DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty‐eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.     

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment

DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome‐wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual‐specific effects that may be caused by other environmental factors.     

Aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity

The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGβ and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene‐related amplicon (CpG1, p ≤ .003, CpG2, p ≤ .0001, CpG3, p ≤ .003 and CpG4, p ≤ .030) and CpG1 in CGβ gene‐related amplicon (p ≤ .0001). Besides, a significant difference was found at two CpGs (CpG1, p ≤ .004 and CpG2, p ≤ .002) tested in the FAM189A1 gene‐related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.     

Alterations in DNA methylation patterns and gene expression in spermatozoa of subfertile males

This study was designed to evaluate the DNA methylation patterns and gene expression in spermatozoa from subfertile males. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then three CpG sites (cg07227024, cg05813498 and cg23081194) have the highest difference in methylation levels and located within ALS2CR12, GAA and UBE2G2 genes respectively; these were selected for further analysis using deep bisulphite sequencing and qPCR in 136 samples (64 proven fertile males “controls” and 72 subfertile males “cases”). A significant difference in the methylation level was found between cases and controls at two CpGs, six CpGs and three CpGs in ALS2CR12, GAA and UBE2G2 gene‐related amplicon respectively. Besides, the qPCR results showed a significant change in the expression levels of GAA, UBE2G2 and ALS2CR12 gene in cases compared to the controls (p ≤ .00001). In conclusion, the methylation levels at CpGs in GAA, UBE2G2 and ALS2CR12 gene amplicons were significantly different in subfertile compared to proven fertile males. In addition, a significantly different was showed in the expression levels of GAA, UBE2G2 and ALS2CR12 genes in subfertile males compared to proven fertile males.     

Association between alterations in DNA methylation level of spermatozoa at CpGs dinucleotide and male subfertility problems

The purpose of this study was to assess the relationship between alterations in sperm DNA methylation levels and sperm count and sperm motility. Five CpG sites underwent deep bisulphite sequencing to validate the observed methylation difference in 78 samples (28 proven fertile males “controls,” and 50 subfertile males “cases”). The results showed that variation in methylation levels was found in more than one CpG: the DNA methylation levels in CpG1, CpG2 and CpG3 of the PRRC2A gene‐related amplicon showed high significant differences in the case group compared to the control group (p ≤ .0001, p ≤ .003, and p ≤ .0001 respectively). Moreover, three CpGs of the four CpGs tested within the ANXA2 gene‐related amplicon (CpG1, CpG3 and CpG4) were significantly different (p ≤ .002, p ≤ .001, and p ≤ .0001, respectively) in the case group compared to the control group. In addition, a significant difference was found in seven CpGs of the twenty‐two CpGs tested within the MAPK8Ip3 gene‐related amplicon, besides six CpGs of the ten CpGs tested within the GAA gene‐related amplicon between case and control groups. In conclusion, this study identifies that CpGs have a significantly different in methylation levels of sperm DNA for subfertile males.     

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS‐IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE‐qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI ≥ 25% was lower than that in the group with DFI < 25% (13.7 ± 3% vs. 31.5 ± 5.3%, p = 0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r = ?0.448, p = 0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r = 0.354, p = 0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.     

Sperm DNA methylation of H19 imprinted gene and male infertility

Infertility affects up to 15% of reproductive‐aged couples worldwide, with male factor being detected in 40%–50% of the cases. Proper sperm production is associated with the establishment of appropriate epigenetic marks in developing germ cells. Several studies have demonstrated the association between abnormal spermatogenesis and epigenetic disturbances with the major focus on DNA methylation. Imprinted genes are expressed in a parent‐of‐origin‐specific manner, and the role of their DNA methylation in proper spermatogenesis has been documented recently. The existing evidence along with the absence of relevant data in south of Iran prompted us to study the methylation of H19 imprinted gene in spermatozoa of idiopathic infertile patients (males with abnormalities in sperm parameters) and healthy controls by Combined Bisulfite Restriction Analysis. According to our results, the lowest methylation percentage of H19 imprinted gene belongs to three cases with sperm characteristics under normal range (two cases Oligoasthenoteratozoospermia and one case Oligoteratozoospermia). However, our results show that the median of methylation percentage for H19 is not statistically significant between case and control groups. Our results and those of others introduce DNA methylation as a potential marker of fertility and should be investigated with more patients in future studies.     

