Sperm Specimens Research Articles

Effectiveness of recombinant follicle-stimulating hormone treatment in patients with oligo-asthenospermia at different levels of DNA fragmentation index: A phase II clinical trial

Abstract Objective This study aimed to perform an evaluation of changes in spermogram parameters after follicle-stimulating hormone (FSH) therapy in infertile males having oligo-asthenospermia at different levels of DNA fragmentation index (DFI). Materials and methods Infertile men with oligo-asthenospermia, no underlying urogenital disease (such as varicocele), and medically fertile partners were enrolled over 1 year. Semen parameters, FSH, luteinizing hormone, and testosterone levels were determined; also, a Sperm DNA Fragmentation Assay Kit (Hamun Teb Pishro, Tehran, Iran) was used for determining sperm DFI at baseline .Participants were categorized into three groups based on DFI: DFI <15% (group 1), DFI of 15%–30% (group 2), and DFI >30% (group 3). All participants received subcutaneous recombinant FSH (150 mg every other day) for 6 months. Sperm specimens were tested 6 months after intervention (a single sperm control test). Results Sixty males whose average age was 28.4 years were enrolled. Only group 3 (poor fertility) exhibited a significant rise in sperm concentration (p = 0.001) and motility (p < 0.05) after FSH treatment. Group 1 (DFI <30 fertility) and group 2 (DFI of 15%–30%) showed increased mean sperm concentration and motility postintervention, although these alterations were not significantly different. Follicle-stimulating hormone levels increased significantly in all three groups after FSH administration. Serum luteinizing hormone and testosterone levels were not significantly increased in any of the groups. Conclusions FSH treatment improves sperm concentration and motility in men with oligo-asthenospermia, with significant improvements observed in men with DFI >30%. DNA fragmentation index can be a predictive indicator of response to FSH treatment in such patients.

Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods.

For sperm cryopreservation, the conventional method, which requires glycerol, has been used for a long time. In addition, the permeable cryoprotectant-free vitrification method has been continuously studied. Although the differences of cryopreservation effects between the two methods have being studied, differences in microRNA (miRNA) profiles between them remain unclear. In this study, we investigated the differences in miRNA expression profiles among conventional freezing sperm, droplet vitrification freezing sperm and fresh human sperm. We also analyzed the differences between these methods in terms of differentially expressed miRNAs (DEmiRs) related to early embryonic development and paternal epigenetics. Our results showed no significant differences between the cryopreservation methods in terms of sperm motility ratio, plasma membrane integrity, DNA integrity, mitochondrial membrane potential, acrosome integrity, and ultrastructural damage. However, sperm miRNA-sequencing showed differences between the two methods in terms of the numbers of DEmiRs (28 and 19 with vitrification using a nonpermeable cryoprotectant and the conventional method, respectively) in postthaw and fresh sperm specimens. DEmiRs related to early embryonic development and paternal epigenetics mainly included common DEmiRs between the groups. Our results showed that the differences between conventional freezing and droplet vitrification were minimal in terms of miRNA expression related to embryonic development and epigenetics. Changes in sperm miRNA expression due to freezing are not always detrimental to embryonic development. This study compared differences in miRNA expression profiles before and after cryopreservation between cryopreservation by conventional and vitrification methods. It offers a new perspective to evaluate various methods of sperm cryopreservation.

Assessment of the In Vivo Reprotoxicity of Isotretinoin in Sprague-Dawley Male Rat.

Isotretinoin (ISO) belongs to a family of drugs called retinoids. It is the most effective drug prescribed by dermatologists for the treatment of the inflammatory disease, acne vulgaris. A significant barrier to the use of ISO has worries regarding its adverse effect profile. Despite the well-recognized reproductive toxicity and teratogenicity in females, there is no warning related to the use by male patients in the medication prospectus. Current data on the effects on human male fertility is contradictory and inconclusive. This study was undertaken to investigate the potential effects of ISO oral doses in the Sprague-Dawley male rat germ cells using the sperm morphology assay. Also, the serum levels of the follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were measured. The rat groups were given varying ISO doses via gastric gavage for seven consecutive days. The epididymis sperm specimens were microscopically examined for the following reproductive toxicity parameters: sperm concentration, examined viability, motility, and morphology. The serum FSH, LH, and testosterone levels were measured by using the corresponding enzyme-linked immunosorbent assay (ELISA) kit. The data were analyzed statistically by one-way analysis of variance (ANOVA) followed by the Tukey test at P ≤ 0.05 significance level. The results indicated that the drug did not significantly increase the sex hormone levels but notably affected both the sperm quantity and quality. These observations suggest that ISO was reprotoxic, and future therapies should be further reassessed.

