INTRODUCTION
The term toxoplasmosis (TOX) is used to describe the pathological and clinical entity of disease caused by Toxoplasma gondii (T . gondii ) which may be asymptomatic primary infection or latent chronic infection.[ 1 ] TOX is divided in clinically into acquired TOX, congenital TOX, and reactivated TOX in immune-deficient patients. However, in any clinical class, the clinical presentations are not specific for TOX, and a broad differential diagnosis must be regarded as a strategy of diagnosis and their interpretations could be differed for each clinical category.[ 2 ]
TOX in pregnancy persists to be the cause of tragic in the offspring due to unfortunate outcome for infants and children in addition to the emotional burdens faced by parents and society. However, most of the infants appear healthy at birth and noteworthy complications and sequelae become noticeable within months or years.[ 3 ] Moreover, TOX in pregnancy is predominantly acquired during gestation; however, in rare cases, TOX has occurred due to reactivation of chronic infection because of immunocompromised complications such as corticosteroid therapy and acquired immunodeficiency syndrome. Besides, most of the infected pregnant women with acquired TOX are asymptomatic and minority of them presented with low-grade fever, malaise, and lymphadenopathy.[ 4 ]
The diagnosis of TOX in pregnancy is considered when there is a history of recurrent abortions, there is presence of fetal abnormalities by ultrasonographic findings, or when the newborn illustrates clinical manifestation of congenital TOX. As well, systemic serological screening including immunoglobulin G (IgG) and immunoglobulin M (IgM) should be done to confirm the diagnosis and determine the type of infection which may be acquired or latent as positive IgG <18 weeks of gestation indicates infection prior to the gestation but positive IgG >18 weeks indicates infection before or after gestation.[ 5 ]
On the other hand, TOX in pregnancy is linked with the development of oxidative stress and systemic inflammatory condition that contribute together in the progress of endothelial and placental dysfunctions (PDs).[ 6 ] Al-Kuraishy et al . reported that acute TOX leads to induction of oxidative stress and lipid peroxidation due to overproduction of free radical and consumption of endogenous antioxidant capacity.[ 7 ] Thus, oxidative stress and lipid peroxidation-induced endothelial dysfunction may contribute into the development of PD.
In addition, melatonin (MEL) is a hormone produced from pineal gland following a circadian rhythm and also produced from extrapineal sites such as innate immune system and gastrointestinal tract. It has been reported that MEL plays an important role in the regulation of immunity and immune response during acute TOX.[ 8 ]
Therefore, the objective of the present study was to elucidate the potential role of inflammatory, pro-inflammatory biomarkers as well as MEL in the development of PD in the pregnant women with acquired TOX.
MATERIALS AND METHODS
Study design
This case–control study was carried out at the Department of Clinical Parasitology in teamwork with the Department of Gynecology and Obstetrics, Al-Yarmouk Teaching Hospital, College of Medicine, Al-Mustansiriya University, Baghdad, Iraq, from September 2018 to February 2019. This study was established and approved by a scientific committee of the Department of Parasitology and certified by the Committee of Medical College, according to the Declaration of Helsinki.[ 9 ]
A screening survey of 75 pregnant women was done in the out-clinic of the Department of Gynecology and Obstetrics for any pregnant women at a gestational age of 20 weeks or less with a history of low-grade fever, recurrent abortion, pregnancy complications, contact with animals, etc., Following a full medical history and complete physical and obstetric examinations, the recruited patients and healthy controls were allocated into two groups after excluding of five patients due to gestational diabetes and hypertension – Group A: pregnant women with acute TOX (n = 45) and Group B: healthy pregnant women (n = 25).
Inclusion criteria
Pregnant women with gestational age <20 weeks with or without AT were included in this study.
Exclusion criteria
Any pregnant women with chronic hypertension, gestational hypertension, cardiovascular complications, endocrine disorders, metabolic disorders, gestational diabetes mellitus, chronic sepsis, urinary tract infections, multiple pregnancy, hepatic disorders, chronic kidney disorders, and psychiatric and mental disorders were excluded from this study.
Biochemical variables
Serum samples
Five milliliters of venous blood was taken from each woman that was separated and stored at 20°C until be used later. Anti-Toxoplasma (Toxo) IgM antibody was tested for each woman included in the study using enzyme-linked immunoassay (ELISA) kit method (Bioactiva Diagnostica, Germany). The serum concentration of MEL was estimated by using ELISA kit method (CUSABIO, Mabtech Company, Germany, and Biosource, Belgium). Interleukins 10 and 12 (IL-10 and IL-12) were estimated by ELISA kit methods (Bioactiva Diagnostica, Germany).
Placental samples
At the time of delivery, 3 ml of placental blood samples was taken from each delivered woman and centrifuged at 3000/rpm and stored at − 20 for assessment of placental MEL, IL-12, and IL-10 by using ELISA kit method (CUSABIO, Mabtech Company, Germany and Biosource, Belgium).
The data of the current study were presented as mean ± standard deviation. Data analysis was done by using SPSS (IBM SPSS Statistics for Windows version 20.0, 2014 Armonk, NY, IBM, Corp, USA). Correlation coefficient was done to explore the correlation between variables. The level of significance was regarded when P < 0.05.
This study was established and approved by a scientific committee of the Department of Parasitology and certified by the Committee of Medical College, according to the Declaration of Helsinki according reference No.23TYY in 2020.