Epigenome‐wide Association of DNA Methylation in Whole Blood With Bone Mineral Density

Genetic and environmental determinants of skeletal phenotypes such as bone mineral density (BMD) may converge through the epigenome, providing a tool to better understand osteoporosis pathophysiology. Because the epigenetics of BMD have been largely unexplored in humans, we performed an epigenome‐wide association study (EWAS) of BMD. We undertook a large‐scale BMD EWAS using the Infinium HumanMethylation450 array to measure site‐specific DNA methylation in up to 5515 European‐descent individuals (N_Discovery = 4614, N_Validation = 901). We associated methylation at multiple cytosine‐phosphate‐guanine (CpG) sites with dual‐energy X‐ray absorptiometry (DXA)‐derived femoral neck and lumbar spine BMD. We performed sex‐combined and stratified analyses, controlling for age, weight, smoking status, estimated white blood cell proportions, and random effects for relatedness and batch effects. A 5% false‐discovery rate was used to identify CpGs associated with BMD. We identified one CpG site, cg23196985, significantly associated with femoral neck BMD in 3232 females (p = 7.9 × 10^?11) and 4614 females and males (p = 3.0 × 10^?8). cg23196985 was not associated with femoral neck BMD in an additional sample of 474 females (p = 0.64) and 901 males and females (p = 0.60). Lack of strong consistent association signal indicates that among the tested probes, no large‐effect epigenetic changes in whole blood associated with BMD, suggesting future epigenomic studies of musculoskeletal traits measure DNA methylation in a different tissue with extended genome coverage. © 2017 American Society for Bone and Mineral Research.     

Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables

OBJECTIVE

To present the first UK data on sperm DNA fragmentation levels in subfertile men and fertile controls, the correlation with semen variables, and to assess the effect on the outcome of intracytoplasmic sperm injection (ICSI).

PATIENTS, SUBJECTS AND METHODS

In all, 56 subfertile men undergoing ICSI (28 with positive and 28 with a negative outcome for paternity) and 10 control fertile semen donors were recruited. The sperm DNA fragmentation index (DFI) was assessed on raw pre‐preparation samples using the sperm chromatin structure assay. A mean of 5212 sperm were analysed per sample and DFI data are presented by fertility status, ICSI outcome and correlated with semen variables (assessed using World Health Organisation criteria).

RESULTS

Total DFI was significantly higher in subfertile men than in fertile controls (mean and median of 22.8% and 17.0% vs 8.4% and 5.0%; P < 0.001), as was the proportion of both moderate DFI (16.4% and 13.0% vs 6.4% and 4.0%; P = 0.001) and high DFI (6.2% and 6.1 vs 2.0% and 1.0%; P = 0.01). This difference remained significant when the control men were compared only with the subfertile men with successful paternity. There was no significant difference in DFI in the subfertile men when analysed by ICSI outcome (mean and median of 24.5% and 17.0% vs 22.3% and 21.0% for successful and unsuccessful cycles, respectively; P = 0.94). There was a positive statistically significant correlation (r = 0.37; P = 0.02) between the DFI and sperm morphology.

CONCLUSIONS

This study confirms a relationship between male subfertility and sperm DFI; we discuss the correct role for genetic testing of sperm in the evaluation of subfertile men. Although DNA fragmentation data might help to decide a suitable treatment, once it is decided to proceed with ICSI, DFI levels have no effect on the outcome.     

