3.1 There was no significant differences in pregnancy outcomes between PE group and PE with IVF group
The 2 groups were closely matched for maternal age, maternal BMI, gestational age, and the disease severity. Table 1 showed the baseline characteristics of enrolled patients for the retrospective study. No significant differences in maternal age, maternal BMI, gestational age, blood pressure, and the number of patients with mild/severe pre-eclampsia were found between the 2 groups. We observed that women in the PE group presented a higher number of deliveries when compared to the PE with IVF group. This happened, because the pre-eclampsia patients in the PE with IVF group were not successfully impregnated or could not complete pregnancy. For IVF treatments condition, since the treatments that our patients received were carried out at different hospitals, the details of IVF for each patient were difficult to obtain. Therefore, we only investigated the use of fresh or frozen embryos in the PE with IVF group. Among the 20 women, 7 received the fresh embryo transfer and 9 received the frozen embryo transfer and 4 missed the data. For pregnancy outcome, 4 (4/20) mothers in the PE group and 3 (3/20) mothers in the PE with IVF group were admitted to the intensive care unit (ICU) for observation after cesarean section. Although the newborns in PE with IVF group showed a slight advantage in birth length, there were no significant differences in the birth weight, placenta weight, Apgar scores between the 2 groups (Table 1). Taken together, our data indicated that IVF experience may be not associated with the worse birth outcome in the pre-eclampsia population.
3.2 Worse results of biochemical blood tests for maternal renal and liver function in the PE with IVF group
The results of liver and kidney function tests obtained at the admission for pre-eclampsia were presented in the Table 2. Prealbumin (PA) was reported to be a sensitive marker for protein-energy and nutritional status [20, 21]. Elevation of maternal serum PA was observed in our PE with IVF group when compared to PE group (P = 0.02). Of note, the level of maternal PA in the PE with IVF group was below the lower limit of the normal reference range, suggesting that these pre-eclampsia patients with IVF treatment experience might have higher likelihood of malnutrition. The results of renal functional assay showed that, the levels of maternal creatinine (Cr) (P = 0.04), and uric acid (UA) (P = 0.03) in the PE-IVF group were significantly higher than those in the PE group. In addition, the UA level in the PE with IVF group exceeded the upper limit of the normal reference range (Table 2). Although there were no significant differences in maternal alkaline phosphatase (ALP) and Cystatin C (CYSC) between the 2 groups, they exhibited anomalies with elevated concentrations in both 2 groups, which were outside the normal range.
3.3 Glucose and lipid metabolism were not significantly different between PE group and PE with IVF group
The maternal glucose and lipid profiles obtained at the admission for pre-eclampsia of each group were summarized in the Table 3. Of interest, no significant differences were noted between the 2 groups for glucose, total triglycerides (TC), total cholesterol (TG), high-density lipoprotein cholesterol (HDL-C), L-density lipoprotein cholesterol (LDL-C) and apolipoproteins with the majority of ApoA1 and B. However, it was noteworthy that the levels of TC in both 2 groups exceeded its normal ranges. This observation was in accordance with previous findings that the pre-eclampsia women population has a higher level of TC than that in the normotensive group . Serum homocysteine (HCY) has been reported to be a predict marker of pre-eclampsia and associated with IVF outcome [22, 23]. In this research, although we observed an elevation of HCY level in the PE with IVF group in comparison to the PE group (P = 0.04), HCY levels in our 2 groups were both in the normal range. Overall, we did not discover significant differences with clinical significance in measures of glucose and lipid profiles between the PE group and the PE with IVF group.
3.4 Dynamic changes of abnormal laboratory data in the 2 groups at the different pregnancy stages of enrolled pre-eclampsia patients
Since our enrolled pregnancies performed the routine examinations during gestation in our hospital, we further investigated the dynamic changes of above abnormal clinical biochemical indexes (ALP, PA, Cr, UA and CYSC) at multiple timepoints of pregnancy, including the results of routine blood tests in the first trimester, the second trimester, and the third trimester of pregnancy. In particular, the third trimester laboratory data referred to the results from the fresh blood samples obtained at their admissions for pre-eclampsia (Figure 2). We discovered that the levels of maternal ALP, PA, Cr, UA and CYSC were all well within the normal range in their early pregnancy. In the second trimester of pregnancy, the levels of maternal ALP, UA and CYSC in both 2 groups were higher than those in the first trimester pregnancy. Of note, the maternal CYSC level in the PE with IVF group that already exceeded the normal range in second trimester of pregnancy, suggesting the minor abnormalities of kidney function might arise before their diagnosis of pre-eclampsia. When women were identified with pre-eclampsia and hospitalized for treatments/cesarean section in the third trimester of pregnancy, almost all above blood indicators exhibited significant changes compared to the second trimester of pregnancy. In addition, the abnormally increased ALP and CYSC levels were discovered in both 2 groups. However, the elevated level of UA, which was over the upper limit of the normal range, and the decreased level of PA, which was below the lower limit of the normal range were only found in the PE with IVF group. Furthermore, representative dynamic alterations (especially including the data from the postpartum period) of these abnormal biochemical indicators of 1 pre-eclampsia patient with spontaneous conception and 3 pre-eclampsia patients who underwent IVF treatment, were shown in the Figure 3. The levels of ALP, PA and CYSC in all 4 patients were back to normal during postpartum recovery period. However, the serum Cr level in 1 pre-eclampsia patient with IVF treatment and serum UA levels in 2 pre-eclampsia patients with IVF treatment and 1 pre-eclampsia patient without IVF treatment still remained in the abnormal range. Although the postpartum recovery data was far more limited, it still indicated that the reassessment of physical condition of mothers would be extremely clinically valuable. Collectively, clinical practitioners should monitor the above indicators to track pre-eclampsia progression and guide personalized treatment of pre-eclampsia.
3.5 Increased NK/γδT cells-derived IFN-γ and TNF-α were at maternal-fetal interface of pre-eclampsia patients who underwent IVF treatment
Generalized activation of the inflammatory response is reported to play a critical role in the pathogenesis of pre-eclampsia . Low-grade pro-inflammatory responses may start in the first trimester pregnancy and excessively increase in women who develop pre-eclampsia .To examine the pro-inflammatory cytokine secretion of 2 classical innate immune cells (NK and γδT cells) in our 2 groups, we collected several fresh decidua tissues enriched in lymphocytes (Figure 4) and cord blood specimens of pre-eclampsia patients with and without IVF treatment for flow cytometry assays, and the gating strategy identifying NK cells and γδT cells was presented in the Figure 4. The results showed that the ratios of both decidual and cord blood γδT cells in the CD3+ T cell population were significantly higher in the PE with IVF group (Figure 5). Besides, statistically significant elevations of IFN-γ and TNF-α secretions were observed in both decidual NK and γδT cells of pre-eclampsia patients who underwent IVF treatment. However, in the matched umbilical cord blood samples, the productions of IFN-γ and TNF-α were only increased in the NK cells of pre-eclampsia patients with IVF treatment when compared to pre-eclampsia patients conceived naturally (Figure 6).