Effects of Pentoxifylline Timing and Storage Temperature on Human Sperm Motility, Morphology, and Viability.

Optimizing incubation conditions for the preservation of sperm motility in processed semen samples

Objective The quality of sperm cells is important role in the success rates of assisted reproduction technology (ART) treatments. The quality of the sperm cells shows variations depending on the temperature of short-term semen storage as well as the methods of semen preparation. Thus, this study aimed to investigate the sperm viability, motility and DNA fragmentation following different sperm preparation methods and short-term storage conditions, respectively. Materials and Methods A total of 25 semen samples were evaluated. In the first part of this study, different incubation temperatures were investigated in two groups, in such the first group involved the semen samples and the second group involved the sperm cells separated by density gradient centrifugation method, respectively. The samples in each group were incubated at 4°C, room temperature (21°C) and 37°C for 24 hours, respectively. The sperm cell qualities were evaluated by mobility analysis, DNA fragmentation by acridine orange staining and sperm cell viability by propidium iodide staining. Results and Discussion The analysis outcome demonstrated that the mobility, DNA fragmentation and viability of the sperm cells were statistically different when incubated at RT (21°C) in both groups. Furthermore, samples prepared by the density gradient centrifugation method were shown to have better quality. The optimum short-term storage temperature was detected to be the room temperature. Conclusion The conclusion of this investigation is crucial to assess storage conditions in ART clinics. This study provides essential data for short-term sperm storage and preparation methods to improve the success rates of ART clinics. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1137-1141

Oxidative stress (OS) contributes to poor sperm parameters and increased sperm DNA fragmentation (sDF), yet effective therapeutic strategies remain limited. This study aimed to evaluate the in vitro efficacy of pentoxifylline (PTX) in improving sperm motility and reducing OS and sDF in men with isolated asthenozoospermia. Thirty semen samples from patients with asthenozoospermia were processed using density gradient centrifugation. Each sample was divided into two aliquots: one treated with PTX at a dose of 3.6 mM and the other without PTX treatment. The sperm viability and motility were assessed at 30 min, 1 h, 2 h, and 24 h post-treatment. OS was evaluated using nitro blue tetrazolium staining and a chemiluminescence assay. sDF was assessed using the alkaline Comet assay. The sperm samples treated with PTX, compared to the controls, exhibited a significant increase in total sperm motility (71.8 ± 23.03% versus 47.47 ± 4.88%, respectively; p < 0.0001). However, no significant difference was observed in the sperm viability. PTX treatment significantly reduced ROS production and sDF levels compared to controls (p < 0.01). These findings suggest that in vitro PTX supplementation enhances sperm motility and reduces the nuclear sperm injury associated with seminal ROS production. Therefore, PTX supplementation in vitro may be beneficial in assisted reproductive technology procedures involving men with asthenozoospermia.

In vitro incubation and centrifugation is known to decrease human sperm quality. In the human body, besides its antioxidant effects, L-carnitine (LC) facilitates the transport of activated fatty acids from the cytosol to the mitochondrial matrix. In this study, we investigated the effect of LC on human sperm motility, viability and DNA oxidation after incubation and centrifugation, following the sperm preparation protocols of assisted reproduction. Normozoospermic semen samples (n = 55) were analysed according to the World Health Organization (WHO) guidelines. LC concentrations that are not toxic to spermatozoa as determined by sperm motility and viability were standardised after 2 and 4 h of incubation at 37 °C. Semen samples to which the optimal LC concentrations were added were also centrifuged for 20 min at 300 g and analysed for sperm motility, viability and DNA oxidation. Sperm motility was improved at 0.5 mg ml(-1) LC after incubation and centrifugation with 5 × 10(6) sperm ml(-1). Higher concentration of LC (50 mg ml(-1)) significantly decreased sperm motility and viability. LC did not alter the baseline of sperm DNA oxidation during both incubation and centrifugation. In conclusion, LC may enhance sperm motility following incubation and centrifugation, while it might not affect sperm viability and DNA oxidation.

Cryopreservation of human spermatozoa is widely used in many assisted reproduction units to preserve male fertility [1]. Vitrification is based on the ultrarapid freezing and is routinely assayed for cryopreservation in assisted reproductive technology. Mohamed [2] showed that cryopreservation significantly affects progressive motility, viability and mitochondrial membrane potential of spermatozoa. Pentoxifylline (PX) is a phosphodiesterase considered to be a sperm movement enhancer, hyperactivation agent, inhibitor of reactive oxygen species and acrosome reaction-improving agent. The aim of our study was to evaluate the effect of in-vitro application of PX on sperm parameters and ultrastructure after vitrification. A total of 30 asthenozoospermic semen samples were selected and divided into two groups after vitrification: control (without PX) and experimental (with PX). A significant decrease in sperm motility, morphology and viability was observed post vitrification, but sperm motility was increased significantly following application of PX. On the other hand, PX did not exert any significant effect on the ultrastructure of the acrosome, plasma membrane and tail of vitrified spermatozoa.

