Molecular pathways of senescence regulate placental structure and function

Abstract The placenta is an autonomous organ that maintains fetal growth and development. Its multinucleated syncytiotrophoblast layer, providing fetal nourishment during gestation, exhibits characteristics of cellular senescence. We show that in human placentas from pregnancies with intrauterine growth restriction, these characteristics are decreased. To elucidate the functions of pathways regulating senescence in syncytiotrophoblast, we used dynamic contrast‐enhanced MRI in mice with attenuated senescence programs. This approach revealed an altered dynamics in placentas of p53 −/−, Cdkn2a −/−, and Cdkn2a −/−;p53 −/− mice, accompanied by histopathological changes in placental labyrinths. Human primary syncytiotrophoblast upregulated senescence markers and molecular pathways associated with cell‐cycle inhibition and senescence‐associated secretory phenotype. The pathways and components of the secretory phenotype were compromised in mouse placentas with attenuated senescence and in human placentas from pregnancies with intrauterine growth restriction. We propose that molecular mediators of senescence regulate placental structure and function, through both cell‐autonomous and non‐autonomous mechanisms.

Appendix Figure S8. SA-β-gal activity in the murine placental labyrinth of Cdkn1a -/and p53 -/mice. Quantification of SA-β-gal activity in the placental labyrinth of Cdkn1a -/and p53 -/mice and in WT mice. Percentages of SA-β-gal positive area are shown. Values are means + SEM of three experiments.
Appendix Figure S9. Human primary trophoblasts differentiate in culture and express markers of syncytiotrophoblast. Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (A, B) On day 3 after seeding, the identity of the seeded population was determined by staining for the trophoblast marker cytokeratin7 (CK7, red) (A) and the ERVWE1 fusogene (ERVWE1, red) (B). (C) On day 3 after seeding, cell fusion was monitored by staining for phalloidin (F-actin; green) and DAPI (blue). Scale bar, 50 µM.
Appendix Figure S10. The βHCG hormone is exclusively expressed in the syncytiotrophoblast of the human placenta. Histological examination of human placenta morphology Sections of human post-partum third trimester placenta were evaluated by A) H&E staining, demonstrating the general structure of the placenta and by (B) Immunohistochemistry for the expression of the syncytiotrophoblast marker βHCG marker. Scale bar, 50 µm. Figure S11. Expression matrix of 204 modulated genes after superparamagnetic clustering (SPC) of differentially expressed genes in human primary trophoblast cultures.

Appendix
The expression data represent trophoblast cultures, derived independently from two human term placentas (labeled p1 and p2). Rows represent genes; columns represent samples. Blue denotes low expression; red denotes high expression. IUGR was defined as fetal weight below the 10th percentile for the gestational age. We included only placental specimens from pregnancies complicated with IUGR attributed to placental insufficiency, after ruling out other reasons for IUGR such as intrauterine viral infections, chromosomal abnormalities congenital anomalies, poor pregnancy dating, or maternal smoking.
Furthermore, in the IUGR group, we included placentas from pregnancies with clinical characteristics of placental insufficiency such as oligohydramnios, umbilical artery abnormalities and non-reassuring fetal heart rates. We also assessed the prevalence of histopathologic characteristics that are typical to placental insufficiency including chronic villitis, perivillous fibrin deposition, villous hypoplasia, and infarcts with fetal and maternal vascular obstruction*, all of which were significantly more abundant in the IUGR placentas included in the study.
We certified that the average gestational age at delivery was similar between the IUGR and the control groups. The control samples were taken from low risk uneventful pregnancies with a thorough follow-up during the pregnancy including normal anomaly scans. Al the fetuses were evaluated as healthy babies during the first neonatal medical evaluation.