Unraveling the link: serological and molecular insights into Toxoplasma gondii infection in women with spontaneous abortion history

Abstract

Background

Spontaneous abortion (SA) associated with infectious pathogens such as Toxoplasma gondii during pregnancy poses a substantial health risk for pregnant women and is linked to transplacental infection of the fetus. This study was conducted to investigate the serological and molecular aspects of T. gondii genotyping in women who have experienced SA at various gestational ages. These women were admitted to the Obstetrics and Gynecology Department of Ali Ibn Abi Talib Zahedan Hospital between September 2021 and May 2024.

Methods

This study examined 163 women with a history of abortion. Blood samples were tested for specific anti-Toxoplasma IgM and IgG antibodies using ELISA. In contrast, tissue samples from their aborted placentas were analyzed for molecular examination using nested PCR targeting the GRA6 gene.

Results

The results indicated that the women in the study ranged in age from 18 to 39 years, with 16% testing positive for anti-Toxoplasma antibodies: 9% had IgG, 4% had IgM, and 3% had both IgM and IgG. Subsequent nested PCR analysis of the placental tissue revealed that 7 cases (4.29%) were positive for the 529 bp fragment of T. gondii. Our data confirmed that five isolates belonged to type I, and two belonged to type II of T. gondii.

Discussion

The findings of this study suggest that screening programs for T. gondii significantly elevate the risk of miscarriage among pregnant women. Examining placental tissue for the molecular epidemiology and genetic variants of T. gondii linked to abortion is advisable to improve detection sensitivity.

Introduction

Spontaneous abortion (SA), defined as the unintended loss of a pregnancy before 20 weeks of gestation, is a significantly health concern and a multifactorial abnormality influenced by a combination of infectious and non-infectious etiologies, contributing to nearly 15% of cases. Pathogens such as viruses, bacteria, fungi, and protozoa, along with genetic abnormalities, hormonal imbalances, nutritional deficiencies, physical stress, toxic substances, and chemical agents, can all contribute to SA. These factors may act independently or in combination, underscording the complexity of the issue [1, 2].

The main infectious agents associated with abortion include Brucella, Salmonella, Mycoplasma, Chlamydia abortus, Coxiella burnetii, and Toxoplasma gondii (T. gondii) [3]. T. gondii is an intracellular parasite causing toxoplasmosis in warm-blooded animals, with cats as the only definitive hosts essential for its life cycle [4, 5].

Human infection primarily occurs through the ingesting of mature sporulated oocysts found in contaminated food, vegetables, and water contaminated by cat feces. Additionally, infection can result from consumption raw or undercooked meat containing tissue cysts, as well as through congenital transmission (CT), organ transplantation, and blood transfusion [6, 7].

The global prevalence of anti-T. gondii antibodies in human serum estimated to be around one-third, with rates ranging from less than 10% to over 90%, depending on geographical location, environmental conditions, and dietary practices. In Iran, seroepidemiological studies conducted across various provinces, particularly Sistan and Baluchistan, have reported an average seropositivity rate of 23% against T. gondii in the general population [8–11].

CT poses a significant health risk for pregnant women in regions with high T. gondii infection rates among both pregnant women and newborns. This condition arises from transplacental infection, with an estimated prevalence of 1–5 cases per 1,000 pregnancies. A systematic review in Iran found that approximately 47.3% of pregnant women test positive for Toxoplasma infection. Fetal gestational age significantly affects the epidemiological impact of maternal Toxoplasma infection, especially concerning maternal anti-toxoplasma antibody status [12]. If a pregnant woman is diagnosed with acute toxoplasmosis, especially in the first or second trimester, treatment may be necessary to reduce fetal transmission and complications. The standard treatment includes medications like pyrimethamine and sulfadiazine, often combined with leucovorin (folinic acid), to decrease the risk of bone marrow suppression associated with pyrimethamine [13].

While most infants CT do not exhibit symptoms at birth, clinical manifestations may develop over time. Maternal infection prior to pregnancy rarely poses a threat to the fetus; thus, CT typically occurs when the mother contracts the infection for first time during pregnancy [14, 15]. The rate of fetal transmission following primary maternal infection ranges from 25% in the first trimester to 65% in the third trimester, with younger fetuses being more susceptible to severe outcomes [16].