The current study was carried out to investigate the protective effects of royal jelly supplementation on sperm motility, viability and pH value during the liquid storage of boar semen at 16 °C and 4 °C, at various periods of time (0, 24, 48, 72 and 96 h). Semen samples were collected from 11 boars, diluted with a long-term extender and supplemented with different concentration of royal jelly (0%, 0.5%, 1% and 2%) at a final concentration of 50 × 106sperm/ml. In the laboratory, the semen was assessed for sperm morphology, viability (eosin-nigrosin staining), subjective motility and objective sperm motility by sperm class analyzer. In total, 396 tests for sperm viability and motility were performed. The longer storage time and the lower incubation temperature showed lower sperm motility and viability results. The results showed that royal jelly supplementation at 1% concentrations protected the functionality of the sperm plasma membrane during the liquid storage time of 96 h at 16 °C. Sperm subjective and objective motility results in samples stored at 4 °C decreased with higher royal jelly concentrations and longer storage time, and differ significantly from the results in samples stored at 16 °C (P< 0.05). Our data showed that royal jelly supplementation at lower concentrations can improve boar semen motility and viability parameters during liquid storage at 16 °C for 96 h.

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

Assessment of sperm motility, viability and apoptosis in human spermatozoa after hydrogen peroxide exposure

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of "sperm maturation" during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is "β-defensin 1" which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500ng/ml) for 1, 2 and 3hr at 37°C under 5% CO2 atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p<.05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.

Clinical guidelines for sperm cryopreservation in cancer patients

Effects of psychotropic drugs on human sperm motility

Study question Are plasticizers bisphenol S (BPS) and bisphenol F (BPF) safer alternatives to bisphenol A (BPA) for human sperm function? Summary answer Unlike BPA, the analogues BPS and BPF do not significantly affect human sperm mitochondrial functions, motility and viability What is known already The widespread distribution of BPA, along with its reputation to be an endocrine disruptor has generated concerns about possible adverse effects for human health, thus prompting the European Food Safety Authority and the Food and Drug Administration to ban the use of this chemical in many plastic products. Following such restrictions, several substitutes have been developed, with BPS and BPF representing the main replacements to BPA. While it has been demonstrated that BPA promotes oxidative damages in spermatozoa from different species, including human, the possible effects exerted by BPS and BPF on human sperm, have not yet been investigated. Study design, size, duration We explored the effect of 4 h in vitro exposure to scalar concentrations of BPS and BPF (from 10 to 400 μM), and to 400 μM BPA on sperm motility, viability, mitochondrial membrane potential (Δm) and mitochondrial generation of reactive oxygen species (ROS). In a set of experiments, the effect of a combination of both BPF (400 μM) and BPS (400 μM) on ΔΨm and mitochondrial ROS generation was also assessed. Participants/materials, setting, methods Sperm Δm was analyzed by flow cytometry with the fluorescent lipophilic cationic dye JC–1. Flow cytometric assessment of mitochondrial generation of ROS was carried out using the lipid soluble cation MitoSOX red (MSR). Sperm motility and viability were evaluated by Computer-Aided Semen Analysis (CASA) and eosin assay, respectively. Main results and the role of chance The exposure to scalar concentration of BPS did not significantly affect sperm motility and viability with respect to untreated controls. A lower, albeit not significant, sperm motility was registered in samples exposed to the highest concentrations of BPF (300 μM and 400 μM). As expected, 400 μM BPA produced a complete sperm immobilization along with a dramatically loss in sperm viability. No significant differences were observed in sperm Δm and ROS generation after exposure to scalar concentration of BPS compared to untreated controls and the trend towards lower Δm and higher mitochondrial ROS generation at the highest concentrations of BPF did not reach statistical significance. On the contrary, after 4 h exposure to 400 μM BPA a significant lower Δm and higher mitochondrial ROS generation were observed. Finally, the exposure to a combination of BPF and BPS at high concentrations (400 μM) did not significantly affect sperm Δm, or mitochondrial ROS generation, when compared to 400 μM BPA, used as positive control. Limitations, reasons for caution: The present study only evaluated BPS and BPF effects, but in daily-life people are exposed to several plasticizers containing different bisphenols at different concentrations. Therefore, adverse effects of synergic exposure to BPA analogues other than BPS and BPF, alone or in combination with BPA, cannot be ruled out. Wider implications of the findings: The analogues BPS and BPF, alone or in combination, appeared to be safer alternatives to BPA on sperm biology as they exert a neutral effect on sperm motility, viability, and mitochondrial functions even at high concentrations. These results could be useful to identify more secure plasticizer components. Trial registration number Not applicable

It is generally acknowledged that lower pH throughout liquid storage can decrease sperm motility and energy production, which may be advantageous for maintaining sperm viability during cryopreservation. Hence, the purpose of this study was to see how pH affected sperm viability, motility, membrane integrity, and oxidative changes after 5 days of storage in chemically specified extenders at refrigerator temperatures to formulate a ready-to-use goat semen diluent. Ejaculates were extended with tris-citric acid-fructose diluent with 20% or without egg yolk and stored at 5°C for 5 days. Sperm motility, live count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, pH, and malondialdehyde all substantially differed significantly (P &lt; 0.05) during different days of liquid storage. The pH of diluted semen decreased considerably (P &lt; 0.05) from its original pH of 6.46 on 0 day to pH 6.06 on the 5th day of liquid storage at refrigeration temperature. Diluted semen at refrigeration temperature having 20% egg yolk had significantly higher (P &lt; 0.05) seminal characteristics compared to liquid semen without egg yolk. The liquid storage of semen quality became worse day by day due to a drop in pH and egg yolks acted as a cryoprotectant against the harmful effects of chilling. Hence, 20% egg yolk may be used to enhance the keeping quality of diluted buck semen at refrigeration temperature for up to 5 days. Based on this result, we formulated a ready to use goat semen diluent for liquid semen storage at room temperature and refrigeration temperature.

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.

Male infertility as a window to health