Histology Atlas of the Developing Mouse Placenta

The use of the mouse as a model organism is common in translational research. This mouse–human similarity holds true for placental development as well. Proper formation of the placenta is vital for development and survival of the maturing embryo. Placentation involves sequential steps with both embryonic and maternal cell lineages playing important roles. The first step in placental development is formation of the blastocyst wall (approximate embryonic days [E] 3.0-3.5). After implantation (∼E4.5), extraembryonic endoderm progressively lines the inner surface of the blastocyst wall (∼E4.5-5.0), forming the yolk sac that provides histiotrophic support to the embryo; subsequently, formation of the umbilical vessels (∼E8.5) supports transition to the chorioallantoic placenta and hemotrophic nutrition. The fully mature (“definitive”) placenta is established by ∼E12.5. Abnormal placental development often leads to embryonic mortality, with the timing of death depending on when placental insufficiency takes place and which cells are involved. This comprehensive macroscopic and microscopic atlas highlights the key features of normal and abnormal mouse placental development from E4.5 to E18.5. This in-depth overview of a transient (and thus seldom-analyzed) developmental tissue should serve as a useful reference to aid researchers in identifying and describing mouse placental changes in engineered, induced, and spontaneous disease models.

Can Prenatal and Postnatal Cell Phone Exposure Increase Adverse Maternal, Infant and Child Outcomes?

Objective To determine the association between maternal mobile phone use and adverse outcomes in infants, children, and mothers. Method In March 202, we conducted a search on the MEDLINE, Embase, and Scopus databases. Data extraction and an assessment of the quality of the studies were performed by two authors. The quality of the studies was assessed using the checklist of the Newcastle-Ottawa scale. Results Studies assessing behavioral problems in infants aged 6 to 18 months reported null findings. However, an increased risk of emotional and behavioral disorders was observed in children aged between 7 and 11 years whose mothers had been exposed to cell phones. The findings regarding the association between maternal cell phone exposure and adverse outcomes in children aged 3 to 5 are controversial. A study found a significant association between the call time (p = 0.002) or the history of mobile phone use (in months) and speech disorders in the children (p = 0.003). However, another study found that maternal cell phone use during pregnancy was not significantly associated with child psychomotor and mental developments. Inconclusive results were observed about the adverse outcomes in fetuses, such as fetal growth restriction or t scores for birth weight in cell phone users as opposed to non-users. On the contrary, the children of mothers who were cell phone users had a lower risk of scoring low on motor skills. Similar results were observed regarding the adverse outcomes of cell phone use in infants, such as fetal growth restriction or low birth weight, and the risk of preeclampsia was lower among subjects with medium and high cell phone exposure, as opposed to those with low exposure. Conclusion Studies on behavioral problems have reported different postnatal results, such as null findings among infants and a positive association in children.

Whole-Genome Methylation Analysis Revealed ART-Specific DNA Methylation Pattern of Neuro- and Immune-System Pathways in Chinese Human Neonates

The DNA methylation of human offspring can change due to the use of assisted reproductive technology (ART). In order to find the differentially methylated regions (DMRs) in ART newborns, cord blood maternal cell contamination and parent DNA methylation background, which will add noise to the real difference, must be removed. We analyzed newborns’ heel blood from six families to identify the DMRs between ART and natural pregnancy newborns, and the genetic model of methylation was explored, meanwhile we analyzed 32 samples of umbilical cord blood of infants born with ART and those of normal pregnancy to confirm which differences are consistent with cord blood data. The DNA methylation level was lower in ART-assisted offspring at the whole genome-wide level. Differentially methylated sites, DMRs, and cord blood differentially expressed genes were enriched in the important pathways of the immune system and nervous system, the genetic patterns of DNA methylation could be changed in the ART group. A total of three imprinted genes and 28 housekeeping genes which were involved in the nervous and immune systems were significant different between the two groups, six of them were detected both in heel blood and cord blood. We concluded that there is an ART-specific DNA methylation pattern involved in neuro- and immune-system pathways of human ART neonates, providing an epigenetic basis for the potential long-term health risks in ART-conceived neonates.

The Influence of Maternal Cell Contamination on Fetal Aneuploidy Detection Using Chip-Based Digital PCR Testing

Prenatal samples obtained by amniocentesis or chorionic villus sampling are at risk of maternal cell contamination (MCC). In traditional prenatal analysis, MCC is recommended to be assayed by special tests, such as the short tandem repeat analysis and, if detected at a high level, may result in failed analysis report. The objective of this study was to test the ability of chip-based digital PCR to detect fetal aneuploidies in the presence of MCC. To determine the level of accuracy of MCC detection, an aneuploid male sample was subjected to serial dilution with an euploid female sample. DNA was extracted from prenatal samples and analyzed with QuantStudio 3D Digital PCR. Digital PCR analysis allowed the detection of trisomy 21, trisomy 18, and X monosomy accurately in samples with 90%, 85%, and 92% of MCC, respectively. Moreover, our results indicated that digital PCR was able to accurately confirm the presence of Y chromosome at up to 95% contamination. The amniotic fluid and chorionic villus sampling (CVS) received in our clinical laboratory was subjected to further analysis of MCC based on the aneuploidy assessment algorithm, resulting in the identification of 10 contaminated samples and four cases of true fetal mosaicism. We conclude that chip-based digital PCR analysis enables the detection of fetal aneuploidy with high levels of accuracy, even in cases of significant MCC. Importantly, the algorithm eliminates the need for maternal DNA and additional MCC tests, which reduces costs and simplifies the diagnostic procedure. The method is easy to set up and suitable for routine clinical practice.

