Abstract
Overview: Hypertensive disorders in pregnancy (HDP), of which the most common is pre- eclampsia (PE), are a leading cause of maternal and foetal morbidity and mortality worldwide. Despite extensive research, no clear cause or mechanism for HDP has been discovered. Human papillomavirus (HPV) has transformative abilities among many cell types and alters key pathways in reproductive cells essential for embryo implantation and placental development in vitro. It has also been consistently found in the placentae, especially in those complicated by PE. These findings suggest HPV may not be only a bystander in complicated pregnancies, but has a direct role in abnormal placental development. Whether HPV genes such as E6/E7 that encode the E6 and E7 proteins, which affect trophoblast function in vitro, are expressed in vivo is unknown. Understanding gene expression profiles in placental tissue is complicated by the wide variety of cell types present and their distinct genetic origins. This study used RNAscope® to investigate whether in situ based techniques could identify HPV E6/E7 expression differences in placentas from a cohort of Japanese women. A second aspect of this study used RNAscope® to investigate if genes found to be differentially methylated in the cohort were associated with HDP. NOTCH2 and CMIP were chosen.
Methods: Fifteen placenta from uncomplicated pregnancies, sixteen placenta from pregnancies complicated by HDP, and six additional cases of non-HDP pathologies, were converted to FFPE slides and examined using RNAScope®, or immunohistochemistry. RNAscope® assays to determine RNA quality, non-specific staining, HPV high-risk E6/E7, HPV low-risk E6/E7 and NOTCH2 were undertaken, along with immunohistochemistry for CMIP. Results were compared between the HDP and control groups.
Results: The majority of placentas from the HDP (n=15/16, 94%) and the control (n=11/15, 73%) cohorts had sufficient RNA quality. In the HDP group, six cases (n=6/15) tested positive for high risk HPV E6/E7 (40%), and five tested positive for low risk HPV E6/E7 (33%). None of the control cases tested positive for high risk HPV (0%) or low risk HPV (0%). Expression of HPV high and low risk E6/E7 was increased in the HDP compared to the control cohort (p<0.05). The NOTCH2 assay was optimised and NOTCH2 was expressed heterogeneously between neighbouring cells in individual cases, but the number of cases tested was too low for statistical power. The CMIP assay did not work, requiring further optimisation.
Conclusion: This study provides evidence that HPV high and low risk E6/E7 is expressed in the placenta of pregnancies complicated by HDP. These results add validity that HPV may affect placental function by showing HPV gene expression is active in the HDP affected placenta. Further research is needed to understand why transcriptionally active HPV E6/E7, of both high and low risk types, would be present in the developing and term placenta. In addition, RNAscope® proved viable for visualising cell types to target for further sequencing, which could be used to determine if one cell population is more differentially methylated within the heterogeneous placenta.