Semiquantitative promoter methylation of MLH1 and MSH2 genes and their impact on sperm DNA fragmentation and chromatin condensation in infertile men

To investigate the semiquantitative methylation alterations of MLH1 and MSH2 and the possible association among methylation of MLH1 and MSH2, sperm DNA fragmentation and sperm chromatin condensation in idiopathic oligoasthenoteratozoospermic men. Seventy-five idiopathic infertile men and 52 fertile and/or normozoospermic men were included in the study. SDF was analysed using the TUNEL assay in semen samples of 100 men. Promoter methylation of MLH1 and MSH2 genes was assessed by semiquantitative methylight analysis in semen samples of 39 and 40 men respectively. Sperm chromatin condensation was evaluated using aniline blue staining in 114 men. MLH1 promoter methylation was positively correlated with the percentage of aniline blue positive spermatozoa (r = 0.401, p = 0.0188). On the other hand, MSH2 promoter methylation was negatively correlated with sperm concentration and total sperm count (r = −0.421, p = 0.0068 and r = 0.4408, p = 0.009 respectively). The percentage of aniline blue positive spermatozoa in the control group was significantly lower than in the OAT group (p < 0.0001) and negatively correlated with total sperm count (r = −0.683, p < 0.0001), progressive sperm motility (r = −0.628, p < 0.0001), total motility (r = −0.639, p < 0.0001) and normal morphology (r = −0.668, p < 0.0001). Promoter methylation profile of MLH1 and MSH2 genes may play role on sperm DNA packaging and conventional semen parameters respectively.     

Methylation patterns of methylenetetrahydrofolate reductase gene promoter in infertile males

Errors of folate/homocysteine pathways which are critical for transferring methyl groups have been suggested to affect male fertility. We aimed to evaluate the methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene in infertile males and to investigate the association between MTHFR promoter methylation and success of sperm retrieval. Thirty-five nonobstructive azoospermic and 46 severe oligozoospermic patients constituted the study group and were compared with 49 fertile and/or normozoospermic men. The methylation status was analysed by methylation-specific polymerase chain reaction. MTHFR promoter methylation was detected in infertile men with NOA and SO in the ratio of 48.6% and 58.7%, respectively. Methylation was also observed in 51% of controls. MTHFR promoter was methylated in 65% of men with viable spermatozoon during TESE. No association was found regarding to the profile of MTHFR promoter methylation between both NOA and SO patients and controls (p = .621). There was no relation between the methylation status of MTHFR promoter and low motility and poor morphology (p = .682 and p = .413, respectively). No association was found between MTHFR promoter methylation and presence of viable spermatozoa (p = .382). Our data indicate that the promoter methylation of MTHFR gene may not be associated with male infertility.     

Analysis of partial azoospermia factor c deletion and DAZ copy number in azoospermia and severe oligozoospermia

Microdeletions of the azoospermia factor (AZF) regions in the Y chromosome are a well‐known genetic cause of male infertility, resulting in impairment of spermatogenesis. However, the partial deletions of AZFc region related to spermatogenetic impairment are controversial. We investigated partial deletion of AZFc region and DAZ copy number in a population of Iranian infertile men and normozoospermic controls. In total, 154 infertile men (113 patients with azoospermia, 41 with oligozoospermia) and 111 normozoospermic controls were analysed using PCR. Gene dosage analysis of the DAZ genes was performed by fragment analysis. Our results showed that the frequencies of gr/gr deletion in the azoospermic, severe oligozoospermic and normozoospermic men were 4.4% (5/113), 7.3% (3/41) and 1.8% (2/111) respectively. In the azoospermic patients, the frequency of b2/b3 was 1.8% (2/113). Partial AZFc deletions were not significantly different between the infertile and normozoospermic men. The frequencies of gr/gr deletions and b2/b3 were not significantly different between the azoospermic/severe oligozoospermic men and normozoospermic controls. Our data suggested that gr/gr deletion was not associated with azoospermia/oligozoospermia in an Iranian population.     

