet-fed at −80 °C. For morphological assessment, samples were obese mouse dams exhibit altered expression of epigenetic fixed in 10% formalin for 48 h, stored in 70% ethanol and machinery genes at term, which could alter the placental then embedded in wax. epigenome and lead to FGR [19]. High fat diet-induced The sex of the fetuses at E19 was determined by visual obesity has also been found to alter the transcriptome of inspection of anogenital anatomy. At E13, DNA extracted placenta progenitor cells at early stages of development and from tail tips was used for PCR sexing as described by is associated with later changes in placental function McFarlane et al. [23], using the SX primer pair. Amplicons resulting in FGR [17]. In our mouse model of maternal diet- were loaded on 2% agarose gels and submitted to electro- induced obesity, in which dams are fed a hypercaloric phoresis together with a 1 kb DNA ladder. Bands were Western-like diet, we have shown that maternal hyper- visualized with SYBR™ Safe DNA gel stain (Thermo insulinemia is strongly associated with offspring insulin Fisher Scientific, Rochford, UK) under UV-illumination resistance and excess placental lipid deposition and hypoxia and the genomic sex of each sample was determined [20]. However, a clear understanding of the molecular according to the number of bands and amplicon size. mechanisms behind these findings is still lacking and war- rants further investigation. RNA extraction It is recognized that the impact of stressors on placental function and offspring health is closely linked to the stage Placenta aliquots were homogenized in 700-µL Qiazol of tissue development, the type of insult and the sex of the using TissueRuptor (Qiagen, Manchester, UK). Total RNA conceptus [21]. Thus, the aim of this study was to identify was isolated with miRNeasy Mini Kit (Qiagen) according to global changes in the placental transcriptome and related the manufacturer’s instructions and including the optional pathways in response to maternal obesity near term at step of DNA digestion with RNase-Free DNAse Set (Qia- embryonic day (E) 19. Furthermore, we investigated whe- gen). Extracted RNA was quantified by spectrophotometry ther the significant transcriptional alterations detected in (Nanodrop Thermo Fisher Scientific) and stored at obese placentae were manifested earlier, i.e., in mid- −80 °C. gestation (E13), and if these alterations translated into a structural phenotype in male and female placentae. RNA sequencing and Ingenuity® Pathway Analysis Total RNA was extracted from E19 male placentae (Control Methods n = 2 and Obese n = 3), as previously outlined. One microgram of total RNA was depleted of ribosomal RNA Animals and diets and PolyA tails of coding RNAs were captured by treatment with Oligo-dT beads. Complementary DNA (cDNA) All experimental protocols were approved by the University libraries were generated after an amplification step, of Cambridge Animal Welfare and Ethical Review Board according to the TruSeq Stranded Total RNA Sample Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1089 Preparation Guide (Illumina, San Diego, CA, USA), and staining. Antigen retrieval was performed with pH9 Target quantified using KAPA Library Quantification Kit. Multi- Retrieval buffer [S2375 (Dako Agilent, Stockport, UK)] and plex single-read sequencing was performed using Illumina nonspecific binding sites were blocked with 3% goat serum. HiSeq 2500 (Illumina). Sequence reads were demultiplexed Sections were incubated overnight at 4 °C with rabbit using the CASAVA pipeline (Illumina) and then aligned to polyclonal primary antibodies against Ki67 [1:200; the Mus musculus genome (GRCm38) using TopHat ver- ab15580 (Abcam, Cambridge, UK)] or CD31 [1:500; sion 2.0.11. Raw read counts and fragments per kilobase of ab124432 (Abcam)]. transcript per million mapped reads (FPKM) were generated Primary antibody binding was visualized by incubation using Cufflinks version 2.2.1. A quality check of mapped at room temperature for 1 h with a fluorescent-conjugated reads was executed using the R package CummeRbund. goat polyclonal anti-rabbit IgG [1:1000; A11008 (Invitro- Databases were trimmed for exclusion of very low detection gen, Warrington, UK)]. In negative control slides, placental or undetectable genes. The resulting data were analyzed sections were incubated in 1.5% goat serum in TBS-T using edgeR by calculating the likelihood ratio, and by instead of primary antibodies (Supplementary Fig. S1). All adjusting P values via Benjamini and Hochberg’s method to sections were incubated with 1:2500 DAPI for 10 min at control the false discovery rate (FDR). Ingenuity Pathway room temperature to stain nuclei. Autofluorescence was Analysis (IPA) was applied to identify biological pathways quenched by incubation with Vector TrueVIEW Auto- related to the genes that were differentially expressed fluorescence Quenching Kit [SP8400 (Vector Laboratories, between Control and Obese E19 male placentae. The pla- Peterborough, UK)]. centa RNA sequencing (RNA-seq) data have been depos- Placental sections were imaged on an AxioScan Slide ited in GEO database under the accession number Scanner (Zeiss, Cambridge, UK) and blindly analyzed using GSE140013. HALO analysis software (Indica Labs, Corrales, NM, USA). The DAPI channel image was used to define nuclear cDNA synthesis and Quantitative real-time PCR outlines using the CytoNuclear FL module. For the analysis (qPCR) of Ki67 staining (Supplementary Fig. S2), nuclear outlines were transposed onto the 488-nm channel image and the Total RNA was extracted from male and female placentae proportion of nuclei positive for Ki67 across the whole of Control and Obese dams at E13 (n = 10/group from section was recorded. Placental sections stained for the separate litters) and E19 (n = 9/group from separate litters). endothelial cell marker CD31 were used to investigate All samples used in the validations were different to those labyrinth zone (LZ) size and structure (Supplementary Fig. used in the RNA-seq and therefore represent biological S3). The border of the LZ was outlined manually at ×40 replicates. Sample size was based on previous data sets/ magnification (depicted in yellow) and total area was power calculations. cDNA was generated from 1-μg RNA recorded using HALO analysis software after canals were using High Capacity cDNA Reverse Transcription Kit excluded. The boundary with the junctional zone was (Applied Biosystems, Foster City, CA, USA). qPCR was determined as the interface between the phenotypically performed on QuantStudio 7 Flex Real-Time PCR System distinct spongiotrophoblast of the junctional zone and the (Applied Biosystems), using 200 nM specific primers fetal capillaries of the LZ. The rest of the boundary was (Sigma-Aldrich, Gillingham, UK), 1× SYBR® Green either at the edge of the tissue image or at the interface with JumpStart™ Taq ReadyMix (Sigma-Aldrich) and cDNA the chorionic plate, which is structurally distinct from the samples at a final dilution of 1:60. For primer sequences see LZ, characterized by smaller nuclei and the absence of Supplementary Table S1. NormFinder software was used to CD31-positive endothelial cells. Indica Labs’ Tissue Clas- select the best combination of two out of four reference sifier module was used to differentiate between fetal blood genes [24]. qPCR results were normalized to the geometric vessels (lumen bound by CD31-positive endothelium) and mean of the reference genes Gapdh and Sdha for E19 other tissue of the LZ. placentae, and Gapdh and Pmm1 for E13 placentae, expression of which did not change between groups. Data Statistical analyses were expressed in arbitrary units relative to Male Control −ΔΔCq average (2 ). Benjamini–Hochberg multiple testing correction [25] was applied to the RNA-seq differential expression data and Structural analyses only genes with FDR < 0.05 were considered significantly different between the two experimental conditions. For E13 and E19 formalin-fixed placentae from males and qPCR validation of RNA-seq differentially expressed females were cut into 5-µm sections. Three serial sections genes, comparisons were made between Control and Obese close to the midline of each placenta were selected for placentae of the