Production and characterization of a new specific monoclonal antibody against A-isoform of SALL4: A novel emerging testicular cancer marker.

SALL4 transcription factor plays an important role to maintain the pluripotent and self-renewal of embryonic stem cells. It contributes to the growth of many cancers and embryonic development. With the exception of spermatogonia, SALL4 expression is silenced in most adult tissues after birth; nevertheless, it is re-expressed in a subset of different solid malignancies. SALL4 is a new, precise biomarker for testicular germ cell cancers that was just introduced. The whole isoform of SALL4 is called SALL4-A. Regarding the lack of antibody against human SALL4 isoforms, the pattern of expression, the role of each isoform remain unknown. Furthermore, in isoform specific evaluations, we aimed, for the first time, to produce and characterize mAb against human SALL4-A. Immunization of mice were performed with a selected 33-mer synthetic peptide of SALL4-A conjugated with KLH. Hybridoma cells were screened by ELISA for positive reactivity with SALL4-A peptide. From the ascites fluid of mice that had been injected with hybridoma cells, anti-SALL4-A mAbs were isolated using a protein G column. Reactivity of the mAbs was evaluated using the peptide and SALL4-A recombinant protein by ELISA and IHC on testicular cancer tissue as positive control, and normal kidney, stomach and prostate tissues as negative control. The produced mAb could well detect SALL4-A in testicular cancer tissues using IHC, while the reactivity was negative in normal kidney, stomach and prostate tissues. Using ELISA, the mAb affinity for the peptide and SALL4-A recombinant protein was assessed, and it was shown to be reasonably high. The mAb detected SALL4-A in nucleus and cytoplasm of several cancer cells and spermatogonia in testicular cancer tissue. In addition, it could recognize SALL4-A recombinant protein. Our produced monoclonal antibody against isoform-A of human SALL4 can specifically recognize SALL4-A using either IHC or ELISA. We hope that this mAb could help researchers in isoform-specific study of human SALL4.

Potential biomarkers for testicular germ cell tumour: Risk assessment, diagnostic, prognostic and monitoring of recurrence.

Testicular germ cell tumour (TGCT) is considered a relatively rare malignancy usually occurring in young men between 15 and 35 years of age, and both genetic and environmental factors contribute to its development. The majority of patients are diagnosed in an early-stage of TGCTs with an elevated 5-year survival rate after therapy. However, approximately 25% of patients show an incomplete response to chemotherapy or tumours relapse. The current therapies are accompanied by several adverse effects, including infertility. Aside from classical serum biomarker, many studies reported novel biomarkers for TGCTs, but without proper validation. Cancer cells share many similarities with embryonic stem cells (ESCs), and since ESC genes are not transcribed in most adult tissues, they could be considered ideal candidate targets for cancer-specific diagnosis and treatment. Added to this, several microRNAs (miRNA) including miRNA-371-3p can be further investigated as a molecular biomarker for diagnosis and monitoring of TGCTs. In this review, we will illustrate the findings of recent investigations in novel TGCTs biomarkers applicable for risk assessment, screening, diagnosis, prognosis, prediction and monitoring of the relapse.

A Association of MTHFR C677T and MTRR A66G Gene Polymorphisms with Iranian Male Infertility and Its Effect on Seminal Folate and Vitamin B12.

BACKGROUND: The relation between key enzymes in regulation of folate metabolism and male infertility is the subject of numerous studies. We aimed to determine whether 5, 10-methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G genotypes are associated with male infertility in Iranian men and to evaluate its effect on seminal levels of folate and vitamin B12. MATERIALS AND METHODS: In this randomized clinical trail study, semen and peripheral blood samples were collected from 254 men with oligoasthenoteratozoospermia (OAT) and 77 normozoospermic men who attended Avicenna infertility clinic. Single nucleotide polymorphism (SNP) analysis was carried out in genomic DNA by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for MTHFR C677T and MTRR A66G gene polymorphisms. RESULTS: In MTHFR C677T, our founding showed that T carrier was conversely lower in OAT than normozoospermic men (χ2-test=7.245, P=0.02) whereas in MTRR A66G, A and G carrier showed no significant difference between the two groups (χ2-test=1.079, P=0.53). The concentration of seminal folate was not different between normozoospermic (18.83 ± 17.1 ng/ ml) and OAT (16.96 ± 14.2 ng/ml) men (P=0.47). The concentration of vitamin B12 was slightly higher in normozospermic men (522.6 ± 388.1 pg/ml) compared to OAT men (412.9 ± 303.6 pg/ml, P=0.058). CONCLUSION: The MTHFR C677T and MTRR A66G have no effect on the concentrations of seminal folate and vitamin B12. The present study showed that two SNPs of MTRR A66G and MTHFR C677T cannot be seen as a risk factor for male factor subfertility.

