Toxoplasmosis is known to cause dangerous variable results during pregnancy. In several previous studies, pregnancy was evaluated as a solitary event but actuality it has various immune phases relied on pregnancy time and probably external agents such as infectious factors. A state of immune homeostasis must be established in addition to the tolerance state for maternal-fetal otherwise, undesirable consequences may occur as a result of an infection [1].

Several data suggest that cytokines play a significant role in the development of appropriate immune responses against T. gondii [2]. Strikingly, cytokines are among a large number of miscarriage-related factors, those involved in abnormal immune reactions [3].

Serological diagnostics are one of the most commonly used methods for T. gondii screening. In humans, IgM antibodies to T. gondii are an early marker of the acute phase of disease and can be detected one week after infection. However, given that IgM antibodies can persist for several months and years, and in pregnant women up to 18 months after an acute disease, this serological finding must be combined with epidemiological data on possible exposure to this pathogen [4, 5]. IgG antibodies to T. gondii appear 1–2 weeks after infection with a peak 1–2 months after infection and have a tendency to survive for life, although there is a gradual decrease in the level of the antibody titer over time [6].

Cytokines are a group of immunomodulatory proteins leading a variety of immune reactions in the human body, which play a significant role in the development of appropriate immune responses against T. gondii [2]. The majority of women infected with

toxoplasmosis have asymptomatic; primary infection during pregnancy can result in disease transmission through the placenta and lead to hazardous consequences such as abortion, stillbirth, mental or physical retardation, hydrocephalus, and blindness [7].

Toxoplasma gondii has an important role in change of cytokines levels and immunoglobulins titer in aborted women that infected with this parasite [8].

Samples

In the present case-control study, 5 ml of venous blood samples were taken from 115 women aged 18-47 years, who were suffering from repeated abortion, and from 30 apparently healthy women as a control group.

The demographic information of patients was recorded in a questionnaire form prepared in advance including age and residency. A written consent was taken from all participants in this study. The study was carried out in Karbala Maternity and Pediatric Hospital and other private laboratories in Kerbala city, Iraq during the period from November 2022 to May 2023.

Immunological assay

The blood samples were put in gel tubes, and centrifuged at 3000 rpm for 10 minutes to obtain serumwhich were stored at -20^oC until use. The levels of serum IgM, IgG, interleukine-4 (IL-4) and interleukine-8 (IL-8) were estimated by ELISA method, using the Human Mini ELISA kits manufactured by ELK Biotechnology Company, USA. The ELISA method adopted theCapture antibody principle.

Statistical analysis

For statistical analysis of data, the Statistical Package for Social Sciences (SPSS-25) was used, which was presented as Mean + SE. Statistical differences between means was estimated by analysis of variances, followed be least difference test or Duncan test. The difference was considered significant at (p<0.05), [9].

The results of the current study showed that the highest rate of infection (56.6%) with T. gondii parasite was among the age groups 18-26 years, and the lowest infection rate was 6 (11.3%) among the age groups 37-47 years, with a statistically highly significant differences (P<0.0001), (Table 1).

Table (1): Distribution of toxoplasmosis patients according to age

The results in table (2) showed that the infection rate with T. gondii parasite was higher in urban areas than in rural areas (60.37%) in comparison with the rural areas (39.6%), but with no significant difference (p=0.131).

Table (2): Distribution of toxoplasmosis patients according to residency

When the sera were examined using the ELISA technique, it was found that 98.1% were positive for IgG antibodies, while only 1.9%

was positive for IgM antibodies, while no IgG or IgM antibodies were detected in the healthy control group, with a highly significant difference p<0.0001 (Table 3).

Table (3): IgG and IgM rates in the study groups

The mean serum level of the cytokine IL-4 increased in the sample of patients aborted due to toxoplasmosis 90.51 pg/ml compared to the control women 21.31 pg/ml, with a highly significant difference (p<0.0001) as demonstrated (Table 4).

Table (4): IL-4 levels in the study groups

The mean serum level of the cytokine IL-8 increased in the sample of patients aborted due to toxoplasmosis 913.30 pg/ml compared to the control women 226.06 pg/ml, with a highly significant difference (p<0.0001) as demonstrated in table (5).

Table (5): IL-8 levels in the study groups

In the current study, it was found that 52 (98.1%) were positive for the IgG antibodies, while it appeared that only 1.9% patient was positive for the IgM antibodies using the ELISA technique. Seroprevalence is the detection of the percentage of individuals in a population having antibodies against an infectious pathogen by testing their blood serum. The samples that come out positive for specified antibodies imply the occurrence of previous exposure to that particular pathogen. The specific IgG and IgM antibodies are indicative of detecting Toxoplasma infection. Repetitive serological screening for IgG and IgM distinguishes between acute and chronic infections [10]. Serological identification of T. gondii- specific IgM indicates recent or current/acute infection, whereas the presence of T. gondii-specific IgG indicates past or latent infection [11].