The Molecular Prevalence of Varicella-Zoster Virus and Human Herpesvirus Types 6 and 7 in Sperm Samples of Infertile Men

Background and Aims: Human herpes virus types 6 and 7 (HHV-6, HHV-7) can remain latent in the urogenital tract epithelial cells. Varicella-Zoster virus (VZV) is among the Alphaherpesviruses infecting most people during childhood. This virus can remain latent in the ganglionic neurons. The latent infection can reactivate through weakness, suppression of the immune system, and aging. Therefore, this study investigated the molecular prevalence of HHV-6, HHV-7, and Varicella-Zoster virus in sperm samples from infertile men.
 Materials and Methods: Sperm samples from 82 infertile patients were collected from infertility centers in Tehran. Chromatin health levels and sperm DNA examinations were carried out using sperm DNA fragmentation index kits. Viral nucleic acids were extracted from the samples. The presence of VZV, HHV-6, and HHV-7 genomes was examined through the conventional polymerase chain reaction assay.
 Results: The average age was 37.3 ± 6.1 years. The mean sperm motility percentage was 33.6 ± 2%. Infertile men's mean DNA fragmentation index was 27.2 ± 1.2. This study shows a 7.3% and 4.9% prevalence for HHV-6 and VZV, respectively, and the absence of HHV-7 in sperm specimens.
 Conclusion: Although the infection rates of these herpesviruses seem relatively low, more comprehensive and multi-center studies are required to obtain more accurate results.

Quality of testicular spermatozoa improves with changes in composition of culture medium

BackgroundSpermatozoa retrieved from the testis and epididymis are deprived of the beneficial effects of seminal fluid. Thus applying an artificial medium with normal seminal fluid characteristics, known as artificial seminal fluid (ASF), may provide an appropriate condition for improving some sperm parameters in azoospermia. The objective was to investigate the impact of in vitro exposure of testicular and epididymal spermatozoa to ASF on sperm quality. The study was conducted on testicular (n = 20) and epididymal (n = 20) sperm specimens obtained from azoospermic men. Each sample was divided into two equal parts: Part I) for processing and incubation with Ham’s F10 medium; Part II) for processing and incubation with ASF.ResultsAfter 2 h incubation, testicular sperm motility was significantly higher in ASF than in Ham’s F10 medium. In comparison to 0 h, mitochondrial membrane potential levels of testicular spermatozoa were significantly higher after 2 h and 24 h in ASF and after 24 h in Ham’s F10 medium. Furthermore, the data indicated significantly lower rates of epididymal spermatozoa with high MMP in both media after 24 h. There were no significant differences in the DNA fragmentation index of testicular and epididymal spermatozoa between ASF and Ham’s F10 medium at different time points.ConclusionThe results demonstrated that in vitro incubation of testicular spermatozoa improved their motility more effectively than Ham’s F10 medium in the short term (2 h), but had no effect on epididymal spermatozoa. Since the physiology of testicular spermatozoa is different from that of ejaculated spermatozoa, it seems that a special environment should be designed and used for each of them.

Immobilization of Multivalent Titanium Cations on Magnetic Composite Microspheres for Highly Efficient DNA Extraction and Amplification.

Magnetic-assisted DNA testing technology has attracted much attention in genetics, clinical diagnostics, environmental microbiology, and molecular biology. However, achieving satisfying DNA adsorption and desorption efficiency in real samples is still a big challenge. In this paper, a new kind of high-quality magnetic composite microsphere of MM@PGMA-PA-Ti4+ was designed and prepared for DNA extraction and detection based on the strong interaction of Ti4+ and phosphate groups. By taking the advantages of high magnetic susceptibility and high Ti4+ content, the MM@PGMA-PA-Ti4+ microspheres possessed remarkable extraction capacity for mimic biological samples (salmon sperm specimens) with saturated loadings up to 533.0 mg/g. When the DNA feeding amount was 100 μg and the MM@PGMA-PA-Ti4+ dosage was 1 mg, the adsorption and desorption efficiencies were 80 and 90%, respectively. The kinetic and equilibrium extraction data were found to fit well with the pseudo-second-order model and Freundlich isotherm model. Furthermore, the MM@PGMA-PA-Ti4+ microspheres were successfully employed for DNA extraction from mouse epithelial-like fibroblasts. The extraction ability (84 ± 4 μg/mg) and DNA purity were superior to the comparative commercial spin kits, as evaluated by electrophoresis assays and qPCR analysis. The experimental results suggest that the MM@PGMA-PA-Ti4+ microspheres possess great potential as an adsorbent for DNA purification from complex biological samples.