Assessing the potential of HSPA2 and ADAM2 as two biomarkers for human sperm selection.

Selection of the best sperm, with the least defects, is a critical factor in the success of ART especially in male factor infertility. This study assessed the potential Heat shock protein (HSPA2) and metallopeptidase domain2 (ADAM2) biomarkers for sperm selection. Sperm were obtained from 72 asthenoteratozoospermic and 42 normospermic ejaculates. The semen characteristic, DNA fragmentation (DFI), chromatin maturation index (CMI), ADAM2 and HSPA2 levels on sperm, and their correlation with embryo quality were assessed in both groups. Results showed the significant reduction in HSPA2 and ADAM2 in asthenoteratozoospermic compared to normazoospermic ejaculates regarding the cut-off value of 14 and 13% for these two biomarkers. The specificity of HSPA2 and ADAM2 separately, and the combination of these two biomarkers, were 95.2, 90.5 and 93.5%, respectively, for sperm from normozoospermic ejaculates. However, they were 48.6, 50.0 and 54.5% for asthenoteratozoospermic ones. A significant correlation was observed with HSPA2, ADAM2 and a combination of these two biomarkers with CMI, DFI and embryo quality. Although a combination of these two biomarkers have the potential to be a good choice for selecting sperm with the lowest level of chromatin damage, it seems that selection according to HSPA2 has priority over ADAM2 or a combination of the two.

Reactive oxygen species-induced alterations in H19-Igf2 methylation patterns, seminal plasma metabolites, and semen quality.

PURPOSE: This study was conducted in order to investigate the effects of reactive oxygen species (ROS) levels on the seminal plasma (SP) metabolite milieu and sperm dysfunction. METHODS: Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/106 sperm]: group 1 (n = 39): low (ROS < 20), group 2 (n = 38): mild (20 ≤ ROS < 40), group 3 (n = 31): moderate (40 ≤ ROS < 60), and group 4 (n = 43): high (ROS ≥ 60). A comprehensive analysis of SP and semen parameters, including conventional semen characteristics, measurement of total antioxidant capacity (TAC), sperm DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out. RESULT(S): The methylation status of H19 and Igf2 was significantly different in specimens with high ROS (P < 0.005). Metabolic fingerprinting of these SP samples showed upregulation of trimethylamine N-oxide (P < 0.001) and downregulations of tryptophan (P < 0.05) and tyrosine/tyrosol (P < 0.01). High ROS significantly reduced total sperm motility (P < 0.05), sperm concentration (P < 0.001), and seminal TAC (P < 0.001) but increased CMI and DFI (P < 0.005). ROS levels have a positive correlation with Igf2 methylation (r = 0.19, P < 0.05), DFI (r = 0.40, P < 0.001), CMI (r = 0.39, P < 0.001), and trimethylamine N-oxide (r = 0.45, P < 0.05) and a negative correlation with H19 methylation (r = - 0.20, P < 0.05), tryptophan (r = - 0.45, P < 0.05), sperm motility (r = - 0.20, P < 0.05), sperm viability (r = - 0.23, P < 0.01), and sperm concentration (r = - 0.30, P < 0.001). CONCLUSION(S): Results showed significant correlation between ROS levels and H19-Igf2 gene methylation as well as semen parameters. These findings are critical to identify idiopathic male infertility and its management through assisted reproduction technology (ART).

Effects of silymarin, cabergoline and letrozole on rat model of endometriosis.

