Human Sperm Viability Research Articles

Evaluation of the effects of hydroxytyrosol on human sperm parameters during cryopreservation

Human sperm cryopreservation is a routine procedure in assisted reproductive technology, but it has detrimental effects on different sperm parameters due to oxidative stress. Our objective was to assess the impacts of hydroxytyrosol (HT), as an antioxidant, on human sperm parameters following cryopreservation. In the first phase, 20 normal human semen samples were cryopreserved using the rapid freezing method with different concentrations of HT including 0, 50, 100, 150, and 200 μg/mL. In the second phase, 20 normal semen samples were collected and cryopreserved with 50 and 100 μg/mL HT. The beneficial effects of HT were determined by evaluation of motility (computer-assisted sperm analysis; CASA), viability (Eosin-nigrosine stain), DNA integrity (sperm chromatic dispersion test, SCD), reactive oxygen species (DCF and DHE staining by flowcytometry) lipid peroxidation (malondialdehyde, MDA test) and mitochondrial membrane potential (JC1 staining by flowcytometry) of sperm after cryopreservation. After thawing, sperm motility had an increasing trend in 50 and 100 μg/mL HT groups in comparison with other groups, althought the difference was not significant. However, sperm viability was significantly increased at 50 and 100 μg/mL HT. Our data also showed that sperm DNA fragmentation was significantly decreased after thawing at 100 μg/mL in comparison with 0 and 50 μg/mL HT. However, the level of intracellular reactive oxygen species, lipid peroxidation and mitochondrial membrane potential were not significantly different between groups. Our results showed that HT may have protective effects on the viability and DNA integrity of human sperm during the freezing-thawing process.

#83 : Human Sperm Cryo-Survival Rate

Background and Aims: Sperm cryopreservation plays an important role in the suite of services available to fertility patients. The ability to preserve the motility and viability of human spermatozoa is a key component in delivering successful assisted reproductive technologies (ART) outcomes. Cryopreservation methods expose sperm to stress, resulting in cellular damage which consequently leads to a reduction in their survival rate. The aim of this study was to evaluate the average cryo-survival rate of semen samples frozen in a clinical setting, to help improve and manage patient expectations prior to accessing this service. Method: Pre and post sperm freeze data from patients who presented at the Genea Sydney clinic for sperm cryopreservation between 2017 and 2022 was analysed. The post-thaw survival rate for 1467 specimens was assessed by averaging the total post-thaw motility with total pre-freeze motility. Only ejaculated samples, frozen on-site, who had a raw motility calculated as well as a post-thaw motility analysis able to be performed were included in the study. Results: The range of post-thaw survival was 57% to 67% per annum over the six years of examined data, with an overall cryo-survival rate of 62%. Conclusions: The data shows a consistent recovery rate of post-thaw sperm motility over the time period analysed. Literature indicates average sperm cryo-survival rates range from 30% to 50%, and previous advice to patients has been to expect around 50% sperm survival. With this new data, Genea can revise its guidance to match the current average figure of around 60%. In addition, future internal freeze optimisation studies can use this figure as a benchmark to improve upon techniques and processes.

Effect of surface profile of superhydrophobic carbon soot coatings on the viability of human spermatozoa upon slow freezing

Effect of surface profile of superhydrophobic carbon soot coatings on the viability of human spermatozoa upon slow freezing

Lysophosphatidic Acid Signalling Regulates Human Sperm Viability via the Phosphoinositide 3-Kinase/AKT Pathway.

Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A2 (iPLA2) releases lysophospholipids such as LPA or arachidonic acid (AA) and that inhibiting PRDX6 iPLA2 activity impairs sperm cell viability. The exogenous addition of LPA bypassed the inhibition of PRDX6 iPLA2 activity and maintained the active phosphoinositide 3-kinase (PI3K)/AKT pathway. Here, we aimed to study PI3K/AKT pathway regulation via LPA signalling and protein kinases in maintaining sperm viability. The localization of LPARs in human spermatozoa was determined using immunocytochemistry, and P-PI3K and P-AKT substrate phosphorylations via immunoblotting. Sperm viability was determined using the hypo-osmotic swelling test. LPAR1, 3, 5 and 6 were located on the sperm plasma membrane. The inhibition of LPAR1-3 with Ki16425 promoted the impairment of sperm viability and decreased the phosphorylation of PI3K AKT substrates. Inhibitors of PKC, receptor-type PTK and PLC impaired sperm viability and the PI3K/AKT pathway. Adding 1-oleoyl-2-acetyl-snglycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), prevented the loss of sperm viability and maintained the phosphorylation of PI3K. In conclusion, human sperm viability is supported by LPAR signalling and regulated by PLC, PKC and RT-PTK by maintaining phosphorylation levels of PI3K and AKT substrates.

Effects of CatSper Stimulation and Inhibition by Progesterone and NNC on Human Sperm

Calcium ion enters the sperm through a specific calcium channel, CatSper. This voltage-sensitive channel is stimulated by intracellular alkalization and progesterone. This study is aimed at investigating the effects of CatSper inhibition or stimulation on sperm motility, viability, and sperm function regulators such as mitochondrial membrane potential (MMP), ATP, and reactive oxygen species (ROS) production. This study was performed on 30 semen samples of fertile volunteers, referred to Shiraz Fertility Center. The semen samples were diluted to 20 × 10 6 sperm/mL. The samples were divided randomly into control, solvent, progesterone (10 μM), NNC (2 μM), and NNC+progesterone groups. Sperm kinematics, viability, MMP, ATP content, and the amount of ROS production were assessed using VT-SPERM3.1, eosin staining, JC1 flow cytometry, bioluminescence, and chemiluminescence methods, respectively. Sperm viability and total and progressive motility were significantly decreased in the NNC and NNC+progesterone groups. The amplitude of lateral head displacement (ALH) and curvilinear velocity (VCL) was reduced in the NNC-containing groups. These parameters did not change in the progesterone group. ROS production by viable spermatozoa in the NNC and NNC+progesterone groups was significantly higher than the controls. MMP and ATP content did not show any significant difference with controls in none of the experimental groups. NNC inhibits the CatSper and reduces human sperm motility and viability. These harmful NNC effects were not due to their impact on MMP or ATP production but are likely because of intracellular calcium reduction and higher ROS production. Progesterone at 10 μM concentration had no significant effect and may not be a considerable stimulator for CatSper.

A case report on the prolonged viability of postmortem human testicular sperm

To report a case of postmortem sperm retrieval with prolonged viability and motility. Case report. Hospital and Medical Examiner Department. A 44-year-old African American male patient with a history of recreational marijuana use and occasional alcohol consumption who died from a cardiac arrest because of drug overdose. Multiple testicular biopsies and sperm analyses. Sperm viability and motility of testicular biopsies at serial time intervals. Sperm obtained from the testis in the morgue remained viable and motile even at 106 hours (>4 days) postmortem. Our study found that sperm obtained from the testis remained viable and motile even after being thawed after cryopreservation, even when obtained up to 100 hours postmortem. This may have implications on the timeframe that postmortem sperm retrieval can be performed successfully several days after death.

Antifertility activity of Oroxylum indicum (L.) Kurz:In vitro and in vivo study on human sperm and male wistar rats

Oroxylum indicum (L.) Kurz, commonly called as Broken bones tree or Indian trumpet flower, belonging to Fam. Bignoniaceae, is traditionally used as a contraceptive by ethnic people of Tripura, North-East India. Here, we investigated the scientific basis for use of O. indicum as male antifertility agent by folklore healers. In vitro spermicidal activity of aqueous (AEOI) and methanolic (MEOI) extracts of O. indicum stem bark were studied on human sperm. The in vivo activity was experimented on male albino rats. The treated animals were allowed to mate and the pups delivered by female rat partners were counted. Phytochemical estimation of test samples was done using HPLC. The AEOI and MEOI treatments significantly decreased human sperm motility and viability. Test extracts have increased the hypo-osmotic swelling of sperm. Both the extracts were significantly declined the weight of reproductive organ. The MEOI treated rats have shown significant decrease in sperm motility and sperm counts. AEOI and MEOI treatment significantly reduced level of testosterone, but sharply raised dihydrotestosterone and prostaglandin in rats. Results testified the traditional claim for use of O. indicum as a male contraceptive agent, where MEOI have shown reversible action on male reproductive system leading to contraception without harming the libido.