Also the results obtained by [12] showed that the prevalence was 38.9% by ELISA IgG in Khartoum state; also [13] found that 73.1% by using ELISA IgG in rural areas in Sudan. It may also disagree with result obtained by [14], who showed that the prevalence using ELISA was 35.1% positive IgG antibodies to T. gondii in Sudanese pregnant women. The result however, agreed with [15], who showed that 20.2% of pregnant women were positive for IgG.

 Another research showed that of 797 studied women of reproductive age, only 23.46% had IgG antibodies against T. gondii. The seroprevalence rate of 23.46% in their study is similar to the 33% prevalence found by a meta-analysis conducted among Iranian women of childbearing age [16].

The lower prevalence of IgM, as an indicator of recent infection, is most likely a consequence of the asymptomatic or oligosymptomatic manifestation of the disease and less testing for T. gondii in the acute phase of the infection [4].

The results of the current study showed that the highest infection rate with T. gondii parasite was within the age group 18-26 years, while the lowest rate was within the age group 37-47 years.The results of the current study are somewhat consistent with the study which stated that seroprevalence is greater within the age group of 15-30 years. Seropositivity does not appear to increase with age in this study. Previous researchers have found that the seroprevalence of parasite infection increases with age, as seen in ages 35 to 38 years and older than 48 years [17, 18]. The observed variation in infection rates can be attributed to the age classification of research participants in the current study. This may also be due to poor personal hygiene. This underscores the importance of continuing to educate women of reproductive age about toxoplasmosis prevention. Seropositivity was not statistically significant [19].

 In contrast to the results of the current study, other researchers have previously reported that the seroprevalence of the parasite has an age-related increase, with lower rates in younger women. This type of association may have occurred due to longer exposure to risk factors associated with infection, such as contact with animals that transmit the parasite, such as cats [20, 21].

The results of the current study showed that the infection rate with T. gondii was higher in urban areas than in rural areas.Most studies showed results different from the results of the current study, as [16] found that the highest seroprevalence rate was found among residents of rural areas. Living in rural areas has been found to be a risk factor for Toxoplasma gondii infection, meaning that low socioeconomic level, difficulties in accessing health services, high exposure, and lack of understanding about disease transmission contribute to the high prevalence of the disease [22].

The mean IL-4 levels in the patients aborted due to toxoplasmosis increased compared to the sample of control women. The results of the current study agreed with the study that found that several cytokines, such as IL-4, IL-5, and IL-10 are increased in patients chronically infected with toxoplasmosis compared to uninfected patients [23].

The IL-4 is intimately involved in the regulation of antibody isotype expression and function. Depending on the surface proteins expressed by neighboring cells and the cytokine environment, activated B cells and plasma cells will secrete different antibody classes. The B cells switch between antibody classes by recombination of the various antibody gene regions [24].

During the early stages of toxoplasmosis i.e. during acute infection, and when the rapidly multiplying phase is present and there is a lack of immune response, it leads to the death of the host, IL-4 plays a protective role to reduce the number of deaths through the secretion of this cytokine by T helper cells. These preventive effects reduce the mortality rate in the short term, but increase the rate of the duration of illness in the long term, due to two factors: the need for different immune states to control the parasite, because it has two life cycles in the final host. Secondly, this cytokine has a direct effect on Th-2 cells, as these cells indirectly inhibit the production of pro-inflammatory cytokines, which inhibits the production of interferon, and IL-4 is known to enhance humoral and cellular immunity [25].

The mean serum level of IL-8 was higher in the sample of patients aborted due to toxoplasmosis, compared to the control women. Many studies agreed with the current results, as both [26 and 27] found an increase in the level of IL-8 in women infected with toxoplasmosis compared to the control group.Interleukin-8 (IL-8) is produced by macrophages and other cell types such as epithelial cells and endothelial cells. Primary function of IL-8 is the induction of chemotaxis in its target cells like neutrophil and granulocytes [5]. IL8 has an important role in the innate immune response. Interleukin-8 is often associated with inflammation. It has been cited as a pro-inflammatory mediator in toxoplasmosis [28].

There is increase in the IL-8 level in the current study this may revealed increasing of inflammatory response in aborted women and this may lead to attract of lymphocyte and neutrophil. This agrees with [26 and 29], who found that IL-8 was significantly increased in acute with early acute inflammation or with a reactive from toxoplasmosis.

It can be concluded from the current study that the levels of IgG, IL-4 and IL-8 were increased in women with repeated abortion who were infected with toxoplasma gondii.

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