OBJECTIVE: Silymarin as an herbal drug has potent antioxidant effects that could make it a good choice for endometriosis therapy. The aim of the current study was to determine the effects of silymarin as an herbal drug on induced endometrial lesion in rat model of endometriosis. MATERIALS AND METHODS: A total of 32 mature, female Sprague-Dawley rats were allocated into 4 experimental groups. The duration of study was about 6 months. Endometriosis implants were surgically prepared and autografted into 32 rats. Three weeks after endometriosis induction, animals were randomly allocated into four groups: Group 1 received cabergoline (CAB group); Group 2 received letrozole (LET group); Group 3 received silymarin (SIL group) and Group 4 received no medication (CONT group). Experimental groups were treated for 3 weeks and then were sacrificed for volume and histopathological evaluation of implants and biochemical assessment. Serum and peritoneal levels of vascular endothelial growth factor (VEGF), total antioxidant activity (TAC) and tumor necrosis (TNF)-α were measured. RESULTS: Mean volume of the implants decreased significantly in silymarin (p < 0.001), letrozole (p < 0.001) and cabergoline (p < 0.001) groups compared to the control. Histopathologic score was significantly lower in silymarin (p: 0.039), letrozole (p: 0.017) and cabergoline (p < 0.001) groups compared to the control. Those receiving silymarin had significantly higher serum TAC compared to control after 21 days of therapy (p < 0.001). CONCLUSION: Silymarin, Letrozole, and Cabergoline administration resulted in decreased size and histopathologic grade of the induced endometrial lesions in a rat model. Silymarin appears to be a virtual novel therapeutic agent for treatment of endometriosis.

Upstream or swim up processing technique: which one is more effective to select human sperm with high chromatin integrity.

BACKGROUND: Sperm processing methods separate motile sperms with good morphology from dead and abnormal forms of sperms, immature germ cells, and non-sperm cells. OBJECTIVE: The propose of this study was to compare the efficacy of upstream and swim-up processing techniques to separate sperms with the high quality especially in relation to sperm chromatin integrity. MATERIALS AND METHODS: This experimental study used semen samples from 60 normozoospermic men. Specimens were divided into equal aliquots for processing by swim up (group A), and upstream (group B) methods and compare with control by raw semen (group C). Sperm concentration, morphology, motility, DNA fragmentation and chromatin maturation were measured in these three groups. RESULTS: The results revealed that sperm concentration in the swim up samples was significantly greater than upstream samples (p≤0.04). as addition, motile sperm recovery including the percentage of progressive motility and a total number of motile sperm was better in the swim-up compared to an upstream method and raw semen (p≤0.001). The cell debris and seminal fluid were equally removed by both methods and the percentage of normal forms was also similar in both procedures (p≥0.4). In addition, sperm DNA fragmentation and chromatin maturation were not significantly different between the three groups (p≥0.1). CONCLUSION: According to results, apparently the upstream method had no significant efficiency to separate good quality sperms compare to swim up. Therefore, swim up seems to be a simple, inexpensive, reliable and widely available method with an efficient yield to separate motile sperm with good morphology and better chromatin integrity for insemination in the infertility clinics.

Transplantation of Spermatogonial Stem Cells Suspension into Rete Testis of Azoospermia Mouse Model.

PURPOSE: The loss of spermatogonia following chemo-or radiotherapy leading to temporary or permanent infertility of the patient is a well known and unwanted side effect of many oncological therapies. MATERIALS AND METHODS: In this study, germ cells were isolated from 4 days old mouse testis cells. Busulfan treatment was used to the eliminate proliferating cells in the testis of recipient mice. The donor cells suspended in DMEM, were introduced into the rete testis of recipient mice via microinjection method. To distinguish the progeny of the transplanted donor stem cells from endogenous germ cells, BrdU-labeled cells were used. In addition, real time PCR was performed to determine expression levels of ngn3 and LIN28 (spermatogonia stem cells markers)before and after transplantation. Western blot analysis was further performed to detect an increase in - ngn3 expression after transplantation. RESULTS: Transplantations of stem cells into rete testis of the recipients was done. Our results clearly showed a significant increase in spermatozoa number in epididymal luman Spermatogonial stem cells (SSCs) did not show alkaline phosphatase activities while ngn3 and LIN28 were clearly expressed. Ngn3 and LIN28 expression were reduced after busulfan treatment compared to untreatmented mice. However, the expression of ngn3 and LIN28 increased after transplantation . BrdU-labeled testis cells were successfully transplanted into rete testis of recipient mice. These cells remained in rete testis of all recipient mice up to two months after transplantation. CONCLUSION: The present study clearly confirme that a regeneration after cytotoxic treatment was based on morphological criteria. We demonstrated the increase in stem cell numbers during regeneration and after transplantation. Transplantation of spermatogonial stem cells suspension by the injection of cells via the rete testis of recipient azoospermia model considerably enhances the efficiency of this procedure.