In vitro investigation of endocrine disrupting effects of pesticides on Ca2+-signaling in human sperm cells through actions on the sperm-specific and steroid-activated CatSper Ca2+-channel.

Ca2+-signaling controls sperm cell functions necessary for successful fertilization. Multiple endocrine disrupting chemicals have been found to interfere with normal Ca2+-signaling in human sperm cells through an activation of the sperm-specific CatSper Ca2+-channel, which is vital for normal male fertility. We investigated 53 pesticides for their ability to interfere with CatSper mediated Ca2+-signaling and function in human sperm cells. Effects of the pesticides on Ca2+-signaling in human sperm cells were evaluated using a Ca2+-fluorometric assay. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function and viability were assessed using an image cytometry-based acrosome reaction assay and the modified Kremer's sperm-mucus penetration assay. 28 of 53 pesticides were found to induce Ca2+-signals in human sperm cells at 10 µM. The majority of these 28 active pesticides induced Ca2+-signals through CatSper and interfered with subsequent Ca2+-signals induced by the two endogenous CatSper ligands progesterone and prostaglandin E1. Multiple active pesticides were found to affect Ca2+-mediated sperm functions and viability at 10 µM. Low nM dose mixtures of the active pesticides alone or in combination with other environmental chemicals were found to significantly induce Ca2+-signals and inhibit Ca2+-signals induced subsequently by progesterone and prostaglandin E1. Our results show that pesticides, both alone and in low nM dose mixtures, interfere with normal Ca2+-signaling in human sperm cells in vitro in low nM concentrations. Biomonitoring of the active pesticides in relevant matrices such as blood and reproductive fluids is very limited and the effects of real time human pesticide exposure on human sperm cells and fertility thus remains largely unknown. To which extent human pesticide exposure affects the chances of a successful fertilization in humans in vivo needs further research.

3D+t feature-based descriptor for unsupervised flagellar human sperm beat classification.

Human spermatozoa must swim through the female reproductive tract, where they undergo a series of biochemical and biophysical reactions called capacitation, a necessary step to fertilize the egg. Capacitation promotes changes in the motility pattern. Historically, a two-dimensional analysis has been used to classify sperm motility and clinical fertilization studies. Nevertheless, in a natural environment sperm motility is three-dimensional (3D). Imaging flagella of freely swimming sperm is a difficult task due to their high beating frequency of up to 25 Hz. Very recent studies have described several sperm flagellum 3D beating features (curvature, torsion, asymmetries, etc.). However, up to date, the 3D motility pattern of hyperactivated spermatozoa has not been characterized. The main difficulty in classifying these patterns in 3D is the lack of a ground truth reference since differences in flagellar beat patterns are very difficult to assess visually. Moreover, only around 10-20% of induced to capacitate spermatozoa are truly capacitated, i.e., hyperactivated. We used an image acquisition system that can acquire, segment, and track spermatozoa flagella in 3D+t. In this work, we propose an original three-dimensional feature vector formed by ellipses describing the envelope of the 3D+t spatio-temporal flagellar sperm motility patterns. These features allowed compressing an unlabeled 3D+t dataset to separate hyperactivated cells from others (capacitated from non-capacitated cells) using unsupervised hierarchical clustering. Preliminary results show three main clusters of flagellar motility patterns. The first principal component of these 3D flagella measurements correlated with 2D OpenCASA head determinations as a first approach to validate the unsupervised classification, showing a reasonable correlation coefficient near to 0.7. Clinical relevance- The novelty of this work is defining a 3D+t feature-based descriptor consisting of a set of ellipses enveloping the flagellar motion of human sperm for its unsu-pervised classification. This is a new promising tool to determine the viability of human sperm to fertilize the egg.

