الفهرس 
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Abstract
Toxoplasmosis is a disease caused by an obligatory intracellular zoonotic protozoan parasite, Toxoplasma gondii. It is estimated to infect more than one third of the world population, it has a complex multi-host life cycle. The sero-prevalnce of Toxoplasma in pregnant women, or in women of reproductive age, or aborted women varies greatly (30% - 60%) among countries and geographical area within the same country.
T.gondii infection occurs by contact with infected feces cats with oocysts during changing a cat litter box or eating raw or under cooked meat containing tissue cysts as, or by ingestion oocyst from soil, or eating unwashed vegetables, or and by acquiring congenital infection.
Acute infection or reactivation of latent infection in pregnant women can be transmitted to fetus and may cause mental retardation, blindness, encephalitis and /or death leads to abortion; women with normal immune function are usually asymptomatic or have mild symptoms such as fever, malaise, and lymphadenopathy.
During pregnancy, the prevalence of toxoplasmosis increase in the last quarter of gestation (2nd and 3rd trimesters) thus it has been suggested that female sex hormones levels as estrogen and progesterone can aggravate or decrease and have a role on Toxoplasma infection by increase or reduce parasite reproduction.
T.gondii induces a robust Th1 immune response which leads to production of pro inflammatory markers (Interleukin12 (IL-12) and Interferon gamma (IFN-γ)). T cell response, which play a major role in cleaning the parasite stage.
The present work was aimed to study female sex hormones and inflammatory markers in aborted and pregnant females infected with Toxoplasma gondii.
The study protocol was reviewed and approved by ethical review committee of MRI, Alexandria University, Egypt, and approval of the ministry of health and the authority of Elshatby hospital. Informed consent was taken from all participants who agreed to participate in this work after explaining the aim of the study.
The study extended from the beginning of May .2019 to mid of February 2020.
The target participants was composed of pregnant (n=50) and /or aborted (n=50) women infected with Toxoplasma, and (50) control pregnant uninfected with T.gondii women, who attended randomly selected Elshatby Hospital for examination and follow up. All women were enrolled in the study fulfilled the inclusion criteria as current pregnancy, or aborted women with toxoplasmosis and response to all questionnaire items, Toxoplasma, hormones and inflammatory markers serological examination.
The enrolled women were classified according to Toxoplasma infection and pregnancy status into three groups:
Gp1: Control Pregnant women were free from toxoplasmosis (n=50).
Gp2: Pregnant women with toxoplasmosis (n=50).
Gp3: Aborted women with positive T.gondii infection (n=50).
Data collection methods
1. Questionnaire
All participants’ women were interviewed using pretested questionnaire to assess the major characteristics data, different risk factors of T.gondii infection.
The questionnaires were applied at face-to-face interviews and recorded by the researcher.
2. Laboratory studies
About 5 ml of venous blood was drawn from each participant. All samples (n=150) were numbered by code numbers they were left to coagulated then centrifuge to obtain sera, then the sera were divided into six parts. 1st and 2nd parts were diagnosed for anti-toxoplasma specific immunoglobulin (IgM , IgG and IgM/IgG ), 3rd and 4th parts were examined for hormones levels (Estrogen and Progesterone), 5th and 6th parts were examined for inflammatory markers levels (IL-12 and IFN-γ ). All sera parts were stored at -20○C until examined by ELISA assays. The procedures of techniques were followed up according to manufacturer’s instructions.
The collected data were entered the computer and analyzed using a statistical version 20.
The association between toxoplasmosis and risk factors were tested by appropriate statistical procedures.
The results of the present study were summarized as follows:
I. Distribution of anti-toxoplasma specific immunoglobulin antibodies among women groups included in the study:
• IgM +ve (Acute active infection) represents in 8% among pregnant women infected with Toxoplasma (Gp2), and 50% among aborted women infected with Toxoplasma (Gp3).
• IgG +ve (Past / chronic infection was found in 72% of pregnant women Gp2 and 30% of aborted women Gp3.
• IgM/IgG were positive of 20% in both studied infected women Gp2 and Gp3 there were a statistically significant in sero-positivity among the both groups.
II. Distribution of toxoplasmosis women according to age and positive specific immunoglobulin antibodies was showed that 36.4% of IgM (acute) positive toxoplasmosis (Gp2 and Gp3) were among women with age between 25 – <30 years, while +ve IgG (chronic) diagnosed among 55.3% of Gp2 and Gp3 with age range between 30 -35 years , IgM/IgG (sub-acute) were found in 26.3% in women with age range 30-35 years.