same sex, by Student’s t test. 1090 D. de Barros Mucci et al. Table 1 Fetal and placental Control Obese P value weights at E13 and E19. Males Females Males Females Maternal Sex obesity E13 Fetal weight (g) 0.17 ± 0.01 0.16 ± 0.00 0.15 ± 0.01 0.14 ± 0.01 0.003 0.187 Placental 94.0 ± 2.2 88.9 ± 3.5 89.7 ± 2.1 78.7 ± 2.9 0.015 0.008 weight (mg) Fetal:placental ratio 1.84 ± 0.06 1.85 ± 0.06 1.66 ± 0.08 1.76 ± 0.08 0.079 0.452 E19 Fetal weight (g) 1.23 ± 0.03 1.17 ± 0.03 1.02 ± 0.03 1.03 ± 0.02 <0.0001 0.339 Placental 93.8 ± 5.1 81.7 ± 5.3 82.8 ± 1.9 73.1 ± 2.7 0.039 0.021 weight (mg) Fetal:placental ratio 13.45 ± 0.76 14.77 ± 0.92 12.38 ± 0.37 14.28 ± 0.78 0.331 0.051 Values are mean ± SEM. P values < 0.05 indicated in bold show significant effect of maternal obesity and sex differences in the studied parameters according to two-way ANOVA followed by Tukey’s multiple comparisons test, using each litter’s average as a single data (Control Male n = 6 and 9, Control Female n = 6 and 9, Obese Male n = 7 and 7, Obese Female n = 7 and 6, respectively, at E13 and E19). Anthropometric parameters and qPCR of selected targets males at both stages of gestation. There was no significant were analyzed by two-way analysis of variance (ANOVA) difference in the ratio of fetal to placental weight, although followed by Tukey’s multiple comparisons test to estimate there was a trend (P = 0.05) toward higher placental effi- the effects of maternal obesity and fetal sex at each ciency in females at E19 (Table 1). time point. For the morphological analyses, the effects of gestational RNA-seq, Ingenuity® Pathway Analysis and qPCR at age (E13 or E19), offspring sex (male or female), and term placentae maternal diet (regular chow or obesogenic diet) on placental phenotype were investigated by three-way ANOVA, and The RNA-seq at E19 detected a total of 350 transcripts backwards stepwise elimination was used to come to a differentially expressed in placentae of Obese compared minimal model. Three-way ANOVA of the proportional to Control males considering a significance threshold of area of the LZ that was fetal capillaries data revealed no P < 0.05 (Fig. 1a, Supplementary Table S2). However, only significant effect of gestational age, maternal diet, or fetal nine genes remained significantly altered after correction for sex. However, there was a borderline significant interaction multiple testing (FDR < 0.05) (Fig. 1a, b). Ingenuity® between maternal diet and fetal sex (P = 0.055). In order to Pathway Analysis (IPA) was used with a less stringent identify if a maternal diet effect was only present in one sex, threshold (P < 0.05) to identify global changes in pathways these data were separated and sex-specific two-way ANO- and biological functions promoted by maternal obesity. The VAs (gestational age/maternal diet) were performed. most significant diseases and bio functions are shown in Only one sample from each litter was used for each Fig. 1c. analysis, except in the case of offspring and placental Genes identified as significantly changed in response to weights, in which each litter’s average was used as a single obesity by RNA-seq (FDR < 0.05) were validated in a larger data point. Data are presented as mean ± standard error of number of samples, all from independent litters, by qPCR the mean (SEM), and the threshold for significance was set (Fig. 2) and results were confirmed in eight out of the nine at P < 0.05, unless stated otherwise. Statistical analyses genes in E19 male placentae (i.e., Pi15, Gabrd, Sez6l, were performed using R (R Core Team 2017) or Prism 6 Nup210, Acta2, Rnf222, Muc15, and Cnn1). These genes (GraphPad Prism, La Jolla, CA, USA). were also examined in female placentae, however, only Pi15, Nup210, Acta2, Rnf222, and Muc15 were sig- nificantly modulated by obesity (Fig. 2). Results Placental gene expression at different gestational Fetal and placental measurements ages Fetal and placental weights were reduced in response to All nine genes found differentially expressed in obese obesity and female placentae were smaller than those of male placentae at E19 were then investigated in E13 Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1091 Fig. 1 RNA-seq identification of differentially expressed genes between Control (C, n = 2) and Obese (O, n = 3) male mouse placentae at E19. a Volcano plot representing all detected transcripts, distributed according to −log10 P value in the y-axis and log2 fold change in the x-axis, with downregulated genes shifted to the left (P < 0.05 in blue) and upregulated genes shifted to the right (P < 0.05 in red). Significantly altered genes after correction for multiple testing (FDR < 0.05) are depicted with a pink diamond. b Heatmap representation of genes significantly regulated by maternal obesity using a cutoff FDR < 0.05, with scaled Z-score color key of normalized counts showing expression levels ranging from blue (lower) to red (higher). Genes are sorted from lowest to highest log2 fold change value.c Top diseases and bio functions from Ingenuity® Pathway Analysis (IPA) of the RNA-seq data with a threshold of P < 0.05, showing the most significant molecular and cellular functions dysregulated in the placenta by maternal obesity, sorted by P value. placentae (Fig. 3a). Pi15, Nup210,and Sez6l were upre- at mid-gestation. Both Hand1 and Pdgfb were down- gulated by maternal obesity in both sexes at mid-gestion. regulated by maternal obesity in both sexes, but no differ- Gabrd mRNA levels, which were upregulated in Obese ences were observed in Prl2c2 (Fig. 3b). male placentae at E19, was downregulated by maternal obesity at E13. Muc15, Nup210,and Acta2 expression was Immunofluorescent staining of placental higher in females compared to males at E13. Rnf222 and morphology Cnn1 were not differentially expressed in either sex at E13, however, their transcript levels were very low com- Phenotypic analyses were also conducted by immuno- pared to E19. fluorescent staining of targets within pathways identified by Since genes involved in spiral artery remodeling (Muc15 IPA. The marker Ki67 was used to investigate cellular and Cnn1) and labyrinthine pericytes (Acta2) that were growth and proliferation. Cellular movement, assembly, and found to be dysregulated in obese placentae at E19 were not organization were assessed through analyses of LZ size and affected at E13, we additionally measured the expression of fetal vasculature structure, using CD31 as a marker of fetal candidate genes recognized as relevant for these processes endothelial cells. 1092 D. de Barros Mucci et al. analyzed by the proportion of the total LZ area that was fetal blood vessels. A reduced model considering males and females separately by two-way ANOVA (gestational age/ maternal condition) detected a reduction in area bound by fetal capillaries within the LZ in female placentae of obese dams (P < 0.05, Fig. 4n–p). Representative images of male placentae are shown in Supplementary Fig. S4. Discussion The RNA-seq analysis revealed a total of 350 transcripts differentially expressed in Obese male placentae at term. Fig. 2 Validation of RNA-seq data by qPCR in E19 male and Among the top downregulated transcripts, Muc15, Cnn1 female placentae. qPCR results were normalized to the reference and Acta2 were of particular significance as these genes are genes Gapdh and Sdha and are expressed as mean ± SEM in arbitrary required for appropriate development of placental vascu- units relative to Male Controls. *P < 0.05, determined by Student’s t test comparing qPCR data of same sex Control vs Obese, n = 9/ lature and related to key pathways identified by IPA such as group. cellular movement, assembly and organization. Previous studies have shown that Muc15 suppresses the migration/ invasion of trophoblast like-cells in vitro, a process impli- cated in blood vessel remodeling in the maternal–fetal interface [26]. Cnn1 is largely expressed by smooth muscle cells [27] which line the uterine blood vessels and are lost in the normal remodeling of maternal spiral arteries during placental development [28]. Acta2 is a marker of pericytes which surround fetal endothelial cells during blood vessel development in the mouse LZ [29]. These data together suggest that exposure to maternal obesity