Successful human spermatogonial stem cells homing in recipient mouse testis after In vitro transplantation and organ culture

Objective: In vitro transplantation [IVT] of spermatogonial stem cells [SSCs] is one of the most recent methods in transplantation in recent decades. In this study, IVT and SSCs homing on seminiferous tubules of host testis in organ culture have been studied

Materials and Methods: In this experimental study, human SSCs were isolated and their identities were confirmed by tracking their promyelocytic leukemia zinc finger [PLZF] protein. These cells were transplanted to adult azoospermia mouse testes using two methods, namely, IVT and in vivo transplantation as transplantation groups, and testes without transplantation of cells were assigned in the control group. Then histomorphometric, immunohistochemical and molecular studies were done after 2 weeks

Results: After two weeks, histomorphometric studies revealed that the number of subsided spermatogonial cells [SCs] and the percentage of tubules with subsided SCs in IVT and in vivo groups were significantly more than those in the control group [P<0.05]. Immunohistochemical studies in the transplantation groups confirmed that the PLZF protein was expressed in the cells subsided on the seminiferous tubule. Quantitative reverse-transcription polymerase chain reaction [qRT-PCR] demonstrated that the PLZF gene expression was only positive in the transplantation groups, but it was not significantly different between the IVT group and the in vivo group [P>0.05]

Conclusion: Testicular tissue culture conditions after SSC transplantation can help these cells subside on the seminiferous tubule basement membrane

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

[ effects of combined docosahexaenoic acid and vitamin E supplements on spermatogram in asthenozoospermic males: a randomized, triple-blind, placebo-controlled clinical trial]

Considering the decrease of omega-3 fatty acids in spermatozoa of asthenozoospermic men, the aim of the present study was to investigate the effects of combined docosahexaenoic acid [DHA] and vitamin E supplements on spermatogram in asthenozoospermic males. In this randomized, triple-blind, placebo-controlled clinical trial, out of 275 men who referred to Avicenna infertility clinic, fifty asthenozoospermic males, defined as less than 50% sperm motility or less than 25% with rapid progressive motility, were randomly assigned to one of two groups according to the stratified blocked randomization. Participants in the intervention group took daily 465 mg of DHA and 600 IU of vitamin E; and those in the control group took daily two placebos for 12 weeks. Sperm characteristics, dietary intakes, anthropometric measurements and physical activity were measured at the baseline and at the end of the study. Statistical analyses were performed using the SPSS software, the statistical tests being analysis of covariance, Student's t-test, paired-samples t-test, Wilcoxon and Mann-Whitney. Out of 50 participants, 22 men in the intervention group and 20 men in the control group completed the protocol of the study. Number of sperms, sperm concentration, percentage of motile sperms and percentage of motile sperms with a straight direction increased in the intervention group, as compared with the control values [p<0.05]. According to this research, combined DHA and vitamin E supplements led to increasing sperm concentration and sperm motility; however no significant changes occurred in sperm morphology and vitality in asthenozoospermic men. DHA and vitamin E, as an antioxidant, may improve sperm motility

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

Tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic men

In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the C with 5% CO[2] in 3% Bovine Serum spermatozoa were incubated up to 6h at 37 Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. The results upon western blotting showed: 1] at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2] The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished tyrosine phosphorylation efficiency in sperm from teratospermic men may be responsible for their compromised capacitation and low fertilization success rates

Effects of sperm chromatin integrity on fertilization rate and embryo quality following intracytoplasmic sperm injection

Sperm chromatin integrity has been being recognized as an important factor in male fertility. During normal fertilization, high quality sperm with intact chromatin are selected through natural selection in journey from vagina to fallopian tube. However, using Assisted Reproductive Techniques, particularly ICSI, the natural selection is bypassed. Therefore sperm with DMA breakage have the opportunity to fertilize the egg which may lead to decreased embryo quality and implantation rate. The aim of this study was to evaluate the effects of sperm chromatin integrity on ICSI outcomes. A total of 200 semen samples were collected from couples undergoing ICSI and were analyzed according to WHO criteria. Each sample was evaluated for sperm chromatin integrity using four cytochemical assays and semen processing by swim up method. The ICSI was carried out according to a long-term pituitary down-regulation protocol. The correlation between sperm parameters, sperm chromatin integrity and ICSI outcomes [fertilization rate and embryo quality] was examined. The mean number of oocyte, fertilization rate and cleavage embryos per cycles was 7.5 +/- 5.0, 74.O6% +/- 25 and 5.4 +/- 3.6, respectively. There was not significant correlation between the results of chromatin assays [AO, AB, TB, and CMA3] and fertilization outcomes following ICSI. The fertilization rate was significantly higher for a group with less than 10% chromatin abnormality [p<0.05]. Sperm chromatin integrity is essential for successful fertilization, embryo development and normal pregnancy. A protamine deficiency appeared to affect fertilization rate and embryo quality. However, the presence of confounding factors such as selection of spermatozoa according to normal morphology may influence the effect of sperm chromatin status on ICSI outcomes