Human Sperm Viability Research Articles

Clinician-induced (iatrogenic) damage incurred during human infertility treatment: Detrimental effects of sperm selection methods and cryopreservation upon the viability, DNA integrity, and function of human sperm

Human infertility is becoming increasingly prevalent and is now estimated to affect 10% of couples worldwide. Although phenomenal development in assisted reproductive technology (ART) has yielded a powerful and sophisticated discipline with which to combat many types of human infertility, success rates vary markedly and rarely exceed 40%. Mounting evidence suggests that laboratory techniques used routinely in ART may inadvertently impart detrimental effects upon gamete and embryo viability and competence. In this mini-review, we discuss how routine cryopreservation methods, commonly used in ART, may exert iatrogenic (clinician-induced) damage upon sperm structure, DNA integrity, and function. It is recommended that future research programmes aim to refine or replace current cryobiology protocols in order that the efficacy of ART can be optimised accordingly.

Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology

The accumulated data suggest that inflammation can increase the level of reactive oxygen species (ROS), which contribute to impaired sperm function and male infertility. Therefore, we propose that inflammation-mediated production of ROS in male and female reproductive tracts hinder sperm fertilisation. To test this hypothesis, phorbol myristate acetate (PMA) with polymorphonuclear leucocytes (PMNs) was applied to generate endogenous ROS. We evaluated the time-dependent effects of ROS on human sperm motility, viability and mitochondrial membrane potential (MMP). The results showed that after treatment with PMA and PMNs, the motility of human spermatozoa significantly decreased to 50% on Day 1 and 15% on Day 4 compared with that of the, respectively, negative controls (P = 0.012). The viability of human spermatozoa decreased on Day 4 of PMA + PMNs treatment (P = 0.028). The MMP of human spermatozoa significantly decreased from Day 2 to Day 4 in the PMA + PMN group compared with that of the controls (P = 0.019). Taken together, the 4-day cultivation approach provided an accurate evaluation of sperm quality, especially sperm motility and MMP. Our findings indicated that endogenous inflammation increased ROS levels, which might induce sperm oxidative damage. Additionally, sperm motility might be one of the earliest and most sensitive indicators of this damage.

N,N'–Dithiobisphthalimide, a disulfide aromatic compound, is a potent spermicide agent in humans

Several studies have shown that users of vaginal preparations containing nonoxynol-9 (N-9) are at a high risk for sexually transmitted diseases, including HIV. Therefore, there is a great interest in identifying compounds that can specifically inhibit sperm without damaging the vaginal lining, possess a powerful spermicide activity, and can be used in contraceptive vaginal preparations to replace N-9. In this work, we studied the spermostatic and/or spermicidal activity of five non-detergent, disulfide compounds on human sperm, HeLa cells, and Lactobacillus acidophilus. The motility and viability of human sperm in semen and culture medium was evaluated after treatment with different concentrations of the disulfide compounds (2.5 – 100 µM). In addition, we evaluated the cytotoxic effect on HeLa cells and L. acidophilus. We identified compound 101, N,N'-dithiobisphthalimide (No. CAS 7764-30-9), as the most effective molecule. It has a half maximal effective concentration (EC50) of 8 µM and a minimum effective concentration (defined as the concentration that immobilizes 100 percent of the sperm in 20 sec) of 24 µM. At these concentrations, compound 101 does not affect the viability of the sperm, HeLa cells, or L. acidophilus. Our results indicate that dithiobisphthalimide has a potent spermostatic, irreversible effect with no toxic effects on HeLa cells and L. acidophilus.

Human sperm DNA oxidation, motility and viability in the presence of l-carnitine during in vitro incubation and centrifugation

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.

Differential expression of the orphan G-protein coupled receptor GPR55 in human spermatozoa

