Early Placenta Research Articles

Natural evolution of a cesarean scar pregnancy up to 34 weeks complicated by an early subserosal uterine rupture and placenta accreta spectrum disorders

Cesarean scar pregnancy (CSP) is an extremely rare form of ectopic pregnancy. A 39-year-old woman with a history of multiple uterine surgeries was diagnosed with a CSP at seven weeks by transvaginal ultrasound. The CSP was misdiagnosed at 13 weeks and 5 days during a second medical opinion. At 22 weeks of gestation, a 3.8 cm subserosal uterine rupture was visualized. Expectant management was decided. A live birth was achieved through an elective cesarean section at 34 weeks of gestation. The complications were early subserosal uterine rupture and placenta accreta spectrum that led to a hysterectomy. Due to risk of uterine rupture, management should consist of early termination of the pregnancy.

Exogenous melatonin ameliorates embryo-maternal cross-talk in early pregnancy in sheep.

Melatonin plays a crucial role in enhancing reproductive performance in small ruminants. This paper reveals the effects of exogenous melatonin on the placental and endometrial rearrangement in early pregnancy in sheep. Early pregnancy losses cause 25% of pregnancy failures in small ruminants because of asynchrony between conceptus and uterine signals. In this context, melatonin plays a crucial role in sheep reproductive dynamics, but little is known about its effects during the peri-implantation period. We hypothesized that melatonin supports embryo implantation by modulating the uterine microenvironment. This study aimed to assess the effects of exogenous melatonin on the endometrial and early placental rearrangement. Ten multiparous ewes either did (MEL, n = 5) or did not (CTR, n = 5) receive a subcutaneous melatonin implant (18 mg) 50 days before a synchronized mating. On day 21 of pregnancy, the sheep were euthanized. MEL ewes exhibited a higher prolificity rate (2.8 vs 2.0 embryos/ewe) and plasma progesterone levels (3.84 vs 2.96 ng/mL, P < 0.05) than did CTR ewes. Groups did not differ significantly in embryo crown-rump length. MEL placentas had significantly (P < 0.001) more binucleated trophoblast cells in the chorion region, and ovine placental lactogen expression was significantly (P < 0.05) more strongly upregulated than in CTR. Exogenous melatonin increased significantly (P < 0.05) gene expression of angiogenic factors (VEGFA, VEGFR1, IGF1R), IFNAR2, and PR in the caruncular endometrium. Expression of the MT2 receptor in the endometrium and placenta was significantly (P < 0.05) higher in the MEL group. These results indicate that melatonin implants acted differentially on uterine and placental rearrangement. Melatonin increases differentiation in the placenta and induces changes that could promote vessel maturation in the endometrium, suggesting that it enhances the uterine microenvironment in the early stage of pregnancy in sheep.

Advanced maternal age induces a reduced activity of FoxO1 and alters the balance between cellular senescence and proliferation in early placenta

Advanced maternal age induces a reduced activity of FoxO1 and alters the balance between cellular senescence and proliferation in early placenta

Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells.

Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5-CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.

P-789 Trophoblast organoids as a 3D model for the study of oxygen concentration during in vitro preimplantation embryo culture