affects the remodeling of maternal spiral arteries and the development of fetal blood vessels within the LZ, both of which are crucial for adequate nutrient and oxygen transfer across the placenta [28] and possibly linked to the uteroplacental hemodynamic alterations present in pre- eclampsia and intrauterine growth restriction [30–32]. Fig. 3 qPCR expression in E13 male and female placentae. Obese women are two to three times more likely to develop a Validated RNA-seq genes.b Hand1, required for trophoblast giant preeclampsia [33] and hypertensive obstetric complications cell (TGC) differentiation; Prl2c2, a marker of spiral artery-associated TGC and canal-associated TGC; Pdgfb, a growth factor that regulates are generally associated with small-for-gestational age placental labyrinthine layer development. qPCR data were normalized neonates [34]. Here, we see significant growth restriction in to the reference genes Gapdh and Pmm1. Results are shown as mean ± the fetus which may result from poor uteroplacental perfu- SEM in arbitrary units relative to Male Control average expression. sion in addition to placental hypoxia previously suggested *Denotes maternal obesity effect (P < 0.05) and # denotes sex differ- ence (P < 0.05), according to two-way ANOVA, n = 10/group. in this model [20]. ªRnf222 and Cnn1 expression levels were low at E13 placentae, with Furthermore, the RNA-seq analysis identified several average Cq values above 31 and 29, respectively. genes that have not been functionally described in placental tissue thus far, but are conserved in humans and rodents. E19 placentae had fewer cells (P < 0.05, Fig. 4a–c) and Pi15 encodes a peptidase inhibitor that may regulate a lower proportion of proliferating cells across the whole extracellular matrix modifications [35] and has been placenta (P < 0.01, Fig. 4d–h) compared to E13. These implicated in vascular defects in rat aorta [36], though its parameters were not affected by offspring sex or maternal role in placental vascularization is unknown. Gabrd gene diet. encodes the delta subunit of gamma-aminobutyric acid type The size of the LZ was significantly reduced in placentae A receptor (GABA ). GABA activation impacts stromal A A from obese dams (P < 0.01, Fig, 4i–m). There was a sig- cell proliferation and apoptosis during decidualization [37] nificant increase in LZ size from mid-gestation to term and increased expression of its pi subunit (GABRP) has (P < 0.01, Fig. 4i). Labyrinthine vascular organization was been detected in preeclamptic placentae [38]. NUP210 is a Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1093 E13 E19 DAPI DAPI 1 mm 1 mm DAPI/Ki67 Ki67 Ki67 DAPI/Ki67 500 μm 500 μm 500 μm 500 μm DAPI/CD31 DAPI/CD31 1 mm 1 mm DAPI/CD31 DAPI/CD31 1 mm 1 mm Control Female Obese Female CD31 CD31 100 μm 100 μm Fig. 4 Immunofluorescent staining of targets related to the top maternal obesity (C n = 10, Ob n = 9, at each time point). n–p The three molecular and cellular functions shown in IPA. All analyses proportion of fetal capillaries within the labyrinth zone was decreased were conducted in both male and female placentae of mothers fed by maternal obesogenic diet in females (C n = 10, Ob n = 9). a, d, i, n either regular chow (C, Control group) or obesogenic diet (Ob, Obese Results are shown as mean ± SEM. Gestational age differences are group), at E13 and E19. a–c The total number of cells in the placenta denoted by *P < 0.05, **P < 0.001, or ***P < 0.0001, and maternal decreased between E13 (n = 20) and E19 (n = 19). d–h The propor- obesity effect is indicated by P < 0.001, according to three-way tion of Ki67-positive cells across the whole placenta decreased ANOVA. §Denotes maternal obesity effect (P < 0.05), determined by between E13 (n = 20) and E19 (n = 19). i–m The size of the labyrinth two-way ANOVA analysis of E13 and E19 female placentae only. zone increased between E13 and E19, and was reduced in response to major component of the nuclear pore complex and is protein-coding gene; however, no functional description has required for regulation of gene expression during differ- been found. entiation and cell fate determination, as demonstrated in Although information on these genes in placentae is myoblasts and embryonic stem cells [39]. Although the currently limited, it must be noted that a large number of function of SEZ6L is not well understood, it has been placental genes and related phenotypes remain unchar- shown both in mice and in vitro that this protein is almost acterized. Recent efforts to systematically identify the genes exclusively processed by β-site APP cleaving enzyme required for normal embryogenesis are still unravelling (BACE) [40]; BACE1 and BACE2 are abundantly expres- many previously underappreciated placental defects [42]. sed in human placentae and are upregulated in pregnancies Thus, our findings might represent novel targets that could complicated with preeclampsia [41]. Lastly, Rnf222 is also a be implicated in the pathophysiology of maternal obesity Ob C 1094 D. de Barros Mucci et al. and associated adverse outcomes in the offspring. More- Next, we used IPA to investigate the mechanism behind over, additional genes might have been identified if a larger this reduction in LZ area. Cellular growth and proliferation sample size was used in the RNA-seq analysis. were pointed out as the main molecular and cellular func- When comparing both timepoints, most transcripts tions affected by obesity. Surprisingly, however, maternal exhibited a different expression pattern, including Muc15, obesity had no effect on the number of Ki67-positive cells Cnn1, and Acta2 which were not affected by obesity at E13. in the placenta. Abnormalities in placental size are often This could be due to low functional relevance of these associated with disruption of cellular growth and/or apop- transcripts at mid-pregnancy rather than absence of altera- tosis [47–49]. Thus, it is possible that other mechanisms tions in related cellular processes, as illustrated by low such as cell death could explain our results, although lim- Cnn1 mRNA levels in our analysis at E13 compared to E19 itations to our analysis, which was not zone-specific, cannot (data not shown). In fact, the mouse placenta undergoes a be discounted. In this regard, it has been shown in a mouse transcriptome transition from the “development phase” of model of high fat diet-induced obesity through phospho- organogenesis to the “mature phase” at mid-pregnancy [43]. histone H3 staining that the proliferating cells in placenta Thus, we next used the IPA data to identify genes which are mostly restricted to the labyrinthine layer and appear were previously shown to be both highly expressed at mid- reduced in response to obesity within this region [50]. pregnancy and pivotal to spiral artery remodeling and for- Despite these morphological disturbances, changes in mation of fetal blood vessels in the LZ. Hand1, which is tissue structure that are expected to occur from mid- required for trophoblast giant cell (TGC) differentiation pregnancy until term seemed preserved in obese placentae. [44], was downregulated in placentae of obese dams. The LZ is well-reported to expand as pregnancy progresses, However, maternal obesity had no effect on the expression so that the transport capacity of the placenta can meet the of Prl2c2, a marker of TGC that line maternal blood canal nutrient demands of the growing fetus [50–52]. Accord- spaces and spiral arteries in the definitive placenta [45]. ingly, we found that labyrinth area increased between E13 Considering the complexity of spiral artery remodeling and and E19, irrespective of maternal diet. the limited number of transcripts that were analyzed here, it We also observed significant differences in placentae remains to be established whether the alterations occur only which are specific to fetal sex. Female placentae were later in development or if other mechanisms are involved. smaller than male counterparts at all timepoints and On the other hand, the growth factor Pdgfb was down- maternal conditions, which is consistent with observations regulated in response to obesity at E13 and could be a from both human cohorts [53] and studies in mice [19, 54]. relevant link to other molecular and