Molecular detection and genotypic characterization of toxoplasma gondii in paraffin-embedded fetoplacental tissues of women with recurrent spontaneous abortion

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

Prevalence of intratubular germ cell neoplasia in cryptorchid testes of infertile men

Cryptorchidism is a common malformation in neonates; surgery or medical treatments are applied during childhood. Untreated cryptorchid testes are in the risk of intratubular germ cell neoplasia [IGCN] and consequently invasive testicular tumors which could be shown by immunohistochemistry staining for placental like acid phosphatase [PLAP] marker. We designed this study to know the prevalence of IGCN in untreated cryptorchid testes of infertile men, in our infertility center as a refferal center. In this cross-sectional study we assessed H and E slides of testicular samples of 13 adult infertile patients with impalpable intra-abdominal testes seeking infertility treatment; then we stained them by PLAP marker. Three [23.08%] samples were positive for PLAP marker means presence of IGCN in testis. One of them showed seminoma besides IGCN. According to the results of this study and the fact that there are adult untreated cryptorchid patients in our country yet, it is suggested to pay more attention in clinical examination, assessment and follow up of such patients for malignancy screening

Production of monoclonal antibody against human nestin

We have employed a peptid-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N-or O-glycosylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays

Thyroid autoantibodies in euthyroid women with recurrent abortions and infertility

Evidences suggest an association between the prevalence of thyroid peroxidase [anti-TPO] and anti thyroglobulin [anti-Tg] with recurrent abortions and infertility. Iodine deficiency was once endemic in Iran and little data is available about the prevalence of these antibodies in different groups of fertile or infertile individuals. This case control study was designed to compare the presence of anti-TPO and anti-Tg in four groups of women to reveal their role in the etiology of recurrent abortion and infertility. Four groups of euthyroid women referring to Avicenna Infertility Clinic in Tehran were selected; 95 cases as fertile controls and 70, 78 and 137 cases with male and female factor infertility and recurrent abortion respectively. TSH, anti-TPO and anti-Tg were evaluated by chemiluminescent immunoassay. The prevalence of the above mentioned autoantibodies in euthyroid controls was about 25% and the percentage of people with an anti-Tg >500 was two times bigger in the abortion group compared to the control group [p<0.05] and the proportion of people with an anti-Tg>500 in younger cases in the abortion group was significantly higher than the rest of the cases [p<0.05]. Anti-TPO distribution had no significant differences. There were no statistically significant differences among four groups. It seems that more comprehensive studies are needed to reach a common conclusion about thyroid autoantibodies in women with recurrent abortions in different groups and different parts of Iran. In addition, dividing the recurrent abortion and infertility groups on the basis of their etiologies could be effective

Comparing seminal plasma biomarkers between normospermic and azoospermic men

Azoospermia affects more than 10% - 15% of infertile male subjects attending infertilty clinics. At present, testicular biopsy is the golden standard procedure for evaluating spermatogenesis status in men with azoospermia. Semen collection and analysis is a non-invasive method and has proven to be valuable in the evaluation of spermatogenesis. Identification of seminal plasma markers with testicular or extra-testicular origins have a great value in predicting the prescence of sperm in testicular tissue and presumptive cause of azoospermia. The aim of this study was to find such markers by comparing the content of seminal plasma using different methods in normospermic and azoospermic men. Semen samples were collected from 200 men attending Avicenna Infertility Clinic [AIC] in Tehran, Iran. Semen samples were analysed according to WHO guidlines. The subjects were divided into two groups: normospermic [n = 100; group one] and azoospermic men [n = 100; group two] according to semen analysis results. Seminal plasma was separated by high speed centrifuagation and stored in -20°C. Four markers including fructose, neutral alpha glucosidase [NaG], inhibin B and anti-Mullerian hormone [AMH] were measured in seminal plasma. Fructose and NaG were evaluated by spectrophotometry, while inhibin B and AMH were assessed by ELISA method. The spermatogenesis status in the azoospermic group was evaluated by histopathological method following testicular biopsy. Fructose concentration showed no difference between the two groups. However, it was significantly correlated with sperm count [p < 0.01, r = -0.408]. Seminal plasma inhibin B [OR: 1.01; 95%: CI: 1.005 - 1.016], AMH [OR: 1.63; 95% CI: 1.17 - 2.28] and N alpha G, [OR: 1.07; 95% CI: 1.04 - 1.1] levels were higher in normospermic subjects compared to azoospermic men. There were significant differences in inhibin B and AMH concentrations between the two groups based on the presence or absence of mature sperm in testicular biopsies [p < 0.01]. Inhibin B concentration was positively correlated with sperm count in the normospermic group, however, N alpha G concentration correlated with sperm count of normospermic men [p < 0.01, r = 0.345] and the subjects'age in both groups. Inhibin B and AMH were correlated with the presence of sperm in testicular tissue samples. According to non-specific changes in inhibin B and AMH concentrations, identification of more specific molecular markers in seminal plasma to definitely evaluate the status of spermatogenesis is recommended