The impact of cryopreservation on both sperm HPV-negative and positive subtypes

It is well known that various human papillomavirus (HPV) genotypes are present in semen specimens. Also, it has been demonstrated that sperm parameters are negatively affected when HPV infection is present in the sperm sample. Besides all these, the effect of cryopreservation on HPV sensitivity and resistance is not known. The aim of the present study is to evaluate first the prevalence of HPV and secondly to elucidate whether cryopreservation of sperm HPV-positive samples has any effect on the viability of HPV. For this purpose, a cohort of 78 sperm specimens was used from a respective number of patients. After giving informed consent, semen analysis was performed. Each sperm sample was divided into four equal aliquots. The first one (fresh) was evaluated for the prevalence of HPV, while the other three aliquots were cryopreserved by adding an equal quantity of cryoprotectant and plunged into the LN. Each of the three aliquots was thawed 3, 6, and 12 months later, respectively, so as to evaluate whether there is a time-resistance period of HPV prevalence. HPV infection was found to be in eleven sperm samples, demonstrating a 14.1% (11/78) HPV prevalence. Among the HPV-positive samples, six of them were high-risk and the remaining were low-risk genotypes. Moreover, the high-risk fresh samples demonstrated higher motility values than the low-risk samples (60% ± 2.7 vs 45.6% ± 3.7, p < .05), while semen volume in the high-risk samples was significantly lower than the respective volume in the low-risk samples (2.26 ± 0.2ml vs 3.5 ± 0.6ml, p < .05). Interestingly, cryopreservation of the HPV-positive samples resulted in the sustainability and time-resistance of HPV in all high-risk HPV-positive samples, something that was not the case with the low-risk HPV-positive samples. Conclusively, sperm samples infected with high-risk HPV, demonstrate lower sperm parameters and time-resistance activity during cryopreservation.

Detection of sperm cells by single-stage and two-stage deep object detectors

Today, infertility is a common health concern that affects approximately 15%–20% of the world population. The evaluation of male patients with infertility includes diverse examination and laboratory tests in comparison to female patients. In the evaluation of male infertility, sperm specimens are examined in terms of morphometry, concentration, and motility. Detection of sperm is the critical step to determine the concentration and motility parameters. In this study, a fusion approach of deep learning-based object detection techniques is utilized in the sperm detection problem for obtaining more accurate and consistent concentration and motility characteristics of sperm specimens. First, 12 sperm specimen videos acquired from different infertile patients were recorded. Then, regions in the video frames were labeled as sperm and non-sperm by the experts. Two different scenarios have been tested over the labeled dataset. Differently arranged train and test data ratios were also utilized for each scenario. In the first scenario, deep learning-based object detection algorithms were individually performed for the sperm detection of patient-oriented videos in terms of different train/test split ratios. Videos with the lowest mAP (mean Average Precision) values resulted in the first scenario were selected for the second scenario as target videos. The rest of the videos were used for model training without any patient-oriented manner. In the second scenario, the detection performance for more challenging videos is aimed to increase by using different videos instead of using the small part of the same video in the model training. Additionally, a fusion idea of the utilized deep learning-based techniques was proposed and performed over these low mAP resulted videos to increase the detection performance. The results are compared regarding the general mAP, class-wise APs, and training time. In the first scenario, YOLOv5 achieved the best results, while the proposed fusion approach achieved the best mAP scores in the second scenario.

Serum Bilirubin and Sperm Quality in Adult Population.