phenotypic observa- Moreover, we found sex differences in a subset of genes, tions in our model. It has been shown that Pdgfb-deficient with females exhibiting slightly increased expression. placentae exhibit defective labyrinthine development, with Similarly, global transcriptomic analysis in normal full-term alterations in fetal blood vessel structure and reduced human placentae revealed higher overall mRNA levels in numbers of pericytes from mid-pregnancy until term, females compared to males [55]. Sexual dimorphism in the leading to growth restriction in PDGFB −/− embryos [29]. context of developmental programming is increasingly Here, lower expression of the pericyte marker Acta2 was commonly reported [56]. How these relate to sex-specific detected by RNA-seq and confirmed by qPCR in obese responses of the placenta to a suboptimal environment placentae at E19. In addition, defects in LZ morphology and remain to be determined. FGR were observed in response to maternal obesity at E13 Overall, we have shown through genome-wide analysis and persisted until E19. that maternal obesity induces a dysregulation of transcripts As shown by our immunofluorescence staining, male and and pathway interactions related to placental vasculature female placentae from Obese dams exhibited reduced LZ and structure. FGR, as well as changes in placental mor- area, which is the primary site of gas, nutrient and waste phology and a gene expression signature associated with exchange between the maternal and fetal circulations in the impaired labyrinthine development, were detectable at mid- mouse [28], and a decrease in the proportion of fetal blood pregnancy, suggesting an enduring negative effect of vessels within the LZ was also evident in females. This is maternal obesity over these processes. The LZ is the further corroborated by recent evidence of lower vascularity exchange region of the murine placenta and reductions in its in placentae of high fat diet-fed dams both at mid- size and vasculature may impair the transport of nutrients pregnancy and near term, which was associated with pla- from the maternal circulation to the developing fetus, thus cental transcriptome alterations in early stages of develop- restricting its growth. Disruption of placental structure ment and FGR [17]. In addition, it has been suggested that could thus represent an important factor contributing to the defects in placental villi vasculature seen in obese human development of FGR in pregnancies complicated by pregnancies could be partly due to obesity-associated tissue maternal obesity. Moreover, novel targets were revealed by hypoxia [46], which is also consistent with our model [20]. our RNA-seq analysis. Characterizing their functional roles Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1095 in the placenta will help us better understand the processes 7. Radulescu L, Munteanu O, Popa F, Cirstoiu M. The implications and consequences of maternal obesity on fetal intrauterine growth mediating the effects of maternal obesity on offspring out- restriction. J Med Life. 2013;6:292–8. comes and potentially inform suitable interventions. 8. Rajasingam D, Seed PT, Briley AL, Shennan AH, Poston L. A prospective study of pregnancy outcome and biomarkers of oxi- Acknowledgements The authors would like to thank Ania Wilczynska dative stress in nulliparous obese women. Am J Obstet Gynecol. and Martin Bushell from the University of Leicester for their helpful 2009;200:395 e391–399. analysis on the RNA-seq data and Claire Custance for technical 9. Howell KR, Powell TL. Effects of maternal obesity on placental assistance. This work was supported by the Biotechnology and Bio- function and fetal development. Reproduction. 2017;153:R97–R108. logical Sciences Research Council (BBSRC—BB/M001636/1) and an 10. Flenady V, Koopmans L, Middleton P, Froen JF, Smith GC, Gibbons MRC Metabolic Diseases Unit award (MC_UU_12012/4). We also K, et al. Major risk factors for stillbirth in high-income countries: a thank BHF, Wellcome Trust, FAPERJ, and CNPq for the financial systematic review and meta-analysis. Lancet. 2011;377:1331–40. support. 11. Moran MC, Mulcahy C, Zombori G, Ryan J, Downey P, McAuliffe FM. Placental volume, vasculature and calcification in pregnancies complicated by pre-eclampsia and intra-uterine growth restriction. Compliance with ethical standards Eur J Obstet Gynecol Reprod Biol. 2015;195:12–17. 12. Dimasuay KG, Boeuf P, Powell TL, Jansson T. Placental Conflict of interest DBM was the recipient of a FAPERJ sandwich responses to changes in the maternal environment determine fetal doctorate scholarship (Carlos Chagas Filho Research Support Foun- growth. Front Physiol. 2016;7:12. dation—FAPERJ—Brazil—E-26/ 200.090/2016). PW is a recipient of 13. Lewis RM, Demmelmair H, Gaillard R, Godfrey KM, Hauguel-de a Wellcome Trust studentship (Wellcome—215242/Z/19/Z). LCP was Mouzon S, Huppertz B, et al. 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Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth restriction in mice

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10.1038/s41366-020-0561-3
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Abstract

Background In utero exposure to obesity is consistently associated with increased risk of metabolic disease, obesity and cardiovascular dysfunction in later life despite the divergence of birth weight outcomes. The placenta plays a critical role in offspring development and long-term health, as it mediates the crosstalk between the maternal and fetal environments. However, its phenotypic and molecular modifications in the context of maternal obesity associated with fetal growth restriction (FGR) remain poorly understood. Methods Using a mouse model of maternal diet-induced obesity, we investigated changes in the placental transcriptome through RNA sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA) at embryonic day (E) 19. The most differ- entially expressed genes (FDR < 0.05) were validated by Quantitative real-time PCR (qPCR) in male and female placentae at E19. The expression of these targets and related genes was also determined by qPCR at E13 to examine whether the observed alterations had an earlier onset at mid-gestation. Structural analyses were performed using immunofluorescent staining against Ki67 and CD31 to investigate phenotypic outcomes at both timepoints. Results RNA-seq and IPA analyses revealed differential expression of transcripts and pathway interactions related to placental vascular development and tissue morphology in obese placentae at term, including downregulation of Muc15, Cnn1, and Acta2. Pdgfb, which is implicated in labyrinthine layer development, was downregulated in obese placentae at E13. This was consistent with the morphological evidence of reduced labyrinth zone (LZ) size, as well as lower fetal weight at both timepoints irrespective of offspring sex. Conclusions Maternal obesity results in abnormal placental LZ development and impaired vascularization, which may mediate the observed FGR through reduced transfer of nutrients across the placenta. Introduction The prevalence of obesity has nearly tripled since 1975 [1]. As a consequence, the number of women who are classified These authors contributed equally: Daniela de Barros Mucci, Laura C. Kusinski as overweight or obese during pregnancy has risen sub- stantially, estimated at 38.9 million in 2014 [2]. This is Supplementary information The online version of this article (https:// especially concerning as offspring born to obese mothers doi.org/10.1038/s41366-020-0561-3) contains supplementary material, which is available to authorized users. are more likely to have poor neonatal outcomes [3] and to * Daniela de Barros Mucci Nutritional Biochemistry Laboratory, Institute of Nutrition Josué danimucci@gmail.com de Castro, Federal University of Rio de Janeiro, Rio de Janeiro, * Laura C. Kusinski Brazil lck34@medschl.cam.ac.uk Nutritional Epidemiology Observatory, Institute of Nutrition Josué de Castro, Federal University of Rio de Janeiro, Rio de Janeiro, Metabolic Research Laboratories and MRC Metabolic Diseases Brazil Unit, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, UK 1234567890();,: 1234567890();,: 1088 D. de Barros Mucci et al. develop obesity, insulin