Methyltetrahydrofolate vs Folic Acid Supplementation in Idiopathic Recurrent Miscarriage with Respect to Methylenetetrahydrofolate Reductase C677T and A1298C Polymorphisms: A Randomized Controlled Trial.

PURPOSE: To determine whether 5-methylenetetrahydrofolate (MTHF) is more effective than folic acid supplementation in treatment of recurrent abortion in different MTHFR gene C677T and A1298C polymorphisms. METHODS: A randomized, double blind, placebo-controlled trial conducted April 2011-September 2014 in recurrent abortion clinics in Tehran, Iran. The participants were women with three or more idiopathic recurrent abortion, aged 20 to 45 years. Two hundred and twenty eligible women who consented to participate were randomly assigned to receive either folic acid or 5-MTHF according to the stratified blocked randomization by age and the number of previous abortions. Participants took daily 1 mg 5-methylentetrahydrofolate or 1 mg folic acid from at least 8 weeks before conception to the 20th week of the pregnancy. The primary outcome was ongoing pregnancy rate at 20th week of pregnancy, and the secondary outcomes were serum folate and homocysteine at the baseline, after 8 weeks, and at the gestational age of 4, 8, 12, and 20 weeks, MTHFR gene C677T and A1298C polymorphisms. RESULTS: There was no significant difference in abortion rate between two groups. Serum folate increased significantly in both groups over time; these changes were significantly higher in the group receiving 5-MTHF than the group receiving folic acid (value = 2.39, p<00.1) and the result was the same by considering the time (value = 1.24, p<0.01). Plasma tHcys decreased significantly in both groups over time; however these changes were not significantly different between the groups (value = 0.01, p = 0.47). CONCLUSION: The results do not support any beneficial effect of 5-MTHF vs. folate supplementation in women with recurrent abortion with any MTHFR C677T and/or A1298C polymorphism. TRIAL REGISTRATION: ClinicalTrials.gov NCT01976676.

Comparison between Platinum-Azidothymidine and Azidothymidine Effects on Bcl-2 and Telomerase Gene Expression in Rats with Hepatocellular Carcinoma.

BACKGROUND: High expression of telomerase and Bcl-2 are reported in hepatocellular carcinoma. Some anticancer drugs show their effects through reduction of these factors. In this study, it was aimed to investigate the effects of a new synthetic compound, platinum azidothymidine, on inhibition of telomerase and Bcl-2 expression in hepatocellular carcinoma compared to azidothymidine. METHODS: To study the effects of Pt-AZT on hepatocellular carcinoma and compare its effects with AZT in inhibition of telomerase and Bcl-2 gene expression, pathogen-free male Wistar rats (n=100) were used. They were randomly divided to 4 groups (n=25). Group A as the control group contained 25 healthy rats; in the rest of animals, preneoplastic lesions were induced in their livers (groups B, C, and D) using Solt-Farber resistant hepatocyte protocol. Cancer development was approved by a pathology laboratory. Group B was negative control (untreated), groups C and D were treated by intraperitoneal injection (IP) of Pt-AZT (0.9 mg/kg/day) and AZT (0.3 mg/kg/day), respectively for 14 days. At the end of the protocol, all rats were sacrificed and Bcl-2 and telomerase gene expression was determined using real-time PCR. RESULTS: No tumor in the livers was found in group A at any point of the study, but it was present in livers of all animals in B, C and D groups. Results showed that telomerase and Bcl-2 expression was significantly lower in group C compared with group B (0.473±0.231 vs. 5.137±5.08, p<0.001, for telomerase expression, and 0.41±0.276 vs. 7.25±11.6, p<0.001, for Bcl-2 expression) and also compared with group D (0.473±0.231 vs. 3.48±4.02, p<0.001, for telomerase expression, and 0.41±0.276 vs. 4.93±18, p<0.001, for Bcl-2 expression). CONCLUSION: For the first time, it was demonstrated that Pt-AZT has more inhibitory effect on telomerase and Bcl-2 expression than AZT. It effectively inhibits the growth of liver tumor in rats by extending apoptosis.

