Molecular Typing of Toxoplasma Gondii Isolated from Infertile Men and Its Effect on the Reproduction

Seminal fluid and blood samples were collated from sixty 20-60 years aged and attended Teba Center for Children and ICSI/ in Babylon Province during the period from 1 June 2016 to 1 February 2017. The same number of samples were also taken from 60 age matched apparently healthy individuals to act as a control group. Anti IgG levels were measured in the sera of both groups to detect the presence of Toxoplasma infection, while seminal fluid samples were examined to detect infertility. Toxoplasma gondii genotype was applied by using nested PCR to detect SAG2 gene. Gene sequencing technique infection was performed for detection occurrence of mutation in the mitochondria of the sperm. Results confirmed that the age group (20-40years) was significantly more prone P<0.05 to infective with T. gondii where the percentage of seropositivty was 65% (39 patients) while it was 35% (21 individuals) in the apparently healthy control group. Concerning the residence, there was a highly significant difference (P < 0.006) where the percentage of seropositivty was 68% in idiveduals living in rural areas while it was 32% among those living in the urban areas. Genotyping showed that presence in two strains of Toxoplasma which has been found in males infected with Toxoplasmosis. Type I was found in 10 persons (17%) while type II was found in 50 persons (83%) and the difference between two strains was significant (P<0.05). Oligospermia has recorded the highest number of positive cases among patients (92%; 55 cases), in comparison with negative cases (8%; 5 cases), Asthenospermia showed significant P<0.05 decrease (35%; Molecular Typing of Toxoplasma Gondii Isolated from Infertile Men and Its Effect on the Reproduction Nazar Sh. Mohammed, Farhan A. Risan and Salwa S. Muhsin 141 Vol: 14 No:1, January 2018 DOI : http://dx.doi.org/10.24237/djps.1401.357C P-ISSN: 2222-8373 E-ISSN: 2518-9255 21 cases) in comparison with negative (65%; 39 cases). Mutation occurs with Oligospermia of both of types I and II of T. gondii strains in 2 cases only, which was found on DN2 gene with a highly significant P<0.01 difference of positive cases (3%; 2 cases), and negative cases (97%; 58 cases). The mutation in single-nucleotide polymorphisms SNP G4580A site, showed that G was converted to A that was recognized at nt 4580 in the ND2 region. This evolution was experiential in oligozoospermic samples (code 010830). This SNP is a synonymous substitution that occurred in the third position of methionine codon, changing the codon from ATG to ATA.


Introduction
Every moment there are new information regarding toxoplasmosis.It; has been found that the infection with this disease is linked with many other illnesses that affecting humans, including deadly diseases, like the caner (1).There have been some many studies about this disease and it is estimated about one third of the world population was infected (2).Mohammed (3) found that 50 of breast cancer, followed by lung, cervical and colon cancer among cancer patients are infected with toxoplasmosis in Iraq.On the other hand, many studies in the world and Iraq in particular, have shown that toxoplasmosis causes a recurrent abortion among pregnant women (4).With the progress of research in the fields of molecular biology it has been found that this parasite, which is transmitted through sexual contact causes serious complications (5).Recent studies conducted in the field of molecular biology has proven the transmission of the parasite through sexual contact leads to a new field of research about the parasite to infect the reproductive organs and causes proliferative device occurs great damage that cannot be cured (6).The symptoms in men infected with cases of toxoplasmosis could cause to permanent infertility (6).Of these symptoms in males are the Oligospermia and Asthenospermia.In a previous study, we have shown that the incidence of toxoplasmosis is one of the causes that lead to infertility (7).Occurrence of mutations, particularly in those cases in the relay for nucleotides sequence of DNA of the mitochondrial sperms (8).This study aimed to prove if infection with T. gondii has an effect on men infertility.

Materials and Methods
Sixty seminal fluids and whole blood samples were collected from infertile male patients who were suffering from toxoplasmosis.Each sample was stored in a sterile container sealed with plastic cover, and incubated at -20 C to conduct the necessary analysis.Sixty seminal fluid samples and sera of apparently healthy individuals were also collected in the current study to serve as a control group the age of the participants was ranging between 20 and 60 years.

Statistical analysis
SPSS Microsoft Office Excel program was used for statistical analysis of data.The values were represented as mean ± SEM (standard error of mean).The comparison of the numeric data for healthy control and patient groups was clone by using the Paired t-test (11).

Results
The distribution of seropsitity rate of toxoplasmosis among age groups among infertile men in comparison with control group was shown in Table 1, the seropsitity rate in the age group (41-60 years), was significantly (P< 0.05) higher (65%) than that of the age group (20-40 years) which was 35%.Concerning the link between the seropositivity and residency, the results showed that 41 subjects (68%) were living in the rural areas while 19 subjects (32%) were living in the urban areas and the difference between the two groups was statistically significant (P < 0.01).Genotyping showed the presence of two strains which have been found in males infected with toxoplasmosis.Type I was found in 10 persons (17%) while type II was found in 50 persons (83%) and the difference between the two strains was significant (P< 0.05) (Table 1).As noted in Table 3, it has been identified that two cases of malformation which is Oligospermia was 8 (80%) out of 10 cases from type I and 47 (97%) out of 50 cases from type II.While the Asthenospermia was 3(30%) out of 10 cases was type I and 18 (36) out of 50 cases was type II.Also there were mutation in each of the Oligospermia and Asthenospermia from both of types I, 1 (10%) out of 10 and II, 1 (2%) out of T. gondii infection which had non-significant different P>0.05.