ObjectiveThe endocannabinoid anandamide (N-arachidonylethanolamide, AEA), reduces human sperm motility and viability by interacting with two G protein-coupled cannabinoid receptors CB1 and CB2. Recent evidence suggests that palmitoylethanolamide signalling through another G-protein coupled receptor GPR55 increases sperm motility and viability. This study was undertaken to explore the presence and differential expression of GPR55 receptor in human spermatozoa.DesignProspective laboratory based clinical study.Materials and MethodsSemen samples were obtained by masturbation from 52 patients (normozoospermia, n = 19 asthenozoospermia, n = 9 teratozoospermia, n = 11 and oligoasthenoteratozoospermia, n = 13). Spermatozoa were separated from seminal plasma by centrifugation and total RNA isolated from spermatozoa. 500ng of total RNA was reverse transcribed to cDNA and GPR55 mRNA were measured by quantitative reverse transcription-polymerase chain reaction using specific primers and SYBR green detection system.ResultsmRNA transcripts were quantified in all 52 samples indicating the presence of GPR55 receptors in human spermatozoa. The GPR55 mRNA transcript levels were significantly lower in spermatozoa from men with astenozoospermia (P<0.001) compared with the normozoospermic controls. There was also a trend towards attenuation of GPR55 mRNA in those with teratozoospermia and oligoasthenoteratozoospermia, although this did not reach statistical significance.ConclusionOur results show that the orphan G-protein coupled receptor GPR55 is expressed in human spermatozoa and the expression is altered in men with abnormal sperm functions suggesting that this signalling pathway may be useful biomarkers for predicting male infertility. ObjectiveThe endocannabinoid anandamide (N-arachidonylethanolamide, AEA), reduces human sperm motility and viability by interacting with two G protein-coupled cannabinoid receptors CB1 and CB2. Recent evidence suggests that palmitoylethanolamide signalling through another G-protein coupled receptor GPR55 increases sperm motility and viability. This study was undertaken to explore the presence and differential expression of GPR55 receptor in human spermatozoa. The endocannabinoid anandamide (N-arachidonylethanolamide, AEA), reduces human sperm motility and viability by interacting with two G protein-coupled cannabinoid receptors CB1 and CB2. Recent evidence suggests that palmitoylethanolamide signalling through another G-protein coupled receptor GPR55 increases sperm motility and viability. This study was undertaken to explore the presence and differential expression of GPR55 receptor in human spermatozoa. DesignProspective laboratory based clinical study. Prospective laboratory based clinical study. Materials and MethodsSemen samples were obtained by masturbation from 52 patients (normozoospermia, n = 19 asthenozoospermia, n = 9 teratozoospermia, n = 11 and oligoasthenoteratozoospermia, n = 13). Spermatozoa were separated from seminal plasma by centrifugation and total RNA isolated from spermatozoa. 500ng of total RNA was reverse transcribed to cDNA and GPR55 mRNA were measured by quantitative reverse transcription-polymerase chain reaction using specific primers and SYBR green detection system. Semen samples were obtained by masturbation from 52 patients (normozoospermia, n = 19 asthenozoospermia, n = 9 teratozoospermia, n = 11 and oligoasthenoteratozoospermia, n = 13). Spermatozoa were separated from seminal plasma by centrifugation and total RNA isolated from spermatozoa. 500ng of total RNA was reverse transcribed to cDNA and GPR55 mRNA were measured by quantitative reverse transcription-polymerase chain reaction using specific primers and SYBR green detection system. ResultsmRNA transcripts were quantified in all 52 samples indicating the presence of GPR55 receptors in human spermatozoa. The GPR55 mRNA transcript levels were significantly lower in spermatozoa from men with astenozoospermia (P<0.001) compared with the normozoospermic controls. There was also a trend towards attenuation of GPR55 mRNA in those with teratozoospermia and oligoasthenoteratozoospermia, although this did not reach statistical significance. mRNA transcripts were quantified in all 52 samples indicating the presence of GPR55 receptors in human spermatozoa. The GPR55 mRNA transcript levels were significantly lower in spermatozoa from men with astenozoospermia (P<0.001) compared with the normozoospermic controls. There was also a trend towards attenuation of GPR55 mRNA in those with teratozoospermia and oligoasthenoteratozoospermia, although this did not reach statistical significance. ConclusionOur results show that the orphan G-protein coupled receptor GPR55 is expressed in human spermatozoa and the expression is altered in men with abnormal sperm functions suggesting that this signalling pathway may be useful biomarkers for predicting male infertility. Our results show that the orphan G-protein coupled receptor GPR55 is expressed in human spermatozoa and the expression is altered in men with abnormal sperm functions suggesting that this signalling pathway may be useful biomarkers for predicting male infertility.

Exogenous glucose improves long-standing human sperm motility, viability, and mitochondrial function

A relevant fraction of human sperm can be maintained in simple phosphate-buffered saline (PBS) for more than 1 week at room temperature, a time that could be doubled if glucose was available. This does not seem to be directly due to sperm mitochondrial activity.