Currently, molecular methods with more specificity and sensitivity than serological tests targeting various genes are employed to detect T. gondii in aborted samples and to study parasite genotypes. Most T. gondii isolates from humans and animals exhibit molecular variations and are categorized into three genotypic groups (I, II, and III), which differ in virulence and clinical outcomes [17]. Notable biological differences exist among these strains; Type I strains are highly virulent and more likely to cause severe disease, particularly in South America and parts of Asia, while types II and III are associated with milder symptoms. Type II strains, which are predominant in Europe and North America [18, 19].

The B1 gene, present in 35 copies across all Toxoplasma species, enhances the sensitivity and specificity of diagnostic test for Toxoplasma infections [20]. Various genetic markers are utilized for Toxoplasma genotyping, including ribosomal DNA, the ribosomal intergenic spacer (IGS), and other gene loci such as SAG1, GRA4 (rough granular antigens), and the beta-tubulin gene. Notably, GRA6 exists as a single copy within the Toxoplasma genome and is used for genotype determination [21].

Given the potential role of T. gondii in causing SA and the uncertain rate of abortion due to CT in regions with high rates of acquired toxoplasmosis, combined with the high fertility rate and ambiguous symptoms of abortion in Sistan and Baluchistan Province, this study was warranted due to the lack of comprehensive data in this area. Since the genotype of T. gondii is a key factor in understanding its pathogenicity and its potential role in SA, this study leverages serological and molecular tools to explore this relationship, providing valuable insights into the epidemiology and pathophysiology of T. gondii infection in pregnant women. The present study bridges critical gaps by integrating serological profiling as a routine complimentary test with molecular investigations to elucidate the role of T. gondii genotyping in women with a history of SA in Zahedan.

Materials and methods

Study area

Zahedan, the capital of Sistan and Baluchistan Province, is one of the largest cities in southeastern Iran. The city is characterized by unique environmental, cultural, and socioeconomic factors that may influence thr prevalence, risk factors, and genotype distribution of T. gondii, distinguishing it from other regions. With an estimated population of 770,800 people residing in 200,200 households. Zahedan is strategically located near the borders of Pakistan and Afghanistan, making it a hub for trade and cultural exchange in the region. Its geographical coordinates are 29°30’45"N and 60°51’25"E (Fig. 1).

Fig. 1.

Map of Sistan and Baluchistan Province as the study area

Zahedan experiences a hot and dry desert climate, with an average annual rainfall of 72 mm, which may impact the survival of T. gondii oocysts in the environment. The average temperature ranges from 42.5^oCin summer to -12.6^oC in winter, shaping the lifestyle and activities of its inhabitants. Agriculture and livestock farmin (cattle, sheep, and goats) are the primary livelihoods for most of the population in this area [22, 23].

Experimental design

In this study, we first collected blood and tissue samples, followed by examining them using serological and molecular methods.

Sampling and demographical data

In this cross-sectional study, 163 women admitted to the Obstetrics and Gynecology Department of Ali Ibn Abi Talib Hospital in Zahedan between September 2021 and May 2024 due to SA were included. After obtaining informed consent, 5 mL of blood and tissue samples from the aborted placentas were collected from each participant. These samples were transferred to the laboratory of the Infectious and Tropical Diseases Research Center at the Research Institute of Cellular and Molecular Sciences in Infectious Diseases, Zahedan University of Medical Sciences. Blood samples were stored at − 20 °C for serological analysis, while placental tissues were preserved at the same temperature for molecular examination.

For each participant residing in the study area, a structured checklist was completed to collect data on serological evidence of T. gondii infection during the pregnancy in which the abortion occurred, maternal age, gestational age, and history of prior abortions. Women with evidence of other infectious or non-infectious causes of abortion were excluded from the study to ensure adherence to the inclusion criteria. All participants provided informed consent prior to their involvement.

Serological survey

The presence of IgG and IgM antibodies against T. gondii was determined using a commercial enzyme immunoassay kit (Pishtaz Teb Diagnostic Company, Tehran, Iran) following the manufacturer’s protocol.

DNA extraction

DNA was extracted from all placental tissue samples using DynaBio™ Blood/Tissue DNA Extraction Kit (Tekapouzist, Iran) according to the manufacturer`s instruction. The quality of the extracted DNA was assessed via spectrophotometry, with quantity measured using absorbance at 260/280 nm and integrity evaluated via gel electrophoresis.

