Levels of pro-inflammatory and anti-inflammatory cytokines and interferons in pregnant women with vaginal infections during vitamin D therapy

Although pregnancy is considered as a unique balanced model of the maternal immunological tolerance to the genetically foreign fetus with transient changes in the mother’s immune system, pregnant women are at risk for the development and exacerbation of infectious diseases [1–4]. Standard antibacterial and antiviral therapy are feasible only in the late stages of gestation. Immunomodulating therapy is also rarely used due to the lack of evidence on its safety in pregnancy and fetal development [5, 6].

Basic antibacterial and antiviral therapy in combination with drugs modulating nonspecific resistance in pregnancy cannot always contribute to the elimination of the pathogen from the body. According to literature, only initially high serum levels of interferon create the conditions to neutralize viruses and prevent the recurrence of infection during pregnancy. One of the immunomodulatory agents used in pregnant women is recombinant human interferon (IFN) alpha-2b. It exerts a regulatory effect on the synthesis of IFN, which provides optimal antiviral protection. A proven decrease in the levels of pro-inflammatory cytokines - interleukins (IL) -1, IL-6, IL-8 during the observation confirms the effectiveness of the drug in the treatment of herpes virus infection in pregnant women [7-10].

Currently, studies on the effects of vitamin D on inflammation and anti-infection immunity deserve special attention. Vitamin D plays a major role in regulating calcium metabolism. However, findings recent studies suggest that vitamin D has physiologic effects much broader than a role in mineral homeostasis. According to some authors, vitamin D supplementation shows superiority over systemic immunomodulators [11–15].

The immunoregulatory effect of vitamin D is due, in particular, to regulation of T-helper cell division, differentiation of B cells, and the effect on the level of pro/anti-inflammatory cytokines and IFN, including tumor necrosis factor (TNF) -α, IL-1, IL-6, IL-8, IL-4, IL-10, and IFN-γ [11, 15–17].

In recent years, studies have been published on the feasibility of using vitamin D for the prevention and treatment of tuberculosis and other infectious diseases including viral hepatitis, influenza, acute respiratory infections (ARI), chronic rhinitis, and rhinosinusitis [12, 13, 15, 18].

The study aimed to investigate the effect of prenatal vitamin D supplementation on the levels of pro/anti-inflammatory cytokines and IFN in pregnant women with vaginal infections.

Materials and methods

The study was conducted at the Moscow Regional Research Institute of Obstetrics and Gynecology and I.I. Mechnikov Research Institute of Vaccines and Sera under the protocol of the study approved at a meeting of the Institute Research Ethics Committee (protocol No. 81 dated 11/05/2015). The study comprised 95 pregnant women aged 20–40 years, who signed informed consent to take part in the study. Patients underwent a standard clinical laboratory examination to diagnose vaginal infections. The study group included 72 pregnant women with vaginal infections thereof 29 patients (group 1) were administered vitamin D supplementation depending on the baseline serum level of 25-hydroxyvitamin D and 43 patients (group 2), who did not receive vitamin D. The comparison group consisted of 23 pregnant women without vaginal infections.

Baseline characteristics of patients regarding vaginal infections in group 1 and 2 were comparable (table 1).

Analysis of vaginal smears showed that among the study participants Candida albicans was the predominant isolated species accounting for 37.9% and 74.4% cases of candidal vaginitis in group 1 and 2, respectively.

The same detection rate was found for sexually transmitted infections; the main causative agents included Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, herpes simplex virus type I and II, and cytomegalovirus. All patients underwent a full course of therapy for vaginal infection. Patients in both groups received similar antibacterial, antiviral therapy, and vaginal debridement. Patients in group 1 were administered vitamin D in prophylactic and therapeutic dosages, depending on the baseline level of 25-hydroxyvitamin D.

The study used oral cholecalciferol in aqueous solution, which is recommended as a treatment for vitamin D deficiency. Dosages of the drug were prescribed depending on the baseline serum level of 25-hydroxyvitamin D: prophylactic (1500-2000 IU per day) or therapeutic (2000-4000 IU per day) dose.

Serum samples were tested three times during pregnancy with visit 1, 2, and 3 at 14-22, 18-26 weeks’ gestation, and before delivery, respectively.

Serum 25-hydroxyvitamin D level was measured by enzyme-linked immunosorbent assay using in vitro 25-OH reagent kits (Vitamin D ELISA catalog number EQ 6411-9601). Interferon status (IFN-α and IFN-γ) and cytokine profile (IL1β, IL4, IL8, TNF-α) were analyzed using the following test systems: Interleukin-1β-IFA-BEST reagent kit A-8766; Interleukin-4-IFA-Best reagent kit A-8754; Interleukin-8-IFA-Best reagent kit A-8762; alpha-TNF-IFA-Best reagent kit A-8756; gamma-Interferon-IFA-Best reagent kit A-8752; α-interferon-IFA-Best A reagent kit. Analysis and interpretation of the results were carried out according to the manufacturer’s instructions.

Also, bacterioscopy of vaginal smears, cervical cultures with antimicrobial susceptibility testing of pathogens, an examination of urogenital scrape samples for detection of sexually transmitted infections by polymerase chain reaction was carried out.

Statistical analysis was performed using Microsoft Excel – 2007and SPSS v.18 for Windows. The distribution of continuous variables was tested for normality using the Shapiro-Wilk and the Kolmogorov-Smirnov tests. Quantitative variables not showing normal distribution were presented as the median (Me) and interquartile range (Q1; Q3) and compared using the Kruskal-Wallis test. Qualitative variables were summarized as counts (n) and percentages and compared by the Chi-square test (χ2) with Yates correction for continuity. Differences between the groups were considered statistically significant at p<0.05.