Studies on the viability and membrane integrity of human spermatozoa treated with essential oil of Trachyspermum ammi (L.) Sprague ex Turrill fruit

The present study aimed at investigating the effects of essential oil of Trachyspermum ammi fruits, an oil-bearing plant of Apiaceae family, on human sperm viability and membrane integrity. Chemical compositions of the oil were analysed by GC-MS. Thirty compounds representing 91.39% of the total oil were identified. The viability and membrane integrity of human spermatozoa were assessed using minimum effective dose (MED) concentration (125 μg ml(-1)) of the oil. Sperm treated with essential oil showed a significant decrease (P < 0.05) in viability assessed by eosin-nigrosin and fluorescence dual staining. Moreover, the treated sperm also showed a significant loss (P < 0.05) of functional mitochondria and antioxidant enzyme, catalase (EC 1.11.1.6, CAT), when compared to control. The cholesterol:phospholipid ratio was also increased (P < 0.05) in treated sperm when compared to control, which is an indicator of loss of binding ability of human spermatozoa to the zona pellucida. The scanning electron microscopic studies demonstrated the loss of membrane integrity in essential oil-treated human spermatozoa, which showed vacuolation, swelling of acrosomal cap, detachment of head portion and tail coiling. Present observations indicate the spermicidal property of essential oil of T. ammi fruits, which could be helpful to develop medicinal preparations as a male contraceptive.

A sperm viability test using SYBR-14/propidium iodide flow cytometry as a tool for rapid screening of primary ciliary dyskinesia patients and for choosing sperm sources for intracytoplasmic sperm injection

Spermatozoa viability tests based on dual-color flow cytometry after staining with Sybr-14/propidium iodide were performed on 44 men with complete asthenospermia for primary ciliary dyskinesia (PCD) screening, and seven were identified with PCD by electron microscopy of ultrastructural ciliary defects. Six PCD patients underwent eight intracytoplasmic sperm injection therapy cycles using ejaculated sperm or testicular sperm, obtaining a mean fertilization rate of 46.6%, with three healthy babies born and one in utero at the time of writing.

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food-grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02-2.5 microg x ml(-1)) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 microg x ml(-1) respectively, while values of nisin found were 0.50 and 1.04 microg x ml(-1). In the LDH assay, the EC50 values were 0.65 and 0.62 microg x ml(-1) for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.

Spontaneously developed tail swellings (SDTS) influence the accuracy of the hypo-osmotic swelling test (HOS-test) in determining membrane integrity and viability of human spermatozoa

To investigate the prevalence of spontaneously developed tail swellings (SDTS) in human sperm samples that are commonly encountered in the laboratory, and their influence on the hypo-osmotic swelling test (HOS-test). Ejaculated, epididymal, and testicular sperm were evaluated for SDTS. Further, HOS-test scores were compared with those of vital stains using column washed sperm maintained in the laboratory. SDTS, at <10%, was present in all types of sperm samples. The highest and lowest occurrences of SDTS were found in cryopreserved sperm, and column-washed sperm respectively. SDTS can inflate the HOS-test score, and so lower the accuracy of the HOS-test. However, the HOS-test efficiency can be improved by assessing SDTS in the sample. HOS-test and vital stain cannot be used interchangeably in all circumstances for sperm viability determination. The accuracy of the HOS-test can be enhanced by incorporating SDTS as a correction factor.

Sperm Viability: A Comparison of Analytical Methods

Two methods of measuring human sperm viability, the stain exclusion assay and the hypoosmotic swelling (HOS) test, were evaluated. Human sperm were pretreated with 2.0% glutaraldehyde or 0.1% Triton X-100 and compared to untreated controls. Approximately one half of the sperm were found to be viable in the control samples and nearly all sperm were non-viable in the Triton X-100 treated samples by both the stain exclusion and HOS assays. After glutaraldehyde pretreatment, presumably inactivating the spermatozoa, the HOS test revealed that most sperm were not viable, while the stain exclusion test found no difference between glutaraldehyde pretreated sperm and control sperm. Investigations with scanning and transmission electron microscopy demonstrated that the HOS test caused the membrane of the sperm tail to swell and the tail fibers to coil several times within the swollen membrane. It is concluded that the stain exclusion assay merely measures the structural integrity of the sperm membrane, whereas the HOS test also provides an indication of the physiological integrity of the sperm membrane.

