Altered Protein Expression Profiles In Umbilical Veins:Insights Into Vascular Dysfunctions Of The Children Born After In Vitro Fertilization

1. ObjectiveAlthough there have been studies showed that IVF offspring will appear vascular metabolic disorders, systemic and pulmonary circulation dysfunctions, and, cardiovascular structures remodeling, the mechanisms remained little known. In our study, we first tested the blood pressure of IVF children and NC children aged3-13year old; using the umbilical veins from IVF children and NC children, we tried to find the differentially expressed proteins in IVF children; someone thought that the IVF treatment or infertility itself led to vascular dysfunctions in IVF children, however there exited no agreement on such subject; but in this study, we attempted to study the clinical and basic research through a combination of to discover the reasons for the differential expression of these proteins, thus digging out the mechanisms of IVF offspring vascular dysfunction2Materials and methods(1) A following up study on the3-13years old children, born in the Women’s Hospital, School of Medicine, Zhejiang University, China, after IVF treatment or NC was performed. The standard IVF singletons were the test group, and the NC singletons were control group. The height, weight, BMI, diastolic blood pressure and systolic blood pressure was analyzed. All the IVF children were tube infertility, and offspring with birth defects, familial vascular disease, pregnancy-induced hypertension or gestational high blood sugar were excluded in both groups.(2)14umbilical vessels from IVF and NC group, and cord blood samples from45IVF and48NC babies were collected after caesarean delivery in Women’s Hospital, School of Medicine, Zhejiang University, China. The inclusion criteria used for mother and children was as follows:maternal mean age between25to35years; full-term delivery; singleton pregnancy; child birth weight between2500to4000g; no indication of pregnancy complications and no birth defect.3umbilical veins from IVF and NC group were used for the proteomic experiment, and the remaining umbilical veins were used for the verification experiment (q-PCR and western blotting). The serum E2level in cord blood was examined according to the protocol of Iodine [125I] Estradiol Radioimmunoassay Kit.(3) The in vitro experiment was performed in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with E2at different concentrations, the expression of lumican and vimentin on mRNA and protein level was detected by quantitative PCR and western blotting.(4) After the treatment of vimentin special RNA interference in HUVECs, the cell morphology, cell cycle and migration was detected by immunofluorescence, flow cytometry and transwell, respectively.3. Results(1) The results of epidemiological investigations revealed that both in3-6years group and6-13years group the SBP and DBP were higher in IVF group.(2) Using iTRAQ, we identified723proteins in umbilical veins between the two group, including355up-regulated proteins and388down-regulated proteins. The Correlation-based Feature Selection (CFS) was employed for the protein selection from the MS intensity values of C (NC) and T (IVF) samples. The selected proteins were further inspected to retain the ones with differential expression ratio of at least over+/-1.2. Compared with the NC babies,47DEPs in umbilical veins of IVF babies were found,20proteins were up-regulated while27proteins were down-regulated. IPA software was used for bioinformatics analysis, and the results showed that these DEPs were related to cardiovascular development and metabolism.(3) To confirm the proteomic results, lumican, an up-regulated protein, and vimentin, a down-regulated protein, in IVF group umbilical veins were verified. The results of q-PCR and western blotting showed that the expression level of lumican was up- regulated and the expression level of vimentin was down-regulated in IVF group. And these results are in line with those of proteomics experiment.(4) The concentration of E2in IVF group was significantly higher than the NC group, indicating the fetal development might be stimulated by high concentration of E2. Therefore, the high level E2might be an important cause for the abnormal expression of these proteins.(5) Treatment of HUVECs with E2dose-dependently increased expression levels of lumican while reduced expression levels of vimentin, which was in line with the proteomic results. All these results revealed the high concentration of E2at pregnancy might be an important cause for these disorders.(6) After the treatment of vimentin special RNA interference in HUVECs, the cell morphology was changed, cell cycle was blocked in S phase and the ability of migration was inhibited, suggesting that the reduced expression of vimentin would affect the normal physiological function of vascular endothelial.4. ConclusionDifferentially expressed proteins which were related with the cardiovascular system development and metabolism were found in IVF offspring, suggesting that these proteins may be an important cause of IVF offspring vascular dysfunction. Besides, high intrauterine estrogen during IVF embryogenesis may be an important factor of these proteins differentially expressed. These results are preliminary screening of IVF-related differences in protein expression in human umbilical veins, providing information for further research on IVF vascular abnormal developmental mechanisms, and, for clarify the relationship between IVF and adverse pregnancy outcomes and the offspring health.