Abstract Study question To determine if trophoblast organoids could represent a relevant robust model to study the impact of oxygen concentration on human preimplantation embryo development. Summary answer Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs differentially expressed. What is known already Our team has previously demonstrated that a biphasic (5-2%) O2 concentration strategy during preimplantation embryo culture (from Day 0 to Day 6) significantly improved blastulation and live birth rates in in vitro fertilization (IVF). We also identified differentially expressed RNAs between monophasic and biphasic O2 strategies through whole transcriptome analysis on human embryos donated for research. However, the number of embryos donated to research is limited, they are all genetically different, and their use iethically controversial, representing a significative drawback to experimentation. Latest studies on trophoblast organoids indicate that they could represent a relevant model for studying preimplantation embryo development. Study design, size, duration A prospective monocentric study approved by the Institutional Review Board (I.R.B.) was conducted between February 2022 and September 2023: four early first-trimester human placentas were collected after surgical abortions and used for derivation of organoids in a 3D culture system. The protocol for organoid culture is described in the study of Sheridan and al, 2020. Organoids were cultured with either a monophasic or a biphasic oxygen concentration strategy. Participants/materials, setting, methods Trophoblast organoids were either exposed to 5% oxygen concentration during 5 days or to 5% O2 for 3 days and to 2% O2 for 2 days. Total RNA extraction of 21 organoids for each condition was performed. After whole transcriptome analysis using human Clariom D arrays (ThermoFisher Scientific®), microarray data were normalized and differentially expressed RNAs were identified using the Transcriptome Analysis Console software; the genetic network was generated with Ingenuity Pathway Analysis (Qiagen®). Main results and the role of chance Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs that were differentially expressed depending on the O2 strategy (fold change &amp;gt;2 or &amp;lt;-2, p &amp;lt; 0.05), with most of them (75.1%, n = 889/1,184) being downregulated in the biphasic (5-2%) O2 group. IPA analysis highlighted downregulation of transcripts involved in cell survival and differentiation as well as upregulation of transcripts involved in embryo angiogenesis and vascularization of extraembryonic tissue. The transcript HIF1a was upregulated in organoids cultured in biphasic strategy compared to monophasic strategy (fold change 2, p = 0.0129). Comparison between differentially expressed transcripts in monophasic and biphasic strategies highlighted 22 transcripts in common between organoids and embryos donated to research from our previous study. Limitations, reasons for caution Our study was limited by a low sample size. Wider implications of the findings This innovative model of trophoblast organoids could help us to overcome the rarity as well as the genetic/epigenetic heterogeneity associated with IVF human embryos donated to the research, thus leading to the realization of large-scale experimentations and promoting the identification of new non-invasive predictive biomarkers for IVF. Trial registration number not applicable

Suppression of GATA3 promotes epithelial-mesenchymal transition and simultaneous cellular senescence in human extravillous trophoblasts

The regulatory mechanism of the transcription factor GATA3 in the differentiation and maturation process of extravillous trophoblasts (EVT) in early pregnancy placenta, as well as its relevance to the occurrence of pregnancy disorders, remains poorly understood. This study leveraged single-cell RNA sequencing data from placental organoid models and placental tissue to explore the dynamic changes in GATA3 expression during EVT maturation. The expression pattern exhibited an initial upregulation followed by subsequent downregulation, with aberrant GATA3 localization observed in cases of recurrent miscarriage (RM). By identifying global targets regulated by GATA3 in primary placental EVT cells, JEG3, and HTR8/SVneo cell lines, this study offered insights into its regulatory mechanisms across different EVT cell models. Shared regulatory targets among these cell types and activation of trophoblast cell marker genes emphasized the importance of GATA3 in EVT differentiation and maturation. Knockdown of GATA3 in JEG3 cells led to repression of GATA3-induced epithelial-mesenchymal transition (EMT), as evidenced by changes in marker gene expression levels and enhanced migration ability. Additionally, interference with GATA3 accelerated cellular senescence, as indicated by reduced proliferation rates and increased activity levels for senescence-associated β-galactosidase enzyme, along with elevated expression levels for senescence-associated genes. This study provides comprehensive insights into the dual role of GATA3 in regulating EMT and cellular senescence during EVT differentiation, shedding light on the dynamic changes in GATA3 expression in normal and pathological placental conditions.

