Placental Axis Dysfunction In Gestational Diabetes Mellitus Is Associated With Trophoblast Autocrine Osteocalcin

Background and objectiveGestational diabetes mellitus(GDM)has a high incidence and complicated etiology.Previous studies have shown that gestational diabetes is similar to type2 diabetes and is related to insulin resistance.Osteocalcin(OCN)has the function of regulating glucose metabolism and has been shown to promote insulin secretion and improve insulin resistance in patients with non-gestational diabetes.Therefore,this study aims to clarify the important relationship between osteocalcin and the development of gestational diabetes,the role and possible mechanism of osteocalcin-placenta,and provide new ideas for the diagnosis and treatment of gestational diabetes.MethodsThis research method is divided into two parts.The first part is a prospective,cross-sectional correlation study and WGCNA analysis of the placental transcriptome.The study was conducted in the Maternal and Child Health Hospital Guangxi Zhuang Autonomous Region from April to June 2018,based on the exclusion criteria A total of 76 pregnant women(36 gestational diabetes groups and 40 normal glucose tolerance groups)were recruited,and 36placental tissues(18 gestational diabetes groups and 21 normal glucose tolerance groups)were collected at the same time.Umbilical vein blood samples of pregnant women and maternal peripheral blood samples were tested for osteocalcin,inflammatory factors and related biochemical indicators;placental tissues were sequenced for mRNA.The continuous variable data between the two groups were compared using two independent sample t-tests,the categorical variables were analyzed byχ~2 test,and the correlation analysis was by partial correlation analysis;the placenta transcriptome was analyzed by WGCNA,GO,KEGG,and protein interaction.The second part was the recruitment of the second batch of 13 pregnant women in the Maternal and Child Health Hospital Guangxi Zhuang Autonomous Region in July 2018 for umbilical vein,umbilical artery,and maternal peripheral blood osteocalcin testing.Immunohistochemical staining of osteocalcin and GPRC6A was performed on the first batch of the placenta.Human trophoblastic choriocarcinoma cells were subjected to osteogenic induction,alizarin red staining and electron microscope scanning analysis.100 ng/mL osteocalcin was used to simultaneously stimulate human trophoblastic choriocarcinoma cells and osteocalcin receptor GPRC6A knockdown human trophoblastic choriocarcinoma cells.And use CCK-8 to detect changes in cell proliferation ability and HCG secretion ability.qRT-PCR detects changes in key aromatization genes,adiponectin,leptin,and inflammatory factors.Results:1.In the third stage of labor,there was no significant difference in the peripheral blood osteocalcin concentration of the mother in the gestational diabetes group and the normal glucose tolerance group(P>0.05);The umbilical vein adiponectin group with diabetes during pregnancy had a lower specific endometrium than the group with normal glucose tolerance(P<0.05).2.In the third stage of labor,umbilical vein blood osteocalcin concentration is negatively correlated with neonatal peripheral blood glucose concentration and leptin,and positively correlated with tumor necrosis factor-α(P<0.05).3.WGCNA analyzed a total of 6 gene modules related to adiponectin,leptin,and found 15 key genes.4.Immunohistochemistry was positive for osteocalcin and GPRC6A protein.Osteogenesis induces calcium nodules in human trophoblastic choriocarcinoma cells.5.Under the stimulation of 100 ng/mL osteocalcin,the proliferation of JAR cells and human chorionic gonadotropin increased.qRT-PCR verified that the expression of aromatase,inflammatory factors and adiponectin genes increased,while the expression of leptin decreased(P<0.05).6.After 100 ng/mL osteocalcin stimulation of GPRC6A knocked down JAR cells,the proliferation ability of JAR cells and human chorionic gonadotropin decreased.qRT-PCR verified that the expression of aromatase,inflammatory factors,and adiponectin genes all decreased,and the expression of leptin increased(P<0.05).Conclusions:1.The concentration of osteocalcin in umbilical vein blood affects the blood glucose concentration of newborns.2.The placenta may promote the synthesis of its own hormones,adipokines,and inflammatory factors through autocrine osteocalcin,and participate in the placental dysfunction of gestational diabetes.