Study The Safety Of The Assisted Reproductive Technology Via Analyzing The Ectopic Pregnancy Risk After ART And Comparing The Proteome Of Human Neonatal Cord Serum Derived From ART

Assisted reproductive technology (ART) has been used in clinic for more than 30 years, and helped thousands of couples conceive children. However ART was applied without long-term comprehensive evaluation of its safety, which has become a major concern worldwide. In recent years, a large number of epidemiological studies reported that ART may increase the short and long term health risk of adverse pregnancy outcomes, including spontaneous abortion, ectopic pregnancy (EP), pre-eclampsia, placenta previa, preterm birth, low birth weight, birth defects, epigenetic disease, cerebral palsy, and cardiometabolic diseases. The research of various complications and future health perspectives of offspring derived from ART will contribute to improving ART treatment greatly. This study assessed the safety of ART from two aspects, one on the EP risk after ART, and the other on offspring health derived from ART.1. EP risk in women with polycystic ovary syndrome (PCOS) after ARTEP is a known complication of ART. The reported incidence of EPs following ART is higher than that among the general population. Several risk factors for EP specifically associated with ART treatment have been proposed, including maternal infertility diseases and hyperphysiological hormonal levels associated with controlled ovarian hyperstimulation (COH). We preliminarily screened EP risk factors in IVF-ET (In vitro fertilization-embryo transfer, IVF-ET) cycles and found that women’s infertility factors PCOS represented a risk factor for EP following IVF. PCOS is a common disorder associated with infertility in women undergoing IVF treatment. Abundant literatures reported increased risk of pregnancy complications in PCOS women following both natural conception and ART, such as miscarriage, and gestational diabetes. However, with regard to EP, a clear association between PCOS and EP following IVF has not been reported yet, although some studies mentioned EP rates of PCOS women who conceived after different ART treatment modalities.To analyze the possible association between PCOS and EP among clinical pregnancies achieved by IVF cycles, we performed a retrospective cohort study, including 5339 women who had clinical pregnancies after IVF treatment, consisting of 205 PCOS and 5134 non-PCOS women at Nanjing Medical University (China) between 2007 and 2011. Fresh and cryo-thawed ET cycles were analyzed respectively. Multivariate logistic regression analysis was used to adjust for important confounders. The results showed that, in fresh ET cycles undergoing controlled ovarian hyperstimulation (COH) (n=3303), women with PCOS had 3.06 times higher risk of ectopic pregnancy compared with those without PCOS (7.0% vs 2.4%, adjusted odds ratio (OR)= 3.06,95% confidence interval (CI) 1.34-6.96, P= 0.008). In cryo-thawed ET cycles without COH (n=2036), the ectopic rates between PCOS and non-PCOS women were comparable (2.2% vs 2.0%,0.94,0.22-4.07, P= 0.941). To identify a possible explanation on how PCOS affected EP’s development, we performed univariate logistic regression analysis to select potential EP risk factors having P value of≤0.05 from the variables in baseline and cycle characteristics among patients recruited. The results showed that the estradiol level on hCG day was significantly associated with EP. Following adjustment for potential confounders by a multivariate logistic regression analysis, women in high estradiol group (>4085pg/ml) had significantly higher risk of EP compared with those in low estradiol group (≤4085pg/ml) [3.5% (29/825) versus 2.2% (54/2478), adjusted OR= 1.83,95% CI 1.12-3.01, P= 0.016]. Then, we stratified the anaylsis by having either PCOS or non-PCOS in the logistic regression model to estimate the possible modification of the variable on the association between PCOS and EP. In particular, in the stratified analysis, for non-PCOS women, the high estradiol group had higher EP rates than the low estradiol group [3.4% (27/789) versus 2.0% (48/2400), adjusted OR= 1.99,95% CI 1.19-3.35, P= 0.009]; for PCOS women, high EP rates were observed in both the groups [5.6% (2/36) versus 7.7% (6/78), adjusted OR= 0.92,95% CI 0.15-5.67, P= 0.928]. This finding showed that PCOS women had high EP rates even under a relatively low estradiol level after COH. This finding is in contrast to the fact that non-PCOS women had higher EP rates only under high estradiol exposure. We postulate that PCOS women hold a lower threshold for hyperphysiologic estradiol level which triggers the occurrence of EP. This mechanism might be explained by higher sensitivity to estrogen action of the PCOS women. This