Effect On The Glucose Metabolism In Offspring Conceived By Mothers With Hyperandrogenism And Mechanism Involved

Part Ⅰ Clinical follow-up study,imprinting gene expression and methylation status of offspring conceived by mothers with pre-conception hyperandrogenismObjective:To investigate the development,blood pressure,blood lipids and glucose levels in offspring conceived by mothers with pre-conception hyperandrogenism at pre-school age.To detect the expression of imprinting genes associated with glucose metabolism and the methylation status of imprinting genes in offspring,in order to evaluate the effect of maternal hyperandrogenism on the phenotype and epigenetics of offspring.Materials and Methods:We recruited offspring conceived by patients diagnosed with hyperandrogenism in Women Hospital,Zhejiang University during the years of 2002-2008.The control group were mathed according to the modes of conception(spontaneously conceived,hyperovulation,IVF-ET),all control offspring were conceived by women with tubal blockage or male factor infertility during the same period.We analyzed the perinatal characteristics,development,blood glucose(fasting and OGTT),blood lipids and blood pressure.We detected the expression of imprinting genes associated with glucose metabolism by Real-time PCR and the methylation status of imprinting genes by Bisulfite sequencing PCR(BSP)in offspring peripheral lymphocyte.We collected abandoned oocyte,detect the expression of IGF2 by immunofluorescence after treating with 10-9M DHT.Results:1.We found that there were no significant differences in the birth weight,gestational age and preterm birh rate between the two groups;2.At follow-up,there were no significant differences in the age,BMI,blood pressure and blood lipids between the two groups,the fasting glucose level of offspring conceived by mothers with hyperandrogenism was significantly higher(4.72 ± 0.04mmol/L vs.4.60±0.03mmol/L,p<0.05),and the fasting insulin level(3.58±0.18μU/ml vs.3.12±0.15μU/ml,p<0.05)and HOMA-IR(0.76±0.04 vs.0.66±0.03,p<0.05)were significantly higher compared with control;3.We conducted OGTT in 74 offspring in hyperandrogenism group and 66 offspring in control group,the 120min glucose and insulin levels of hyperandrogenism offspring were significantly higher compared to control;4.The expression of IGF2 and GRB20 significantly increased in lymphocyte of hyperandrogenism offfspring,the methylation levels of IGF2 DMR2 and GRB10 DMR were significantly decreased compared with control;In-vitro treatment of DHT significantly upregulated IGF2 expression in oocyte.Conclusions:Offspring conceived by mothers with pre-conception hyperandrogenism manifested significantly increased fasting glucose and fasting insulin levels,however,there was no one with abnormal blood glucose;Altered expression of IGF2 and GRB10 was associated with altered blood glucose level in offspring conceived by mothers with hyperandrogenism while the decreased methylation level was the mechanism of increased expression,indicating that the hyperandrogenism of mothers altered the epigenetics of their offspring.Part Ⅱ Phenotype of rat offspring conceived by mothers with pre-conception hyperandrogenism,expression and methylation status of imprinting gene in FO oocyte and offspring pancreatic isletObjective:To study the glucose and insulin metabolism in rat offspring conceived by mothers with pre-conception hyperandrogenism;to explore the effect of hyperandrogenism on the Igf2 expression and Igf2 DMR2 methylation level in FO oocyte and F1 pancreatic islet.Materials and Methods:We established a pre-conception hyperandrogenism rat model,and detect the testosterone and free androgen index level of FO at 6-weeks old.At 7-weeks of age,FO was intercoursed with normal male rat to obtain HA-F1 and normal 7-weeks old female rat was intercoursed with normal male to obtain Ctrl-F1.We detected the offspring’s birth weight,body weight at 3-and 8-weeks old,daily energy intake and daily water intake,and we invested fastig glucose level and GTT glucose using blood glucose meter,fasting and GTT insulin level using ELISA kit.Further,we separated the pancreatic islets from FO and MⅡ oocyte from F1,to detect the expression of imprinting genes by real-time RT-PCR and methylation status by BSP.Results:1.We found out that the birth weight didn’t differ between the two groups;2.Offspring conceived by hyperandrogenism mothers showed significantly increased daily energy and water intake in childhood which persisted into adulthood;3.In 3-weeks old,the fasting glucose level of HA-offspring was significantly higher,while 27%HA-offspring were with diabetes.In 8-weeks old,although there was no significant difference in fasting glucose level,the glucose levels of HA-offspring at 30min and 60min after glucose injection were significantly increased,76%HA-offspring showed diabetes in adulthood;4.The fasting insulin level in HA-offspring was significantly lower in 3-weeks old,in 8-weeks old,the fasting insulin didn’t show significance which was consistent with fasting glucose,however,the GTT insulin was significantly decreased compared with control;5.Igf2 expression was significantly increased in HA-offspring pancreatic islet in accordance with decreased methylation Igf2 DMR2 level;6.Igf2 expression was also significantly increased in HA-FO oocyte in accordance with decreased methylation Igf2 DMR2 level,interestingly,CpG sites 4,7 and 9 were both hypomethylated in FO oocyte and offspring islet.Conclusions:1.Maternal hyperandrogenism predisposed off-spring to diabetes;2.Offspring conceived by mothers with hyperandrogenism showed islet secretion dysfunction,which led to diabetes;3.One potential candidate underlying the islet dysfunction was the altered expression of Igf2,attributed to hypomethylation of Igf2 DMR2;5.Excessive androgen exposue altered expression of Igf2 stemed from Igf2 DMR2 hypomethylation;6.Hyperandrogenism altered IGF2 methylation in mother’s germ cells which is subsequently inherited and maintained in offspring somatic cells,leading to diabetes in the offspring.Part Ⅲ Effect of hyperandrogenism on DNMT3a expression in rat oocyte and human granulosa cellsObjective:To investigate the effect of hyperandrogenism on DNMT3a in vivo and in vitro,and to explore the possible molecular mechanism.Materials and Methods:We obtained MⅡ oocyte,compared the expression of DNM’T3a between HA and Ctrl groups by immunofluorescence;We treated human primary granulosa cells and KGN granulosa cells with DHT(10-11M-10-8M),and treated cells with androgen receptor(AR)SiRNA,then detected the expression of DNMT3a in mRNA level(real-time PCR)and protein level(western blot);STAT3 is a transcription factor,we detected the effect of DHT on STAT3 expression in KGN granulosa cells using real-time PCR and western blot;Chromatin immunoprecipitation(ChIP)experiments were performed to detect the binding of STAT3 on the STAT3 response element site of DNMT3a DNA.Results:1.In vivo study showed that hyperandrogenism increased DNMT3a expression in MⅡ oocyte;2.In vitro study showed that DHT down-regulated the expression of DNMT3a and up-regulated the expression of IGF2 in human primary granulosa cells.Similarily,DHT down-regulated DNMT3a mRNA in a dose-dependent manner in KGN cells,the down regulation of DNMT3a by DHT was partially abolished by AR knock down;3.DHT down-regulated STAT3 mRNA and protein in dose-dependent manner in KGN cells;4.STAT3 binds to DNMT3a promoter at-1118bp upstrem from the transcriptional start site.Conclusions:1.Hyperandrogenism decreased the expression of DNMT3a of rat MⅡoocyte in vivo which could disturped the reestablishment of maternal imprinting,this could be a underlying mechanism for the hypomethylaion of Igf2;2.DHT down-regulated the expression of DNMT3 a in vitro in human granulosa cells via AR pathway;3.There was a STAT3 response element at the promoter of DNMT3a,the down-regulation of STAT3 by DHT could alter the transcription of DNMT3a mRNA.