Preparation and observation methods can produce misleading artefacts in human sperm ultrastructural morphology

The aim of this study was to evaluate the production of artefacts during preparation and observation of human sperm for ultrastructural morphology and analyse the possible reasons of causing these artefacts. Under the scanning electron microscopy, damaged sperm heads (crack or/and rupture), necks (head‐neck or head‐midpiece separation) and midpiece (disassembled and denuded axoneme ultrastructures, bent midpiece) were analysed to be the consequence of exogenous effects. Thirty infertile men with teratozoospermia revealed more spermatozoa with damage to head, neck and midpiece than did thirty fertile males (p < .01). After the samples from fertile males underwent five repeated observations, most sperm heads and necks in the samples were destroyed when compared with the single observation (p < .01). Destroyed sperm heads were full of cracks and peelings, even sperm tails were broken and fragmented, and separations of the sperm head‐neck or head‐midpiece became common. Spermatozoa from fertile males with centrifugation of 600 g for washing sperm exhibited more damage to the midpiece than those with the 300 g (p < .01). These results demonstrate that preparation and observation methods can damage sperm ultrastructures, leading to producing artefacts of ultrastructural morphology. The artefacts of sperm ultrastructural morphology may be associated with sperm structural fragility, preparation conditions and electron beam damage.     

The presence of human papillomavirus in semen does not affect the integrity of sperm DNA

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO‐LiPA test PCR‐based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.     

Investigating the relationship between BRCA1 and BRCA2 genes methylation profile and sperm DNA fragmentation in infertile men

The purpose of the study was to investigate whether the promoter methylation status of BRCA1 and BRCA2 DNA repair genes is associated with sperm DNA fragmentation (sDF) in infertile men with oligoasthenoteratozoospermia (OAT) which emerges due to various reasons and is effective in male infertility. Seventy‐three infertile men with OAT and 20 normozoospermic volunteers participated in the study. To investigate sDF and methylation patterns of BRCA1 and BRCA2 gene promoters, TUNEL assay and methylation‐specific PCR (MS‐PCR) were used. The mean sDF ratio for the patients was calculated as 22.50%. The calculated cut‐off value for sDF ratio was 17.0% in ROC curve analysis. Regarding sDF, a significant difference between the normozoospermic group and the OAT group with abnormal semen parameters (p < 0.001) was found. sDF demonstrated a significant effect on the semen parameters and negative correlations on sDF ratios and sperm motility, concentration and morphology. There was no statistically significant association between sDF and the methylation status of the promoter of either BRCA1 or BRCA2 genes. In routine clinical practice, sperm DNA integrity should be investigated before applying assisted reproductive techniques. To understand better the relationship between epigenetic regulation of DNA repair genes and male infertility, additional studies are required.     

Comparison of sperm morphology and nuclear sperm quality in SPATA16‐ and DPY19L2‐mutated globozoospermic patients

The aim of this study was to compare the sperm morphology and nuclear sperm quality (sperm aneuploidy and DNA fragmentation) in two groups of globozoospermic patients: DPY19L2‐mutated patients (n = 6) and SPATA16‐mutated patients (n = 2). Results for these two groups were also compared to a group of fertile men (n = 25). Fluorescence in situ hybridisation was performed for chromosomes X, Y and 18. Sperm DNA fragmentation was evaluated by TUNEL assay. Sanger sequencing was performed for mutations screening of DPY19L2 and SPATA16 genes. Sperm analysis revealed a classic phenotype of total globozoospermia in DPY19L2‐mutated group and a particular phenotype characterised by a predominance of double/multiple round‐headed (39.00 ± 4.2%) and multi‐tailed spermatozoa (26.00 ± 16.97%) in SPATA16‐mutated group. FISH analysis showed a significantly higher aneuploidy rate in globozoospermic patients compared to controls (p < 0.05), and a higher rate was observed in SPATA16‐mutated group compared to DPY19L2‐mutated group (p < 0.05). DNA fragmentation index was significantly higher in globozoospermic men compared to controls (p < 0.001), and there is no statistically significant difference between the two globozoospermic groups. We showed that SPATA16 defects could be associated with an abnormal meiosis leading to a particular morphological sperm defect of double/multiple round‐headed and multi‐flagella and a higher sperm aneuploidy rate than in case of DPY19L2‐defects in classic globozoospermia.     