Association of XRCC1 and ERCC2 promoters’ methylation with chromatin condensation and sperm DNA fragmentation in idiopathic oligoasthenoteratozoospermic men

The aim of the study was to investigate whether the promoter methylation of XRCC1 and ERCC2 genes is associated with sperm DNA fragmentation and chromatin condensation in idiopathic oligoasthenoteratozoospermic men. This study involved 77 infertile men with idiopathic oligoasthenoteratozoospermia and 51 normozoospermic controls. The methylight method, TUNEL assay and aniline blue staining were used for the evaluation of XRCC1 and ERCC2 genes’ methylation, SDF and sperm chromatin condensation, respectively. SDF (p = .004) and XRCC1 methylation (p = .0056) were found to be significantly higher in men with idiopathic OAT than in the controls, while mature spermatozoa frequency was higher in controls as compared to infertile men (p < .0001). No significant association was found between SDF and methylation of XRCC1 and ERCC2 genes (p = .9277 and p = .8257, respectively). However, compared to the cut-off point obtained by receiver operating characteristic analysis, a significant association was found between SDF and XRCC1 methylation, positive and negative methylation groups, generated according to the cut-off value for XRCC1. XRCC1 methylation was found to have a significant effect on chromatin condensation (p = .0017). No significant difference was detected among ERCC2 methylation, male infertility and SDF. In conclusion, XRCC1 methylation may have a role in sperm chromatin condensation and idiopathic OAT.     

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.     

Exploring the relationship between the severity of oligozoospermia and the frequencies of sperm chromosome aneuploidies

The study was aimed to investigate the association between the degree of oligozoospermia and sperm chromosome aneuploidy frequencies in male infertility and to determine whether chromosomal profiles of sperm nuclei would be used for a supportive test before additive reproduction technics. The meiotic segregation profiles of chromosomes X, Y, 13, 18 and 21 were compared by fluorescent in‐situ hybridisation (FISH) on the spermatozoa of 30 normally karyotyped oligozoospermic (10 mild, 11 moderate, nine severe) cases without Y‐microdeletions, and 10 normozoospermic cases. The results showed significantly higher frequencies of chromosomes 13, 18, 21 disomies (P < 0.001) in the group of patients with moderate and severe oligozoospermia compared with the disomy frequencies of normozoospermic group. The statistically significant differences were also determined in disomy frequencies of sex chromosomes (XY, XX and YY) in between oligozoospermic and normozoospermic groups (P < 0.001, P < 0.001, P < 0.040, respectively). Because oligozoospermic patients are the ones consulted the most for assisted reproductive techniques, identification of sperm aneuploidy rates in men could be considered as an appropriate supportive test before the reproductive implementations. Furthermore, the patients should be counselled with respect to genetic screening results for the potential risk of aneuploid embryo and pre‐implantation genetic diagnosis or prenatal diagnosis.     

Oxidative stress‐induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch‐like ECH‐associated protein 1 (keap1)‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1‐Nrf2 system in several oxidative stress–associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n = 39, ROS < 20 RLU/s per 10⁶ spermatozoa), group B (n = 38, 20 ≤ ROS < 40 RLU/s per 10⁶ spermatozoa), group C (n = 31, 40 ≤ ROS < 60 RLU/s per 10⁶ spermatozoa) and group D; (n = 43, ROS ≥ 60 RLU/s per 10⁶ spermatozoa). Keap1 methylation status was assessed using methylation‐specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.     

Association between methylenetetrahydrofolate reductase (MTHFR) gene promoter hypermethylation and the risk of idiopathic male infertility

Epigenetics has become a major field of reproductive medicine after the epigenetic regulation of gene expression was discovered. The aim of this study was to find out whether or not methylenetetrahydrofolate reductase (MTHFR) gene promoter hypermethylation in the spermatozoa of men who were offered assisted reproduction is associated with idiopathic male infertility. Sperm DNAs from 40 idiopathic infertile men with normozoospermia and 40 controls consisting of healthy fertile men were isolated. Following the modification of DNAs by sodium bisulphite, the methylation status of the MTHFR gene promoter was quantified by pyrosequencing. No significant differences were observed between the clinical characteristics of patients and controls. The percentage of MTHFR promoter methylation in infertile men with normozoospermia (11%) was significantly higher than that in the healthy control (4.3%) group (p = .01). A 9.5% of methylation level was determined via receiver operator characteristic (ROC) analysis as the cut‐off value. There were 21 (53%) hypermethylated men among the infertile men and 2 (5%) in the control group (p = .0001). The intragroup analysis of the infertile group did not reveal any statistically significant differences in terms of overall clinical characteristics between hyper‐ and normo‐methylated infertile men. Our results suggest that epigenetic silencing (hypermethylation) of MTHFR could result in an elevated risk of male infertility.     

