Tricky TRIC: A replication study using trophoblast retrieval and isolation from the cervix to study genetic birth defects.

OBJECTIVE: Noninvasive Prenatal Diagnosis has recently been introduced for a limited number of monogenetic disorders. However, the majority of DNA diagnostics still require fetal material obtained using an invasive test. Recently, a novel technique, TRIC (Trophoblast Retrieval and Isolation from the Cervix), has been described, which collects fetal trophoblast cells by endocervical sampling. Since this technique has not been successfully replicated by other groups, we aimed to achieve this in the current study. METHOD: Pregnant women referred for transvaginal chorionic villous sampling (CVS) were asked for an endocervical sample prior to CVS. The TRIC samples were processed to isolate trophoblast DNA. TRIC DNA was used in ForenSeq to determine the amount of maternal DNA contamination, and for Sanger sequencing in case of a monogenic disorder. RESULTS: 23%-44% of samples had a sufficiently high fetal DNA fraction to allow genetic testing, as calculated by Sanger sequencing and ForenSeq, respectively. CONCLUSION: We have been able to successfully replicate the TRIC protocol, although with a much lower success rate as described by the original study performing TRIC. As we obtained the samples in the actual clinical setting envisioned, the method in its current setup is not advisable for use in prenatal diagnostics.

Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching.

Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome.

Peptide hormone ELABELA enhances extravillous trophoblast differentiation, but placenta is not the major source of circulating ELABELA in pregnancy.

Preeclampsia is a frequent gestational hypertensive disorder with equivocal pathophysiology. Knockout of peptide hormone ELABELA (ELA) has been shown to cause preeclampsia-like symptoms in mice. However, the role of ELA in human placentation and whether ELA is involved in the development of preeclampsia in humans is not yet known. In this study, we show that exogenous administration of ELA peptide is able to increase invasiveness of extravillous trophoblasts in vitro, is able to change outgrowth morphology and reduce trophoblast proliferation ex vivo, and that these effects are, at least in part, independent of signaling through the Apelin Receptor (APLNR). Moreover, we show that circulating levels of ELA are highly variable between women, correlate with BMI, but are significantly reduced in first trimester plasma of women with a healthy BMI later developing preeclampsia. We conclude that the large variability and BMI dependence of ELA levels in circulation make this peptide an unlikely candidate to function as a first trimester preeclampsia screening biomarker, while in the future administering ELA or a derivative might be considered as a potential preeclampsia treatment option as ELA is able to drive extravillous trophoblast differentiation.

In silico analysis of the Mus musculus uterine gene expression landscape during pregnancy identifies putative upstream regulators for labour.

BACKGROUND: The molecular pathways involved in the transition from uterine quiescence to overt labour are mapped and form the currently established pharmacological targets for both the induction and inhibition of human labour. However, both spontaneous premature labour and functional dystocia occur and are difficult to treat adequately. The identification of upstream regulators involved in the onset and orchestration of labour pathways is essential to develop additional therapies that will contribute to the regulation of the timing of birth. OBJECTIVES: To define uterine biological processes and their upstream activators involved in the transition from uterine quiescence to overt labour. STUDY DESIGN: The uterus of non-pregnant and pregnant FVB M. musculus is collected at embryonic days (E) 6.5, 8.5, 10.5, 12.5, 15.5 and 17.5 and the uterine transcriptome is determined using the Illumina mouse Ref8v2 micro-array platform. K-means clustering and Ingenuity Pathway Analysis are applied to further dissect the transcriptome data. RESULTS: From E6.5 to E17.5, 5405 genes are significantly differentially expressed and they segregate into 7 unique clusters. Five of the 7 clusters are enriched for genes involved in specific biological processes that include regulation of gene-expression, T-cell receptor activation, Toll-like receptor signalling and steroid metabolism. The identification of upstream activators for differentially expressed genes between consecutive time points highlights the E10.5 to E12.5 window during which the role from progesterone switches from an activated state to the inhibited state reflecting the process of functional progesterone withdrawal essential for the transgression from myometrial quiescence to synchronized contractions. For this time window in which 189 genes are differentially expressed we define 22 putative upstream activators of which NUPR1 and TBX2 are the most significant with respectively an activated and an inhibited status. CONCLUSIONS: Gene expression profiling of mice uterus from E6.5 to E17.5 results in 7 unique gene expression clusters from early to late pregnancy that define the landscape of molecular events in ongoing pregnancy. In the current dataset progesterone is predicted as an activated upstream regulator and maintainer of myometrial quiescence and is active till E10.5. Progesterone is predicted as an inhibited upstream regulator at E12.5. We identify 22 upstream regulators in the E10.5 to E12.5 time window where the switch to progesterone withdrawal occurs. They are putative relevant upstream activators of labour.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

Total bile acids in the maternal and fetal compartment in relation to placental ABCG2 expression in preeclamptic pregnancies complicated by HELLP syndrome.

OBJECTIVE: To investigate total bile acid (TBA) levels in maternal (MB) and umbilical cord blood (UCB) in normotensive, preeclamptic (PE), and PE pregnancies complicated by hemolysis elevated liver enzymes and low platelets (HELLP) syndrome in the context of ABCG2 placental gene expression levels, a recently reported placental bile acid transporter. METHODS: TBA levels were determined in 83 paired MB and UCB samples of normotensive, PE and PE/HELLP pregnancies and in 22 paired arterial and venous UCB samples from uncomplicated term pregnancies. ABCG2 gene expression was measured in 104 human placentas by reverse transcriptase quantitative polymerase chain reaction. RESULTS: Overall, TBA levels in MB are higher compared to levels in UCB (p<0.0001), but this comparison looses statistical significance for the 11 PE/HELLP cases. TBA levels in maternal blood are increased in PE/HELLP compared to PE pregnancies (p=0.016). TBA levels in arterial and venous UCB from 22 normotensive pregnancies are not statistically different. ABCG2 expression is reduced in pregnancies where preeclampsia is further complicated by HELLP syndrome. ABCG2 expression in human placenta is not correlated with TBA levels in either the maternal or fetal compartment. CONCLUSION: Increased maternal TBA levels in PE/HELLP pregnancies indicate a relation between bile acids in the maternal circulation and HELLP syndrome. As overall TBA levels in maternal blood are increased compared to UCB, we conclude that the placenta partly protects the fetus from increased maternal TBA levels. This consistent difference in TBA levels between the maternal and fetal compartment is unrelated to the placental expression of ABCG2.