Effect of varicocelectomy on sperm functional characteristics and DNA methylation

In individuals with varicocele, DNA is damaged due to high level of oxidative stress, and varicocelectomy can overcome this effect. Damaged DNA is less liable to DNA methylation, and antioxidant therapy appears to have the potential to reduce sperm oxidative stress and DNA damage and thereby maintain DNA methylation, while effect of varicocelectomy on DNA methylation patterns has remained unclear. In the light of these considerations, we aimed to examine the effect of varicocelectomy on sperm DNA methylation and functional characteristics. Fifty‐two men with left‐sided varicocele (grade II &III) were included. Sperm parameters, DNA fragmentation, protamine deficiency, oxidative stress and global DNA methylation were evaluated before and 3 months after surgery. Our data show that sperm concentration, percentages of spermatozoon with abnormal morphology, DNA fragmentation, protamine deficiency and oxidative stress significantly improved after surgery. Percentage of sperm motility, global DNA methylation and intensity of DNA methylation also improved after surgery, although the differences were not significant when compared with before surgery. Categorisation of individuals to subgroups revealed that improvement of DNA methylation appears to take place in oligozoospermic individuals, which are more severely affected by state of varicocele. However, this is a preliminary study, and further studies are required to solidify this conclusion.     

Characterisation of a subpopulation of sperm with massive nuclear damage,as recognised with the sperm chromatin dispersion test

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7‐dibrom‐4‐hydroxy‐mercury‐fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two‐dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double‐ and single‐strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele.     

Association between total globozoospermia and sperm chromatin defects

Globozoospermia is a severe sperm morphological anomaly leading to primary infertility and low fertilisation following intracytoplasmic sperm injection (ICSI). This phenotype is observed in less than 0.1% of infertile men and is determined by small, round‐headed spermatozoa with absence of an acrosomal cap, acrosome protease and also cytoskeletal proteins. Failure of oocyte activation is considered as the main cause of fertilisation failure in these individuals post‐ICSI. Therefore, artificial oocyte activation (AOA) along with ICSI is commonly implemented. However, based on previous report, fertilisation rate remains low despite implementation of ICSI‐AOA. Therefore, other mechanisms like sperm chromatin packaging and DNA fragmentation may account for low fertilisation and development post‐ICSI‐AOA. Therefore, this study aims to assess and compare the degree of sperm protamine deficiency and DNA fragmentation in large population of infertile men with total globozoospermia (30 globozoospermic men presenting with 100% round‐headed spermatozoa) with 22 fertile individuals using chromomycin A3 and TUNEL assay respectively. Results clearly show that mean of sperm concentration and percentage of sperm motility were significantly lower, while percentage of sperm abnormal morphology, protamine‐deficient and DNA‐fragmented spermatozoa were significantly higher in infertile men with globozoospermia compared to fertile men. Therefore, increased sperm DNA damage in globozoospermia is likely related to defective DNA compaction and antioxidant therapy before ICSI‐AOA could be recommended as an appropriate option before ICSI‐AOA.     

DNA integrity and methylation changes of mouse spermatozoa following prolonged incubation

