Assessment of the Impact Induced by Different Incubation Time, Storage Time, Storage Medium and Thawing Methods on Sperm DNA Fragmentation Assay: A Before-After Study.

Background: The sperm DNA fragmentation has been considered an important index in the field of male infertility. Aims: Our study aims to evaluate the impact of different factors, including incubation time, storage time, storage medium and method of thawing, on DNA fragmentation of semen samples. Settings and Design: This study was designed as a before-after study in five experiments. Materials and Methods: Experiment 1 was conducted to assess the effect of storage time in liquid nitrogen on 15 semen samples. In experiment 2, DNA fragmentation was performed on 10 semen samples with different incubation times before freezing. In experiments 3, 4, two different storage media and thawing methods were applied respectively in two separate groups, each containing 30 samples and the DNA fragmentation index (DFI) was measured using the sperm chromatin structure assay method. Statistical Analysis: Data were analysed using Stata version 11. Results: There was a significant increase in sperm DNA fragmentation of samples stored in liquid nitrogen for 1 month. This increase occurred in the first 2 weeks. Furthermore, our results showed a significant increase in the DFI after 120 min of incubation at room temperature (RT) and also thawing in RT separately. Conclusion: It is better to use fresh samples to measure DNA fragmentation up to 2 h after ejaculation to achieve more accurate results. Furthermore, if sperm freezing is inevitable, the use of a water bath (37°C) to thaw will be the most appropriate option, as it can lead to less DNA damage.

The influence of ginger (Zingiber officinale) on human sperm quality and DNA fragmentation: A double-blind randomized clinical trial.

BACKGROUND: Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. OBJECTIVE: This study was designed to investigate the effects of ginger (Zingiber officinale) on sperm DNA fragmentation (SDF) in infertile men. MATERIALS AND METHODS: This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group). Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. RESULTS: There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02) in cases and (56.75, 95%CI: 40.01-73.5) in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39) was lower compared with placebo (40.54, 95%CI: 23.94-57.13) after three month of treatment (p=0.02). In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009). There were no significant differences between two groups regarding to semen parameters. CONCLUSION: The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men.

Predictive factors of successful microdissection testicular sperm extraction in patients with presumed sertoli cell-only syndrome.

BACKGROUND: To evaluate predictive factors of successful microdissection-testicular sperm extraction (MD-TESE) in patients with presumed Sertoli cell-only syndrome (SCOS). MATERIALS AND METHODS: In this retrospective analysis, 874 men with non-obstructive azoospermia (NOA), among whom 148 individuals with diagnosis of SCOS in prior biopsy, underwent MD-TESE at Department of Andrology, Royan Institute, Tehran, Iran. The predictive values of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels, testicular volume, as well as male age for retrieving testicular sperm by MD-TESE were analyzed by multiple logistic regression analysis. RESULTS: Testicular sperm were successfully retrieved in 23.6% men with presumed SCOS. Using receiver operating characteristic (ROC) curve analysis, it was shown that sperm retrieval rate in the group of men with FSH values >15.25% was 28.9%. This was higher than the group of men with FSH ≤15.25 (11.8%). CONCLUSION: Sperm retrieval rate (SRR) was 23.6% in men with presumed SCOS and FSH level can be a fair predictor for SPR at MD-TESE. MD-TESE appears to be recommendable in such cases (SCOS with high FSH concentration) with reasonable results.

Relationship between absence of annulus and asthenozoospermia in Iranian men.

PURPOSE: To find a relationship between absence of annulus and asthenozoospermia in Iranian men. METHODS: In the present study, semen samples from 100 asthenozoospermic and 20 normozospermic patients were analyzed for sperm concentration and motility. Spermatozoa were immunostained for the two septin subunits Sept4 and Sept7. The absence of the annulus structure was confirmed by transmission electron microscopy and western blot analysis for septin 4. DNA sequencing for all coding exons of SEPT12 was performed for a patient using peripheral blood sample. RESULTS: Specific antibodies for SEPT4 and SEPT7 consistently labeled the annuli in spermatozoa from all of the 20 normozospermic men, while in one of 100 patients with asthenozoospermia, 75% of sperms lacking septin 4 or septin 7 proteins at the annulus. It was shown that the structural defect in annulus formation is not caused by point mutation of SEPT12 gene. CONCLUSIONS: In conclusion, the results of this study demonstrated that the frequency of the absence of annulus in asthenozoospermic sample of Iranian population has a low frequency and could not be assume as a diagnostic marker for classifying asthenozoospermic patients.