Sperm nuclear DNA fragmentation and its association with semen quality in Greek men

Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non‐normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non‐normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility.     

Seminal oxidation–reduction potential levels are not influenced by the presence of leucocytospermia

Oxidative stress (OS) is characterised by an excessive amount of reactive oxygen species (ROS) which negatively affect sperm functions. In this study, the influence of leucocytes on seminal oxidation–reduction potential (ORP) and sperm DNA fragmentation (SDF) was investigated in 1,068 men. Seminal leucocyte concentration did not correlate with SDF, unadjusted ORP, ORP normalised for sperm concentration (sORP), ORP normalised for total motile sperm concentration (motORP) or total motile sperm count (TMSC-ORP). Although receiver operator characteristic (ROC) curve analyses show that leucocytospermia does not predict high sORP values (>1.34 mV/10⁶ spermatozoa/ml), the motORP (AUC: 0.666) and TMSC-ORP (AUC: 0.683) predict the rate of leucocytospermia significantly (p = .0195 and p = .0085 respectively). Moreover, SDF can significantly predict leucocytospermia (AUC: 0.679; p = .011) and vice versa (AUC: 0.657, p = .0298). Our data confirm the association between OS and SDF. In conclusion, motORP and TMSC-ORP may be better predictive factors of leucocytospermia, probably because sperm motility, included in motORP and TMSC-ORP calculation, is the first seminal parameter to be affected by OS. Although all these parameters are indicative of OS, ORP values, SDF and leucocytospermia should be considered independently for the evaluation of redox seminal status, as they probe distinct seminal features.     

Implications of mismatch repair genes hMLH1 and hMSH2 in patients with sporadic renal cell carcinoma

OBJECTIVES

To analyse the implications of DNA mismatch repair genes hMLH1 and hMSH2 in sporadic renal cell carcinoma (RCC).

MATERIALS AND METHODS

Specimens of tumour and healthy renal tissue were collected from 89 patients treated for sporadic RCC. Another 95 blood samples taken from individuals with no history of cancer were also analysed. After DNA extraction and PCR amplification, microsatellite instability (MSI) was determined using the Bethesda microsatellite panel, two exonic microsatellites of the TGFbRII and BAX genes, and the microsatellite D3S1611. The promoter methylation status of hMLH1 was investigated using the HpaII and MspI restriction enzymes. In addition, a sequencing analysis of complete coding region of hMLH1 and hMSH2 genes was performed.

RESULTS

MSI and promoter hypermethylation of hMLH1 were not detected. Interestingly, loss of heterozygosity (LOH) was common among patients with RCC, particularly in microsatellite D3S1611 (34.9%). Mutations were identified in eight patients: K618A and V716M in gene hMLH1; and I145V, G322D, and the novel mutation P349A, in gene hMSH2. The mutations also appeared in healthy renal tissue and therefore, were considered as germline DNA sequence variations. There were G322D and K618A changes in >1% of the healthy control subjects, suggesting that they are DNA polymorphisms.

CONCLUSIONS

Our data show that loss of function of both hMLH1 and hMSH2 is not involved in sporadic RCC, either by promoter methylation or mutation in their exons. However, LOH indicated that chromosomal instability affecting large fragments of DNA was the main genetic alteration we detected associated with RCC.     

Oxidative stress status and sperm DNA fragmentation in fertile and infertile men

Evidence suggests that disturbing the balance between reactive oxygen species levels and antioxidant contents in seminal plasma leads to oxidative stress resulting in male infertility. This study was carried out to identifying clinical significance of seminal oxidative stress and sperm DNA fragmentation in treatment strategies of male infertility in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in patients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma were significantly (p < .001) lower in infertile men. Significant negative correlations were observed between MDA levels and sperm motility and normal morphology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men and significantly correlated with MDA levels and SOD and GPx activities. MDA of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut‐off limits to discriminate infertile patients from fertile men. The results show the leading role of oxidative stress in aetiology of male infertility in southwest Iran and indicate that evaluation of seminal antioxidant status and DNA integrity can be helpful in men attending infertility clinics during fertility assessment.     

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.     