Sperm quality can be affected by different factors including the length of incubation time between sperm preparation and intracytoplasmic sperm injection. Here, we have evaluated the level of DNA methylation and expressions of related genes in mice spermatozoa. The spermatozoa were divided into three groups: fresh, spermatozoa incubated at room temperature (RT) and 37°C for 24 hr. The sperm chromatin structure assay was used to determine the DNA fragmentation index (DFI), and DNA methylation was analysed by flow cytometry. The expression levels of DNA methylation‐related genes were determined by quantitative real‐time PCR (qRT‐PCR). According to the results, we observed significantly higher sperm progressive motility and viability in the group incubated at RT compared to the spermatozoa incubated at 37°C (p < 0.05). Spermatozoa incubated at 37°C had a higher DFI compared to the other groups (p < 0.05), but the DNA methylation level significantly decreased (p < 0.05). qRT‐PCR analysis showed increased Dnmt‐1 expression in spermatozoa after 24‐hr incubation at 37°C. However, there were significantly higher expression levels of Dnmt‐3l, Dnmt‐3a and Dnmt‐3b after incubation at both RT and 37°C compared to the fresh group (p < 0.05). The 24‐hr incubation period affected both sperm DNA methylation and integrity. This study indicated that incubation at RT resulted in better sperm quality.     

Aneuploidy and DNA fragmentation in morphologically abnormal sperm

In light of the relative success of ICSI in the treatment of male infertility, much importance has been made to the selection of morphologically viable sperm. However, correlation between specific sperm morphology and chromosomal abnormalities is still relatively limited and less is known about the connection between sperm morphology and DNA integrity. Sperm obtained from isolated teratozoospermic men ( n  = 10) and control men ( n  = 9) were analysed using FISH (for chromosomes 13, 18, 21, X and Y) and TUNEL assays to determine the level of aneuploidy and DNA fragmentation. Sperm morphology was evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. Sperm from teratozoospermic men, compared with fertile men, had higher rates of total chromosomal abnormality ( p  < 0.05), total aneuploidy ( p  < 0.01) and chromosome 13 disomy ( p  < 0.01). Associations between particular types of sperm morphology and chromosomal abnormalities were observed in both control (tapered heads) and teratozoospermic (amorphous heads and tail abnormalities) samples. Levels of DNA fragmented sperm were higher in teratozoospermic men than in the control men (60.28 ± 21.40% vs. 32.40 ± 17.20%, p  < 0.05) and positively correlated to sperm with bent necks in control samples and round heads in teratozoospermic samples ( p  < 0.05). Sperm of isolated teratozoospermic men have higher rates of chromosomal abnormalities and DNA fragmentation than that of the fertile controls. Specific abnormal sperm morphology can be correlated to chromosomal abnormalities and level of DNA fragmentation in sperm.     

Relationship between sperm telomere length and sperm quality in infertile men

Telomeres, noncoding and repetitive DNA sequences play a significant function in chromatin integrity. Telomere length is age-dependent in somatic cells, while it increases in sperm cell with age. Therefore, we aimed to assess sperm chromatin, leucocyte and sperm telomere length (LTL, STL) in spermatozoon of 38 infertile and 19 fertile men aged between 20 and 50 years. Protamine deficiency (chromomycin A3 test), DNA fragmentation (TUNEL assay), lipid peroxidation (Bodipy probe) and telomere length (quantitative real-time PCR) were assessed. A significant decrease in mean of sperm concentration and motility and a significant increase in means of sperm abnormal morphology, DNA fragmentation, lipid peroxidation and protamine deficiency were observed in infertile compared with fertile men. In addition, the mean of LTL and STL were significantly shorter in infertile men compared with fertile individuals. We observed significant associations between telomere length with sperm concentration, DNA fragmentation and lipid peroxidation. We hypothesised that increased oxidative stress in spermatozoa of infertile men can result in abnormal packaging of chromatin, damage of DNA and shorter sperm telomere length. Together, these anomalies may account for fertility failure in these individuals.     

Famotidine does not affect human sperm fertility characteristics (motility,viability and DNA integrity) in vitro

Famotidine, a histamine‐2 receptor antagonist, is commonly used to relieve the acid‐related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10 mM concentrations in vitro. Forty‐nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin–nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10 mM had insignificant effect on human sperm motility (progressive, p = .9594; and total, p = .8420), sperm viability (p = .6471), and content of DNA breaks in sperm (p > .05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10 mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.