Sperm quality improvement after date seed oil in vitro supplementation in spontaneous and induced oxidative stress

In vitro supplementation with date seed oil (DSO) can protect spermatozoa against hydrogen peroxide (H2O2)-mediated damage and can improve sperm function, possibly owing to antioxidant properties. We tested the antioxidant effects of DSO on human sperm motility, sperm viability, reacted acrosome and lipid peroxidation assessed in vitro after H2O2-mediated oxidative damage in spermatozoa. Sixteen patients (mean age: 35 years; range: 25-45 years) referred to the Histology-Embryology Laboratory of the Medicine Faculty of Sfax for semen analysis after 12-24 months of sexual intercourse without conception were selected. After spermiogram, sperm selection by two-interface discontinuous Sill Select gradient was performed, and selected spermatozoa were used in four experimental assays: control; incubation with 100 microm H2O2; incubation with 0.1% DSO; and co-incubation with 0.1% DSO and 100 microm H2O2. Motility and viability were determined using World Health Organization criteria. Acrosome reaction and lipid peroxidation were assessed by staining with fluorescein isothiocyanate-Pisum sativum and spectrophotometric measurement of malondialdehyde, respectively. Results showed that incubation with H2O2 alone led to a significant increase in lipid peroxidation (57.83%, P<0.05) associated with a significant decrease in sperm motility, sperm viability (after 30 min and 24 h) and percentage of reacted acrosome (P<0.05). Date seed oil improved sperm motility after 24 h of incubation (P<0.05) and protected spermatozoa against the deleterious effects of H2O2 on motility, viability, acrosome reaction and lipid peroxidation. We conclude that supplementation with DSO may have a function in antioxidant protection against male infertility.

115. WHOLE BODY HEAT EXPOSURE INDUCES APOPTOSIS IN MOUSE CAUDAL EPIDIDYMAL SPERMATOZOA

Introduction: The aim of the present study was to determine the immediate effects of whole body heating on sperm numbers, motility and apoptosis. Material and Methods: C57BL/6 mice (n=7) were exposed to 37-38oC (8 hours/day), for three consecutive days while control mice (n=7) were kept at 23-24oC. Caudal epididymal spermatozoa were collected from control and heat treated mice 16 hours after the last heat treatment to determine the sperm number and motility using a Neubauer haemocytometer and sperm apoptotic changes by dual colorflow cytometry using Annexin V/PE (Annexin V conjugated with phycoerythrin) and 7AAD (7-amino-actinomycin D) stains. Results: There were no significant differences (p&gt;0.05) in sperm numbers between heat treated and control mice, however heating did result in a significant reduction in sperm motility (p&lt;0.05). Apoptosis staining identified four different subpopulations of spermatozoa: (a) live spermatozoa (Annexin V-/7AAD-), (b) early apoptotic spermatozoa with exteriorized phosphotidylserine (PS) receptor and intact plasmalemma (Annexin V+/7AAD-), (c) late apoptotic spermatozoa with PS receptor translocation and leaky plasmalemma (Annexin V+/7AAD+) and (d) dead spermatozoa with damaged plasmalemma with no detectable PS receptor (Annexin V-/7AAD+). Heating resulted in significant reduction in the percentage of live spermatozoa (p&lt;0.05), an increase in early apoptotic (p&lt;0.05), late apoptotic (p&lt;0.05), and dead spermatozoa (p&lt;0.05). Conclusion: This study shows that mice exposed to whole body heat exposure of 37-38oC for 8 hours per day for three consecutive days exhibited early and late apoptotic changes to epididymal spermatozoa. These findings suggest possible adverse effects of exposure to high temperature on the viability of human spermatozoa in the epididymides. In addition, these findings reinforce the importance of temperature during sperm preparation procedures in infertility clinics, and research laboratories.

Effect of Bisphenol A, 4-octylphenol and Phthalic acid on human sperm

OBJECTIVE: Exposure to environmental compounds can be a major factor leading to infertility. We evaluated the in vitro effects of three common environmental chemicals, Bisphenol A, 4-Octylphenol, and Phthalic acid on motility, viability, and chromatin integrity of human sperm.

Insulin and leptin enhance human sperm motility, acrosome reaction and nitric oxide production

To investigate the in vitro effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production. Washed human spermatozoa from normozoospermic donors were treated with insulin (10 microIU) and leptin (10 nmol). Insulin and leptin effects were blocked by inhibition of their intracellular effector, phosphotidylinositol 3-kinase (PI3K), by wortmannin (10 micromol) 30 min prior to insulin and leptin being given. Computer-assisted semen analysis was used to assess motility after 1, 2 and 3 h of incubation. Viability was assessed by fluorescence-activated cell sorting using propidium iodide as a fluorescent probe. Acrosome-reacted cells were observed under a fluorescent microscope using fluorescein-isothiocyanate-Pisum sativum agglutinin as a probe. NO was measured after treating the sperm with 4,5-diaminofluorescein-2/diacetate (DAF-2/DA) and analyzed by fluorescence-activated cell sorting. Insulin and leptin significantly increased total motility, progressive motility and acrosome reaction, as well as NO production. This study showed the in vitro beneficial effects of insulin and leptin on human sperm function. These hormones could play a role in enhancing the fertilization capacity of human spermatozoa.