EFFECT OF CONCENTRATION OF PHOSPHOLIPIDS
 AND HUMIC COMPOUNDS IN DILUENT COMPOSITION ON BIOLOGICAL PARAMETERS OF BULL AND HUMAN SPERM

In the course of the experimental work, it was found that the concentration of phospholipids and an aqueous solution of activated potassium humic substances affects the viability of bovine and human sperm.
 Phospholipids included in the composition of the diluent up to 10.0 and 20.0 mg, after 5 hours of incubation at a temperature of (37±1) °C, provide the survival rate of bull and human sperm at 34.4±0.86–35.5±0.86 % and 36.2±0.86–32.4±0.86 % germ cells, respectively.
 Diluents containing 0.0025 % aqueous solution of activated potassium humate and 10.0 and 20.0 mg of phospholipids provide the highest survival rate relative to the baseline in bovine and human spermatozoa, respectively, up to 72.9–78.8% and 73. 4–76.2 %.

Interferon γ, IL-17, and IL-1β impair sperm motility and viability and induce sperm apoptosis.

Urogenital inflammation is a known cause of male infertility. Increased levels of inflammatory cytokines, leukocyte counts and oxidative stress are highly detrimental for sperm quality thus compromising male fertility. Although cytokines affect sperm by recruiting and activating leukocytes consequently inducing tissue inflammation and oxidative stress, scarce to absent data have been reported about the putative direct effects of inflammatory cytokines on spermatozoa. Herein, we analyzed whether IFNγ, IL-17A, IL-1β, and IL-8 can alter human sperm motility and viability per se. Fractions of viable and motile spermatozoa from normospermic healthy donors were in vitro incubated with recombinant human IFNγ, IL-17A, IL-1β or IL-8 and sperm ROS production, motility, viability and apoptosis were analyzed. Sperm exposed to different concentrations of IFNγ, IL-17A and IL-1β, or a combination of them, for either 1 or 3h showed significantly increased levels of mitochondrial ROS production and reduced motility and viability with respect to sperm incubated with vehicle. Moreover, the exposure to IFNγ, IL-17A and IL-1β resulted in significantly higher levels of early and/or late apoptotic and/or necrotic spermatozoa. Interestingly, no significant differences in sperm motility, viability and apoptosis were observed in sperm incubated with the concentrations of IL-8 analyzed, for either 1 or 3h, with respect to sperm incubated with vehicle. In conclusion, our results indicate that IFNγ, IL-17A and IL-1β per se impair sperm motility and decreases viability by triggering increased mitochondrial ROS production and inducing sperm apoptosis. Our results suggest that screening inflammatory cytokines in semen would be an additional helpful tool for the diagnostic workup of male infertility.

Impact of metabolic syndrome on the viability of human spermatozoa: a cross-sectional descriptive study in men from infertile couples

BackgroundA direct association between metabolic syndrome (MetS) and sperm production/function has been proposed. In this cross-sectional study, we aimed to determine the impact of MetS on sperm survival. Men from infertile couples treated at Hue University Hospital, Vietnam, were enrolled in this study, which spanned the October 2018 to October 2020 period. The general characteristics of the patients, including body mass index (BMI), waist-to-hip ratio (WHR), the levels of different biochemicals, and semen parameters were determined, and sperm survival tests (SSTs) were performed. The modified National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III for the Asian population was used for MetS diagnosis.ResultsMen with an abnormal waist circumference (≥ 90 cm) showed a higher rate of abnormal SST results (30.1% vs. 16.7%, p = 0.012). The frequency of abnormal SST results in patients with MetS (72.3%) was significantly higher than that in individuals without MetS (53.4%) (p = 0.02). Furthermore, the percentage of abnormal SST results in patients with MetS and with BMI ≥ 23 was significantly higher than those in individuals without MetS (77.1% vs. 55.2%, p = 0.03). Weak negative correlations were also observed between the patients’ age and the SST results.ConclusionSperm viability was lower in men with MetS. We also observed that age and BMI were independent factors associated with abnormal SST.

A cryoprotectant supplemented with pentoxifylline can improve the effect of freezing on the motility of human testicular sperm.