Acute response to pathogens in the early human placenta at single-cell resolution

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T.gondii. Finally, we revealed how P.falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

Sex differences in DNA methylation across gestation: a large scale, cross-cohort, multi-tissue analysis

Biological sex is a key variable influencing many physiological systems. Disease prevalence as well as treatment success can be modified by sex. Differences emerge already early in life and include pregnancy complications and adverse birth outcomes. The placenta is a critical organ for fetal development and shows sex-based differences in the expression of hormones and cytokines. Epigenetic regulation, such as DNA methylation (DNAm), may underlie the previously reported placental sexual dimorphism. We associated placental DNAm with fetal sex in three cohorts. Individual cohort results were meta-analyzed with random-effects modelling. CpG-sites differentially methylated with sex were further investigated regarding pathway enrichment, overlap with methylation quantitative trait loci (meQTLs), and hits from phenome-wide association studies (PheWAS). We evaluated the consistency of findings across tissues (CVS, i.e. chorionic villus sampling from early placenta, and cord blood) as well as with gene expression. We identified 10,320 epigenome-wide significant sex-differentially methylated probes (DMPs) spread throughout the epigenome of the placenta at birth. Most DMPs presented with lower DNAm levels in females. DMPs mapped to genes upregulated in brain, were enriched for neurodevelopmental pathways and significantly overlapped with meQTLs and PheWAS hits. Effect sizes were moderately correlated between CVS and placenta at birth, but only weakly correlated between birth placenta and cord blood. Sex differential gene expression in birth placenta was less pronounced and implicated genetic regions only marginally overlapped with those associated with differential DNAm. Our study provides an integrative perspective on sex-differential DNAm in perinatal tissues underscoring the possible link between placenta and brain.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions

IntroductionSpontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. MethodologyThe study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10–12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12–15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. ResultsMembrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%).Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. ConclusionOur data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA–C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

Identifying the Causality between Early Placenta Abruption and Its After-Effects

Identifying the Causality between Early Placenta Abruption and Its After-Effects

VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification

In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.

Ovine Trophoblast Cells: Cell Isolation and Culturing from the Placenta at the Early Stage of Pregnancy.

Embryo development is dependent upon the exchange of oxygen and nutrients through the placenta, mainly composed of peculiar epithelioid cells, known as trophoblast cells. Normal trophoblast functionality plays a key role during the whole pregnancy, especially in the first stage of placentation. This chapter explains the techniques to obtain sheep primary trophoblast cells from the early placenta. Overall, procedures for cell isolation, culture, characterization, and cryopreservation are described.

Spatial Localization of Eubacterial 16S rRNA in Early Pregnancy Placenta and Decidua.

Bacteria derived from the maternal circulation have been suggested to seed the human placenta during pregnancy leading to development of an intrinsic placental microbiome; however, other data indicates these bacteria are artifactual contaminants. Limited research on the localization of bacteria in human placental tissue is available, which may help differentiate resident placental bacteria from contaminants. This study spatially localizes bacteria in situ in normal late first to early second trimester human placenta by 16S rRNA chromogenic in situ hybridization and demonstrates patterns consistent with both contaminants and intraparenchymal signals. These results suggest that placental microbiome studies may benefit from spatial strategies that can exclude surface contamination.

NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as pre-eclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of placental epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. The NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator mastermind-like 1 (MAML1). Doxycycline-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, which are the differentiated hormone-producing cells of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs, whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development by promoting self-renewal of CTB progenitors.

The clinical value of combining shear wave elastography, VOCAL technique, and T2* MRI of early gestation placenta to predict pre-eclampsia.

To investigate the clinical value of combining shear wave elastography (SWE) with the Volumetric Organ Computer-Aided AnaLysis (VOCAL) technique and T2* magnetic resonance imaging (MRI) to predict pre-eclampsia (PE). From December 2022 to March 2023, we recruited 31 pregnant women diagnosed with PE at our hospital as the observation group and 85 normal pregnant women as the control group. Differences in placental elasticity, vascularization index (VI), flow index (FI), vascularization flow index (VFI), and T2* MRI perfusion fraction (f) were compared between the two groups. Received operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of placental elasticity, VI, FI, VFI, f, and their combination for predicting PE. Placental elasticity was higher in the observation group than in the control group, while VI, FI, VFI, and f were lower in the observation group (all p < 0.05). The area under the curve (AUC) for placental elasticity, VI, FI, VFI, f, and their combination for predicting PE were 0.85, 0.77, 0.78, 0.84, 0.65, and 0.94, respectively. The sensitivity was 71%, 55%, 94%, 65%, 55%, and 81%. The specificity was 92%, 91%, 60%, 92%, 79%, and 98%. The combined prediction model had a higher AUC than the individual predictors (p < 0.05). SWE combined with VOCAL technique and T2* MRI has high value for predicting PE and can provide reference information for clinical diagnosis.