hypothesis provides an explanation for the increased risk of EP observed among those women undergoing fresh ET cycles. The finding suggested that PCOS patients after COH need increased surveillance on EP at clinic, and these patients might benefit from frozen-thawed ET cycles in reducing the risk of EP. In non-PCOS patients, controlling estradiol level in COH treatment for non-PCOS women may help prevent EP.2. Comparative proteomic analysis of neonatal cord serum derived from ARTAs to the safety of ART offspring, the ART procedures such as hormonal stimulation, IVF, micromanipulation of gametes, exposure to embryo culture medium and cryopreservation of embryos may influence early fetal and placental development, which leads to the adaptation changes of fetus "programmed" in utero for the risk of developing a host of diseases in later adult life, such as cardiovascular disease. Recent studies have reported the influence of IVF on placenta with alterations in ultrastructure, DNA methylation, gene and protein expressions is related to placental functions. Furthermore, alterations in DNA methylation and gene expression levels were measured in cord blood from ART pregnancies. However, different protein profiles in UC serum between ART and normal neonates have not yet been studied. Umbilical cord blood (UCB) represents the fetal blood compartment, directly reflecting the physiological or pathological states of neonates. Proteomic investigation of different protein expressions in UC serum has provided novel biomarkers for fetal abnormalities. Thus, studying UC serum between infants from ART and normal conceptions may reveal enduring biological changes indicative of fetal responses to environmental stress caused by ART procedures and the potential long-term adverse health outcomes, which cannot be detected by fetal appearance after birth.To ensure that samples reflect the fetal state accurately, the UC arterial blood was collected for proteomic analysis. We obtained the UC arterial blood belonging to fetus before placental balances, best describing fetus’states. To focus on the influences of ART procedures per se, we strictly limited ART patients with only tubal factors to minimize the possible interference from maternal factors. Thirty-nine neonates were recruited in the study, grouped by IVF procedures into Fresh-IVF group (born after fresh IVF embryo transfers), Cryo-IVF group (born after cryopreserved/thawed IVF embryo transfers) and control group (born after natural conception). Each group consisted of thirteen neonates UCB samples were used for subsequent analysis. After depleting 14 high abundant proteins,2D/MS/MS strategy was used to compare the protein expression profiles between those groups. We identified 19 differently expressed protein spots in the serum of neonates in fresh-IVF group; 16 in the cryo-IVF group compared with control group, corresponding to 15 and 12 proteins respectively. Western blotting was used to confirm the expression of the selected proteins. The results were consistent with the proteomic analysis. According to the protein profiles, seven biological processes may be involved in fetal responses to environmental stress caused by ART procedures, including metabolism, innate immunity, inflammation, vascular dysfunction, anti-oxidative mechanism, brain development and regulation of steroid response. Specifically, ART-derived neonates appear to have inhibited innate immunity of complement cascade. Biomarkers of cardiometabolic disorders are only shown in serum from neonates after fresh-IVF treatment, suggesting those after Cryo-IVF treatment may have a better outcome.Conclusions:(1) PCOS was associated with an increased risk of ectopic pregnancy after COH in fresh ET cycles, but not in cryo-thawed ET cycles. A possible explanation is that compared with non-PCOS women, PCOS women appear to hold a lower threshold of hyperphysiologic estradiol level that triggers the occurrence of ectopic pregnancy after COH.(2) ART offspring from both Fresh-IVF and Cryo-IVF groups may have adverse health risks. Proteins involved in complement cascade showed inhibition of complement activities in both ART groups; proteins involved in vascular dysfunction and lipid metabolism were only differently expressed in fresh-IVF group, suggesting offspring born after fresh-IVF may have a risk of long-term cardiometabolic disease.(3) The results of this study suggested that considering from these two aspects above-mentioned, frozen-thawed treatment cycles may reduce the risk of EP in women with PCOS after ART and health of offspring in part.(4) This study provides theoretical basis for the ART practice and improvements, has a certain significance for clinical work, and adds evidence to the study of ART safety from a different point of view.