(363) Understanding IVF-Induced Placental transcriptional Changes at Single-Nucleus Resolution

Reza K. Oqani¹, Emin Maltepe¹, Paolo Rinaudo¹ and Daniel E. Wagner¹

¹University of California San Francisco, San Francisco, CA

Abstract Text:

Background:

In vitro fertilization (IVF) is associated with increased risks of placental dysfunction, fetal growth abnormalities, and pregnancy complications, yet the molecular mechanisms underlying these changes remain poorly understood. The placenta, a highly specialized organ regulating maternal-fetal exchange, hormone production, and immune tolerance, undergoes complex lineage differentiation. This study aimed to investigate how IVF alters placental development at the transcriptomic level using single-nucleus RNA sequencing (snRNA-seq) to profile gene expression changes across distinct placental cell types.

Methods:

IVF conceptuses were generated by superovulating CF-1 female mice with PMSG (5 IU) followed by hCG (5 IU) 48 hours later, then retrieving MII oocytes from ampullae. Sperm were collected from B6D2F1 males, capacitated in HTF medium, and co-incubated with oocytes for 4 hours. Zygotes were cultured in KSOMaa medium until the blastocyst stage. For the FB control group, naturally fertilized E3.5 blastocysts were collected from superovulated CD-1 females mated with B6D2F1 males. Blastocyst-stage embryos were transferred into one uterine horn of pseudo-pregnant CD-1 females (16 embryos per recipient).

At E12.5, placentas from three recipients per group were collected, weighed, flash-frozen, and subjected to single-nucleus isolation using the Singulator 100 system (S2 Genomics). Automated nuclei dissociation was performed, and nuclear suspensions were assessed via AO/PI staining (Cellaca MX, Nexcelom). Libraries were prepared using the 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kit v3.1 and sequenced on an Illumina NovaSeq 6000 (S4 flow cell, 500M reads per GEX library).

snRNA-seq data were processed using STARsolo, ScanPy, and Seurat. Nuclei with < 1,000 detected UMIs or >5% mitochondrial transcripts were excluded. Batch correction (Harmonypy), clustering (Leiden algorithm, resolution = 1.5), and UMAP visualization were performed. Differential gene expression analysis was conducted using the Wilcoxon rank-sum test with Benjamini-Hochberg FDR correction, and pseudobulking followed by DESeq2 analysis. GO and KEGG pathway enrichment were performed using ShinyGO.

Results:

We identified 38 cell clusters, including trophoblasts (11 subtypes), fetal mesenchyme, pericytes, endothelial cells, and immune cells. While cell-type proportions remained largely unchanged, we detected 199 differentially expressed genes (DEGs) in IVF placentas, particularly in parietal trophoblast giant cells (P-TGCs). The placental lactogen genes Prl3d1, Prl2c3, and Prl7a1 were upregulated, suggesting altered endocrine regulation. Additionally, H19, an imprinted gene critical for placental and fetal growth, was significantly overexpressed in P-TGCs, hinting at epigenetic dysregulation. GO and KEGG analyses revealed enrichment in pathways linked to lactation, JAK-STAT signaling, and hypoxia responses, while genes involved in trophoblast differentiation and metabolic regulation were downregulated. RT-qPCR confirmed upregulation of Prl3d1, Prl7a1, Prl2c3, and H19, and downregulation of Fam13a, a marker of trophoblast differentiation.

Conclusion:

Our findings indicate that IVF induces distinct transcriptional changes in the placenta, particularly in hormone-regulating trophoblast subtypes. The upregulation of placental lactogens and imprinted genes suggests potential alterations in placental function and fetal adaptation. These findings provide insights into how IVF influences placental biology at a molecular level, emphasizing the need for further research into optimizing ART protocols to minimize placental dysfunction and associated pregnancy risks.