The Generation of Insulin Producing Cells from Human Mesenchymal Stem Cells by MiR-375 and Anti-MiR-9.

BACKGROUND: MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. A number of studies have led to the notion that some miRNAs have key roles in control of pancreatic islet development and insulin secretion. Based on some studies on miRNAs pattern, the researchers in this paper investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. METHODOLOGY: After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and ß cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose so extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. Although derived IPCs by miR-375 alone were capable to express insulin and other endocrine specific transcription factors, the cells lacked the machinery to respond to glucose. CONCLUSION: It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. Although the roles of miR-375 and miR-9 are well known in pancreatic development and insulin secretion, the use of these miRNAs in transdifferentiation was never demonstrated. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

Exploring the human seminal plasma proteome: an unexplored gold mine of biomarker for male infertility and male reproduction disorder.

BACKGROUND: The human seminal fluid is a complex body fluid. It is not known how many proteins are expressed in the seminal plasma; however in analog with the blood it is possible up to 10,000 proteins are expressed in the seminal plasma. The human seminal fluid is a rich source of potential biomarkers for male infertility and reproduction disorder. METHODS: In this review, the ongoing list of proteins identified from the human seminal fluid was collected. To date, 4188 redundant proteins of the seminal fluid are identified using different proteomics technology, including 2-DE, SDS-PAGE-LC-MS/MS, MudPIT. However, this was reduced to a database of 2168 non-redundant protein using UniProtKB/Swiss-Prot reviewed database. RESULTS: The core concept of proteome were analyzed including pI, MW, Amino Acids, Chromosome and PTM distribution in the human seminal plasma proteome. Additionally, the biological process, molecular function and KEGG pathway were investigated using DAVID software. Finally, the biomarker identified in different male reproductive system disorder was investigated using proteomics platforms so far. CONCLUSION: In this study, an attempt was made to update the human seminal plasma proteome database. Our finding showed that human seminal plasma studies used to date seem to have converged on a set of proteins that are repeatedly identified in many studies and that represent only a small fraction of the entire human seminal plasma proteome.

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

BACKGROUND: The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. METHODS: Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). RESULTS: The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). CONCLUSION: Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

Molecular analysis of testis biopsy and semen pellet as complementary methods with histopathological analysis of testis in non-obstructive azoospermia.

PURPOSE: Non-obstructive azoospermia (NOA) is one the many causes of male infertility (10 %) resulting from testicular failure. Multiple testicular biopsies fail to find mature sperm in at least 50 % of cases Therefore; hunting for sensitive and specific biomarkers of spermatogenesis that could better determine the fertility status in NOA can lead to improved management of male infertility. Therefore, we evaluated sperm production through analyses of germ cell-specific transcripts (DAZ, TSPY1, SPTRX3 and SPTRX1) in semen and testicular biopsies of men with azoospermia. METHODS: We collected semen (N=83) and testis biopsies (N=31) from men with non-obstructive azoospermia. We later extracted RNA and synthesized cDNA using washed semen precipitate and testicular tissues. We also performed semi-nested PCR with designed specific primers. Using H&E method, an expert pathologist performed the histopathological evaluation. Having categorized the patients into three groups based on histopathological results, we calculated the agreement between molecular results of semen and tissues with histopathological findings for each patient using Kappa statistical test. RESULTS: Molecular findings of precipitated semen and testicular tissues were in disagreement with histopathological results in most cases. Molecular analysis of testis biopsies showed significant difference (Kappa coefficient=0.009, P value=0.894) with histopathological results; TSPY1, DAZ, SPTRX3 and SPTRX1 were respectively detected in 94 %, 94 %, 17.6 % and 52.9 % of men diagnosed with germ cell aplasia. CONCLUSIONS: Molecular analysis of semen does not provide sufficient sensitivity and specificity to be used as a screening test at the present time, but it is a useful adjunct to histopathological methods in men with NOA. Spermatid/sperm specific transcripts indicated the possibility to find mature sperm following repeated multiple testicular sperm extraction (TESE) or microdisection TESE (mTESE).