Study of the effect of varicocelectomy on sperm proteins expression in patients with varicocele and poor sperm quality by using two-dimensional gel electrophoresis.

PURPOSE: To compare proteomic profiles of spermatozoa from patients with varicocele and poor sperm quality before and after varicocelectomy. METHODS: This work was designed as a prospective and observational study. The study was based on 20 men with varicocele grade 3 and poor sperm quality undergoing varicocelectomy at the Fertility Unit of Royan institute in 2009. Two semen samples were collected, one before varicocelectomy and the other after surgery. Protein separation was done by two-dimensional protein electrophoresis, and analyzed by gel densitometry and mass spectrometry. Differential sperm protein expression levels were measured by gel densitometry. RESULTS: Comparison of the sperm parameters showed that sperm motility and concentration were increased after varicocelectomy. At the level of protein, a total of 3 protein spots were identified whose expression was significantly lower in sperm samples before varicocelectomy compared with after surgery including heat shock protein A5 (HSPA5), superoxide dismutase 1 (SOD1) and δ-subunit of the catalytic core of mitochondrial adenosine triphosphate synthase (ATP5D). CONCLUSIONS: High grade varicocoele affects sperm protein expression presumably because of increasing testicular temperature. These proteins play essential roles in sperm production, DNA integrity protection, and sperm motility. This novel study demonstrates that varicocelectomy can improve both sperm quality and proteins expression.

Comparison of sperm retrieval and intracytoplasmic sperm injection outcome in patients with and without Klinefelter syndrome.

OBJECTIVE: To evaluate microsurgical testicular sperm extraction (micro-TESE) and intracytoplasmic sperm injection (ICSI) treatment in patients with non-mosaic Klinefelter syndrome (KS). METHODS: We retrospectively evaluated the micro-TESE/ICSI performance in 134 patients with classic KS and 537 men with nonobstructive azoospermia (NOA) and normal karyotype referred to Royan Institute between 2009 and 2011. The patients were divided into 2 groups according to micro-TESE outcome. Several factors, including patients' age, level of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone, were compared between the 2 groups. RESULTS: Sperm retrieval rate by micro-TESE in the KS and control groups were 28.4% (38/134) and 22.2% (119/537), respectively. In the KS group, the mean age of patients with successful sperm retrieval of spermatozoa was significantly lower than those with negative results (30.0 ± 0.65 vs 33.68 ± 0.6 year, P = .001) and comparison of laboratory parameters between the 2 groups showed that the level of testosterone was significantly higher in patients with successful sperm retrieval (3.4 ± 0.48 vs 2.33 ± 0.23 ng/mL, P <.001). The fertilization rate was significantly higher in the KS group than the control group (28% vs 21%, P = .038). Also, live birth rate per embryo transfer was higher in the KS group, but it was not significant (13% vs 3%, P = .05). CONCLUSION: This study of sperm recovery and ICSI outcome in men with and without KS shows that micro-TESE/ICSI is a successful intervention for the majority of men with KS.

Assessment of deoxyribonucleic acid fragmentation index, testicular volume, semen parameters, and hormone profile in gonadotropin-treated men with hypogonadotropic hypogonadism.

OBJECTIVE: To study the sperm deoxyribonucleic acid (DNA) fragmentation index (DFI), testicular volume, semen parameters, and hormone profile in human chorionic gonadotropin (hCG)- and human menopausal gonadotrophin (hMG)-treated patients with hypogonadotropic hypogonadism (HH) with and without a successful pregnancy. METHODS: This is a cross sectional study. The study initially included 81 patients with HH and azoospermia at the Infertility Unit of Royan Institute between 2010 and 2012. Fifty-eight of 81 patients achieved >1 × 10(6) sperm/mL during hCG and hMG therapy. These 58 patients were divided into the following 2 groups: 20 patients with HH who achieved pregnancy in response to hCG/hMG (responders, 16 naturally and 4 by intrauterine insemination) and 38 gonadotropin-treated patients with HH with failed pregnancy (nonresponders, 29 naturally, 5 by intrauterine insemination, 1 by in vitro fertilization, and 3 by intracytoplasmic sperm injection). Sperm DNA fragmentation was visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: Average of DFI (responders: 13.45 ± 0.64; nonresponders: 21.92 ± 0.86), age, body mass index, testis volume semen parameters, and follicle-stimulating hormone, luteinizing hormone, and testosterone levels in the 2 groups were calculated. Cut-off point for DFI was determined by receiver operating curve analysis (17.5%). CONCLUSION: It was shown that DFI in responders is significantly lower than DFI in nonresponders (P <.001), and duration of hCG and hMG therapy in responders is significantly higher than those of nonresponders (P <.05). DFI could be predictive of conception (P <.001; odds ratio 0.57; 95% confidence interval 0.417-0.778). It can be concluded that despite low sperm quality, especially sperm concentration in these patients, decreasing sperm DNA damage may result in successful fertilization.