The neurotoxicity of bilirubin has been extensively reported in numerous studies. However, the association between bilirubin and male fertility has not yet been studied. The main goal of this study was to investigate the association between serum total bilirubin and sperm quality in an adult population. In this cross-sectional study, 9057 participants who attended the MJ health examination (2010–2016) were enrolled. Sperm specimens were collected by masturbation, and sperm quality was analyzed in accordance with the WHO criteria. Serum total bilirubin levels were measured by an automatic biochemical profile analyzer. Thereafter, the associations between serum total bilirubin and sperm quality were determined by a multivariable linear regression. Serum total bilirubin was inversely associated with sperm concentration and normal morphology with β values of −13.82 (95% CI: −26.99, −0.64) and −18.38 (95% CI: −30.46, −6.29) after adjusting for covariables. The highest levels of serum total bilirubin were significantly associated with sperm concentration and normal morphology with β values of −14.15 (95% CI: −28.36, 0.06) and −21.15 (95% CI: −33.99, −8.30). Our study highlighted the potential impact of serum bilirubin on sperm quality in a male population. Additional longitudinal research is necessary to explore these findings and underlying mechanisms.

Effects of Color Space Transformations on Classification Performance of Sperm Morphology

Infertility is defined by the World Health Organization as the inability of a woman to become pregnant even though the couple had sexual intercourse for one year without any protection. Male and/or female factors might be the reasons for infertility. When diagnosing the male factors, sperm specimens are analyzed in a laboratory environment under certain conditions. The morphological abnormality, characteristic motility and concentration of sperm are examined in the analysis called spermiogram. Spermiogram tests can be done manually by doctors, as well as by using computer-assisted sperm analyzing systems. The importance of computer aided analysis is increasing day by day because visual inspection can give different results from person to person and is costly. In this study, the effect of different color spaces as a preprocessing step is investigated to increase the classification performance of a computer based analyzing approach for sperm morphology. Three sperm morphology data sets abbreviated as SMIDS, HuSHeM and SCIAN-Morpho were used in the experimental tests. Data augmentation was applied on the data sets due to the unbalanced distribution of sperm images among the classes and insufficient data. Then, data sets were converted to two well-known color spaces, LAB and HSV to observe the effects of color space in the classification. MobileNetV2 was utilized as the classification model. In order to indicate the effects of color spaces, results were compared with previously published study where no color transform was implemented. The classification of images in LAB and HSV color spaces had better results than RGB images trained under the same conditions. The maximum classification accuracies of 89%, 85% and 68% were obtained for SMIDS, HuSHeM, SCIAN-Morpho data sets by using the color space transform idea, respectively.

P–120 Selecting spermatozoa with the highest genomic integrity in order to enhance clinical outcomes in men with high DNA fragmentation levels

