Comparison of serum levels of interleukin 33 in combination with serum levels of C-reactive protein, Immunoglobulin G, Immunoglobulin A, and Immunoglobulin M in recurrent pregnancy loss: A case-control study

Abstract Background One of the critical cases of recurrent pregnancy loss is immunological factors, whereas obtaining effective prevention or treatment is necessary for cognition of reasons. Objective In this study, we tried to evaluate some immunological factors related to recurrent pregnancy loss. Materials and Methods This case-control study was conducted on 66 women at the age of 18–35 yr who were referred to the Clinic of Gynecology and Obstetrics, Ali Ibn Abi Taleb hospital, Zahedan, Iran, from August-December 2019. Interleukin 33 (IL-33) serum levels were measured using enzyme-linked immunosorbent assay. Immunoglobulin G, Immunoglobulin A, Immunoglobulin M (IgM), and C-reactive protein levels were measured by serology and hematology methods. Results The mean age of total participants was 30.8 ± 3.80 yr. The mean serum IL-33 in the case group was 318.5 ± 254.1 pg/ml and was lower than the control group (354.2 ± 259.9 pg/ml), which was not statistically significant (p = 0.52). The level of C-reactive protein in the case and control was not significantly different (p = 0.27), and Immunoglobulin A and Immunoglobulin G in the case and control were also not significantly different (p = 0.46, and p = 0.16, respectively), but there were significant differences (p = 0.003) between the level of the IgM in the case and control groups. Conclusion No statistically significant difference was observed in the IL-33 serum level, for at least 4–6 months after the last abortion in the case group and the final live birth in the control group. In contrast, serum levels of IgM were statistically significant. Finally, the need for more studies is felt according to the different results of the previous studies in this field.

For the medical and scientific communities, RPL is challenging.In reality, only about 50% of RPL cases can be defined (3).The causes of RPL are chromosomal defects of the fetus, uterine disorders, infections, hormonal or endocrine causes, disorders of immunological factors, hereditary and acquired thrombophilia, or environmental and nutritional factors.However, in more than 50% of cases, the reasons for abortion are etiologically unknown (2).Studies have shown that chromosomal abnormalities in miscarriage in the first trimester are well established and that about 5% of couples with 2 or more miscarriages carry chromosomal structural abnormalities or may have multiple problems simultaneously (4)(5)(6).
The innate and adaptive immune system plays a significant role in endometrial remodeling and maternal tolerance towards the embryo (7).
On the other hand, a successful pregnancy depends on the role of inflammatory markers, as mentioned above, which can be protective or harmful to fertilization.It has been suggested that a balance between pro-inflammatory and anti-inflammatory factors such as C-reactive protein (CRP), Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), and cytokines is essential for successful pregnancy (8,9).Interleukin (IL) 33, which is a member of the IL-1, plays an important role in host defense, immune regulation, neuronal damage, and inflammation (10,11).Most of the markers reported in previous studies showed that the role of regulatory T cells in RPL in Iranian women is highlighted by their association with them.
Endothelial, epithelial, T helper 2-activated, and mast cells are the main cells that express IL-33 (9,12).Endothelial cells and smooth muscle cells within the placenta, chorioamniotic membranes, and umbilical cord are where the expression of IL-33 occurs.Also, some studies have shown that IL-33 serum levels in recurrent miscarriages are different compared to normal pregnancies (10,13).Another study reported that IL-33 serum levels were significantly lower in women with recurrent miscarriages than in control group (14).It also shows what cellular and molecular mechanisms may be involved in the dysregulation of IL-33 signaling and poor pregnancy outcomes in women (11).This complexity further fuels the ongoing controversy about which immunological factors play a role in the pathogenesis of RPL (15).This study aimed to determine the association of the serum levels of IL-33, CRP, IgG, IgM, IgA with RPL.

Sample size
A preliminary estimate of the study's sample size was calculated from a previous study (16).
Mean IL-33 serum levels were used, with a type I error of 0.05 and a type II error of 0.2.According to the comparison of the 2 mean formulas, each group necessarily had at least 12 participants.
During the study, this number increased to 33 patients in each group.