FABP9 Mutations Are Not Detected in Cases of Infertility due to Sperm Morphological Defects in Iranian Men.

BACKGROUND: Fatty acid binding proteins (FABPs) are members of the intracellular li- pid binding protein (iLBPs) family and most of them show tissue specific expression. FABP9/PERF15 (Perforatorial15) is the male germ cell-specific fatty acid-binding pro- tein. It was first identified as the major constituent of the murine sperm perforatorium and perinuclear theca. To date, investigations in mice have demonstrated that this protein has a role in the male reproductive system, especially in spermatogenesis. Also, it has been reported that FABP9 can protect sperm fatty acids from oxidative damage. Recently it was shown that it can affect sperm morphology in mice. Based on these findings, we designed a study to evaluate if mutations of this gene can affect sperm morphology in humans. MATERIALS AND METHODS: In this case-control study, DNA was extracted from peripheral blood of 100 infertile males with normal sperm count but with a number of morphologi- cally abnormal sperms in their semen that was above normal. Four exons and one intron of the FABP9 gene were amplified by polymerase chain reaction (PCR), re-sequenced and then analyzed for mutation detection. RESULTS: We did not detect any mutation in any area of the four exons, intron 3 and splice sites of FABP9 gene in any of the studied 100 samples. CONCLUSION: There was no mutation in the exonic regions and the poor sperm mor- phology. However, we didn't analyze the promoter, intron 1 and 2 to establish conclusions regarding the association of these genic regions and sperm dysmor- phology.

Differentiation potential of menstrual blood- versus bone marrow-stem cells into glial-like cells.

Menstrual blood is easily accessible, renewable, and inexpensive source of stem cells that have been interested for cell therapy of neurodegenerative diseases. In this study, we showed conversion of menstrual blood stem cells (MenSCs) into clonogenic neurosphere- like cells (NSCs), which can be differentiated into glial-like cells. Moreover, differentiation potential of MenSCs into glial lineage was compared with bone marrow stem cells (BMSCs). Differentiation potential of individual converted NSCs derived from MenSCs or BMSCs into glial-like cells was investigated using immunofluorescence staining and real-time polymerase chain reaction.The fibroblastic morphology of both MenSCs and BMSCs was turned into NSCs shape during first step of differentiation. NSCs derived from both BMSCs and MenSCs expressed higher levels of Olig-2 and Nestin markers compared to undifferentiated cells. The expression levels of myelin basic protein (MBP) mRNA up regulated only in BMSCs-NSCs no in MenSCs-NSCs. However, outgrowth of individual NSCs derived from both MenSCs and BMSCs into glial-like cells led to significant up regulation of glial fibrillary acidic protein,Olig-2 and MBP at mRNA and protein level accompanied with down regulation of Nestin protein.This is the first study demonstrating that MenSCs can be converted to NSCs with differentiation ability into glial-like cells. Accumulative data show different expression pattern of glial markers in differentiated MenSCs compared to BMSCs. The comparable differentiation potential, more accessibility and no invasive technique for sample collection of MenSCs in comparison with BMSCs introduce MenSCs as an apt, consistent and safe alternative to BMSCs for cell therapy of neurodegenerative diseases.

The association of seminal plasma antioxidant levels and sperm chromatin status with genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) in infertile men with oligoasthenoteratozoospermia.