resistance, hypertension and dys- and were carried under the Home Office Animals (Scientific lipidemia later in life [4]. Interestingly, maternal obesity Procedures) Act 1986. The model has been described in leads to divergent birth weight outcomes; while it is often detail previously [20, 22]. Briefly, female C57BL/6J mice, shown to increase the risk of macrosomia [5, 6], a higher proven breeders, were randomly assigned either a standard incidence of low birth weight is also documented [6–8]. chow RM1 diet [7% simple sugars, 3% fat (wt/wt), Although both are similarly associated with metabolic dis- 10.74 kJ/g] or an energy-rich highly palatable obesogenic ease in later life, distinct placental alterations seem to diet [10% simple sugars, 20% animal lard (wt/wt), 28.43 kJ/g] mediate these contrasting offspring phenotypes [9]. supplemented with sweetened condensed milk [55% simple Changes in placental function are thought to be pivotal in sugar, 8% fat (wt/wt); Nestle, Croydon, UK], and fortified the development of pregnancy complications [10, 11] and with mineral and vitamin mix AIN93G. Both diets were fed could also be a key link between the maternal and intrau- ad libitum and purchased from Special Dietary Services terine milieu and long-term health of the offspring [12, 13]. (Witham, UK). Body composition was monitored (TD- Alterations in placental function and structure in response NMR, Bruker Minispec) and females were set up to breed if to obesity and their underlying molecular mechanisms body fat was between the thresholds of 10–12% or 35–40% have been explored both in humans and in animal models for Control and Obese dams, respectively. After mating for [9, 14–17]. Yet, even though fetal growth restriction (FGR) the second time with RM1 fed males, dams were killed at is recognized as a placenta-related disorder [18], the impact either E13 or E19 by rising CO concentration. Fetal and of maternal obesity on the placental transcriptome in this placental weights were recorded. Placentae for molecular context remains largely unknown. analysis were immediately snap frozen on dry ice and stored It has been shown that placentae from high fat diet-fed at −80 °C. For morphological assessment, samples were obese mouse dams exhibit altered expression of epigenetic fixed in 10% formalin for 48 h, stored in 70% ethanol and machinery genes at term, which could alter the placental then embedded in wax. epigenome and lead to FGR [19]. High fat diet-induced The sex of the fetuses at E19 was determined by visual obesity has also been found to alter the transcriptome of inspection of anogenital anatomy. At E13, DNA extracted placenta progenitor cells at early stages of development and from tail tips was used for PCR sexing as described by is associated with later changes in placental function McFarlane et al. [23], using the SX primer pair. Amplicons resulting in FGR [17]. In our mouse model of maternal diet- were loaded on 2% agarose gels and submitted to electro- induced obesity, in which dams are fed a hypercaloric phoresis together with a 1 kb DNA ladder. Bands were Western-like diet, we have shown that maternal hyper- visualized with SYBR™ Safe DNA gel stain (Thermo insulinemia is strongly associated with offspring insulin Fisher Scientific, Rochford, UK) under UV-illumination resistance and excess placental lipid deposition and hypoxia and the genomic sex of each sample was determined [20]. However, a clear understanding of the molecular according to the number of bands and amplicon size. mechanisms behind these findings is still lacking and war- rants further investigation. RNA extraction It is recognized that the impact of stressors on placental function and offspring health is closely linked to the stage Placenta aliquots were homogenized in 700-µL Qiazol of tissue development, the type of insult and the sex of the using TissueRuptor (Qiagen, Manchester, UK). Total RNA conceptus [21]. Thus, the aim of this study was to identify was isolated with miRNeasy Mini Kit (Qiagen) according to global changes in the placental transcriptome and related the manufacturer’s instructions and including the optional pathways in response to maternal obesity near term at step of DNA digestion with RNase-Free DNAse Set (Qia- embryonic day (E) 19. Furthermore, we investigated whe- gen). Extracted RNA was quantified by spectrophotometry ther the significant transcriptional alterations detected in (Nanodrop Thermo Fisher Scientific) and stored at obese placentae were manifested earlier, i.e., in mid- −80 °C. gestation (E13), and if these alterations translated into a structural phenotype in male and female placentae. RNA sequencing and Ingenuity® Pathway Analysis Total RNA was extracted from E19 male placentae (Control Methods n = 2 and Obese n = 3), as previously outlined. One microgram of total RNA was depleted of ribosomal RNA Animals and diets and PolyA tails of coding RNAs were captured by treatment with Oligo-dT beads. Complementary DNA (cDNA) All experimental protocols were approved by the University libraries were generated after an amplification step, of Cambridge Animal Welfare and Ethical Review Board according to the TruSeq Stranded Total RNA Sample Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1089 Preparation Guide (Illumina, San Diego, CA, USA), and staining. Antigen retrieval was performed with pH9 Target quantified using KAPA Library Quantification Kit. Multi- Retrieval buffer [S2375 (Dako Agilent, Stockport, UK)] and plex single-read sequencing was performed using Illumina nonspecific binding sites were blocked with 3% goat serum. HiSeq 2500 (Illumina). Sequence reads were demultiplexed Sections were incubated overnight at 4 °C with rabbit using the CASAVA pipeline (Illumina) and then aligned to polyclonal primary antibodies against Ki67 [1:200; the Mus musculus genome (GRCm38) using TopHat ver- ab15580 (Abcam, Cambridge, UK)] or CD31 [1:500; sion 2.0.11. Raw read counts and fragments per kilobase of ab124432 (Abcam)]. transcript per million mapped reads (FPKM) were generated Primary antibody binding was visualized by incubation using Cufflinks version 2.2.1. A quality check of mapped at room temperature for 1 h with a fluorescent-conjugated reads was executed using the R package CummeRbund. goat polyclonal anti-rabbit IgG [1:1000; A11008 (Invitro- Databases were trimmed for exclusion of very low detection gen, Warrington, UK)]. In negative control slides, placental or undetectable genes. The resulting data were analyzed sections were incubated in 1.5% goat serum in TBS-T using edgeR by calculating the likelihood ratio, and by instead of primary antibodies (Supplementary Fig. S1). All adjusting P values via Benjamini and Hochberg’s method to sections were incubated with 1:2500 DAPI for 10 min at control the false discovery rate (FDR). Ingenuity Pathway room temperature to stain nuclei. Autofluorescence was Analysis (IPA) was applied to identify biological pathways quenched by incubation with Vector TrueVIEW Auto- related to the genes that were differentially expressed fluorescence Quenching Kit [SP8400 (Vector Laboratories, between Control and Obese E19 male placentae. The pla- Peterborough, UK)]. centa RNA sequencing (RNA-seq) data have been depos- Placental sections were imaged on an AxioScan Slide ited in GEO database under the accession number Scanner (Zeiss, Cambridge, UK) and blindly analyzed using GSE140013. HALO analysis software (Indica Labs, Corrales, NM, USA). The DAPI channel image was used to define nuclear cDNA synthesis and Quantitative real-time PCR outlines using the CytoNuclear FL module. For the analysis (qPCR) of Ki67 staining (Supplementary Fig. S2), nuclear outlines were transposed onto the 488-nm channel image and the Total RNA was extracted from male and female placentae proportion of nuclei positive for Ki67 across the whole of Control and Obese dams at E13 (n = 10/group from section was recorded. Placental sections stained for the separate litters) and E19 (n = 9/group from separate litters). endothelial cell marker CD31 were used to investigate All samples used in the validations were different to those labyrinth zone (LZ) size and structure (Supplementary Fig. used in the RNA-seq and therefore represent biological S3). The border