O-237 A non-invasive approach for aneuploidy analysis in clinical miscarriages

Study question Is maternal cell-free DNA (cfDNA) testing a feasible alternative to the analysis of the product of conception (POC) in clinical miscarriages? Summary answer This study demonstrates that genome-wide cfDNA testing in the maternal bloodstream constitutes a reliable tool to analyse chromosome aneuploidies in clinical miscarriages. What is known already It is well established that 50-70% of clinical miscarriages are caused by numerical chromosomal anomalies (aneuploidies), mostly trisomies. To date, conventional cytogenetic and advanced molecular techniques are used for the analysis of POC to identify the genetic cause of miscarriage, providing valuable information for genetic counselling. However, both approaches are based in the direct analysis of the abortive tissue, which entails several limitations due to the risk of culture failure and/or maternal cell contamination. To solve these drawbacks, maternal cfDNA testing emerges as a promising alternative due to the accumulated evidence. Study design, size, duration This was a retrospective study conducted in a reference genetic laboratory from January to December 2020. Before carrying out the foetal tissues collection that precludes the POC analysis, a blood sample was drawn to evaluate possible aneuploidies by cfDNA testing. Using NGS+STR POC results as the gold standard, results derived from both studies were compared to assess the percentage of concordance and the cases of non-informativeness (foetal fraction (FF) <2%), false positives, and false negatives. Participants/materials, setting, methods A total of 12 cases were included in the study. cfDNA testing in the mother’s blood was performed by using Illumina’s technology platform. Genetic testing for POC was done using an NGS technology (Thermo Fisher Scientific, USA) for 24 chromosome aneuploidy screening. Short-tandem repeat (STR) analysis allowed us to detect or rule out maternal cell contamination (MCC) and some types of polyploidies. Main results and the role of chance The non-informative rate for both analysis techniques was 9.1% (1 out of 12 cases: 1 low FF for cfDNA testing and maternal cell contamination for POC analysis). The median cfDNA FF was 9.0%. Using the molecular POC analysis as gold standard, the concordance rate between both studies was 90.0% (9 out of 10 cases;1 monosomy X, 1 trisomy (T) 21, 1 T22, 1 T11 and 5 patients with no alteration detected). No mosaics or structural rearrangements were identified by either of the two analysis techniques. The only discordant result was a case in which cytogenetics of POCs identified a triploidy. This discordancy is expected since triploidies are outside the scope of cfDNA testing. Also, foetal sex was correctly assigned in all informative cases. The sensibility and specificity of the study were estimated at 80.0 (4/5) and 100.0% (6/6), respectively. Statistics analysis suggested that no significant difference was found between both techniques regarding the aneuploidy detection ability (P=0.5). These promising results indicate that genome-wide cfDNA-based screening provides a non-invasive approach for determining whether foetal aneuploidy could explain the loss in patients experiencing early o recurrent pregnancy loss (RPL). Limitations, reasons for caution The sample size prevents drawing more significant conclusions regarding the diagnosis power similarity between both testing techniques. Therefore, a larger cohort will be essential to improve confirm the cfDNA testing performance. Current cfDNA testing technology fails in polyploidy identification, which is a potential cause of pregnancy loss. Wider implications of the findings CfDNA testing could be an alternative to POC analysis in clinical miscarriage. If optimized, cfDNA testing could be used contingently with the molecular POC analysis in cases where maternal cell contamination is present. As a result, the overall success rate in the POC program could be substantially improved. Trial registration number NA

O-046 Implantation and beyond, the endometrial perspective

text Early pregnancy failures are spread throughout the first few weeks, suggesting that impairment of normal biological processes can occur at sequential stages: epithelial attachment and breaching, early stromal and then decidual invasion, glandular and vascular invasion. Biopsy-based transcriptomics and proteomics have failed to demonstrate a reproducible or interpretable molecular signature for endometrial receptivity, but single cell RNAseq suggests a step change in the epithelial transcriptome in the mid secretory phase, consistent with the appearance of new cell phenotype(s). Previously unseen heterogeneity in both epithelial and stromal cell populations has become evident, but gene signatures have not yet achieved a level of resolution that allows insights clear enough to decode the biology of the receptive state. However, methodology for propagating and recombining endometrial cell populations into 3D engineered tissue models has advanced, so that new mechanistic questions can be asked. Initial acquisition of receptivity to implantation is followed by the development of a supportive environment for embryos that possess the capacity to progress, with maternal cell populations (epithelial, stromal, immune and vascular) acting cooperatively within a remodelling extracellular matrix (ECM). A balance must be achieved in the ECM between breakdown, with opening of hydrated spaces to allow expansion of the embryonic sac while maintaining a substrate for stable physical attachment to allow invasion by extravillous trophoblast. The extent and nature of cellular trafficking to and from the uterus is important before and during early pregnancy. There is evidence that regulation of a resident senescent cell subpopulation by uterine NK cells occurs in concert with ECM remodelling to achieve a functionally supportive environment in the early first trimester. Access of bone marrow-derived cells to vessel walls is regulated by placental-endothelial signalling to initiate remodelling for the increased blood flow to the conceptus that is required in later pregnancy. Thus ‘receptivity’ and ‘supportiveness’ require normal cell proportions and functional phenotypes in multiple endometrial cell populations and their physical environment in order to allow a well-calibrated sequential developmental response in the conceptus.

