The presence, severity, and onset of preeclampsia is associated with maternal interleukin-23 level: A case-control study

Abstract Background Scientific evidence support that imbalance between inflammatory and anti-inflammatory cytokines play a critical role in preeclampsia (PE). Objective To investigate the relationship between the maternal serum level of interleukin (IL)-23, a pro-inflammatory cytokine, PE and its severity risk was investigated. Materials and Methods The case-control study included a total of 145 women counting 75 PE cases, 35 healthy pregnant and 35 healthy non-pregnant controls from Zahedan, southeast of Iran. The maternal levels of IL-23 in circulation were determined via enzyme-linked immunosorbent assay. Results The maternal serum levels of IL-23 were increased in PE and its 2 subgroups severe PE and mild PE, so that these increases were significant in PE and severe PE, but not in mild PE compared with the controls (p < 0.001 and p < 0.001, p = 0.08, respectively). Besides, the maternal IL-23 serum level was statically significant in the early onset PE, but not in the late onset-PE group compared to healthy pregnant controls (p < 0.001, p = 0.46 respectively). Conclusion The results of our study showed a positive association between IL-23 level and PE, especially in severe type and early onset PE, which suggests that IL-23 may be involved in the pathogenesis of this systemic syndrome.


Introduction
In pregnancy, preeclampsia (PE) is considered to be a specific disorder that can threaten the life of both the mother and the fetus in

Material and Methods
25 participants were considered as the minimum number of participants that was computed through a comparison of the mean difference between the 2 groups regarding the following assumptions: 80% power, 95% CI, 1:1 PE: controls ratio. The present case-control study was done on 145 subjects referring to Imam Ali hospital, Zabol, Iran from July 2017 to November 2018. The PE and controls were 112 and 70 subjects, respectively. 37 cases were excluded from the study because of the following reasons that were described previously (20): twin or multiple pregnancies, tuberculosis, leprosy, tularemia, sepsis, lyme disease, liver diphtheria, renal disease, diabetes, hydrops fetalis, hydatidiform mole.
The control group had a normal pregnancy without any fetal disorders, pathological problems, and no history of multiple births, as well. None of the control subjects shown PE after giving birth.
The age-matched healthy controls were excluded due to common autoimmune diseases. No history of hypertension was detected in the PE and control groups.
The women with systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 90 mmHg, and also, proteinuria > 300 mg in a 24 hr period or > 1+, after 20 W of pregnancy sorted in PE group. Both cases and controls contain different subgroups, so that, on one hand, PE is divided into 46 severe and 29 mild PE groups. The controls comprise 35 healthy pregnant and 35 healthy non-pregnant women ( Table I). The diagnostic criteria of severe PE, mild PE, early and late-onset were described in our previous works (6,20). The PE subjects which have high blood pressure ≥ 160 mmHg systolic or ≥ 110 mmHg diastolic and also, protein excretion ≥ 5 g/24 hr were characterized as severe PE. Moreover, the case group was assigned into 2 other subgroups early-onset PE (≤ 34 wk of gestation, n = 34) and late-onset PE (> 34 wk of gestation, n = 41) (Tables II,  III). Blood samples were stored at -80°C until further usage. IL-23 serum levels were measured in PE and control women using an ELISA kit (Bio Legend, San Diego, CA, USA), pursuant to the manufacturer's instruction. The sensitivity of the IL-23 test was 4.27 pg/mL in our laboratory.

Ethical considerations
The

Statistical analysis
All the records obtained were mean ± SD. The Kolmogrov-Smirnov test was recruited to test the normal distribution of data. The ANOVA and Student t tests statistical analysis were performed for normally distributed data, and/or Kruskal-Wallis and Mann-Whitney U tests were utilized for non-normally distributed data via GraphPad Prism (version 7.01) software package (Graph Pad, CA, USA). The p < 0.05 was contemplated statistically significant.

Results
The clinical properties of cases and controls are summarized in weekly pregnancy in the patient group was 33.71 ± 5.36, which was not significantly different from the control group. In terms of age and body mass index, no significant difference was observed between healthy and pregnant women. However, proteinuria, systolic blood pressure, and diastolic blood pressure were significantly different in the healthy group and the patient group (p < 0.001). In addition, the birth weight of the newborns in the healthy group was higher than that of the PE group, the result of which is shown in table I.
The maternal serum levels of IL-23 significantly increased in PE and healthy pregnant groups which compared to non-pregnant controls (p < 0.001 and p < 0.001, respectively). Moreover, a significantly elevated amount of IL-23 was observed in PE women compared with healthy pregnant women, with the median of 24.461 ± 8.027 pg/ml compared with median of 17.622 ± 6.310 pg/ml, respectively (p < 0.001). Also, when the PE women were divided into different severe and mild PE subgroups, the IL-23 serum levels remained significantly higher in severe PE than in healthy pregnant women (p < 0.001). No significant association was observed between mild PE and healthy pregnant women (p = 0.08).

Discussion
The results of the current study showed a high level of IL-23 in severe PE women, which is in assent with previous reports that showed higher levels of inflammatory cytokines in women with severe PE. Moreover, the highest level of IL-23 was seen in early severe PE as compared to gestation-matched healthy group, which is consistent with some studies, but not with others in the relationship between inflammatory cytokines and onset of PE (4-6).
In addition, we found a significant association Several studies consider IL-23 as an inflammatory cytokine that mediated various immune disorders (10)(11)(12)(13)(14)(15)(16)(17)(18); thus we hypothesized that the serum IL-23 level might be associated with PE or its severity. In this study, we detected a significant association between IL-23 levels of PE and healthy pregnant women compared with non-pregnant controls (Table II). Furthermore, it can be proposed that the serum IL-23 levels were positively correlated with the severity and onset of PE. Accordingly, elevated IL-23 levels were observed in severe type as well as early onset PE (Table III).
IL-23, as a member of the family IL-12, released by macrophages and dendritic cells activated and can induce and produce IFNγ, but IL-23 has a distinctive capacity at IL-17 production induction in comparison with IL-12, which happened in inflammatory diseases (7). In addition, IL-23 is essential in the development of Th17, as well as has a pivotal role in the survival and expansion of pathological Th17 cells (7,21). Through IFN-γ production, IL-23 causes increased activity of trophoblast CD4 + cells, these cells produce IL-17, IL-6, TNF-α, and other inflammatory cytokines (21,22). Previous studies have recognized IFN-γ as a major reason for PE, which has a critical role in the expansion of inflammatory cytokines. Accumulating data propose that IFN-γ-producing cells have a pivotal impact on immune system disorders such as PE (8,23). In addition, it has been confirmed that IL-23-dependent T cells are highly pathogenic and has an essential role in inflammatory responses (21,23).
The participation of TH17 cells in pregnancyrelated disorders including PE has been revealed lately. In these conditions, TH17 cells have been adjusted so that the prevalence of TH17 cells in PE women can be seen (8,19,24,25).

Conclusion
IL-23 serum levels in PE, and also in the severe,