Pregnancy Outcome And Placenta Proteomic Analyses Of Offspring Conceived By Assisted Reproductive Technology

Assisted reproductive technology (ART) has helped thousands of couples conceive children in the past 30 years. Early studies suggested ART might be safe and children born from ART developped normally. However, in recent years, more and more well-designed studies have consistently documented associations of ART with an increased risk of birth defects, low birth weight, chromosome abnormalities, childhood cancer and epigenetic defects. As IVF was introduced into practice without formal evaluation of its effects on the health of children conceived with this procedure, there is little information about the mechanisms underlying the increased health defects. It is also unclear whether the infertility background of the parents or which stage(s) from the controlled ovarian hyperstimulation, gametes and embryo manipulation, in vitro culture to embryo transfer to the baby's birth may raise the risk of health defects. Although the precise significance and origin of ART and offspring safety still require confirmation and clarification, available evidence has suggested the concerns for the long-term safety of ART procedures.In rencent years, more and more epidemiological investigations have been carried out by the reproductive associations or ART centres for the ART offsprings. However, most researches focused on the overall pregnancy outcomes such as birth weight and number of multiple gestations when evaluating the safety of ART. Limited data are available to describe patterns of adverse obstetric and delivery complications and their relationship with prenatal outcome, both in China and abroad. Pregnancy complications are the diseases raised during the pregnancy or delivery process. They are not only the clinical manifestations of abnormal pregnancy, but also the main reasons of adverse outcome of mother and newborn. Therefore, assessment of the influence of ART on the pregnancy complications and newborn outcome will benefit the knowledge of existed information on safety evaluation of ART offspring, in order to well analyze the mechanisms of adverse health of ART offspring.The placenta is a temporary organ that consists of a fetal component and a maternal component and it is a structure unique to pregnancy functioning to sustain and protect the fetus until birth. Placenta vascular formation is important for fetal growth and development. Proper development of placenta ensures the exchange of oxygen/nutrients and blood flow necessary for fetal growth. Abnormal placenta vasculogenesis has been consistently associated with different pregnancy complications and worse perinatal outcome. Abnormal placentation has been observed following in vitro embryo production in animals. It was also reported that human placenta derived from ART were associated with more frequent pathological findings. These results of abnormal placentation in assisted reproduction related procedures in animals and human being reveal partial evidence for the possibility of worse outcome of ART offspring. To overall evaluate the specific gene products in placenta following ART, global screening of protein expression in placenta is needed.In this study, we first analysis the pregnancy complications and petinatal outcomes associated with ART. Then comparative proteomic analysis of ART placenta was carried out using two-dimensional electrophorosis and mass spectrometry. At last, role of two differentially expressed proteins, annexin A3 and a-SNAP, which were detected by proteomics as differentially expressed in ART placenta, was further studied in ART placenta of newborn with low birth weight. PartⅠLate pregnancy complications and perinatal outcomes analyses of assisted reproductive technologyObjective:To determine whether the use of ART is associated with an increase incidence of maternal late pregnancy complications, labor and delivery complications or adverse perinatal outcomes.Patients and methods:A retrospective cohort study was conducted on the 1013 cases of pregnancy conceived through ART (containing standard IVF and ICSI) during 2003-2009 in Zhejiang Women's Hospital.3069 cases of natural conceived pregnancy with matched maternal age were considered as control group. Maternal late pregnancy complications, labor and delivery complications and perinatal outcomes were evaluated. Then, these indicators were evaluated respectively after ART group and control group were separated into singleton pregnancy and multiple pregnancy.Results:The multiple pregnancy rate was 37.8% in ART group, significantly higher than control group. ART was associated with higher incidence of maternal pregnancy and delivery complications [containing gestational hypertension, pre-eclampsia, gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), placenta previa, placental abruption, preterm premature rupture of memberane (PPROM), placental conglutination and postpartum hemorrage] and worse perinatal outcomes [containing preterm labor, low birth weight (LBW), small for term labor and newborn asphyxia]. ART singleton pregnancy was associated with higher incidence of pre-eclampsia, GDM, placenta previa, PPROM, abnormal placental umbilical cord insertion (UCI), placental conglutination, postpartum hemorrhage, preterm labor, LBW and newborn asphyxia. ART multiple pregnancy was associated with lower incidence of small for term labor and twin discordance. Low birth weight newborn is mainly associated with preterm labor.73.2% of preterm labor was induced by different pregnancy complications.Conclusion:Patients who undergo ART are at increased risk for pregnancy complications and worse perinatal outcomes. Multiple pregnancy is closely related to these outcomes. ART singleton pregnancy was also associated with an increased risk for some pregnancy complications, which indicated that ART itself might contribute to the worse outcome of offspring. Low birth weight newborn is mainly associated with preterm labor and prenancy complications, especially for the placenta-related diseases. Therefore, promote single embryos transfer, strict control ART indication, improve ART manipulation, minimize the non-physiological interference, prevent and treat the pregnancy complications, will benefit to improve the ART outcome.Part II Comparative proteomic analysis of human placenta derived from assisted reproductive technologyObjective:To examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy using global proteomics-based approach.Methods:We applied a proteomic approach on six placenta tissues from patients underwent standard IVF, six from ICSI and six from natural conceived pregnancy. Two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF Mass Spectrometry (MS) were performed to describe differential protein expression profiles in ART placenta and control placenta. Western blot analysis was used to validate the result of 2-DE.Results:Using 2-DE we found that, compared with the control group,12 spots in standard in vitro fertilization (IVF) group and 18 spots in intracytoplasmic sperm injection (ICSI) group were significantly differentially expressed. Among them,6 spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally,20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, a-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot analysis. Conclusion:Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.Part III Roles of annexin A3 and a-SNAP in placenta derived from ART offspring with low birth weightObjective:To investigate the expression of annexin A3 and a-SNAP, the density of microvessels in placenta derived from ART offspring with low birth weight (LBW), discuss the relationship of abnormal expression of annexin A3, a-SNAP and microvascular density (MVD) in ART/LBW offspring placenta.Methods:12 placentas derived from ART/LBW offspring,12 from ART normal birth weight (NBW) offspring and 12 placentas from normal pregnancy were examined. Western blot analysis was performed to analyze the expression level of annexin A3 and a-SNAP. Immunohistochemistry (IHC) for annexin A3 andα-SNAP together with quantitative CD34 IHC used for detecting MVD was carried out to analyze the relativity between expression levels of annexin A3 or a-SNAP and MVD.Results:Western blot results showed that expression levels of annexin A3 and a-SNAP in ART/LBW group were significantly lower than control group, which is opposite with result in ART/NBW group. IHC results also showed significantly down-regulated expression of annexin A3 and a-SNAP in ART/LBW group, together with significantly lower density of microvessels. There were significant correlation between the expression of annexin A3 or a-SNAP and MVD.Conclusion:The significantly lower expression of annexin A3 andα-SNAP in placenta may play an important role in the pathology of low birth weight in ART derived pregnancy by affecting placenta angiogenesis.