Disorders Of Lipid Metabolism Regulate Abnormal Endometrial Decidualization Caused By High-fat Diet In Early Pregnancy Mice

Objective: According to the World Health Organization,between 1975 and 2016,the prevalence of obesity worldwide almost tripled,accompanied by a significant increase in infertility rates and abortion risks among obese women.In the early pregnancy,the blastocyst adhered to and invaded the endometrium on day 5 of gestation,the stromal cells proliferated and differentiated extensively,and decidualization took place in the endometrium on day 6 and day 7 of gestation.This process is an important basis to ensure the normal formation of the placenta,which plays an important role in protecting and nourishing the embryo before the formation of the placenta,and is essential for the establishment and maintenance of pregnancy.Rhee JS team used artificial induced decidualization model of pseudopregnant mice and human endometrial stromal cells decidualization induction in vitro model to find that high fat may cause abnormal endometrial decidualization through autophagy disorder.Although the current study found that obesity may be related to endometrial decidualization injury,but whether obesity leads to abnormal decidualization of endometrial stromal cells during pregnancy and the specific mechanism is not clear.Therefore,this study intends to explore whether maternal obesity induced by high-fat diet can cause abnormal endometrial decidualization through a series of animal and cell models,and further explore the possible mechanism of high-fat diet induced decidualization injury in early pregnancy mice from the perspective of lipid metabolism.Methods: 1.Construction of experimental model and material collection: 1)Establishment of high-fat obese mice model: Four-week-old female C57BL/6J mice were fed with high-fat diet(60% kcal)/control diet(10% kcal)for 12 weeks,and the data of body weight were recorded weekly.The weight increasing curve was drawn in 12 weeks;Dual-energy X-ray absorptiometry(DEXA)was used to determine the composition of body fat;The fasting serum triglyceride(TG)and total cholesterol(TC)were measured by automatic biochemistry analyzer;The content of fasting insulin was measured by ELISA;Glucose tolerance(GTT)was measured and the area under the curve was calculated.Through these tests to verify whether the high-fat obese mouse model is successfully constructed.2)Establishment of normal pregnant mice model: The HFD mice/CFD mice were mated with normal male C57BL/6J mice,the vaginal plug was examined on the next morning,and the vaginal plug appeared was marked as the first day of pregnancy(D1),and the uterine tissues at the key stage of decidualization(D5-D7)were collected for detection.3)Establishment of artificial induced decidualization of pseudopregnant mice: The HFD/CFD mice were mated with vasectomized male C57BL/6J mice,the vaginal plug was checked on the next morning,and the plug appeared on the first day of pseudopregnancy(PD1),corn oil was injected into one side of uterine horn in PD4 to induce decidualization of endometrium in mice.The contralateral uterine horn was used as control.Uterine decidualization was induced in mice on the eighth day of pseudopregnancy(PD8).4)Establishment of an artificial induced decidualization of mouse endometrial stromal cells(m ESCs): Primary mouse endometrial stromal cells were isolated and treated with estrogen(E2)and progesterone(P4)to induce decidualization.Morphology of decidual cells was observed by phalloidin staining,and the expression of decidual-related protein BMP2 was detected by Western blot.2.Effect of high-fat obesity on decidualization of endometrium in mice: 1)Count and analyze the number of implantation sites of embryos from D5 to D7 pregnant mice.Observe the fibrous morphology of endometrium and decidual cells by HE staining,detect the expression of MMP2,BMP2,PR and other decidualization related proteins by Western blot.2)Weigh the uterus of induced side and non-induced side of decidualization in the two groups,and detect the expression of decidualization related proteins by Western blot.3)Phalloidin staining was used to observe the decidualization of stromal cells,and Western blot was used to detect the expression of decidualization related proteins.3.Effects of high fat diet on the function of endometrial mitochondria in mice: The morphology of endometrial mitochondria was observed by transmission electron microscopy(TEM)and the content of ATP was detected by gas chromatography-mass spectrometry(GC-MS)in D7 pregnant mice.The expression levels of genes related to oxidative