Attenuation of placental pyruvate kinase M2 promotes oxidative imbalance and enhances inflammatory- apoptosis cross talk in rats with hyperhomocysteinemia associated pregnancy loss

Abstract

Abstract 10-15% of clinically recognized pregnancies end in miscarriage. Hyperhomocysteinemia in pregnant women has been associated with deep venous thrombosis, recurrent miscarriage, preeclampsia to name a few. Impaired placental function due to overt oxidative stress is one of the key mechanisms in development of pregnancy loss. Paucity of pathway–based microarray approach in embryonic–endometrial communication warrants elucidation of distinct profile of miRNAs in hyperhomocysteinemia-associated pregnancy loss (HAPL). Hyperhomocysteinemia was induced at a dose of 100mg/kg body-weight/day for D1-D18 of pregnancy. Placental histology by haematoxylin-eosin staining documented thrombus with reduced area of spongiotropoblasts in chorionic plate vessel. Placental mRNA was subjected to microarray analysis followed by pathway-analysis using Ingenuity Pathway Analysis (IPA). Genes involved in reproductive physiology, inflammatory pathways, immune responses, homocysteine metabolism, glucose metabolism, and oxidative stress were differentially expressed in HAPL. 21 pathways documented by IPA, were skewed to 10 by recursive feature elimination highlighting possible deregulation/s. Expression/s was re-confirmed by quantitative real- time PCR (qRT-PCR), western blot and flow cytometric analysis (FACS). Nine priori molecules (PKM2, AKT, PI3K, NF-κB, COX-2, sflt-1, HIF-1α, bax, caspase 9) were specifically modulated in HAPL as demonstrated by protein and mRNA expression. A parallel increase in insulin signaling (PI3K+,AKT+), inflammation (COX2+,NF-κB+), hypoxia (sflt-1+,HIF-1α+), apoptosis (bax+,caspase9+) with concomitant decrease in pyruvate kinase M2 in hyperhomocysteinemic placental cells by FACS with CD56, a marker for pregnancy loss was documented. The findings provide evidence that an oxidative stress-mediated placental damage perhaps represents the pathogenesis of HAPL, which may explore pathway-based therapeutic options for recurrent miscarriage.10–15% of clinically recognized pregnancies end in miscarriage. Hyperhomocysteinemia in pregnant women has been associated with deep venous thrombosis, recurrent miscarriage, preeclampsia to name a few. Impaired placental function due to overt oxidative stress is one of the key mechanisms in development of pregnancy loss. Paucity of pathway–based microarray approach in embryonic–endometrial communication warrants elucidation of distinct profile of miRNAs in hyperhomocysteinemia-associated pregnancy loss (HAPL). Hyperhomocysteinemia was induced at a dose of 100mg/kg body-weight/day for D1-D18 of pregnancy. Placental histology by haematoxylin-eosin staining documented thrombus with reduced area of spongiotropoblasts in chorionic plate vessel. Placental mRNA was subjected to microarray analysis followed by pathway-analysis using Ingenuity Pathway Analysis (IPA). Genes involved in reproductive physiology, inflammatory pathways, immune responses, homocysteine metabolism, glucose metabolism, and oxidative stress were differentially expressed in HAPL. 21 pathways documented by IPA, were skewed to 10 by recursive feature elimination highlighting possible deregulation/s. Expression/s was re-confirmed by quantitative real- time PCR (qRT-PCR), western blot and flow cytometric analysis (FACS). Nine priori molecules (PKM2, AKT, PI3K, NF-κB, COX-2, sflt-1, HIF-1α, bax, caspase 9) were specifically modulated in HAPL as demonstrated by protein and mRNA expression. A parallel increase in insulin signaling (PI3K+,AKT+), inflammation (COX2+,NF-κB+), hypoxia (sflt-1+,HIF-1α+), apoptosis (bax+,caspase9+) with concomitant decrease in pyruvate kinase M2 in hyperhomocysteinemic placental cells by FACS with CD56, a marker for pregnancy loss was documented. The findings provide evidence that an oxidative stress-mediated placental damage perhaps represents the pathogenesis of HAPL, which may explore pathway-based therapeutic options for recurrent miscarriage.