CORRELATION BETWEEN INTERLEUKIN 17 AND TRANSFORMING GROWTH FACTOR BETA WITH HABITUAL ABORTION IN WOMEN INFECTED WITH CYTOMEGALOVIRUS

Qasim mohammed banja 1 , thamer mutlk 1 , samah a. Jassam 1 , mohamed kadum al-araji 1 and qays ahmed alkafaji 2 . 1. Department of Clinical Laboratory Sciences, College of Pharmacy, AL-Mustansiriyah University. Baghdad, Iraq. 2. Kamal Al-Samarraee Teaching Hospital for Infertility and IVF. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


IL-17 and TGFβ and recurrent pregnancy loss:-
IL-17 (also known as IL-17A) plays a crucial role in immunity against extracellular pathogens and intracellular pathogens, also, it is involved in the pathogenesis of many autoimmune and inflammatory diseases (Happel et al 2005, Kuwabara et al 2017. Different immune cells, such cells γδT-cell and T-cytotoxic cell produce IL-17 (Huang et al 2004). Dysregulation of immune response suppresses Th1-cell, Th17-cells but not Th2-cells, and that increases the incidence of autoimmune diseases (Kuwabara et al 2017). Previous studies showed that, increased level of IL-17 is correlated with over-generation of Th17-cells, combined with autoimmune diseases ( IL-17 is known as a potent pro-inflammatory cytokine; recent studies manifest the role of IL-17 in developing inflammation process during autoimmune diseases and graft rejection (Gaffen 2009). Moreover, IL-17 influences the maturation process of dendritic cell; also, it prohibits the immune response induced by Th-cells, leading to immune tolerance (Gaffen 2009).
Transforming growth factor-β (TGF-β) is produced by platelets, lymphocytes, neutrophils, and macrophages (Gaffen 2009). It is a pleiotropic polypeptide, it influences different biological activities, TGF-β inhibits Blymphocytes, T-lymphocytes and natural killer cells proliferation, wound healing, inflammation, immune regulation, embryonic development and differentiation of adult stem cell (Gaffen 2009). In addition, TGB-β reduces the output of pro-inflammatory cytokines, and it prevents the formation of major histocompatibility complex class II (MHC II) on cell membrane of antigen presenting cells (Li et al 2006). Moreover, TGB-β regulates the entry of cells to apoptosis (Walshe et al 2009). TGB-β1 is the most studied member of TGB-β family; it acts as an immunosuppressive factor on B and T lymphocytes, absence of this cytokine is correlated with autoimmune diseases as lupus and scleroderma (Letterio, Roberts 1998).
The actual role and the underlining mechanisms of CMV infection effect in RPL are not fully understood yet. Thus, this study focuses on the correlation between cytomegalovirus infection, levels of IL-17 and TGFβ with the recurrent pregnancy loss. 234 control group was included 37 healthy pregnant women without history of recurrent abortion, 27 women with history of one abortion, 24 women with history of two, or more recurrent abortions. Samples of peripheral blood were collected (non-fasting), into sterile gelatine tube, samples were incubated for one hour, then serum was separated by centrifuging at 4000 rpm for 15 minutes. The Ethical committee of the Baghdad medical city hospital approved this study. The immunoenzymatic assay was used to measure levels of IL17, TGFβ1 , and anti-CMV IgG, IgM in collected samples, using the following kits: CMV LgG, IgM avidity kit (Bioactive diagnostic, Germany), human TGFβ1 kit (Demeditec diagnostics, Germany) and human IL17 is manufactured by Biological Technology, China.

Statistical analysis:-
The statistical analysis was conducted using Anderson Darling test to test the distribution of continuous variables. Discrete variables presented using there number and percentage used to present the data, chi square test used to analyse the discrete variable, and statistical difference between studied groups was rated using one way ANOVA and Tukey`s multiple comparison. All experiments were carried out three times independently and the data was expressed as ±SE. Binary logistic regression analysis used to calculate the odd ratio (OR) and their 95% confidence intervals, when the outcome can be categorized into 2 binary levels, and if appropriate probability plot used to present the relationship. SPSS 20.0.0 software package was used to make the statistical analysis; p value was considered a significant, when less than 0.05.

Levels of Anti-Cytomegalovirus (CMV) IgM and IgG:-
Results showed no significant difference in levels of anti-CMV IgM and IgG in patients with single or multi abortion, compared with the healthy control group. Anti-CMV IgM was negative in all included groups. In addition, anti-CMV IgG showed a different levels in each group: 31.5% in group with single abortion, 39.1% in multiple abortions compared with 18.5% in healthy control group, although differences were statistically non-significant as in figure 1. IgG avidity:-Avidity is known as the degree of immune activity in which the polyclonal IgG molecules binds to multiple epitopes of the antigen (Hazell 2007). IgG validity results showed 0.65 ± 0.25 in healthy control group, while 0.60 ± 0.06 of validity was seen in women with single abortion. 0.68 ± 0.05 IgG validity was noted in patients with recurrent abortion. However, no significant difference was found in the avidity of studied groups. One way ANOVA was used in statistical analysis. Data was presented as mean ± SD, p<0.05.

A B
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Correlation between IL17 and CMV infection:-
Control group showed a distinctive correlation between anti-CMV IgM and IL17, on the other hand, a weak correlation between IL17 and anti CMV IgG was seen, as shown in table 2 and figure 2A. Single abortion group  revealed inverse, weak correlation between IgG avidity and IL17, however no correlation was noted between IL17,  IgG and IgM as illustrated in table 2 and figure 2B. Recurrent abortion group declared a direct, but weak correlation between IgG avidity and IL17. Although, there was a mild, direct correlation of IgM and IL17, as in figure 2 A, B and C.