This study examined the effect of a cryoprotectant with and without pentoxifylline supplementation on the motility and viability of human testicular sperm, both before and after freezing. Testicular samples were obtained from 68 patients with azoospermia who came to the Andrology Service of West China Second University Hospital, Sichuan University, for testicular biopsies from December 2019 to April 2020. All patients were assigned randomly to two groups: experimental, whose testicular sperm were added to the cryoprotectant with pentoxifylline, and the control, whose testicular sperm were added to the cryoprotectant without pentoxifylline. Both groups used the same freezing and thawing methods. Testicular sperm motility in the experimental group was significantly higher than that of the control group, both before and after cryopreservation. The recovery rate of sperm motility in the experimental group was significantly higher than that of the control group. The percentage of samples with motile testicular sperm in the experimental group was significantly higher than that of the control group after thawing. Sperm viability was unchanged between the experimental and control groups, both before and after freezing. Overall, a pentoxifylline-supplemented cryoprotectant can significantly improve the motility of testicular sperm before and after cryopreservation.

Physiologically detectable bisphenol A impairs human sperm functions by reducing protein-tyrosine phosphorylation

BackgroundBisphenol A (BPA), a widely used plastic monomer and plasticizer, is detectable in blood, urine and semen of a healthy people, with concentrations ranging from 0.1 nM to 10 nM. It has been shown that in vitro exposure of BPA as low as 0.001 nM could significantly inhibited mouse sperm motility and acrosome reaction. However, it is still unclear whether BPA at those physiologically detectable concentration affects human sperm. MethodsThe effects of different concentrations of BPA (0, 10−3, 10−2, 10−1, 10, 103 nM) on sperm functions were examined, including human sperm viability, kinematic parameters, hyperactivation and capacitation. ResultsBPA caused a remarkable decline in human sperm viability, motility and progressive motility, hyperactivation, capacitation and progesterone-induced acrosome reaction. Mechanism studies showed that BPA could suppress the protein tyrosine phosphorylation level of human sperm, but had no effect on sperm calcium signaling. ConclusionsPhysiologically detectable concentrations of BPA may impair human sperm functions via suppressing protein tyrosine phosphorylation of human sperm, implying that environmental pollution of BPA might be a factor contributing to male infertility.

Quantitative evaluation of human sperm viability using MTT assay: A laboratory study.

Background3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay which evaluates cellular mitochondrial activity is widely used for the assessment of cell proliferation and viability.Objective This study was performed to assess human sperm viability using MTT assay.Materials and MethodsIn this laboratory study, human-ejaculated semen samples (n = 56 from different donors) were used. The sperm viability was determined using quantitative MTT assay and the sperm motility was assessed according to World Health Organization guidelines. Sperm viability and the correlation between sperm viability and motility were analyzed.ResultsData revealed a marked positive correlation between MTT reduction rate and the percentage of viable spermatozoa. The Pearson's correlation coefficients also showed a significant correlation between sperm viability and motility.Conclusion MTT assay which is based on mitochondrial functionality is a reliable method for evaluating human sperm viability and could be used as a diagnostic test for predicting sperm fertilization ability in clinical settings.

The effect of vitamin B12 supplement on post‐thaw motility, viability and DNA damage of human sperm

Sperm cryopreservation may lead to adverse effects on sperm structure and function. Cyanocobalamin (vitamin B12) has antioxidant potential and can protect DNA from free radical-induced damages. Recent studies have shown that vitamin B12 preserves glutathione that leads to modulate oxidative stress responses. Also, vitamin B12 might act directly as a scavenger of ROS. The aim of this study was to investigate the effects of vitamin B12 supplementation on human sperm parameters during the cryopreservation process. Thirty semen samples were obtained from normozoospermic men. Using cryopreservation medium supplemented with different concentrations of vitamin B12 (0, 0.5, 1, 2, 2.5mg/ml), the semen samples were cryopreserved. After thawing, all samples were evaluated for motility and viability. Based on results, 2mg/ml was considered as the optimal concentration of vitamin B12 for evaluating sperm DNA fragmentation. The results showed that 1 and 2mg/ml vitamin B12 significantly increased post-thawing motility and viability compared with the 0mg/ml vitamin B12 (p<.05). Also, by supplementing with 2mg/ml vitamin B12, DNA fragmentation decreased when compared to the control. The present study showed that cryopreservation medium supplemented with vitamin B12 at 2mg/ml couldimprove sperm quality after freeze-thaw process.