RESULTS
The findings of the present study illustrate that there was a significant difference in the number of controls and pregnant women with acute TOX (P = 0.02). However, there were insignificant differences regarding the maternal age, gestational age, and gravidity (P > 0.05). The parity was lower in the pregnant women with acute TOX compared with controls (P = 0.001). In the pregnant women with acute TOX, anti-Toxo IgM serum level was higher compared with controls (P = 0.001), but anti-Toxo IgG serum level was not significantly different (P = 0.15). Both MEL and IL-10 serum levels were lower in the pregnant women with acute TOX compared with controls (P = 0.002 and P = 0.002), respectively. However, IL-12 serum level was higher in the pregnant women with acute TOX compared with controls (P = 0.001). Besides, both MEL and IL-10 placental levels were lower in the pregnant women with acute TOX compared with controls (P = 0.001). As well, placental IL-12 level was higher in the pregnant women with acute TOX compared with controls (P = 0.001) [Table 1 ].
Table 1: Demographic characteristics of the present study
Moreover, anti-Toxo IgM serum level was positively correlated with serum and placental levels of IL-12 serum level and negatively correlated with serum and placental MEL and IL-10 serum level [Table 2 ].
Table 2: Pearson correlations of anti-toxoplasma antibody titer with melatonin and pro-inflammatory and inflammatory biomarkers in the pregnant women with acute toxoplasmosis
Furthermore, the present study illustrated that serum–placental (SP) ratio of MEL was higher (5.79) in the pregnant women with acute TOX compared with controls (2.25) (P = 0.001). However, SP ratio of IL-10 and IL-12 was not significantly differed in the pregnant women with acute TOX compared with controls (P > 0.05) [Figure 1 ].
Figure 1: SP-MEL: serum–placental ratio of melatonin. SP-IL-10: serum–placental ratio of IL-10. SP-IL-12: serum–placental ratio of IL-12. *P < 0.001 compared to the control
DISCUSSION
The present study showed that acute TOX is considerably associated with inflammatory burden and PD through reduction of endogenous antioxidant potential of MEL and anti-inflammatory potential of IL-10 with augmentation of the effect of pro-inflammatory IL-12. Pinto-Ferreira et al . confirm the association between PD and acute TOX through induction of localized inflammatory reactions in the placenta.[ 10 ] As well, Zhang et al . showed that IL-10 has an immune-reactive role in the attenuation of poor feto-maternal outcomes and PD due to its potent anti-inflammatory effect.[ 11 ] A recent study by Machado et al . shows that MEL serum level is reduced in acute TOX due to oxidative stress and inflammatory reactions that are induced by T . gondii .[ 8 ]
It has been reported that acquired TOX during pregnancy leads to placental infection and injury with subsequent increase of placental permeability to the T . gondii and development of congenital TOX.[ 12 ]
It has been shown that MEL serum level is increased and reached to its maximum level during pregnancy and return to the normal level after delivery. Besides, MEL serum level is higher in twin than in single pregnancies.[ 13 ] MEL as well improves placental function and feto-placental development through its direct antioxidant effect or indirectly through enhancement of the activity of placental antioxidant enzymes with scavenging of free radicals.[ 14 ] In TOX-induced PD, there is a significant alteration in the placental antioxidant enzymes with augmentation of oxidative stress due to reduction of the synthesis and release of placental MEL.[ 15 ]
MEL improves placental perfusion through activation of the synthesis and release of endothelial nitric oxide (NO); however, in acquired TOX in pregnancy, higher placental free radicals reduce the bioavailability of NO with subsequent placental ischemia. Therefore, MEL therapy in the pregnant women with acute TOX improves placental function and fetal outcomes through improvement of placental transport of glucose and amino acids as well as oxygen and nutrients delivery to the fetus.[ 16 ]
Moreover, MEL regulates placental pro-inflammatory/anti-inflammatory axis in the pregnant women with acute TOX. Different studies show that acute TOX is associated with elevation of pro-inflammatory IL-12 and reduction of anti-inflammatory IL-10 serum and placental levels.[ 17 ] Thus, reduction of placental and serum MEL level is associated with significant decrease and elevation of IL-10 and IL-12, respectively, as confirmed by our findings.
Therefore, most of the pregnancy-related complications such as acute TOX and preeclampsia are correlated with PD as evaluated by alteration in the MEL, IL-10, and IL-12 serum and placental levels.[ 18 ]
More to these points, the findings of the present study disclosed that SP ratio was higher in the pregnant women with acute TOX as compared to the controls. This suggests a higher reduction in the placental MEL concentration due to development of PD as revealed in different research lines.[ 19 ] Nevertheless, SP ratio of IL-10 and IL-12 was not significantly differed in the pregnant women with acute TOX as compared to the controls, suggesting that placental pro-inflammatory/anti-inflammatory axis was not linked with PD. Thus, MEL but not IL-10 or IL-12 is regarded as a surrogate biomarker of PD in the pregnant women with acute TOX.
During pregnancy, physiological inflammation is important in the development feto-placental unite; however, placental inflammation is augmented in complicated pregnancies contributing to reduction of the viability of placental trophoblasts.[ 20 ] MEL regulates placental inflammation and autophagy which are increased in acute TOX during pregnancy.[ 21 ]
Therefore, early estimation of MEL, IL-10, and IL-12 in pregnant women with acute TOX may reflect the placental function and risk of congenital TOX.
The present study has several limitations which were small sample size, not prospective study; also histopathological examination of placental tissue was not evaluated. Nevertheless, this study is regarded as a preliminary study confirms that MEL is a link between acute TOX and PD.
CONCLUSION
MEL and anti-inflammatory IL-10 are reduced in the pregnant women with acute TOX, while IL-12 is increased. SP ratio of MEL but not of IL-10 or IL-12 is elevated in the pregnant women with acute TOX reflecting the risk of PD.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
We are grateful to Prof. Dr. Sadiq M. Al-Hamash for his great support.
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