Detection of cytogenomic abnormalities by OncoScan microarray assay for products of conception from formalin-fixed paraffin-embedded and fresh fetal tissues

Background The OncoScan microarray assay (OMA) using highly multiplexed molecular inversion probes for single nucleotide polymorphism (SNP) loci enabled the detection of cytogenomic abnormalities of chromosomal imbalances and pathogenic copy number variants (pCNV). The small size of molecular inversion probes is optimal for SNP genotyping of fragmented DNA from fixed tissues. This retrospective study evaluated the clinical utility of OMA as a uniform platform to detect cytogenomic abnormalities for pregnancy loss from fresh and fixed tissues of products of conception (POC). Results Fresh specimens of POC were routinely subjected to cell culture and then analyzed by karyotyping. POC specimens with a normal karyotype (NK) or culture failure (CF) and from formalin-fixed paraffin-embedded (FFPE) tissues were subjected to DNA extraction for OMA. The abnormality detection rate (ADR) by OMA on 94 cases of POC-NK, 38 cases of POC-CF, and 35 cases of POC-FFPE tissues were 2% (2/94), 26% (10/38), and 57% (20/35), respectively. The detected cytogenomic abnormalities of aneuploidies, triploidies and pCNV accounted for 50%, 40% and 10% in POC-CF and 85%, 10% and 5% in POC-FFPE, respectively. False negative result from cultured maternal cells and maternal cell contamination were each detected in one case. OMA on two cases with unbalanced structural chromosome abnormalities further defined genomic imbalances and breakpoints. Conclusion OMA on POC-CF and POC-FFPE showed a high diagnostic yield of cytogenomic abnormalities. This approach circumvented the obstacles of CF from fresh specimens and fragmented DNA from fixed tissues and provided a reliable and effective platform for detecting cytogenomic abnormalities and monitoring true fetal result from maternal cell contamination.

Characterisation of two unusual cases of haemoglobin Bart’s hydrops foetalis caused by –SEA and large novel α-globin gene cluster deletions

Background We describe 2 unusual haemoglobin (Hb) Bart’s hydrops cases that could not be explained by traditional factors. Case presentation: Two families with a diagnosis or history of foetal hydrops were enrolled. A suspension-array system was used to detect the 23 most frequent mutations in southern China. Multiplex ligation-dependent probe amplification (MLPA) was used to screen for possible deletions. Precise characterisation of the breakpoints of the novel variants and uniparental disomy analysis were performed using a single nucleotide polymorphism (SNP) array. Quantitative fluorescence PCR was used to eliminate maternal cell contamination and nonpaternity. In case 1, the suspension-array system indicated a maternal heterozygous (–SEA/) deletion, and the paternal sample was negative. The foetal hydrops was caused by the maternal (–SEA/) deletion and a de novo α-globin gene deletion (–193). In case 2, the paternal sample had a heterozygous (–SEA/) deletion, and MLPA and SNP array analysis revealed a large maternal deletion (–227) that encompassed the α-globin gene, which explained the history of Hb Bart’s foetal hydrops. Conclusions Our cases describe 2 new α0-thalassaemia deletions and illustrate the importance of using a combination of methods to detect rare types of α-thalassaemia.

Pregnant Females as Historical Individuals: An Insight From the Philosophy of Evo-Devo

Criticisms of the “container” model of pregnancy picturing female and embryo as separate entities multiply in various philosophical and scientific contexts during the last decades. In this paper, we examine how this model underlies received views of pregnancy in evolutionary biology, in the characterization of the transition from oviparity to viviparity in mammals and in the selectionist explanations of pregnancy as an evolutionary strategy. In contrast, recent evo-devo studies on eutherian reproduction, including the role of inflammation and new maternal cell types, gather evidence in favor of considering pregnancy as an evolved relational novelty. Our thesis is that from this perspective we can identify the emergence of a new historical individual in evolution. In evo-devo, historical units are conceptualized as evolved entities which fulfill two main criteria, their continuous persistence and their non-exchangeability. As pregnancy can be individuated in this way, we contend that pregnant females are historical individuals. We argue that historical individuality differs from, and coexists with, other views of biological individuality as applied to pregnancy (the physiological, the evolutionary and the ecological one), but brings forward an important new insight which might help dissolve misguided conceptions.