of the LZ was outlined manually at ×40 replicates. Sample size was based on previous data sets/ magnification (depicted in yellow) and total area was power calculations. cDNA was generated from 1-μg RNA recorded using HALO analysis software after canals were using High Capacity cDNA Reverse Transcription Kit excluded. The boundary with the junctional zone was (Applied Biosystems, Foster City, CA, USA). qPCR was determined as the interface between the phenotypically performed on QuantStudio 7 Flex Real-Time PCR System distinct spongiotrophoblast of the junctional zone and the (Applied Biosystems), using 200 nM specific primers fetal capillaries of the LZ. The rest of the boundary was (Sigma-Aldrich, Gillingham, UK), 1× SYBR® Green either at the edge of the tissue image or at the interface with JumpStart™ Taq ReadyMix (Sigma-Aldrich) and cDNA the chorionic plate, which is structurally distinct from the samples at a final dilution of 1:60. For primer sequences see LZ, characterized by smaller nuclei and the absence of Supplementary Table S1. NormFinder software was used to CD31-positive endothelial cells. Indica Labs’ Tissue Clas- select the best combination of two out of four reference sifier module was used to differentiate between fetal blood genes [24]. qPCR results were normalized to the geometric vessels (lumen bound by CD31-positive endothelium) and mean of the reference genes Gapdh and Sdha for E19 other tissue of the LZ. placentae, and Gapdh and Pmm1 for E13 placentae, expression of which did not change between groups. Data Statistical analyses were expressed in arbitrary units relative to Male Control −ΔΔCq average (2 ). Benjamini–Hochberg multiple testing correction [25] was applied to the RNA-seq differential expression data and Structural analyses only genes with FDR < 0.05 were considered significantly different between the two experimental conditions. For E13 and E19 formalin-fixed placentae from males and qPCR validation of RNA-seq differentially expressed females were cut into 5-µm sections. Three serial sections genes, comparisons were made between Control and Obese close to the midline of each placenta were selected for placentae of the same sex, by Student’s t test. 1090 D. de Barros Mucci et al. Table 1 Fetal and placental Control Obese P value weights at E13 and E19. Males Females Males Females Maternal Sex obesity E13 Fetal weight (g) 0.17 ± 0.01 0.16 ± 0.00 0.15 ± 0.01 0.14 ± 0.01 0.003 0.187 Placental 94.0 ± 2.2 88.9 ± 3.5 89.7 ± 2.1 78.7 ± 2.9 0.015 0.008 weight (mg) Fetal:placental ratio 1.84 ± 0.06 1.85 ± 0.06 1.66 ± 0.08 1.76 ± 0.08 0.079 0.452 E19 Fetal weight (g) 1.23 ± 0.03 1.17 ± 0.03 1.02 ± 0.03 1.03 ± 0.02 <0.0001 0.339 Placental 93.8 ± 5.1 81.7 ± 5.3 82.8 ± 1.9 73.1 ± 2.7 0.039 0.021 weight (mg) Fetal:placental ratio 13.45 ± 0.76 14.77 ± 0.92 12.38 ± 0.37 14.28 ± 0.78 0.331 0.051 Values are mean ± SEM. P values < 0.05 indicated in bold show significant effect of maternal obesity and sex differences in the studied parameters according to two-way ANOVA followed by Tukey’s multiple comparisons test, using each litter’s average as a single data (Control Male n = 6 and 9, Control Female n = 6 and 9, Obese Male n = 7 and 7, Obese Female n = 7 and 6, respectively, at E13 and E19). Anthropometric parameters and qPCR of selected targets males at both stages of gestation. There was no significant were analyzed by two-way analysis of variance (ANOVA) difference in the ratio of fetal to placental weight, although followed by Tukey’s multiple comparisons test to estimate there was a trend (P = 0.05) toward higher placental effi- the effects of maternal obesity and fetal sex at each ciency in females at E19 (Table 1). time point. For the morphological analyses, the effects of gestational RNA-seq, Ingenuity® Pathway Analysis and qPCR at age (E13 or E19), offspring sex (male or female), and term placentae maternal diet (regular chow or obesogenic diet) on placental phenotype were investigated by three-way ANOVA, and The RNA-seq at E19 detected a total of 350 transcripts backwards stepwise elimination was used to come to a differentially expressed in placentae of Obese compared minimal model. Three-way ANOVA of the proportional to Control males considering a significance threshold of area of the LZ that was fetal capillaries data revealed no P < 0.05 (Fig. 1a, Supplementary Table S2). However, only significant effect of gestational age, maternal diet, or fetal nine genes remained significantly altered after correction for sex. However, there was a borderline significant interaction multiple testing (FDR < 0.05) (Fig. 1a, b). Ingenuity® between maternal diet and fetal sex (P = 0.055). In order to Pathway Analysis (IPA) was used with a less stringent identify if a maternal diet effect was only present in one sex, threshold (P < 0.05) to identify global changes in pathways these data were separated and sex-specific two-way ANO- and biological functions promoted by maternal obesity. The VAs (gestational age/maternal diet) were performed. most significant diseases and bio functions are shown in Only one sample from each litter was used for each Fig. 1c. analysis, except in the case of offspring and placental Genes identified as significantly changed in response to weights, in which each litter’s average was used as a single obesity by RNA-seq (FDR < 0.05) were validated in a larger data point. Data are presented as mean ± standard error of number of samples, all from independent litters, by qPCR the mean (SEM), and the threshold for significance was set (Fig. 2) and results were confirmed in eight out of the nine at P < 0.05, unless stated otherwise. Statistical analyses genes in E19 male placentae (i.e., Pi15, Gabrd, Sez6l, were performed using R (R Core Team 2017) or Prism 6 Nup210, Acta2, Rnf222, Muc15, and Cnn1). These genes (GraphPad Prism, La Jolla, CA, USA). were also examined in female placentae, however, only Pi15, Nup210, Acta2, Rnf222, and Muc15 were sig- nificantly modulated by obesity (Fig. 2). Results Placental gene expression at different gestational Fetal and placental measurements ages Fetal and placental weights were reduced in response to All nine genes found differentially expressed in obese obesity and female placentae were smaller than those of male placentae at E19 were then investigated in E13 Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1091 Fig. 1 RNA-seq identification of differentially expressed genes between Control (C, n = 2) and Obese (O, n = 3) male mouse placentae at E19. a Volcano plot representing all detected transcripts, distributed according to −log10 P value in the y-axis and log2 fold change in the x-axis, with downregulated genes shifted to the left (P < 0.05 in blue) and upregulated genes shifted to the right (P < 0.05 in red). Significantly altered genes after correction for multiple testing (FDR < 0.05) are depicted with a pink diamond. b Heatmap representation of genes significantly regulated by maternal obesity using a cutoff FDR < 0.05, with scaled Z-score color key of normalized counts showing expression levels ranging from blue (lower) to red (higher). Genes are sorted from lowest to highest log2 fold change value.c Top diseases and bio functions from Ingenuity® Pathway Analysis (IPA) of the RNA-seq data with a threshold of P < 0.05, showing the most significant molecular and cellular functions dysregulated in the placenta by maternal obesity, sorted by P value. placentae (Fig. 3a). Pi15, Nup210,and Sez6l were upre- at mid-gestation. Both Hand1 and Pdgfb were down- gulated by maternal obesity in both sexes at mid-gestion. regulated by maternal obesity in both sexes, but no differ- Gabrd mRNA levels, which were upregulated in Obese ences were observed in Prl2c2 (Fig. 3b). male placentae at E19, was downregulated by maternal obesity at E13. Muc15, Nup210,and Acta2 expression was Immunofluorescent staining of placental higher in females compared to males at E13. Rnf222 and morphology Cnn1 were not differentially expressed in either sex at E13, however, their transcript levels were very low com- Phenotypic analyses were also conducted by immuno- pared to E19. fluorescent staining of targets within pathways identified by Since genes involved in spiral artery remodeling (Muc15 IPA. The marker Ki67 was used to investigate cellular and Cnn1) and labyrinthine pericytes (Acta2) that were growth and proliferation. Cellular movement, assembly, and found to be dysregulated in obese placentae at E19 were not organization were assessed through analyses of LZ size and affected at E13, we additionally measured the expression of fetal vasculature structure, using CD31 as a marker of fetal candidate genes recognized as relevant for these processes endothelial cells. 