Abstract Study question What are the best methods of selecting spermatozoa with the highest genomic integrity in order to improve embryo implantation and term pregnancy rates with ICSI? Summary answer Testicular or ejaculate spermatozoa isolated by microfluidic sperm selection (MFSS) were characterized by superior genomic integrity with improved clinical pregnancy and delivery rates. What is known already In couples with unexplained infertility, a subtle male factor can often be identified. Both single-strand (ss) and double-strand (ds) DNA nicks and breaks hinder the ability of the male gamete to support embryonic development. Surgical retrieval of spermatozoa from the proximal male genital tract can prevent their exposure to oxidative stress. Moreover, use of membrane-based microfluidics chips has been shown to allow for selection of the most progressively motile spermatozoa with higher genomic integrity. Study design, size, duration Over the course of 48 months, 86 consenting men presenting with high sperm chromatin fragmentation (SCF) in their ejaculate with prior ART failure underwent a subsequent cycle with specimens retrieved by testicular biopsy or ejaculate processed by MFSS. A concurrent TUNEL assay was performed on samples collected or selected by each method. Sperm specimens of both origins were utilized for ICSI cycles. Semen parameters, chromatin integrity, and pregnancy outcomes were compared between the two methods. Participants/materials, setting, methods Fresh ejaculates from consenting men were collected for standard semen analysis (WHO 2010). Testicular biopsy and MFSS were used to isolate spermatozoa with a higher genomic integrity after previous ART failure. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) on at least 500 spermatozoa under a fluorescent microscope with a threshold of ≥ 15%. MFSS was carried out by Zymot® chips. ICSI was performed in the standard fashion. Main results and the role of chance A total of 86 men (36.5±5 years) had the following semen parameters: volume of 2.6 ±1mL, concentration of 27.0±33 x 106/mL, 35.6±15% motility, and high SCF (24.1±10%). They underwent 146 ICSI cycles with their partners (maternal age, 33.7±3) resulting in a high incidence of pregnancy loss (100%; 13/13). Of those who failed to conceive, 22 couples used surgically retrieved spermatozoa (SRS) with a concentration of 1.8 ± 4 x 106/mL (P &amp;lt; 0.01), 5.0±11% motility (P &amp;lt; 0.01), and an SCF of 12.6 ± 6% (P &amp;lt; 0.0001). SRS was used in 37 ICSI cycles, yielding a fertilization rate of 61.6% (204/331, P &amp;lt; 0.01), an implantation rate of 10.6% (9/85, P &amp;lt; 0.01), a CPR of 23.5% (8/34, P &amp;lt; 0.01), and a delivery rate of 17.6% (6/34, P &amp;lt; 0.01). Another 24 couples underwent ICSI cycles with ejaculated spermatozoa processed by MFSS with a concentration of 1.8±3 x 106/mL (P &amp;lt; 0.01), but an increased motility of 99±1% (P &amp;lt; 0.01) and an SCF of 1.2 ±1%, lower than both the raw and testicular specimens (P &amp;lt; 0.0001). MFSS-processed specimens resulted in a fertilization rate of 76% (335/441, P &amp;lt; 0.01), an implantation rate of 26.3% (15/57, P &amp;lt; 0.05), and a CPR of 67.9% (19/28, P &amp;lt; 0.01), of which 15 patients delivered and 2 pregnancies are ongoing (89.5%; P &amp;lt; 0.01). Limitations, reasons for caution This is a preliminary study on a small number of subjects. A randomized prospective study conducted on a larger cohort would be required to confirm our findings. Wider implications of the findings: SCF severely affects pregnancy by impairing embryonic development, consequently promoting implantation failure. While retrieving spermatozoa from the germinal epithelium is a viable option, MFSS provides an alternative. Although MFSS requires an adequate number of sperm with good kinetic characteristics, it provides a more palatable option, reducing surgical risk and costs. Trial registration number Not applicable

Vital Analysis of Cryopreserved Sperm of Marbled Flounder, Pseudopleuronectes yokohamae.

The marbled flounder (Pseudopleuronectes yokohamae) is a commercial flatfish in East Asia. The aim of this study was to improve its sperm cryopreservation protocol based on the vitality assessment of 7-day and 1-year cryopreserved sperm. Four extenders (extender-1: sucrose solution; extender-2: glucose solution; extender-3: fish Ringer's solution; and extender-4: modified fish Ringer's solution) were tested with a combination of five cryoprotectants (CPAs) (dimethyl sulfoxide: Me2SO; glycerol: GLY; ethylene glycol: EG; propylene glycol: PG; and methanol: MeOH) at four different concentrations (5, 10, 12, and 15%). Fluorescent technique was applied to detect the plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and DNA integrity of fresh and cryopreserved sperm specimens. Fresh sperm was diluted at a ratio of 1:2 (sperm:extender). Post-thaw motility of sperm cryopreserved using 15% Me2SO along with either extender-1 (86.0 ± 5.2%) or extender-2 (85.7 ± 7.1%) was similar (p > 0.05) to that of fresh sperm. Sperm cryopreserved using 12% GLY combined with extender-1 (83.67 ± 6.7%) or extender-2 (83.3 ± 4.7%) showed a similar motility to those cryopreserved with 15% Me2SO, but significantly lower from fresh sperm. The type of straw (0.25 or 0.50 mL) did not show any significant difference (p > 0.05) in post-thaw sperm motility. The highest values of PMI and MMP were observed for 7-day cryopreserved sperm using extender-1 in combination with 15% Me2SO (91.0 ± 2.9% and 90.0 ± 2.0%, respectively) or 12% GLY (90.0 ± 1.3% and 90.0 ± 4.6%, respectively). These results were similar to those of fresh sperm (95.3 ± 2.1% and 92.9 ± 2.5%, respectively). PMI and MMP of 1-year cryopreserved sperm using extender-1 in combination with 15% Me2SO (90.3 ± 2.5% and 89.3 ± 2.1%, respectively) or 12% GLY (90.0 ± 4.4% and 88.7 ± 2.2%, respectively) were significantly similar (p > 0.05) to those of fresh sperm. Sperm DNA integrity did not reveal any significant difference (p > 0.05) between fresh and cryopreserved (7-day and 1-year) sperm. Based on the assessed sperm vitality indicators, a cryopreservation protocol using extender-1 in combination with 15% Me2SO or 12% GLY has potential for hatchery as well as to create a germplasm bank.