Nested-PCR for B1 gene

The nested PCR assay targeted the B1 gene, a repeated genomic sequence, to detect T. gondii DNA in placental tissues [21]. Two primer pairs of the B1 gene (531 bp) were used:

First amplification: S1 (5´-CGACAGAAAGGGAGCAAGAG-3´) and AS1 (5´ ACGCTGTGTCTCCTCTAGGC-3´).

Second amplification: S2 (5´-TCTTCCCAGACGTGGATTTC-3´) and AS2 (5´-CTCGACAATACGCTGCTTGA-3´).

The first amplification was performed in 20 µl of reaction mixture containing 1 µL of each primer (S1 and AS1), 10 µL of Master mix (Ampliqon Company, Denmark), 2 µL extracted of extracted DNA, and 6 µL of sterile distilled water. The PCR conditions included initial denaturation at 94 °C for 3 min; followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 2 min, and a final extension step at 72 °C for 1 min.

The second amplification used a 1:40 dilution of the PCR product as a template. The 20 µl reaction mixture contained 1 µL of each primer (S2 and AS2), 8 µL of Master mix, 1 µL of the diluted template and 9 µL sterile distilled water. The second PCR was performed under the same conditions as the first.

Nested PCR for GRA6 gene

Positive samples from the B1 gene nested PCR were further analyzed using nested PCR of the GRA6 gene, a highly polymorphic gene suitable for distinguishing between T. gondii types I, II and III. The primer pairs were:

First amplification: GRA6FO (5´GGCAAACAAAACGAAGTG-3´) and GRA6RO (5´-CGACTACAAGACATAGAGTG-3´).

Second amplification: GRA6R (5´-GTAGCGTGCTTGTTGGCGAC-3´) and GRA6 (5´TACAAGACATAGAGTGCCCC-3´) [24].

The first amplification was performed in a 25 µL reaction mixture containing 1 µL of each primer (GRA6FO and GRA6RO), 8 µL of Master mix, 5 µL of extracted DNA, and 10 µL of sterilized distilled water. The PCR conditions included initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 60 s, extension at 72 °C for 90 s and a final extension at 72 °C for 7 min.

The second amplification used a 1:10 dilution of the PCR product as a template. The 25 µL reaction mixture contained 1 µL of each primer (GRA6R and GRA6), 8 µL Master mix, 1µL of the diluted template, and 14 µL of sterile distilled water. The second PCR was performed with an annealing temperature of 60 °C for 60 s [16].

PCR products were electrophoresed on a 1.5% agarose gel in 0.5X TBE buffer and stained with ethidium bromide. Positive samples were further analyzed using PCR-RFLP to differentiate between T. gondii genotypes.

PCR-RFLP assay

The GRA6 PCR products were digested with MseI restriction endonuclease (10 U/µl, 300 units, Fermentas, Thermo Scientific, USA). A 30 µL reaction mixture containing 10 µL of GRA6 PCR product, 1 µL of MspI, 2 µL of 10x Tango buffer (Fermentas, Lithuania), and 17 µL of distilled water was incubated at 37 °C for 2–3 h. The digested products were analysed on a 2% agarose gel. MseI differentiates T. gondii genotypes by producing fragments of 168 bp and 544 bp for type I, 75 bp and 623 bp for type II, and 97 bp and 544 bp for types III [25].

Ethics statement

This study adheres to the principles of the principles of the Helsinki Declaration of 1975, as revised in 2000. Ethical approval was obtained from the Ethical Committee of Zahedan University of Medical Sciences, Zahedan, Iran (IR.ZAUMS.REC.1399.191).

Results

Demographic data

The age range of the participants was 18–39 years, with a mean age of 29 years. Thirty women were under 20 years old, 92 were between 20 and 34 years old, and 41 were over 35 years old. Most cases (72.4%) were in the first trimester of pregnancy, while 27.6% were in the second trimester (Table 1). Additionally, 9.37% of the participants had no prior history of abortion.

Table 1.