III. Determination of hormones levels among women groups
a) Estrogen and progesterone levels among Gp1 and Gp2 showed no statistical significant difference
b) Estrogen and progesterone levels among Gp2 and Gp3. Estrogen levels estimated among Gp3 was 547.5 pg/ml less by three times than recorded in Gp2 (1600.0pg/ml), while progesterone were approximately equal levels in Gp2 and Gp3. No statistical significant difference was found regarding levels of estrogen and progesterone.
c) Estrogen and progesterone levels among Gp3 and control Gp1. Estrogen levels detected among Gp3 was 547.5 pg/ml less than levels estimated among Gp1 (1382.5pg/ml), while levels of progesterone in two groups Gp1 and Gp3 were nearly a similar, with no statistical significant difference was found
IV. Determination of inflammatory markers among women groups.
A. Determination of IL-12 and IFN-γ levels among Gp2 and control Gp1. It was no statistical significant difference was found between the Gp1 and Gp2 regarding level of IL-12 and IFNγ.
B. Determination IL-12 and IFN-γ levels among Gp2 and Gp3 illustrated no statistical significant difference regarding levels of IL-12. However, IFNγ level among Gp3 was highly (12pg/ml) than diagnosed among Gp2 (7 ng/ml). IFNγ was statistical significant between Gp2 and Gp3.
C. Determination of IL-12 and IFN-γ levels among Gp3 and control Gp1. The comparison between the levels in two groups was statistically significant regarding levels of IL-12. No statistical significant difference was found between the two groups regarding levels of IFNγ.
V. Relation between hormonal, inflammatory markers levels and age categories among different women groups.
A. Relation between age categories, hormonal and inflammatory markers levels among control pregnant uninfected with T.gondii group (Gp1), revealed that no statistical significant difference were found (Gp1) between different age categories and levels of estrogen and progesterone, and levels of IL-12 (p=0.217). IFNγ showed significant difference in (Gp1) and different age categories with highest level (12 ng/ml) found in the age group between 25 – <30 years.
B. Relation between age categories, hormones and inflammatory marker levels among pregnant toxoplasmosis women (Gp2). No statistically significant difference were found between different age categories and levels of estrogen and progesterone, IL-12 and IFNγ.
C. Relation between age categories, hormones and inflammatory markers levels among aborted women infected with Toxoplasma (Gp3). No statistically significant difference were found between different age categories regarding levels of estrogen and progesterone, and level of IL-12. As regarding to IFNγ levels, showed significant difference in the Gp3 between different age categories, and highest level (18.0 ng/ml) found in the age category between 25 – <30 years.
VI. Relation between toxoplasmosis specific immunoglobulin antibodies, hormonal and inflammatory markers levels among pregnant and aborted women infected with Toxoplasma (Gp2 and Gp3).
a) Relation between toxoplasmosis immunoglobulin antibodies and hormonal levels in Gp2 and Gp3. No statistically significant difference were found in Gp2 and Gp3.
b) Relation between toxoplasmosis specific immunoglobulin antibodies and inflammatory markers levels among Gp2 and Gp3. No statistically, significance differences were found.
VII. Correlation between anti-toxoplasma specific immunoglobulin antibodies and the different parameters in toxoplasmosis infected groups (Gp2 and Gp3).
a) Correlation between the different parameters and specific immunoglobulin in pregnant women infected with Toxoplasma (Gp2). It show negative correlation between IL-12 vs progesterone and positive correlation between IFN-γ vs estrogen in IgM\IgG +ve sub-acute. IFNγ vs. Progesterone showed positive correlation (rs=0.351, p=0.036) in the IgG +ve (chronic) group.
b) Correlation between immunoglobulin antibodies and the different parameters among Gp3. No correlation were found between examined parameters among the aborted women infected with Toxoplasma Gp3.
c) Correlation between the specific immunoglobulin antibodies and the different parameters in all women infected with Toxoplasma (Gp2 and Gp3). Significant positive correlation between IL-12 vs estrogen in the IgM\IgG +ve sub-acute were detected.