phosphorylation in the endometrium of high-fat pregnant mice were detected by RT-PCR.Mitotracker were used to detect the cell membrane potential of m ESCs after decidualization.4.Effects of high fat diet on lipid metabolism of endometrium in mice: 1)Oil red O,transmission electron microscopy and GC-MS were used to detect lipid droplet deposition and the content of medium-long-chain fatty acids in endometrium of pregnant mice(D6-D7).Triglyceride(TG)and malondialdehyde(MDA)detection kit were used to detect the content of TG and MDA in endometrium of pregnant mice(D7).The expression of protein and gene related to lipid metabolism was detected by Western blot and RT-PCR.2)Oil red O was used to detect the deposition of lipid droplets in decidualized m ESCs.TG/MDA assay kit was used to detect the contents of TG and MDA in cells.Western blot was used to analyze the expression of lipid metabolism-associated proteins.On The basis of control group and high-fat diet group,the high-fat diet and CPT1 A inhibitor group was set up(Etomoxir,a CPT1 A inhibitor,was added into the high-fat diet group),BODIPY probe was used to detect the deposition of lipid droplets in m ESCs with or without decidualization.The lipid metabolism of m ESCs and the expression of decidualization related proteins were detected by Western blot.5.Effects of high-fat obesity on the development of fetus and placenta in mice: Observe the development of fetus and placenta in pregnant mice D12,and weigh the weight.Results: 1.Obese mice induced by high-fat diet was successfully established: body weight and body fat ratio of mice in high-fat diet group were significantly increased,fasting serum insulin,TG and total cholesterol levels were significantly increased,and GTT was significantly decreased.2.High fat diet induced impaired decidualization of early pregnancy endometrium in obese mice: High fat diet induced a significant decrease in the number of embryos implanted on D5-D7 in normal pregnant mice,and a significant decrease in the weight of the uterus in artificial induced decidualization in pseudopregnant mice.Phalloidin staining showed that the decidualization induced by primary stromal cells in high fat group was impaired.The expression of decidualization marker such as BMP2,MMP2 and PCNA was significantly decreased in the HFD group by Western blot in the uterus tissue of pregnant mice on day 7.The results of Western blot in the model of artificial decidualization and the model of m ESCs decidual induction in vitro were the same.The HE staining of the uterus of pregnant mice on day 7 showed that the high-fat diet caused abnormal morphology of decidual cells.3.Endometrial mitochondrial function of HFD mice was impaired: The mitochondria of endometrium of high-fat diet induced obese mice was swollen on day 6 of pregnancy by TEM,and the level of ATP in decidual tissue was increased on day 7 of pregnancy by GC-MS.The results of Mitotracker showed that the mitochondrial transmembrane potential of decidual cells was decreased in the high fat pregnant group.The expression of Ndufb6,Atp5 b and other oxidative phosphorylation markers was increased in the endometrium of the high fat pregnant mice on day 7 by RT-PCR.4.During the decidualization of early pregnancy,the lipid metabolism of the endometrium of HFD mice was disorder: Compared with control mice,the results of Oil Red O and TG determination kit and TEM detection showed that lipid droplets were obviously accumulated in the endometrium of HFD mice,which was consistent with the results of Oil Red O and BODIPY staining of m ESCs induced cells in vitro.The results of GC-MS showed that the content of medium and long chain fatty acids changed in the endometrium of HFD pregnant mice on day 6.The results of Western blot and RT-PCR showed that the expression of CPT1A(fatty acid oxidation),FAS(fatty acid synthesis)and other important molecules of lipid metabolism increased significantly.In vitro cell model,western blot results showed that the intervention of CPT1 A inhibitor(Etomoxir)can significantly reduce the high-fat intervention group in the expression of lipid metabolism-related molecules,and the original decidual injury has been improved to some extent.5.The fetus of obese pregnant mice induced by high fat diet did not develop well.The statistical results showed that the volume and weight of the fetus and placenta of obese pregnant mice D12 induced by high fat diet were significantly reduced.Conclusions: Abnormal decidualization of endometrium in obese pregnant mice induced by high-fat diet may be related to the disorder of lipid metabolism of endometrium.