Molecular Detection and Genotypic Characterization of Toxoplasma gondii in Paraffin-Embedded Fetoplacental Tissues of Women with Recurrent Spontaneous Abortion

Abstract

Background: Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii) in women with recurrent spontaneous abortion (RSA). The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs) of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran.

Materials and methods: This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples.

Results: T. gondii DNA was detected in 3.8% (8/210) of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient.

Conclusion: The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA.

Keywords: Abortion; Genotype; Iran; Molecular Detection; Toxoplasma gondii.

Fig.1

PCR products of the GRA6 positive samples. Toxoplasma gondii (T. gondii) positive samples give a 344-bp band. M; 100 bp DNA marker, Pos; Positive control, Neg; Negative control, and Lanes 1-8; Positive samples.

Fig.2

Genotyping of positive samples with the GRA6 marker. The products were digested with Tru1I enzyme. A, B, C. Patterns of three types of Toxoplasma gondii (T. gondii) genotype, and D. Agarose gel electrophoresis of PCR products digested with Tru1I enzyme. M; 100 and 50 bp DNA marker, Lanes 1 and 10; Undigested positive samples, and Lanes 2-9; T. gondii genotype type III.

Fig.3

Genotyping of positive samples with the SAG3 marker. The products were digested with BcnI enzyme. A. Patterns of Toxoplasma gondii (T. gondii) type III genotype and B. Agarose gel electrophoresis of PCR products digested with BcnI enzyme. M; 100 and 50 bp DNA marker, Lanes 1 and 8; Uundigested positive samples, and Lanes 2-7; T. gondii genotype type III.