Transcriptomic analysis of the chorioallantois of equine cloned pregnancies

Abstract

Despite notable accomplishments over the last 25 years, somatic cell nuclear transfer (SCNT) still suffers from high pregnancy loss rate and a high incidence of abnormal offspring. The problems could be related to placental anomalies, which in turn may originate in defective epigenetic reprogramming. It is well known that a high percentage of SCNT pregnancies are associated with placental abnormalities including hydrallantois, reduced vascularization, and edematous placental membranes. To date, there is little known about the underlying mechanisms of the placental pathologies associated with cloned equine fetuses. Our study aimed to (1) characterize the transcriptome and biological pathways of placentas from cloned pregnancies at term, and (2) compare the transcriptome of placentas from cloned pregnancies that result in normal foals with placentas of cloned pregnancies resulting in non-viable foals. Chorioallantois (CA) was collected from the pregnant horn of placentas after normal parturition (mean gestational age of 339 days). A total of 19 samples were analyzed from 5 healthy, age-matched control pregnancies by artificial insemination (CON), 7 cloned pregnancies resulting in a healthy live foal (SCNT-L), and 7 cloned pregnancies resulting in a foal that died within the hour after birth (SCNT-D). Total RNA was extracted and sequenced, generating ∼33 million reads/sample. The raw reads were trimmed (Trimmgalore) and mapped (STAR) to the current equine reference genome. Mapped reads were quantified (FeatureCounts), and differentially expressed genes (DEGs) were evaluatedusing DESeq2, followed by gene ontology (GO) analysis. Macroscopically, 50% of the placentas from SCNT derived pregnancies showed edema. We identified 1332 DEGs (FDR <0.05, FoldChange>2) between the SCNT-L and CON groups and 1651 DEGs between the SCNT-D and CON groups. Pathway analysis of DEGs demonstrated that these genes are involved with extracellular matrix/ structure organization, response to growth factor, angiogenesis, and integrin signaling pathway independent of the outcome of the cloned pregnancies. When comparing the SCNT-L and SCNT-D groups, we identified 44 DEGs, mainly involved in inflammatory processes, even though histopathological examinations of the CA samples did not reveal any ongoing inflammatory process. Overall, our data highlights the underlying molecular pathways associated with abnormal placental development in cloned equine fetuses. Moreover, our data demonstrated the disruption of angiogenesis in the cloned placenta, manifested as excessive edema in the placenta. Similarly, in a previous study, we demonstrated the association between abnormal angiogenesis and placental edema and hydrallantois in equine pregnancy, highlighting the importance of adequate angiogenesis in placental function. This study was funded by the Center for Equine Health at UC Davis, FAPESP 2020/10260-3, the BOF Special Research Fund at Ghent University, a Fellowship of the Belgian American Educational Foundation, and the L'Oréal-UNESCO Fellowship.