Study of sperm protein profile in men with and without varicocele using two-dimensional gel electrophoresis.

OBJECTIVE: To compare the sperm protein profile between men with and without varicocele. METHODS: The present study was designed as a case-control study. The research patients were recruited from the Infertility Unit of the Royan Institute in 2009. We included 20 sperm samples from normozoospermic men without varicocele (control group) and 20 sperm samples from oligozoospermic patients with varicocele, grade 3 (varicocele group) in the present study. The sperm protein profile in the 2 groups was characterized using 2-dimensional gel electrophoresis. Differences in protein expression were established using gel analysis software, and protein identification was performed using mass spectroscopy analysis. RESULTS: In the varicocele group, we noted 15 consistent differences in protein expression (1, spots missing; 12, less abundant; and 2, more abundant) compared with the control group (P < .01). The findings revealed that heat shock proteins, mitochondrial proteins, and cytoskeleton proteins are the proteins mainly affected by varicocele disease. CONCLUSION: To our knowledge, the present study is a novel study, with few studies describing the correlation between sperm protein in men with and without varicocele obtained using a 2-dimensional proteomic approach. It could be an important prerequisite to the development of diagnostic tests to predict varicocelectomy outcomes in patients with varicocele and abnormal findings on a spermogram in the clinical environment.

Enzymatic digestion improves testicular sperm retrieval in non-obstructive azoospermic patients.

BACKGROUND: In patients with non-obstructive azoospermia (NOA), vital spermatozoa from the tissue is obtained from testes by enzymatic treatment besides the mechanical treatment. OBJECTIVE: To increase the sperm recovery success of testicular sperm extraction (TESE), with enzymatic digestion if no sperm is obtained from testis tissue by mechanical method. MATERIALS AND METHODS: Tissue samples were collected from 150 men who presented with clinical and laboratory data indicating NOA by means of TESE and micro dissection TESE methods. Initially, mature spermatozoa were examined for by mechanical extraction technique shredding the biopsy fractions. In cases whom no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was followed by enzymatic digestion using DNaseI and collagenase type IV. Surgery type, pathology, AZF, karyotype, hormones and testis size were compared in patients. RESULTS: Of 150 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery in 13 (about 9%) patients. Comparison of parameters revealed that level of FSH and LH were significantly different (p=0.04 and 0.08 respectively) between two groups that response negative and positive to enzymatic digestion. CONCLUSION: The combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa. The benefit of the mincing combined with enzyme to sperm retrieval for NOA firstly shorten the mechanical searching time, leading to minimizing further cellular damage as well as exposure to external conditions, and secondly reduce the number of cases with sperm recovery failures. Also, the serum level of FSH and LH are factors that influence the chance of sperm retrieval.

Achieving high survival rate following cryopreservation after isolation of prepubertal mouse spermatogonial cells.

PURPOSE: Isolating spermatogonia cells with high purity and viability and achieving better survival rate following cryopreservation METHODS: Isolating the cells by Magnetic Activating Cell Sorting (MACS) method using anti CD49f (alpha6 integrin) antibody and Dynabeads and freezing in DMSO-based freezing mediums containing three different FBS concentrations of 50%, 60% and 70%. RESULTS: The mean (+/-SD) purity of the isolated cells was 92.52+/-3.57 (range 92.43-98.25). The cells frozen in group I, II and III had mean 39.60+/-1.48 (range 37.98-41.62), 89.05+/-3.83 (range 80.83-90.33) and 90.52+/-1.71 (range 89.07-92.52) viability, respectively. CONCLUSION: Higher viable cell counts and purity can be attained by the use of alpha6 integrin and magnetic beads. After the thawing of spermatogonial cells, optimum viability was achieved in freezing media containing 60% FBS.