Serum leptin levels in women with immunological recurrent abortion

Recurrent abortion [RA] may be a consequence of aberrant expression of immunological factors during pregnancy. Although the relative importance of immunological factors in human reproduction remains controversial, substantial evidence suggests that autoantibodies contribute to reproductive failure. Production of such antibodies is under the control of cytokines; and leptin, besides its role in reproductive success, has a profound effect on directing the cytokine profile toward Th[1] [cellular] pattern. Therefore, the present study was performed to assess serum leptin levels in women with immunological recurrent abortion. In this prospective study, 250 women who attended Avicenna Infertility Clinic with RA were screened for known causes of abortion from July to December 2008 in Tehran, Iran. Eighty-one patients with normal karyotypes and hormonal profile with normal ovaries and uterus and no signs of infection were categorized as patients with immunological [IRA, n = 39] or unexplained [URA, n = 42] recurrent abortion based on presence or absence of autoantibodies. After blood sampling, levels of anti-nuclear antibody [ANA], anti-double stranded DNA antibody [anti-dsDNA], lupus anti-coagulant antibody [LACAb], anti-phospholipid antibody [APA], anti-cardiolipin antibody [ACA], anti-thyroglobulin antibody [TgAb], anti-thyroperoxidase antibody [TPOAb] and anti-thrombin III antibody [ATIIIAb] were measured by enzyme-linked immunosorbent assay [ELISA] or chemiluminescent enzyme immunoassay [CLEIA]. In IRA group, 9 [23.1%], 24 [61.5%], 25[64.1%] and 1 [2.6%] women were above the normal cut-off point for ANA, TgAbs, TPOAbs and AT-III Abs, respectively. IRA patients had normal values of LACAbs, APA and ACA. With normal level of fasting blood sugar [FBS], IRA and URA groups had similar serum leptin levels [23.7 +/- 13.2 ng/ml vs. 22.7 +/- 12.5 ng/ml, respectively]. Serum leptin concentrations showed a positive correlation with weight and BMI in both groups. This study suggests that serum leptin levels are higher in IRA and URA patients than normal women. The findings of this study suggest the need for a more comprehensive study and comparison of leptin levels in IRA and URA patients to women with no history of miscarriages

Ectopic expression of sortilin 1 [NTR-3] in patients with ovarian carcinoma

Gene expression profiling of ovarian carcinoma tissues has shown an increase of four-fold expression of SORTl gene. Sortilin 1 [NTR-3] is a 95-100 kDa protein normally expressed in heart, brain, placenta, skeletal muscle, spinal cord, thyroid, and testis. However, its expression has never been reported in normal ovary. Here, we report expression of sortilin 1 in ovarian carcinoma tissues both at gene and protein levels. Sortilin 1 was expressed in all ovarian carcinoma patients [n=15] as well as ovarian carcinoma cell lines [n=5] regardless of their phenotypic characteristics. Non-malignant ovaries [n=6] did not express sortilin 1. The molecular basis for this ectopic expression is not yet clear. Our results showed a major cell surface expression of sortilin 1 rather than ER-Golgi compartment where it is mainly expressed. This finding may introduce sortilin 1 as a novel tumor marker for diagnosis of ovarian carcinoma and may signify its therapeutic value in targeted therapy