Detection of mitochondrial DNA
In order to performe to amplify the ND2 genes from the extracted DNA samples, 47 samples showed the proper PCR product size (1125 bp) with ND2 gene (Figures 1)  4).

SNP G4580A
A G to A conversion was observed at nt 4580 in the ND2 region, and this evolution was   4.

Discussion
Toxoplasmosis has become a life-threatening infection and many human diseases have accompanied this infection.It was found that toxoplasmosis is associated with more serious illnesses such as cancer (12,13).This study has been conducted for the first time in Iraq.The results showed that the highest seropositivity rate was found in the age groups 20-40 years in comparison with the other age groups.These results are consistent with the results of Wilking et al (14), who assessed of seropositivity of T.oxoplasma gondii among adults.in Germany.
The present study has shown that there are two types of T. oxoplasma gondii strains distributed among infertile males, type I and type II.The rate of type II was significantly higher (83%), than type I (17%).This result is in agreement with the results of the study which has been conducted by Al-Azawi (4), who which identified the genotyping of T.
gondii strains and found that 48 men (93.3%) showed type II and while 9 men (15.25%) have been identified with type I, in Baghdad city.This study demonstrated that T. gondii had clear impact on the fertility of as evidenced by the identification of both; Oligospermia and Asthenospermia.Oligospermia refer to semen with a low number of sperms and is a common finding in male infertility.Asthenospermia is the condition in which sperms has poor motility.

Vol: 14
No:1, January 2018 DOI : http://dx.doi.org/10.24237/djps.1401.357CP-ISSN: 2222-8373 E-ISSN: 2518-9255 Macroscopic and microscopic examination of seminal fluid were done to determine the sperm count and motility.Enzyme liked immunosorbent (ELISA) was applied for detection the Anti-Toxoplasma IgG antibodies parameter (ElISA TOXO IgG Biotik, SUA).DNA isolation kit (QIAGEN) was used to extract genetic DNA of T. gondii from blood of infected infertile men.SAG2 gene, genotyping was used for nested PCR using DNA Isolation Kit Genotyping for the nested PCR genetic markers.Amplification of Mitochondrial DNA; extraction and isolation of mtDNA were carried out by using mitochondrial isolation kit ( Biovision).DNA Sequencing by Bio System Big Dye TM termination V 3.1 cycle sequencing kit ( Biovision) was used to perform automated DNA sequencing.The mitochondrial DNA samples of different fertility groups were sequenced for ND2 gene.
of Toxoplasma gondii Primers had been selected for amplification of ‫׳5‬ and ‫׳3‬ ends of the SAG2 locus separately.The size of fragment that splatted was 241 bp and 221bp.The splitting process was performed by application of two types of endonucleases enzymes.Sau3A1 which digest the 3 rd allele (Type III) at ‫׳5‬ end, while Hhal enzyme acts to digest the 2 nd allele (Type II) in which the splitting takes place at ‫.׳3‬ If the fragmentation or splitting does not occur by any of these two enzymes it will be indication for the presence of type I strain.It has been identified a clonally (Type II) in 50(83%) and nine cases of type I 19(17%) were found.Sixty samples were taken from all categories and after the completion of the reaction stage within the thermal polymer (Thermo-cycler) were deported reaction solution electrically (agarose gel) with concentration of 2.5% with the use of Volumetric Guide DNA ladder (M) 100bp size (Figures 1).
experiential in one oligozoospermic (sample code 010830) sample (Fig 4).This SNP is a synonymous substitution that teak place which resulted in the change of the codon from ATG to ATA. (Figure 2):

Figure2: A :
Figure2: A: Chromatogram of a control sample at nt 4580 in the ND2 region, and B show the G4580A transition in an oligozoospermic (010830)

Table 1 :
Distribution of seropositivity of anti-Toxoplasma gondii IgG antibodies according to age,

Table 2 ,
shows that a highly significant difference regarding the levels of anti-Toxoplasma

Table 2 :
Estimation of Anti-Toxoplasma IgG antibody, among toxoplasmosis patients Not: P 1 = A.H. Control Vs Genotype I, P 2 = A.H. Control Vs Genotype II & P 3 = Genotype I Vs Genotype II.

Table 3 :
Detection of Oligospermia and Asthenospermia and genotypes of Toxoplasma gondii

Table 4 :
DNA sequence analysis and mutations in different groups of seminal fluid samples in ND2 * Indicates the mutations of different individuals of seminal fluid samples.