Demographic characteristics of mothers and samples

No.				ELISA				PCR results in placenta samples
								Positive Negative
			IgM+		IgG+	IgG+&IgM+	IgG-
Age groups (year)	≤ 20	30	1		3	1	25	2	28
	20–34	92	4		5	1	82	4	88
	≥ 35	41	2		6	3	30	1	40
	Total	163	7		14	5	137	7	156
Gestational age	1st trimester	118	6		5	4	103	5	113
	2th trimester	45	1		9	1	34	2	43
	Total	163	7		14	5	137	7	156
History of abortion	YES	54	1		5	1	47	1	53
	NO	109	6		9	4	90	6	103
	Total	163	7		14	5	137	7	156

Serological findings

Table 1 shows the results of serological tests for 163 patients. Of these patients, 137 (84%) had no detectable antibodies against T. gondii, while 26 (16%) tested positive. Among those who tested positive, 14 individuals (9%) had anti-T. gondii IgG, seven individuals (4%) had anti-T. gondii IgM, and five individuals (3%) were positive for both IgG and IgM.

Nested-PCR for the B1 gene

Out of 163 placental samples, T. gondii DNA was detected in 7 cases (Fig. 2a). All positive results were confirmed through duplicate testing to ensure accuracy.

Fig. 2.

(a) Agarose gel separation of representative nested PCR products of the B1 gene (531 bp). Lane 1–7, positive placenta sample; c⁻: negative control, c⁺: positive control, DNA ladder 100 bp. (b) Electrophoretic pattern of the Nested PCR products of GRA6 gene from placenta samples. Lanes 1–7: samples from B1 positive samples M: DNA Ladder marker. (c) PCR-RFLP analysis of GRA6 gene coding region with MseI endonuclease., Lanes 1–5, Line 6–7 are Toxoplasma gondii, type I and II patterns respectively

Nested-PCR for the GRA6 gene and PCR-RFLP

Figure 2b shows the results of nested PCR for the GRA6 gene performed on two positive B1 gene-positive samples. Figure 2c presents the PCR-RFLP results used to determine T. gondii genotypes. The data confirmed that five isolates belonged to types I and two isolates to type II of T. gondii.

Discussion

Toxoplasmosis is recognized as a global infection associated with the TORCH pathogens syndrome (an acronym for a group of infectious diseases that can cause significant complication during pregnancy, potentially leading to congenital infections in the fetus). It is linked to SA in both humans and animals [26].

In certain regions worldwide, including parts of Iran, various studies have indicated that the seroprevalence rate among the general population and pregnant women can reach 39.3% and 41.0%, respectively. This high prevalence may be attributed to factors such as geographical location, habitat, dietary habits, and lifestyle [12, 27]. Infection with T. gondii during pregnancy poses significant risks, including the timing of maternal infection (gestational age), the status of the maternal immune system, and the type and number of pathogens transmitted to the embryo. These factors can lead to miscarriage, stillbirth, or severe fetal complications [28].

Previous studies have shown that maternal infection with the active form of Toxoplasma during the first trimester (5%) and second trimester (2%) of pregnancy may result in fetal demise [29, 30]. Accordingly, the global annual incidence rate of congenital toxoplasmosis and associated anomalies in approximately 1.5 cases per 1,000 live births and 3% of live births, respectively [31, 32].

Specifically, the seroprevalence of toxoplasmosis in pregnant women who experienced an abortion during their current pregnancy or had a history of abortion was found to be 33% and 43% respectively, across various continents and countries [33]. Previous reports indicate that the prevalence of toxoplasmosis among pregnant women in Iran is relatively high, with rates of 27% in Zahedan and 43.8% in the southeast and northern regions of Iran [34, 35]. This suggests that in northern Iran where the climate is humid, seroprevalence rates are often higher due to favorable conditions for oocyst survival. In contrast, Zahedan’s arid climate may result in lower environmental contamination but could still exhibit high infection rates due to other risk factors such as dietary habits. The unique combination of cultural practices and environmental conditions in this region may create distinct risk profiles.

