Effect Of Commonly Used ART On Glucose And Lipid Metabolism Of Mouse Offspring And The Underlying Mechanisms

1.Effect of ART on general growth and development on mouse offspringObjective: To observe the effect of the most commonly used assisted reproductive technologies(ARTs),including embryo transplantation(ET),superovulation(SO),in vitro fertilization(IVF),in vitro maturation of oocytes(IVM),on the general growth and development of the mouse offspring from birth to aged.Methods: The control group(natural conception,CON)and four groups of ART mice offspring,including transplantation group(natural conception + embryo tubal transplantation,ET),superovulation group(hormones stimulate ovulation + natural conception + embryos tubal transplantation,SO),in vitro fertilization group(hormones stimulate ovulation + in vitro fertilization + embryo tubal transplantation,IVF)and in vitro maturation group(in vitro maturation of oocytes + in vitro fertilization + embryo tubal transplantation,IVM)were modeled.Body weight,body length and tail length of mice were measured per week from birth to 40 weeks old,and the increase ratio of 40 weeks compared with birth weight and Lee "s index were calculated.Results: 1)Compared with the same sex control group,the ARTs have the trend of lower birth weight,especially the male mice of IVF and IVM groups(P < 0.05).Average body weight of 40 weeks old mice in each ART group was more than the control group,in which the male and female IVM mice got significant weight gain(P < 0.05).The weight gain ratio of the ART groups increased,especially the male IVF and IVM group increased significantly(P = 0.004).Repeated measurement statistical method is used to compare of weight growth curve from birth to 40 weeks old,the increase trend in each ART is greater than the control group,especially the female SO group,the change trend of body weight growth curve has a statistically significant.2)Mice body length and tail length had no difference in birth.Compared with the same sex control group,the body and tail length of the male IVF and IVM group were increased(P < 0.05).Compared with SO groups,the body length of the male ET,IVF and IVM groups and the tail length of the male IVF group obviously increased(P < 0.05).The ET,IVF and IVM groups body length growth curve increased faster than the control group in male and female.The tail length growth curve of male IVF and IVM groups increased faster than the control group.3)Lee 's Index of SO group significantly greater than the other groups.Compared with the male and female control,IVF,IVM and female ET groups,Lee' s Index of the SO group increased significantly(P < 0.05).Conclusion: ART reduced the birth weight of mice offspring,but didn't affect their birth body and tail length.Low birth weight made ARTs mice had a “catch-up growth” pattern after birth,and resulted in each ART group weighing more than normal mice in adulthood.The weight of the IVF and IVM offspring gain but did not lead to obesity in adulthood,because the body length were longer than normal mice too.But superovulation didn't change the offspring mice body length,while they got obesity in aged.2.Effect of ART on glouse and lipid metabolism on mouse offspringObjective: By comparing the glucolipid metabolic state of ART offspring mice in aged,to evaluate the ART in long-term security of glucolipid metabolism,and analysis the key factors may result in the glouse and lipid metabolic disorder.These may provide a theoretical basis for clinical ART improvement.Methods: Fasting blood glucose and Introperitoneal glucose tolerance test(IPGTT)were detected and the area under the IPGTT curve(AUC)were computed to analysis of ability of glucose metabolism in ART mice.ELLSA method was used to detecte fasting serum insulin and insulin resistance index(HOMA-IR),insulin sensitive index(ISI)and insulin secretion index(HOMA-beta)were computed to analysis of insulin secretion and resistance status.Fasting blood lipids(T-CHO,TG,HDL and LDL)levels were detected to analysis of lipid metabolic stateResults: 1)Fasting blood glucose levels of the male IVM and female SO groups mice wrer significantly higher.Compared with the same sex control group,AUC of IPGTT in male and female mice of SO and IVF group were obviously reduced,glucose metabolism ability significantly decreased(P < 0.05).Compared with the ET group,glucose metabolism ability of the male IVF,female SO and IVF group were significantly decreased also(P < 0.05).2)Compared with the same sex control group,serum insulin levels in each ART group had a trend of increased,especially the male SO and IVM group(P < 0.05).HOMA – IR of the female ET group,female and male SO,IVF and IVM group increased significantly