1092 D. de Barros Mucci et al. analyzed by the proportion of the total LZ area that was fetal blood vessels. A reduced model considering males and females separately by two-way ANOVA (gestational age/ maternal condition) detected a reduction in area bound by fetal capillaries within the LZ in female placentae of obese dams (P < 0.05, Fig. 4n–p). Representative images of male placentae are shown in Supplementary Fig. S4. Discussion The RNA-seq analysis revealed a total of 350 transcripts differentially expressed in Obese male placentae at term. Fig. 2 Validation of RNA-seq data by qPCR in E19 male and Among the top downregulated transcripts, Muc15, Cnn1 female placentae. qPCR results were normalized to the reference and Acta2 were of particular significance as these genes are genes Gapdh and Sdha and are expressed as mean ± SEM in arbitrary required for appropriate development of placental vascu- units relative to Male Controls. *P < 0.05, determined by Student’s t test comparing qPCR data of same sex Control vs Obese, n = 9/ lature and related to key pathways identified by IPA such as group. cellular movement, assembly and organization. Previous studies have shown that Muc15 suppresses the migration/ invasion of trophoblast like-cells in vitro, a process impli- cated in blood vessel remodeling in the maternal–fetal interface [26]. Cnn1 is largely expressed by smooth muscle cells [27] which line the uterine blood vessels and are lost in the normal remodeling of maternal spiral arteries during placental development [28]. Acta2 is a marker of pericytes which surround fetal endothelial cells during blood vessel development in the mouse LZ [29]. These data together suggest that exposure to maternal obesity affects the remodeling of maternal spiral arteries and the development of fetal blood vessels within the LZ, both of which are crucial for adequate nutrient and oxygen transfer across the placenta [28] and possibly linked to the uteroplacental hemodynamic alterations present in pre- eclampsia and intrauterine growth restriction [30–32]. Fig. 3 qPCR expression in E13 male and female placentae. Obese women are two to three times more likely to develop a Validated RNA-seq genes.b Hand1, required for trophoblast giant preeclampsia [33] and hypertensive obstetric complications cell (TGC) differentiation; Prl2c2, a marker of spiral artery-associated TGC and canal-associated TGC; Pdgfb, a growth factor that regulates are generally associated with small-for-gestational age placental labyrinthine layer development. qPCR data were normalized neonates [34]. Here, we see significant growth restriction in to the reference genes Gapdh and Pmm1. Results are shown as mean ± the fetus which may result from poor uteroplacental perfu- SEM in arbitrary units relative to Male Control average expression. sion in addition to placental hypoxia previously suggested *Denotes maternal obesity effect (P < 0.05) and # denotes sex differ- ence (P < 0.05), according to two-way ANOVA, n = 10/group. in this model [20]. ªRnf222 and Cnn1 expression levels were low at E13 placentae, with Furthermore, the RNA-seq analysis identified several average Cq values above 31 and 29, respectively. genes that have not been functionally described in placental tissue thus far, but are conserved in humans and rodents. E19 placentae had fewer cells (P < 0.05, Fig. 4a–c) and Pi15 encodes a peptidase inhibitor that may regulate a lower proportion of proliferating cells across the whole extracellular matrix modifications [35] and has been placenta (P < 0.01, Fig. 4d–h) compared to E13. These implicated in vascular defects in rat aorta [36], though its parameters were not affected by offspring sex or maternal role in placental vascularization is unknown. Gabrd gene diet. encodes the delta subunit of gamma-aminobutyric acid type The size of the LZ was significantly reduced in placentae A receptor (GABA ). GABA activation impacts stromal A A from obese dams (P < 0.01, Fig, 4i–m). There was a sig- cell proliferation and apoptosis during decidualization [37] nificant increase in LZ size from mid-gestation to term and increased expression of its pi subunit (GABRP) has (P < 0.01, Fig. 4i). Labyrinthine vascular organization was been detected in preeclamptic placentae [38]. NUP210 is a Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1093 E13 E19 DAPI DAPI 1 mm 1 mm DAPI/Ki67 Ki67 Ki67 DAPI/Ki67 500 μm 500 μm 500 μm 500 μm DAPI/CD31 DAPI/CD31 1 mm 1 mm DAPI/CD31 DAPI/CD31 1 mm 1 mm Control Female Obese Female CD31 CD31 100 μm 100 μm Fig. 4 Immunofluorescent staining of targets related to the top maternal obesity (C n = 10, Ob n = 9, at each time point). n–p The three molecular and cellular functions shown in IPA. All analyses proportion of fetal capillaries within the labyrinth zone was decreased were conducted in both male and female placentae of mothers fed by maternal obesogenic diet in females (C n = 10, Ob n = 9). a, d, i, n either regular chow (C, Control group) or obesogenic diet (Ob, Obese Results are shown as mean ± SEM. Gestational age differences are group), at E13 and E19. a–c The total number of cells in the placenta denoted by *P < 0.05, **P < 0.001, or ***P < 0.0001, and maternal decreased between E13 (n = 20) and E19 (n = 19). d–h The propor- obesity effect is indicated by P < 0.001, according to three-way tion of Ki67-positive cells across the whole placenta decreased ANOVA. §Denotes maternal obesity effect (P < 0.05), determined by between E13 (n = 20) and E19 (n = 19). i–m The size of the labyrinth two-way ANOVA analysis of E13 and E19 female placentae only. zone increased between E13 and E19, and was reduced in response to major component of the nuclear pore complex and is protein-coding gene; however, no functional description has required for regulation of gene expression during differ- been found. entiation and cell fate determination, as demonstrated in Although information on these genes in placentae is myoblasts and embryonic stem cells [39]. Although the currently limited, it must be noted that a large number of function of SEZ6L is not well understood, it has been placental genes and related phenotypes remain unchar- shown both in mice and in vitro that this protein is almost acterized. Recent efforts to systematically identify the genes exclusively processed by β-site APP cleaving enzyme required for normal embryogenesis are still unravelling (BACE) [40]; BACE1 and BACE2 are abundantly expres- many previously underappreciated placental defects [42]. sed in human placentae and are upregulated in pregnancies Thus, our findings might represent novel targets that could complicated with preeclampsia [41]. Lastly, Rnf222 is also a be implicated in the pathophysiology of maternal obesity Ob C 1094 D. de Barros Mucci et al. and associated adverse outcomes in the offspring. More- Next, we used IPA to investigate the mechanism behind over, additional genes might have been identified if a larger this reduction in LZ area. Cellular growth and proliferation sample size was used in the RNA-seq analysis. were pointed out as the main molecular and cellular func- When comparing both timepoints, most transcripts tions affected by obesity. Surprisingly, however, maternal exhibited a different expression pattern, including Muc15, obesity had no effect on the number of Ki67-positive cells Cnn1, and Acta2 which were not affected by obesity at E13. in the placenta. Abnormalities in placental size are often This could be due to low functional relevance of these associated with disruption of cellular growth and/or apop- transcripts at mid-pregnancy rather than absence of altera- tosis [47–49]. Thus, it is possible that other mechanisms tions in related cellular processes, as illustrated by low such as cell death could explain our results, although lim- Cnn1 mRNA levels