Криоконсервирование сперматозоидов человека в ПВП и сахарозе

В данном исследовании изучали уровень жизнеспособности спермиев человека после криоконсервирования с непроникающими криопротекторами, которые не требуют удаления из суспензии клеток перед применением сперматозоидов во вспомогательных репродуктивных технологиях (ВРТ). В результате проведенных исследований установили, что использование сахарозы и поливинилпирролидона (ПВП) в различных комбинациях в качестве криозащитных агентов при замораживании сперматозоидов позволяет получить их выживаемость на уровне 60–80 %. Таким образом, криоконсервирование спермиев человека с использованием сахарозы и ПВП является перспективным для ВРТ, поскольку спермии можно использовать сразу после отогрева для оплодотворения ооцитов in vitro

Effect of recombinant β-defensin 1 protein on human sperm motility and viability.

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.

Peroxiredoxin 6 regulates the phosphoinositide 3-kinase/AKT pathway to maintain human sperm viability.

Peroxiredoxins (PRDXs) are antioxidant enzymes proven to control the levels of reactive oxygen species (ROS) and to avoid oxidative damage in the spermatozoon. Previously, we have shown that low amounts of PRDXs are associated with male infertility and that PRDX6 is the primary antioxidant defense in human spermatozoa, maintaining survival and DNA integrity (Gong et al., 2012, Fernandez and O'Flaherty, 2018). Oxidative stress can trigger different pathway cascades in the spermatozoa, including truncated apoptosis. It has been reported that the phosphorylation status of phosphoinositide 3-kinase (PI3K) and its target AKT (protein kinase B) prevent the spermatozoon from entering the truncated apoptotic cascade. Here, we aim to study the regulation of the PI3K/AKT pathway by PRDX6 and assess its role in maintaining sperm viability. Human semen samples were obtained over 1year from 20 healthy non-smoking volunteers aged 22-30years. Sperm viability, lipid peroxidation and apoptosis-like changes were determined by flow cytometry while phosphorylation of PI3K and AKT substrates were assessed by immunoblotting using anti-phospho-PI3K and anti-phospho-AKT substrates antibodies. We found that the addition of arachidonic acid and lysophosphatidic acid, products of PRDX6 calcium-independent phospholipase A2 (Ca2+-iPLA2), prevented loss of sperm viability and maintained the phosphorylation of PI3K. Antioxidant compounds such as D-penicillamine partially prevented the oxidative damage on spermatozoa that led to a reduction of their viability. Thus, other pathways can also participate in sperm survival and be regulated by PRDXs. In conclusion, PRDX6 contributes to the regulation of ROS production and the PI3K/AKT pathway for the maintenance of sperm survival.

Human Sperm Motility, Viability, and Morphology Decreased after Cryopreservation

The aim of this study was to analyze human sperm motility, viability, and morphology before and after cryopreservation. This true laboratory experimental study had pre and post randomized one group design. The study was conducted at the Embryology, Andrology, and Genetics Laboratory, Department of Medical Biology, Faculty of Medicine, Universitas Airlangga from August to November 2017. The eighteen samples of fresh semen were collected from male volunteers who agreed and signed the informed consent of the study. Samples were analyzed their motility, viability, and morphology before and after cryopreservation. Results of this study indicated differentiation between motility before and after cryopreservation. Cryopreservation process decreased progressive motility (42.22 + 9.46%; 17.83 + 6.24%; p&lt; 0.0001) and increased the number of immotile spermatozoa (35.44 + 10.15%; 60.11 + 12.53%; p&lt; 0.0001). Cryopreservation also decreased human sperm viability (73.78 + 8.91%; 40.83 + 12.89%; p&lt; 0.0001) and morphology (10.94 + 4.96%; 7.39 + 3.90%; p&lt; 0.0001). Cryopreservation of human spermatozoa caused the decreased of motility, viability, and morphology.