Hypothermic storage in salt-free preservative solution alter motility duration in sterlet sperm

Together with concentration, motility is one of the most important characteristics of sturgeon sperm, determining its quality and suitability for insemination. After activation in water, the duration of progressive sperm motility is also important, and this time should not be less than that required for fertilization. Motility of spermatozoa depends on their physiological state, maturity, age and intracellular reserves of macroergic substances. During hypothermic storage, the percentage of spermatozoa that can be activated decreases progressively due to depletion of ATP supply or cell death. To improve the hypothermic storage of sterlet sperm, we have developed salt-free preservative solution ISGT-80 based on glucose and trehalose. During storage of sterlet sperm specimens from 20 males in ISGT-80 for 18 days, we observed, along with a progressive decrease in the percentage of motile spermatozoa, an alteration in the duration of their motility. On the 3rd to 6th day of storage, the time of half-loss of motility (τ50) increased significantly by approximately 1 min on average compared with fresh samples, then gradually decreased, however, not descending to the initial value. The reasons for this prolongation of motility are not clear, but we do not exclude the predominant death of spermatozoa with a short motility duration in the first days of storage and selection in favor of long-lived spermatozoa. Such gametic selection can lead to a shift in allele frequencies at heterozygous loci in the offspring. Thus, hypothermic storage of sperm could become an attractive subject for genetic research with the aim of developing new selection tools in sturgeon breeding.

The Role of Mitochondrial Impairment and Oxidative Stress in the Pathogenesis of Lithium-Induced Reproductive Toxicity in Male Mice.

Lithium (Li+) is prescribed against a wide range of neurological disorders. Besides its excellent therapeutic properties, there are several adverse effects associated with Li+. The impact of Li+ on renal function and diabetes insipidus is the most common adverse effect of this drug. On the other hand, infertility and decreased libido is another complication associated with Li+. It has been found that sperm indices of functionality, as well as libido, is significantly reduced in Li+-treated men. These adverse effects might lead to drug incompliance and the cessation of drug therapy. Hence, the main aims of the current study were to illustrate the mechanisms of adverse effects of Li+ on the testis tissue, spermatogenesis process, and hormonal changes in two experimental models. In the in vitro experiments, Leydig cells (LCs) were isolated from healthy mice, cultured, and exposed to increasing concentrations of Li+ (0, 10, 50, and 100 ppm). In the in vivo section of the current study, mice were treated with Li+ (0, 10, 50, and 100 ppm, in drinking water) for five consecutive weeks. Testis and sperm samples were collected and assessed. A significant sign of cytotoxicity (LDH release and MTT assay), along with disrupted testosterone biosynthesis, impaired mitochondrial indices (ATP level and mitochondrial depolarization), and increased biomarkers of oxidative stress were detected in LCs exposed to Li+. On the other hand, a significant increase in serum and testis Li+ levels were detected in drug-treated mice. Moreover, ROS formation, LPO, protein carbonylation, and increased oxidized glutathione (GSSG) were detected in both testis tissue and sperm specimens of Li+-treated mice. Several sperm anomalies were also detected in Li+-treated animals. On the other hand, sperm mitochondrial indices (mitochondrial dehydrogenases activity and ATP levels) were significantly decreased in drug-treated groups where mitochondrial depolarization was increased dose-dependently. Altogether, these data mention oxidative stress and mitochondrial impairment as pivotal mechanisms involved in Li+-induced reproductive toxicity. Therefore, based on our previous publications in this area, therapeutic options, including compounds with high antioxidant properties that target these points might find a clinical value in ameliorating Li+-induced adverse effects on the male reproductive system.