VIII. Risk factors for toxoplasmosis
a) Socio-demographic data (major characteristics of studied women)
As regards to sero-prevalence of Toxoplasma infection in relation to major characteristics demographic data. Among pregnant and aborted women. Maximal positive age range were 30-35 (38%) with median (28.0 years) not significantly, and show that most positive residential area were urban (51%) but not significantly, the greater positive participants were housewife (76%) and significantly. As regards to educational background, most of the infected studied women (29%) were preparatory educational level, while women with university educational level showed (14%). The association was statistically significant regards to the sero prevalence of T.gondii infection in relation to the knowledge about toxoplasmosis among the studied women. The majority of the sero positive participants (56%) had knowledge about toxoplasmosis and the route of transmission of infective stage (47%) but about half of participants had no knowledge about prevention toxoplasmosis all results of knowledge about prevention were statistically significant result.
b) Animal rearing risk factors
It was found only 38% of sero-positive toxoplasmosis (Gp2 and Gp3) infected women had reared cats in their household. The majority of infected cases (52.8%) were feeding their cats with ordinary food also 39.5% of them were removing the cats stool without using gloves.66% sero positive cases of Gp2 and Gp3 were rearing other animals (chicken , ducks ,rabbits and birds ) in their house holds.42% of sero positive toxoplasmosis were contact with soil. There was statistically significant relation between the studied women groups and cat rearing.
c) Variables related to source of infection (food, and ways hygiene in kitchen). About 58% of sero-positive toxoplasmosis cases were used tap water for drinking and used in cooking. The majority of sero-positive the studied women (51%) were consume ready to eat processed meat as luncheon and pastrami, about 49% and 61% were eating under cooked and drink fresh milk product directly, respectively. Only 53% of positive toxoplasmosis infected women were eating raw vegetables and fruits outdoor there was statistically significant between positive toxoplasmosis women (Gp2 and Gp3) and negative group (Gp1) with this variable, majority of them (77%) were washing vegetables before eating. 47% of sero-positive Toxoplasma infected women were washing vegetables by running tap water only. While 18% of them not wash and eat directly from field. All of toxoplasmosis-infected women were washing their hands, utensils and knives before cooking. About 63% of them using the same knife for meat and vegetables. Only 49% of sero-positive Toxoplasma infection among studied women were using a cutting board. 50% of them were not applied separate cutting board for meet or vegetables and not washed the cutting board between cuttings. There was no statistically significant relation between toxoplasmosis and these risk variables between the studied infected women (Gp2 and Gp3).
6.2. Conclusion
Egypt is not having a basic action to prevent toxoplasmosis in pregnant women. There is no definite and precise figure for:
• The infection rate of congenital toxoplasmosis during pregnancy among Egyptian women.
• The serological screening for specific immunoglobulin antibodies, female sex hormones and inflammatory markers levels are not a routine checkup for any pregnant women in each monthly clinical examination until delivery.
• Determination of IFN-γ level changes occurs during pregnancy. If sero-conversion occurs during 1st trimester of pregnancy, pregnant women must be treated with anti-parasite medication.
Modulation of the anti-T.gondii immune response in pregnant women and significant correlation between abortion caused by Toxoplasma and IgM sero-positivity of toxoplasmosis in women
This study support earlier observation that estrogen and progesterone may act as an immune regulator through effect on inflammatory markers.
Estrogen may have immune modulatory effect on Toxoplasma infection (total infection) where it showed positive correlation with interferon gamma (IFNγ) in infected pregnant group.
Progesterone may have immune protective effect against Toxoplasma infection as it showed negative correlation with IL-12 and positive correlation with Interferon gamma (IFNγ) in pregnant infected group.
Estrogen and progesterone effect on immune modulation was not profound in aborted group, as their level did not show significant different between studied groups.
6.3. Recommendations
Based on the results of the present study, the following suggesting items could be recommended:
1. Estimation of the burden of toxoplasmosis. This will be achieved through the applicability of a nation-wide toxoplasmosis screening program of obtaining population –based data regarding incidence of new infections and number of pregnant women with a diagnosis of Toxoplasma sero-conversion is expected to increase and the number of cases by mode of transmission risk factors.
2. Improving immune diagnosis for toxoplasmosis emphasing upon the use of more accurate screening diagnosis and confirmatory tests for Toxoplasma sero-conversion, for pregnant women.
• Role of sex hormones in anti-toxoplasma immunity during last trimester of gestation and its mechanism of action is still needed
• Future studies on the interaction of sex hormones with other factors responsible for immune response against T.gondii.
• Use of modern molecular methods are required to define the mechanistic role of hormones in the regulation of toxoplasmosis
• Further studies on wider cohort is needed to prove the role of female sex hormones on immune modulatory effect and cytokines level on occurrence of abortion in toxoplasma positive pregnant females.