compared with the same sex control group(P < 0.05).And ISI of all the male and female ART groups were significantly decreased(P < 0.05),which meant insulin resistance levels significantly increased.Compared with the same sex control and ET groups,HOMA-beta of the male SO and IVF,female IVF and IVM groups significantly increased,which meant the ability of beta cells secreting insulin improved.While HOMA-beta of the female SO mice was significantly reduced(P < 0.05),which meant the ability of beta cells secreting insulin decreased obviously.3)Compared with the same sex control group,T-CHO of fasting serum in female SO group were significantly higher(P < 0.05).TG in male SO,IVF,and IVM groups and female SO and IVF groups obviously increased(P < 0.05).HDL in male SO group obviously increased(P < 0.05).LDL in male SO and IVF groups and female SO and IVM groups obviously decreased(P < 0.05).Conclusion: ART had made impairing of glucose scavenging ability,insulin resistance and lipid metabolism disorders in aged mice offspring.Glouse and lipid metabolic disorder caused by different ART technology had different levels,the degree of which caused by ET is the lightest,followed the IVM procedure,while it was most serious made by the SO and IVF procedure.At the same time,the effects had gender differences still.3.Explore the mechanism of glouse and lipid metabolism disorders in ART mice offspring preliminaryObjective: By detecting the imprinting or non imprinted genes which were closely related to the glucolipid metabolism in blood,liver,muscle,fat,and pancreatic tissue of offspring mice,and the key gene methylation status,to explore the mechanism preliminary.And it can provide the experimental basis of molecules to further clarify the pathogenic factors,and to prevent and control of disease occurrence.Methods: ELLISA method was used to detect the leptin and adiponectin levels in serum.Real-time fluorescent quantitative polymerase chain reaction(q RT-PCR)was adopted to detect the m RNA expression level of genes H19/Igf2,Grb10,Dlk1,Mest,Serpinf1,and Nnat related to metabolism in liver,muscle,adipose,and pancreatic tissue and Western blot method was adopted to test the corresponding protein expression levels further.Pyrophosphate sequencing method was used to detect the methylation level of the Cp G locis in the key gene Nnat.Results: 1)Compared with the same sex control group,serum leptin levels of ART groups had no difference,while adiponectin levels of male SO and female IVF groups significantly decreased(P < 0.05).2)Compared with the same sex control group,Serpinf1 gene m RNA and protein expression levels in the muscle tissue of SO and IVF group were significantly lower(P < 0.05),no matter male or female mice.No other genes expressed differently.3)Compared with the same sex control group,H19 m RNA expression level in adipose tissue of male IVF,female IVF and IVM groups decreased obviously(P < 0.05).Dlk1 gene m RNA and protein expression levels of female and male mice SO group significantly decreased(P < 0.05).No other genes expressed differently.4)Compared with the same sex control group,H19 m RNA expression level in liver tissueof the male and female mice ET,SO and female IVF groups significantly decreased(P < 0.05),while it of the IVM group,no matter male or female,increased significantly(P < 0.05).And compared with control and ET groups,Igf2 gene m RNA expression levels of the SO,IVF and IVM groups increased significantly,and the protein expression levels of the SO and IVF groups increased significantly(P < 0.05)too.The rest of the genes expressed no difference.5)Compared with the same sex control group,the H19 and Grb10 genes m RNA expression levels in pancreas of the male and female ART groups were significantly lower(P < 0.05).But GRB10 protein levels have no difference.Serpinf1 gene m RNA and protein expression levels of male and female SO group significantly decreased(P < 0.05).Nnat gene m RNA expression levels of male and female SO,IVF and IVM groups increased significantly,while the protein expression levels of SO and IVF group also increased significantly(P < 0.05).The rest of the genes expressed no difference.Cp G locies methylation levels in Nnat of the SO group were significantly lower(P < 0.01)and the other four groups had no difference.Conclusion: The glucolipid metabolic abnormalities of aged ART offspring may be associated with the abnormal expression of cytokines adiponectin and SERPINF1,imprinted gene H19/Igf2,Dlk1,and especially the metabolic key genes Nnat.The methylation level of Cp G island in the upstream of Nnat which regulats gene expression may be involved in the mechanism of abnormal gene expression pattern of ART offspring.