In this study we aimed to examine the effects of genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) on GST activity, seminal oxidative stress and sperm chromatin status in infertile men with oligoasthenoteratozoospermia (OAT). The study population (n=121) consisted of 95 infertile men with OAT and 26 controls with normozoospermia. Multiplex polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were utilized to detect the aforesaid genetic variants. We measured GST activity and total antioxidant capacity (TAC) of seminal plasma by spectrophotometry. Sperm chromatin integrity and maturity were assessed using toluidine blue and chromomycin A3 (CMA3-positive sperm) staining, respectively. The analysis showed that subgroups of GSTM1 null and GSTP1 C/T+T/T genotypes in comparison with GSTM1 present and GSTP1 wild type (C/C) genotypes did not have statistically significant differences in both OAT or normozoospermic men considering sperm concentration and motility, percentage of CMA3-positive sperm, seminal plasma TAC, sperm chromatin integrity and GST activity. Thus, the findings of our study suggest that there are no significant associations between GSTM1 and GSTP1 polymorphisms and sperm parameters at conventional or at molecular levels including OS status, sperm chromatin integrity or maturity in Iranian infertile men with OAT and normozoospermia. However, these polymorphisms could be related to the fertility status of the studied population but not evaluated in this study.

Effects of very rapid versus vapor phase freezing on human sperm parameters.

The aim of the present study was to compare the effects of two freezing methods, vapor phase and very rapid freezing, with and without cryoprotectant on semen parameters in men with normal semen criteria. Cryopreservation was done on semen samples from 31 men by two methods of vapor phase freezing and very rapid freezing, with and without Test Yolk buffered glycerol (TYBG) as cryoprotectant. The motility, viability, acrosome and DNA integrity were evaluated on fresh and post-thaw samples. Post-thaw sperm progressive motility was significantly higher in the presence of TYBG in the vapor phase cryopreservation (%6.30 ± 3.74) compared with the very rapid freezing method (%2.2 ± 1.97 and %4.00 ± 2.42 in the presence and absence of TYBG, respectively). There was no significant difference in viability, acrosome status and DNA integrity between two methods in presence or absence of TYBG. The very rapid freezing method in the absence of TYBG showed better sperm motility but viability, acrosome and DNA integrity were similar to the presence of TYBG. The results show that cryopreservation of human spermatozoa together with seminal plasma by using vapor phase method is better than very rapid freezing method to preserve sperm progressive motility; however very rapid freezing method is quick and simple and do not require special cryoprotectant. It can be used for cryopreservation of small number of spermatozoa in IVF centers.

Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility.

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n = 81) and non-male factor (NMF, n = 52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p < 0.001). Sperm chromatin damage was correlated with percentage of CMA(3)- positive sperm (r = 0.64, p < 0.001) and with sperm concentration (r = -0.36, p < 0.001), motility (r = -0.21, p < 0.05), and morphologically normal spermatozoa (r = -0.29, p < 0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA(3)- positive sperm (p < 0.001) in ungrouped patients. It was related to both percentage of CMA(3)- positive sperm and serum DHEA in the MF group (p < 0.001 and p < 0.05, respectively). Sperm chromatin maturity assessed by CMA(3) test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.

Seminal molecular markers as a non-invasive diagnostic tool for the evaluation of spermatogenesis in non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is currently evaluated by the use of conventional histopathological methods. In some cases, focal spermatogenesis is present in the testes of patients with NOA which may be almost undetectable by routine histopathological examinations. Application of molecular markers in semen to predict the spermatogenesis status in the testis will emphasize the probability of finding sperm in NOA testis through further search using TESE or mTESE. Detection of germ cell-specific transcripts in semen is a signal of germ cells present in the testis. In this study, we used molecular methods to evaluate spermatogenesis status in azoospermic men. Semen samples were collected from 203 men with azoospermia. Total RNA was extracted from the semen precipitates. First-strand complementary deoxyribonucleic acid (cDNA) was synthesized by reverse transcriptase then, (RT)-PCRs were carried out using primers for testis stage-specific genes (DAZ, AKAP4, PRM1, and PRM2). Testicular tissue biopsies were used for evaluating spermatogenesis status in testis. Histopathological examination and LH, FSH, and testosterone level measurements (chemiluminescence assay) were performed. The presence of DAZ and PRM2 transcripts in semen significantly indicated the presence of spermatogonia and spermatids in the testicular tissues. Absence of all four markers in semen confirmed the histopathological results corresponding to sertoli cell only syndrome (SCO). Although TESE should not be excluded solely on this criteria, using PRM1, PRM2, AKAP4, and DAZ transcripts in semen would provide a non-invasive molecular diagnostic tool to better counsel patients before undergoing TESE.