Method-Related Estimates of Sperm Vitality

Comparison of methods that estimate viability of human spermatozoa by monitoring head membrane permeability revealed that wet preparations (whether using positive or negative phase-contrast microscopy) generated significantly higher percentages of nonviable cells than did air-dried eosin-nigrosin smears. Only with the latter method did the sum of motile (presumed live) and stained (presumed dead) preparations never exceed 100%, making this the method of choice for sperm viability estimates.

SYTO dyes and histoproteins—Myriad of applications

SYTO dyes and histoproteins—Myriad of applications

TNF-α and IL-6 affect human sperm function by elevating nitric oxide production

Many studies have reported the effects of cytokines on human sperm function, even though their role and the mechanisms involved remain unclear. The effects of increasing concentrations of the cytokines TNF-α and IL-6 on human sperm motility and viability were assessed, and the possible mechanisms involved were investigated. TNF-α and IL-6 significantly reduced progressive motility at higher concentrations in a dose- and time-dependent manner. No differences were observed in cell viability. Both cytokines increased nitric oxide production in a dose-dependent manner. TNF-α and IL-6 did not statistically differ in their detrimental effects on human spermatozoa. These results indicate that TNF-α and IL-6 have an effect on sperm function. This effect is possibly mediated via an increase in nitric oxide production.

Affects of high frequency electromagnetic fields on human sperm cellular function

Previous studies have suggested that electromagnetic fields (EMF), such as those found in several common pieces of equipment in the assisted reproductive laboratory (ART), have detrimental effects on gametes and embryos, yet little controlled data exists. Last year at this meeting we presented what appears to be one of the first controlled studies of EMFs in an ART setting, using a porcine sperm model. In that study, high-frequency EMF (roughly three times that of the top shelf of a culture incubator; ∼80 mGuass) disrupted sperm cellular function. The objective of the present study was to determine if EMFs had similar affects on human semen samples. An in vitro based study of EMF affects on sperm cell function using a human model. Semen samples were collected from proven donors and prepared with a modified human tubal media using standard techniques. Ejaculates were then split between four EMF fields based upon levels consistent with the selves of a standard culture incubator (2, 30 and 90 mGuass), or a a high-frequency (HF) EMF (>250 mGuass) control. The samples were cultured under conditions of 37°C and room air. To assess the effects of EMF, standard semen parameters were assessed by CASA at times 30, 60, 90, 120, 150, 180 minutes and 24 hours. Resulting data were compared using ANOVA with repeated measures. Additional samples were collected and fixed with gluteraldehyde for later biochemical analysis. As expected, motility and forward progression (FP) decreased with time (P<.001). However, ejaculates exposed to the HF-EMF suffered a much more rapid decrease in both motility (P<.001) and FP (P<.04). While samples exposed to low EMF lost ∼20% of their motility in the first 24 hrs, HF-EMF exposed cells lost over 70% of their initial motility. Further, cells exposed to the HF-EMF demonstrated first a rapid increase then decline in FP. Biochemical assays remain pending at this time. As in the earlier animal study, exposure to extremely high EMF appears to have a negative impact on the functional viability of human sperm by decreasing motility and forward progression. However, at this point, it is unclear if the lower levels of EMF, consistent with the levels seen in an assisted reproductive laboratory (ART), also have deleterious effects. Given the ubiquitous nature of EMF in the ART laboratory, further research is warranted to determine if EMF in this environment does decrease cell function, and therefore pregnancy rates.

Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients

The DNA damage in human spermatozoa is a relevant predictor of prognosis in male infertility, whereby increased sperm DNA damage impairs the outcomes of artificial reproduction. Theoretically, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Nevertheless, intracytoplasmic sperm injection (ICSI) has been reported to alleviate such negative outcomes due to DNA damage. This study investigated the relationship between DNA fragmentation and acrosome reaction as well as viability in ICSI patients. The study enrolled 42 men undergoing ICSI due to poor sperm parameters. The DNA fragmentation indexes (DFI) were 4–10% in 38% of the cases, and ≥10% in 19% of the cases. The results of both acrosome reaction and viability assays showed negative correlations with DFI values in all cases and especially in cases with fertilization rates <60% (P < 0.05). However, such correlations were not found in cases with fertilization rates >60%. There were no live deliveries in patients with high DFI levels (>10%). In conclusion, negative correlations were identified between increased DNA damage, and acrosome reaction and/or viability of human spermatozoa, especially in cases with reduced fertilization rates.