9765 Uterine Conserving Surgical Management of Early Placenta Accreta Syndrome (PAS)

9765 Uterine Conserving Surgical Management of Early Placenta Accreta Syndrome (PAS)

Alternative splicing in the transitional period underpinning the onset of maternal blood flow in the human early placenta.

Alternative splicing in the transitional period underpinning the onset of maternal blood flow in the human early placenta.

Porcine-specific expression ofthe three functional CYP19paralogs in early conceptus, placenta, and gonads.

Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expression was detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both conceptuses and placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of Day 21 pregnant, as well as Days 12 and 17 pseudopregnant females, we did not detect expression of Cyp19a1, Cyp19a2 or Cyp19a3. In our study we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling secretion of estrogen from the conceptus and the placenta.

A placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility and differentiation impairment in syncytiotrophoblasts

SARS-CoV-2 infection causes COVID-19. Several clinical reports have linked COVID-19 during pregnancy to negative birth outcomes and placentitis. However, the pathophysiological mechanisms underpinning SARS-CoV-2 infection during placentation and early pregnancy are not clear. Here, to shed light on this, we used induced trophoblast stem cells to generate an in vitro early placenta infection model. We identified that syncytiotrophoblasts could be infected through angiotensin-converting enzyme 2 (ACE2). Using a co-culture model of vertical transmission, we confirmed the ability of the virus to infect syncytiotrophoblasts through a previous endometrial cell infection. We further demonstrated transcriptional changes in infected syncytiotrophoblasts that led to impairment of cellular processes, reduced secretion of HCG hormone and morphological changes vital for syncytiotrophoblast function. Furthermore, different antibody strategies and antiviral drugs restore these impairments. In summary, we have established a scalable and tractable platform to study early placental cell types and highlighted its use in studying strategies to protect the placenta.

EP300 facilitates human trophoblast stem cell differentiation

Early placenta development involves cytotrophoblast differentiation into extravillous trophoblast (EVT) and syncytiotrophoblast (STB). Defective trophoblast development and function may result in severe pregnancy complications, including fetal growth restriction and pre-eclampsia. The incidence of these complications is increased in pregnancies of fetuses affected by Rubinstein-Taybi syndrome, a developmental disorder predominantly caused by heterozygous mutations in CREB-binding protein (CREBBP) or E1A-binding protein p300 (EP300). Although the acetyltransferases CREBBP and EP300 are paralogs with many overlapping functions, the increased incidence of pregnancy complications is specific for EP300 mutations. We hypothesized that these complications have their origin in early placentation and that EP300 is involved in that process. Therefore, we investigated the role of EP300 and CREBBP in trophoblast differentiation, using human trophoblast stem cells (TSCs) and trophoblast organoids. We found that pharmacological CREBBP/EP300 inhibition blocks differentiation of TSCs into both EVT and STB lineages, and results in an expansion of TSC-like cells under differentiation-inducing conditions. Specific targeting by RNA interference or CRISPR/Cas9-mediated mutagenesis demonstrated that knockdown of EP300 but not CREBBP, inhibits trophoblast differentiation, consistent with the complications seen in Rubinstein-Taybi syndrome pregnancies. By transcriptome sequencing, we identified transforming growth factor alpha (TGFA, encoding TGF-α) as being strongly upregulated upon EP300 knockdown. Moreover, supplementing differentiation medium with TGF-α, which is a ligand for the epidermal growth factor receptor (EGFR), likewise affected trophoblast differentiation and resulted in increased TSC-like cell proliferation. These findings suggest that EP300 facilitates trophoblast differentiation by interfering with at least EGFR signaling, pointing towards a crucial role for EP300 in early human placentation.