Comparative Study of Sperm DNA Isolation Method for Forensic Analysis

Forensic is a multi-discipline science that is used to obtain evidence of various criminal cases, such as rape. DNA analysis on sperm specimen is needed to identify the rapist. However, the success of this analysis depends on the DNA isolation method used. Several methods of DNA isolation from human sperm have been developed, but no method has been proven effective for the forensic analysis need. This study aimed to determine the effective sperm DNA isolation method for forensic analysis. In this study, the DNA of sperm specimens was isolated using three methods: Boiling Water, modified TRIzol, and Chelex-100. The DNA isolation result was visualized using agarose gel electrophoresis method. The concentration and purity of isolated DNA were measured using a Nanodrop by comparing the absorbance of DNA at λ 260 nm and protein at λ 280 nm. The effectiveness of the sperm DNA isolation method was determined based on the concentration and purity of DNA, the specimen volume, the implementation time, and the costs involved. The result showed that the successful methods for isolating sperm DNA were TRIzol and Chelex-100. The quantity of DNA isolated using the modified TRIzol method was 1,5 times higher than Chelex-100 but equired 120 times more specimen volume than Chelex-100. From 25 µl sperm specimens, the concentration of DNA isolated using the Chelex-100 method was 612.6 ng/µl with a purity of about 1.7. Therefore, it can be concluded that the Chelex-100 is the most effective method for isolating sperm DNA for forensic analysis.

The effect of sperm miR-34c on human embryonic development kinetics and clinical outcomes

AimsWe aimed to investigate the effect of sperm miR-34c on early human embryonic development kinetics and clinical outcomes of in vitro fertilization (IVF) patients. Materials and methodsAfter oocyte insemination, residual sperm specimens were collected from 58 patients undergoing IVF. miR-34c expression levels in sperm, oocytes, zygotes, and embryos/blastocysts were detected with qRT-PCR, and embryonic development kinetics were observed using time-lapse technology. To confirm the role of miR-34c in regulation of early embryonic development, miR-34c siRNA was injected into zygotes obtained from in vitro-matured oocytes. A ROC curve was used to determine the cutoff value. Comparisons of embryonic development kinetics and clinical outcomes were performed according to the cutoff value. Key findingsThe miR-34c expression level was highest in 3PN zygotes, but was not expressed in human oocytes. In the miR-34c siRNA group, embryonic development kinetic parameters t2, t3, t4, and t5 were significantly prolonged, but the cleavage rate and high-quality embryo rate were lower than in the control group. The levels of sperm miR-34c were negatively correlated with t5 and positively correlated with rates of blastocyst formation, high-quality blastocysts, and pregnancy. The miR-34c levels and the blastocyst formation rate were higher in the pregnancy group (p < 0.05). Logistic regression analysis showed that sperm miR-34c level was significantly correlated with pregnancy (OR: 5.056, 95% CI: 1.560–16.384; p = 0.007). SignificanceThe sperm miR-34c expression level is associated with embryonic development kinetics and clinical outcomes. Thus, miR-34c expression is beneficial to embryonic development and may be used as an indicator of IVF outcomes.

Preliminary observations on embryo morphokinetics and sperm DNA fragmentation assessed using the halo sperm kit

Sperm DNA fragmentation has been suggested to be a contributor to male infertility and lower pregnancy outcomes. The degree to which DNA fragmentation influences outcomes in IVF/ICSI cycles is however still controversial. Integration of time-lapse microscopy (TLM) in to IVF labs allows more in depth analysis of embryo developmental kinetics than was previously possible with conventional microscopy. This study examines sperm DNA fragmentation and its relationship to embryo morphokinetics, multinucleation and cleavage anomalies. Pilot study looking at DNA fragmentation (DF) and embryo morphokinetics. This study encompassed 77 couples undergoing IVF. Sperm specimens were prepared for ICSI using density gradient centrifugation. Mature oocytes were fertilized by ICSI and immature oocytes were incubated overnight with sperm. Normally fertilized oocytes were cultured in the EmbryoScope time lapse chamber. Timing of specific events from the point of insemination was determined using time lapse (TL) imaging and expressed in hours. The following kinetic markers were assessed: time to syngamy (t-syn), time to 2c (t2), 3c (t3), 4c (t4), 5c (t5), 8c (t8), morula (Mor-t), start of blastulation (tSB), blastocyst (BL-t), and expanded blastocyst (EBL-t). Durations of the second (cc2) and third (cc3) cell cycles as well as time to complete synchronous divisions s2 and s3 were calculated. Embryos were also observed for presence or multinucleation and cleavage anomalies. Sperm DNA fragmentation testing was performed on an aliquot of the unused ICSI sperm preparation using the Halosperm kit (HaloTech/Spectrum Technologies). The kit enabled microscopic detection of sperm chromatins dispersion. Sperm were embedded in agarose coated slides and exposed to acid denaturation and a lysis buffer. Following alcohol dehydration and staining with DiffQuik, slides were examined to detect presence of halos surrounding sperm. Sperm cells with large/medium sized halos characteristic of dispersed DNA loops were considered to have intact DNA. Sperm with small, degraded or absent halos were considered to have fragmented DNA. Cases were divided according to % DF in ICSI specimens. Morphokinetic parameters were compared. P value of <0.05 were considered to be statistically significant. Only parameters that were significantly different are shown in the table.Tabled 1Morphokinetics and DNA fragmentationDNA Fragmentation <15%DNA Fragmentation>=15%P ValueTotal embryos63354Multinucleation47%69%<0.01Irregular chaotic division25%46%<0.001Morula formation79%66%<0.05Start of blastulation74%60%<0.05Early blastocyst formation66%48%<0.01Expanded blastocyst formation52%32%<0.01t439.6+ 0.342.2 + 1.2<0.01t4-t32.7 + 0.24.8 + 0.7<0.01t859.5 + 0.564.7 + 2.2<0.01 Open table in a new tab The Halosperm kit provided an easy inexpensive means to assay DNA fragmentation. Further study is needed on the association between sperm DNA fragmentation and embryo quality markers made possible thru time lapse imaging systems.