Statistical analysis
The

Results
In this study, 66 women were divided into

Conclusion
In conclusion, in this study, IgM was found to be significantly increased in patients with recurrent miscarriage.This relationship was not observed in IL-33 and other immunological factors.Therefore, the role of IgM as an acute inflammatory marker in recurrent miscarriage has been confirmed.
It seems that different aspects of the effects of IL-33 as a predictive biomarker in PRL, should be evaluated in more comprehensive studies.
Due to no information available and the role of immune function, we aimed to evaluate and compare the IL-33 serum level some related immunological factors in patients with RPL and normal individuals referred to the gynecology and obstetrics clinic of Ali Ibn Abi Taleb hospital in Zahedan, Iran.
This case-control study was conducted on 66 women aged between 18 and 35 yr, who referred to the Clinic of Gynecology and Obstetrics, Ali Ibn Abi Taleb hospital, Zahedan, Iran from August-December 2019.Participants were selected by an easy and accessible method and divided into 2 groups of 33 as a case (women International Journal of Reproductive BioMedicine Volume 22, Issue no. 4. https://doi.org/10.18502/ijrm.v22i4.16392IL-33, IgG, IgA, IgM, CRP serum levels, and RPL with a history of RPL) and 33 as the control group (including healthy women).Inclusion criteria were having a history of recurrent miscarriage (3 consecutive miscarriages before 20 th wk), at least 4-6 months after the last miscarriage.The control group included 33 healthy women aged between 18 and 35 yr without a history of abortion and who have at least one child (at least 4-6 months have passed since the last delivery).Those women having uterine anatomical disorders, such as double uterus, arcuate uterus, unicornuate uterus, bicornuate uterus, septate uterus, thyroid disorders, systemic diseases such as antiphospholipid syndrome, systemic lupus erythematosus, rheumatoid arthritis, hyperprolactinemia, chromosomal problems of parents, and allergic diseases were excluded from the study.6 women were excluded according to exclusion criteria (autoimmune diseases, history of allergic reactions, hypothyroidism).In this study, repeated abortions meant losing pregnancy products before the 20 th wk of pregnancy on at least 3 consecutive or non-consecutive occasions.5 cc of peripheral blood was taken from each participant to determine the serum levels of IL-33, CRP, IgG, IgA, and IgM and sent to the laboratory of Ali Ibn Abi Taleb hospital, Zahedan, Iran.The blood samples were centrifuged according to the kit instructions.The serum was separated and stored in the laboratory freezer until the end of the study.Finally, after completing the number of samples, the IL-33 serum level in 2 groups was measured by enzyme-linked immunosorbent assay method (EASTBIOPHARM CO., China).
Kolmogorov-Smirnov test was used to evaluate the normal distribution of data.Due to the existence of abnormal distribution in quantitative variables, Mann-Whitney non-parametric test was used to compare the level of CRP and antibodies between case and control groups.P < 0.05 was considered significant.IBM SPSS Statistics for Windows, version 22.0 (IBM Corp), was used for the analysis, 2013, IBM Corp., Armonk, New York.

Finally, it can
be summarized that the IL-33 serum level, at least 4-6 months after the last abortion (in the case group) and the previous live delivery (in the control group), did not show a significant difference between the 2 groups.However, due to the different results of earlier studies in this field, there is still a need for further studies.The results of the present study showed no significant difference in the IL-33 serum level between normal women without recurrent miscarriages and women with a history of recurrent miscarriages.However, the mean IL-33 serum level was slightly lower in women with a history of recurrent miscarriage, which could be a significant difference in larger samples due to the limited sample size in this study.A protein phenotype may be related to the ethnicity and racial status of the patient; hence more extensive studies in this field are essential.Other suggestions include repetition of studies similar to the follow-up period of most patients to identify the IL-33 serum level more accurately in different conditions and times, designing new studies to identify pathways affecting changes in IL-33 serum levels outside of pregnancy.Moreover, determination and comparison of changes in IL-33 serum levels up to the 20 th wk of pregnancy, determining the cytokine phenotype in patients according to their demographic conditions, determining the genotype of this cytokine in patients according to their demographic conditions, they could be suitable research fields in the future.

Table I .
Comparison of the mean and standard deviation of IL-33 serum levels in case and control groups Data presented as Mean ± SD (IQR).Mann-Whitney test.IL-33: Interleukin 33, CI: Confidence interval

Table II .
The observed serologic patterns and the mean differences of CRP, IgG, IgM, and IgA between study groups