Figure 2:
The correlation between IL17 and CMV infection . A: Scatter plot for the correlation between IL17 and IgM in all the studied groups, B: represents the correlation between IL17 and IgG in all the studied groups. C: shows the correlation between IL17 and IgG avidity in all the studied groups. Plots represent linear regression analysis. Blue represents the control group, green represents single abortion group and the orange represents multiple abortion groups.

The correlation between CMV infection and TGF:-
Results revealed an inverse, weak correlation with anti-IgM and IgG CMV with TGF in healthy control group as shown in figure 3A. While, no correlation was seen in single abortion group neither IgM nor IgG avidity with TGF, as illustrated in figure 3B. Recurrent abortion group also, showed no correlation between IgG, IgM and IgG avidity with TGF as in figure 3C. Figure 3.A: Scatter plot for the correlation between TGF and IgM in all the studied groups, B: scatter plot for the correlation between TGF and IgG in all the studied groups, C: scatter plot for the correlation between TGF and IgG avidity in all the studied groups. Blue represents the control group, green represents single abortion group and the orange represents multiple abortion group.

History of past CMV infection:-
There was a significant high proportion of patients had past infection in both recurrent and single abortion groups (83.3% and 96.3%) compared to control group (56.8%). Recurrent abortion associated 3.8 folds compared to healthy control, with past CMV infection, while single abortion group associated 19.8 folds compared to control with past CMV infection.

Is there any correlation Relationship between IL17 and TGF?:-
Results showed that there was a weak (non-significant) inverse correlation between IL17 and TGF in both control and single abortion group. While, in RPL group the relationship remain weak but it become direct relationship between them as illustrate in figure 4. In addition, the lowest level of IL17 was seen in patients with multiple recurrent abortion, compared with patients with single abortion and healthy control group, (40.05%, 42.47% and 48.40% respectively). However, no statistical significance was found. Furthermore, TGFβ results did not show any statistical differences between different groups, figure 4.  . However, IgG alone to diagnose CMV infection was not highly recommended, it must combine with IgG avidity to determine the onset of infection and its chronicity. In the current study IgG avidity was 0.65 ± 0.25 IU/ml for the healthy control pregnant women, 0.60 ± 0.06 IU/ml for single abortion group and 0.68 ± 0.05 IU/ml for RSA group, there was no significant difference between them p<0.05. That revealed that most of the infected patients with CMV had chronic infection.
In addition, the frequency of CMV infection was significantly higher in the recurrent abortion group (83.3%) compared to healthy control group (56.8%). All the infected CMV were positive to past CMV infection (defined as negative IgM, positive IgG and high titre IgG avidity).
An Egyptian study showed the frequency of CMV infection was 12%, and it represented acute CMV infection using IgM PCR for diagnosis; however this study did not report the frequency of chronic CMV infection in included cases, since the focus was on CMV IgM only (Zaki, Goda 2007 it has lower risk to cause damage, making the primary CMV infection to be the most severe, the primary infection usually occur during childhood with higher prevalence in rural areas compared to urban (Hazell 2007 Najafi et al (2014). The study revealed that IL17F did not exert a statistical difference p < 0.01, while another form of IL17F (re763780) showed a significant difference between the two groups. However, the same study revealed that some Iraqi women were positive to IL17A (re2275913) form, which was not associated with RPL and there was not significant difference between RPL and healthy control group (Najafi et al 2014).
There was ongoing interest in literature to find the role of the immunological effect of cytokines in avidity, particularly in female marginal plasma and in the tissues of uteroplacental, for the homeostasis of the immune system, there must be an equilibrium among the cells effector and between the controller cells, ( The effect of Treg is to maintain the embryo-maternal acceptance, however the effect of IL17 producing T-helper seventeen cells in the normal and abnormal gravidity particularly in recurrent pregnancy lose need more study, Th17 cells considered a mediator of tissue distraction and inflammation (Fischer 2008 The current study found a direct association between IL17 and TGFβ (p <0.05). In addition TGFβ was noted to increase IL17 level in serum. However, our study did not find a significant difference in TGFβ levels between RPL and healthy pregnant women (p <0.05), with a median of TGFβ in control group 162.5 ng/ml and in RPL group 145 ng/ml.

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Although, Ozkan et al 2014 results disagreed with our findings, he found that TGFβ was significantly higher in RPL women compared to healthy pregnant women control group 46.8±9.3 vs. 2.9±0.5 ng/ml respectively, p<0.01, with an optimal cut point of ≥5.5 ng/ml to predict RPL (Ozkan et al 2014). This disagreement with our findings could be caused by the RPL cases were included in both studies ).
In the current study IL17 was seen to be correlated directly with CMV IgM level in both healthy control and RPL groups at a similar degree. Furthermore, IL17 was correlated directly with CMV IgG avidity in the RPL group only (p<0.05), but no correlation with CMV IgG. TGFβ was noted to be inversely connected with IgM in the control group (p<0.05), and no correlation was recorded in the RPL group. In addition, TGFβ was not corresponded with CMV IgG or CMV IgG avidity in all studied groups. In conclusion, we found that CMV infection was directly correlated with high levels of IL17 in serum, but no correlation was seen between CMV infection and levels of TGFβ.