in our analysis at E13 compared to E19 itations to our analysis, which was not zone-specific, cannot (data not shown). In fact, the mouse placenta undergoes a be discounted. In this regard, it has been shown in a mouse transcriptome transition from the “development phase” of model of high fat diet-induced obesity through phospho- organogenesis to the “mature phase” at mid-pregnancy [43]. histone H3 staining that the proliferating cells in placenta Thus, we next used the IPA data to identify genes which are mostly restricted to the labyrinthine layer and appear were previously shown to be both highly expressed at mid- reduced in response to obesity within this region [50]. pregnancy and pivotal to spiral artery remodeling and for- Despite these morphological disturbances, changes in mation of fetal blood vessels in the LZ. Hand1, which is tissue structure that are expected to occur from mid- required for trophoblast giant cell (TGC) differentiation pregnancy until term seemed preserved in obese placentae. [44], was downregulated in placentae of obese dams. The LZ is well-reported to expand as pregnancy progresses, However, maternal obesity had no effect on the expression so that the transport capacity of the placenta can meet the of Prl2c2, a marker of TGC that line maternal blood canal nutrient demands of the growing fetus [50–52]. Accord- spaces and spiral arteries in the definitive placenta [45]. ingly, we found that labyrinth area increased between E13 Considering the complexity of spiral artery remodeling and and E19, irrespective of maternal diet. the limited number of transcripts that were analyzed here, it We also observed significant differences in placentae remains to be established whether the alterations occur only which are specific to fetal sex. Female placentae were later in development or if other mechanisms are involved. smaller than male counterparts at all timepoints and On the other hand, the growth factor Pdgfb was down- maternal conditions, which is consistent with observations regulated in response to obesity at E13 and could be a from both human cohorts [53] and studies in mice [19, 54]. relevant link to other molecular and phenotypic observa- Moreover, we found sex differences in a subset of genes, tions in our model. It has been shown that Pdgfb-deficient with females exhibiting slightly increased expression. placentae exhibit defective labyrinthine development, with Similarly, global transcriptomic analysis in normal full-term alterations in fetal blood vessel structure and reduced human placentae revealed higher overall mRNA levels in numbers of pericytes from mid-pregnancy until term, females compared to males [55]. Sexual dimorphism in the leading to growth restriction in PDGFB −/− embryos [29]. context of developmental programming is increasingly Here, lower expression of the pericyte marker Acta2 was commonly reported [56]. How these relate to sex-specific detected by RNA-seq and confirmed by qPCR in obese responses of the placenta to a suboptimal environment placentae at E19. In addition, defects in LZ morphology and remain to be determined. FGR were observed in response to maternal obesity at E13 Overall, we have shown through genome-wide analysis and persisted until E19. that maternal obesity induces a dysregulation of transcripts As shown by our immunofluorescence staining, male and and pathway interactions related to placental vasculature female placentae from Obese dams exhibited reduced LZ and structure. FGR, as well as changes in placental mor- area, which is the primary site of gas, nutrient and waste phology and a gene expression signature associated with exchange between the maternal and fetal circulations in the impaired labyrinthine development, were detectable at mid- mouse [28], and a decrease in the proportion of fetal blood pregnancy, suggesting an enduring negative effect of vessels within the LZ was also evident in females. This is maternal obesity over these processes. The LZ is the further corroborated by recent evidence of lower vascularity exchange region of the murine placenta and reductions in its in placentae of high fat diet-fed dams both at mid- size and vasculature may impair the transport of nutrients pregnancy and near term, which was associated with pla- from the maternal circulation to the developing fetus, thus cental transcriptome alterations in early stages of develop- restricting its growth. Disruption of placental structure ment and FGR [17]. In addition, it has been suggested that could thus represent an important factor contributing to the defects in placental villi vasculature seen in obese human development of FGR in pregnancies complicated by pregnancies could be partly due to obesity-associated tissue maternal obesity. Moreover, novel targets were revealed by hypoxia [46], which is also consistent with our model [20]. our RNA-seq analysis. Characterizing their functional roles Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth. . . 1095 in the placenta will help us better understand the processes 7. Radulescu L, Munteanu O, Popa F, Cirstoiu M. The implications and consequences of maternal obesity on fetal intrauterine growth mediating the effects of maternal obesity on offspring out- restriction. J Med Life. 2013;6:292–8. comes and potentially inform suitable interventions. 8. Rajasingam D, Seed PT, Briley AL, Shennan AH, Poston L. A prospective study of pregnancy outcome and biomarkers of oxi- Acknowledgements The authors would like to thank Ania Wilczynska dative stress in nulliparous obese women. Am J Obstet Gynecol. and Martin Bushell from the University of Leicester for their helpful 2009;200:395 e391–399. analysis on the RNA-seq data and Claire Custance for technical 9. Howell KR, Powell TL. Effects of maternal obesity on placental assistance. This work was supported by the Biotechnology and Bio- function and fetal development. Reproduction. 2017;153:R97–R108. logical Sciences Research Council (BBSRC—BB/M001636/1) and an 10. Flenady V, Koopmans L, Middleton P, Froen JF, Smith GC, Gibbons MRC Metabolic Diseases Unit award (MC_UU_12012/4). We also K, et al. Major risk factors for stillbirth in high-income countries: a thank BHF, Wellcome Trust, FAPERJ, and CNPq for the financial systematic review and meta-analysis. Lancet. 2011;377:1331–40. support. 11. Moran MC, Mulcahy C, Zombori G, Ryan J, Downey P, McAuliffe FM. Placental volume, vasculature and calcification in pregnancies complicated by pre-eclampsia and intra-uterine growth restriction. Compliance with ethical standards Eur J Obstet Gynecol Reprod Biol. 2015;195:12–17. 12. Dimasuay KG, Boeuf P, Powell TL, Jansson T. Placental Conflict of interest DBM was the recipient of a FAPERJ sandwich responses to changes in the maternal environment determine fetal doctorate scholarship (Carlos Chagas Filho Research Support Foun- growth. Front Physiol. 2016;7:12. dation—FAPERJ—Brazil—E-26/ 200.090/2016). PW is a recipient of 13. Lewis RM, Demmelmair H, Gaillard R, Godfrey KM, Hauguel-de a Wellcome Trust studentship (Wellcome—215242/Z/19/Z). LCP was Mouzon S, Huppertz B, et al. The placental exposome: placental the recipient of a CNPq Science Without Borders Post-Doctoral Fel- determinants of fetal adiposity and postnatal body composition. lowship (National Council of Technological and Scientific Develop- Ann Nutr Metab. 2013;63:208–15. ment—CNPq—Brazil—PDE/204416/2014–0). The authors declare 14. Altmae S, Segura MT, Esteban FJ, Bartel S, Brandi P, Irmler M, that they have no conflict of interest. et al. Maternal pre-pregnancy obesity is associated with altered placental transcriptome. PLoS ONE. 2017;12:e0169223 Publisher’s note Springer Nature remains neutral with regard to 15. Saben J, Kang P, Zhong Y, Thakali KM, Gomez-Acevedo H, jurisdictional claims in published maps and institutional affiliations. Borengasser SJ, et al. RNA-seq analysis of the rat placentation site reveals maternal obesity-associated changes in placental and off- Open Access This article is licensed under a Creative Commons spring thyroid hormone signaling. Placenta. 2014;35:1013–20. 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International Journal of Obesity – Springer Journals

Published: May 13, 2020

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