Results

All pregnant women were tested for the baseline serum 25-hydroxyvitamin D level at the time of enrollment into the study. In 41 patients the level of vitamin D was within the normal range. Thirty-seven, 13, and 4 women had vitamin D deficiency (< 20 ng/ml), insufficiency (<10 ng/ml), and severe insufficiency (<10 ng/ml), respectively. At baseline, the study groups had comparable concentrations of serum 25-hydroxyvitamin D. After the assessment of vitamin D status, pregnant women in group 1 were administered vitamin D depending on its baseline level.

A preliminary analysis of the study results showed that there were no significant changes in the concentration of cytokines in patients with vaginal infections at the time of visit 2 (18-26 weeks of gestation). Due to the lack of information at the time of visit 2, the analysis of interferon status (IFN-α and IFN-γ) and the cytokine profile (IL1β, IL4, IL8, TNF-a) was carried out only at the time of enrollment into the study (visit 1) and before delivery (visit 3).

At the time of enrollment into the study (visit1), concentrations of IL-8 in group 1 and 2 was higher than in the comparison group (p <0.001) (Table 2).

Among women with vaginal infections (groups 1 and 2), patients with high concentrations of this proinflammatory cytokine made up a higher proportion than in the comparison group, which included women without vaginal infections (p <0.001) (Table 3).

Before delivery (visit 3), levels of IL-8 among pregnant women receiving vitamin D supplementation (group 1) were lower than among pregnant women without specific treatment (group 2) ( p <0.001). A similar pattern was observed when comparing the proportions of patients with elevated IL-8 levels: the proportion of patients with IL-8 concentration above the normal range was greater among women who did not receive 25-hydroxyvitamin D than in the group supplemented with vitamin D (p = 0.01 ) and in the comparison group (p = 0.01). Serum concentrations of IL-8 at visit 3 were higher among pregnant women in groups 1 and 2 than in the comparison group (p <0.001).

The concentrations of pro-inflammatory cytokine IL-1β were similar in all study groups both at baseline and before delivery (Table 2). However, in group 1, the level of IL-1β at visit 3 was lower than at baseline (p <0.01).

The concentrations of pro-inflammatory cytokine TNF-α were similar in all study groups (Table 2). At visit 3, levels of TNF-α were higher among pregnant women without vitamin D supplementation than in group 1 participants (p <0.01); women without vitamin D supplementation also had higher concentrations of this cytokine compared with baseline levels (p <0.01).

No significant differences between the study groups were observed in concentrations of IL-4, IFN-α, including in level changes during observation.

At the time of enrollment into the study, concentrations of IFN-γ did not differ significantly between the groups, but before delivery, they were significantly higher in women supplemented with vitamin D (group 1) than in the comparison group (p <0.01).

The incidence rate of ARI among pregnant women with vaginal infections supplemented with vitamin D (group 1) was lower (3.4%) compared both with those who did not receive vitamin D (group 2) (p <0.03) and women without vaginal infections (comparison group) (p <0.04).

Discussion

Despite the established role of vitamin D in immunogenesis, various mechanisms of the inflammation process continue to be an area of intense research. In particular, this study investigated the effect of 25-hydroxyvitamin D supplementation on the changes in the levels of pro/anti-inflammatory cytokines in pregnant women with vaginal infections. The study findings showed that the baseline concentration of IL-8 was higher in pregnant women with vaginal infections than in the group of healthy women (p <0.01). In the group of pregnant women supplemented with vitamin D, concentration of IL-8 decreased by the time of delivery. The level of this cytokine was significantly lower than in women with vaginal infections who did not receive vitamin D (p <0.01) and was comparable with the values ​​obtained in the group of healthy pregnant women (comparison group). Also, pregnant women supplemented with 25-hydroxyvitamin D had lower levels of pro-inflammatory cytokine IL-1β before delivery than at baseline (p <0.01), which may additionally indicate the importance of vitamin D’s role in the synthesis of this cytokine.

Concentrations of IFN-γ increased in women supplemented with vitamin D (group 1) over the study period, and at visit 3, they were significantly higher than in the comparison group (p <0.05). This increase may indicate activation of the class-II Major Histocompatibility Complex by tissue cells [19]. Given the fact that IFN-γ also has a substantial effect on adaptive immunity, it can be assumed that 25-hydroxyvitamin D supplementation is accompanied by transient stimulation of specific antibodies, making pregnant women less susceptible to infectious agents. Similar effect was observed after influenza vaccination in adults with low baseline levels of IFN-γ, which increased significantly by day 56 after immunization [20, 21].

The incidence rate of ARI among pregnant women with vaginal infections not supplemented with vitamin D (44.2%) was 13 times higher than in women supplemented with vitamin D (3.4 %), and also five times higher than in the comparison group (8.7%) (p <0.05). Therefore, the immunoprotective effect of prenatal vitamin D supplementation of women with vaginal infections was manifested by a decrease in ARI incidence rate, a lower incidence rate of upper respiratory tract bacterial complications, and a lower need for antibacterial therapy. That is, a timely 25-hydroxyvitamin D supplementation for infectious pregnancy complications may be preventive intervention for ARI, along with other drugs that prevent the onset and development of respiratory diseases [22–27].

Conclusion

The use of prenatal vitamin D supplementation in the comprehensive management of vaginal infections demonstrated its superiority over basic therapy because it results in a change in the levels of pro, anti-inflammatory cytokines, which is clinically manifested by a decrease in the incidence rate of ARI during pregnancy.