Our findings revealed that out of 163 serum samples, 26 cases (16%) were positive for T. gondii antibodies, while 137 cases (84%) were negative for IgG, and only 7 cases (4%) were positive IgM. The observed seroprevalence rate in our study is lower than those reported in various studies from Iran, which range from 56 to 75.02% in the northern regions, 31.1% in Tehran, 41.8% in Gorgan, 32% in Yazd, 33% in Bojnord and 18.4% in Urmia [36–42]. In contrast, other studies conducted in Iran on women with SA reported IgG seroprevalence of 7% in Yasuj (southwest), 43% in Ardabil (northwest), 32.6% in Urmia (northwest) [22], 24.6% in Ahvaz (southwest), and 25.5% in Tehran, the capital of Iran [43–46]. IgM seroprevalence rates in these studies were 3% in Yasuj, 4% in Ardabil, 1.4% in Urmia, 1% in Ahvaz, and 2.7% in Tehran. In two studies conducted in Turkey and Iraq, neighboring countries of Iran, the seroprevalence of anti-toxoplasma IgG antibodies in women with SA was reported as 30.6% and, 6.6%, respectively, while IgM seroprevalence was 37.9% and 5.2%, respectively [47, 48].

A global study reported that the seroprevalence of IgG and IgM antibodies in women with a history of miscarriage and miscarriage during the current pregnancy was 43%, 3%, 33% and 1%, respectively [49]. In Iran, the estimated seroprevalence of IgG and IgM antibodies against toxoplasmosis in pregnant women was 38% and 4%, respectively. The highest and lowest prevalence rates of toxoplasmosis in Iran were observed in the south (53%) and east (33%) [12]. Additionally, the rate of T. gondii infection in the placenta of aborted fetuses in our study was 4.3%, which is consistence with rates reported in other global studies [50].

Regarding the T. gondii isolated from abortion samples, molecular detection and identification of T. gondii in placental tissues with acute toxoplasmosis were performed using the GRA6 gene. Among various genetic markers, GRA6, a single-copy gene, is highly polymorphic and can differentiate between three genotypes (I, II, and III) using a single PCR reaction, followed by digestion with the MseI endonuclease [51]. Accurate genotyping of T. gondii isolates is crucial for optimizing epidemiological studies in different regions, predicting clinical outcomes, guiding treatment strategies, and developing targeted prevention measures for toxoplasmosis [52, 53]. Identifying T. gondii genotypes is essential for understanding the pathogenesis of CT and its association with abortion. It provides insights into virulence of different strains, helps predict health outcomes, and informs public health strategies to reduce the burden of toxoplasmosis in pregnant women and their fetuses [53–56].

In our study, 5 out of 7 IgM-positive samples (72%) were identified as Type I, while 2 out of 7 samples (28%) were Type II. Studies from other regions of Iran have confirmed that all three genotypes of T. gondii circulate among pregnant women or are identified in congenital toxoplasmosis cases [40, 43–45, 57–60]. It is important to note that Types I and II of T. gondii are predominantly found in various other hosts, including humans, in Iranian studies [25, 61–63].

Similar to our research, a study conducted on 38 female patients who experienced miscarriage and intrauterine fetal demise in Egypt reported a predominance of type II genotype, identified in 87% of the cases, while type I accounted for the remaining 13% of the strains [64]. In contrast to our findings, type II is predominantly associated with miscarriage in several countries, including the United States, France, Serbia, Romania, Argentina, and Portugal. Conversely, type I is more commonly linked to CT in regions such as the United States, France, Tanzania, and Portugal [65].

Conclusion

Despite the growing body of research on various infectious diseases, the detrimental impact of toxoplasmosis in Iran has received relatively little attention. Our research indicates that toxoplasmosis significantly elevates the risk of miscarriage among pregnant women. Given these findings, healthcare practitioners must emphasize the importance of including Toxoplasma screening in the TORCH panel, both preconception and during pregnancy. Such measures could be instrumental in preventing adverse pregnancy outcomes and improving clinical guidelines for managing suspected cases.

Author contributions

HM: Conceptualization and data collection. NA: methodology, writing - original draft. AH: software and research approve. NF: writing original draft preparation, and writing – review & editing. RS: conceptualization, data curation, methodology, writing – review & editing, and supervision. All authors have read and agreed to the published present manuscript version.

Funding

The research leading to these results was financially supported by a grant from the project NO. IR.ZAUMS.REC.1399.191 from Zahedan University of Medical Sciences, Zahedan, Iran.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics declarations

All methods were carried out in accordance with relevant guidelines and regulations.

Ethics approval and consent to participate

The present work is approved by the ethical committee of Zahedan University of Medical Sciences, Zahedan, Iran. The ethical approval Code is IR.ZAUMS.REC.1399.191.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.