Correlation of the hyaluronan binding assay to fertilization in vitro

Correlation of the hyaluronan binding assay to fertilization in vitro

THE ADDITION OF CAFFEINE IN EARLE’S BALANCED SALT SOLUTION MEDIA WITH WASHING UP METHOD INCREASE MEMBRANE INTEGRITY AND ACROSOMAL SPERM

Background: caffeine, a methylxanthine derivate, appears to inhibit phosphodiesterase, thereby inhibiting the break down of cAMP and increasing its concentration inside cell. This study aims to assess the effect of caffeine addition in Earles’s Balanced Salt Solution ( EBSS) on the increase in membrane integrity and acrosome reaction of spermatozoa using swim up method Methods: This study was carried out at the Clinic of Sexology and Andrology, Sanglah Public Hospital at Denpasar Bali-Indonesia. This study was an experimental study using the design of pre and post test paired control group design. The samples were sperm specimens of eighteen infertile couple male or volunteers who were infertile with age ranged between 20-40 years old. The samples were divided into two groups: treatment group (caffeine + EBSS) and control group (EBSS). The data were analysed statistically by normality test (Kolmogorov - Smirnov Goodness of Fit Test), Homogeneity test, and Paired Student’s t test. Results: The results showed that the caffeine addition in EBSS medium could increase significantly (p<0.05). The integrity of the sperm membrane obtained were from 81.30 % to 86.60 % and acrosomal reaction from 82.60% to 89.60% evaluated by hypo-osmotic swelling test (HOS). The conclusion of this study is that addition of caffeine in EBSS medium increases significantly membrane integrity and acrosomal reaction of the human sperm.

Association of DNA methylation and mitochondrial DNA copy number with human semen quality.

Whether there is a relationship between quality, DNA methylation, and mitochondrial DNA (mtDNA) copy number in human-derived sperm specimens is unknown. A cohort (n = 118) of male partners of couples who were undergoing fertility assessment because of an idiopathic inability to conceive were recruited. Sperm motility parameters were determined by computer-aided sperm analysis (CASA), while sperm quality was assessed using World Health Organization criteria, mtDNA copy number was measured by real-time PCR, and DNA methylation patterns were analyzed employing high-melting resolution PCR and bisulfite sequencing PCR. The mtDNA copy number negatively correlated with semen parameters, including sperm motility, concentration, morphology, progression, and motion characteristics (r for -0.19 to -0.54; P < 0.05 for all). As a surrogate marker for global DNA methylation, LINE-1 negatively correlated with sperm motility (r = -0.25; P = 0.009). Meanwhile, after adjustment for age, length of abstinence, smoking, and alcohol intake, there was a suggested association for increased LINE-1 methylation and mtDNA copy number tertiles versus sperm motility (odd ratios were 1.0, 2.6, and 4.7, and 1.0, 2.5, and 4.9, respectively). Altered mtDNA copy number and DNA methylation may serve as genetic and epigenetic markers to assess human sperm quality together with CASA parameters.