Caffeine increased progressive motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples and enhanced activity of seminal creatine kinase

Even though the effect of caffeine on humans' health has been revealed in various research studies, its effect on semen quality has yet to be well explained. Here, we measured the effect of caffeine at 1, 5, 10 and 20 mM on motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples, level of seminal nitric oxide, chelation of seminal calcium ions and activity of seminal creatine kinase. Fifty-one normozoospermic and ten asthenozoospermic semen samples were recruited in this study. Sperm motility was evaluated by Makler counter, and seminal nitric oxide, seminal-free calcium and activity of seminal creatine kinase were measured spectrophotometrically. Caffeine at 10 mM significantly (p < .05) increased progressive motility of spermatozoa in both tested groups. Also, caffeine significantly increased (p < .05) activity of creatine kinase and insignificantly (p > .05) altered nitric oxide and free calcium levels in seminal plasma. In conclusion, progressive motility of human spermatozoa was found to be higher in the presence of caffeine at 10 mM in normozoospermic and asthenozoospermic semen samples; this increase, albeit partially, could be due to increased activity of seminal creatine kinase, but not to increased production of nitric oxide or chelation of free calcium ions.     

Assessment of seminal granulysin in infertile men with varicocele

Varicocele has a common association with male infertility, but its exact role is still debated. Apoptosis has been suggested as one of the mechanisms of varicocele‐associated infertility. Granulysin is a molecule that plays a role in apoptosis with no previous study about its role in male infertility. This case‐controlled study aimed to assess seminal plasma granulysin level in infertile patients with varicocele. This study involved 90 men that were allocated into fertile normozoospermic men (n = 20), infertile men without varicocele (n = 30) and infertile men with varicocele (n = 40). These men were subjected to history taking, clinical examination, semen analysis and estimation of seminal granulysin. In general, seminal granulysin level was significantly elevated in infertile men compared with fertile men. Infertile men with varicocele showed significantly higher seminal granulysin compared with infertile men without varicocele, in bilateral varicocele cases and in grade III varicocele. Seminal granulysin level was negatively correlated with sperm concentration, sperm motility, sperm normal forms percentage and testicular volumes. It is concluded that increased seminal granulysin has a negative impact on spermatogenesis in infertile men in general and in infertile men associated with varicocele in particular.     

"Null pattern" of immunoreactivity in a Lynch syndrome-associated colon cancer due to germline MSH2 mutation and somatic MLH1 hypermethylation

Lynch syndrome accounts for approximately 3% of newly diagnosed colorectal cancers and is caused by germline defects in DNA mismatch repair genes. Screening of patients for Lynch syndrome can be done by immunohistochemical staining for a panel of mismatch repair proteins and/or DNA testing for microsatellite instability. We describe a unique "null" immunophenotype in a Lynch syndrome associated colon cancer in a 71-year-old woman who also had a personal history of ureteral cancer and a strong family history of various malignancies. Immunohistochemical stains for MLH1, MSH2, PMS2, and MSH6 were completely negative in the tumor cells, with positive staining in stromal and inflammatory cells. Mutation analysis using peripheral blood showed a germline G587R mutation in the MSH2 gene. Further testing revealed the tumor to be positive for MLH1 promoter hypermethylation. Normal colonic mucosa adjacent to the tumor was negative for MLH1 promoter methylation. The lack of immunostaining for any of the 4 DNA mismatch repair proteins in this extremely unusual case was, therefore, related to a germline MSH2 mutation and somatic MLH1 promoter hypermethylation.     

Immunohistochemical staining for mismatch repair proteins, and its relevance in the diagnosis of hereditary non-polyposis colorectal cancer

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) arises mostly from germline mutations of the mismatch repair genes MSH2 and MLH1. The diagnosis of HNPCC is based on a set of clinical criteria that may be too restrictive to identify all affected patients. Immunohistochemical staining (IHC) for the mismatch repair proteins, MutS homologue 2 (MSH2) and MutL homologue 1 (MLH1), reliably identifies the microsatellite instability phenotype. This study evaluated the ability of IHC to detect germline mutations in an unselected group of patients with colorectal cancer (CRC). METHODS: All patients with CRC operated on between July 2000 and March 2003, and demonstrating a loss of protein, were contacted. Following informed consent, searchs for germline mutation and methylation of the promoter were performed on normal and tumoral DNA. RESULTS: Thirty patients agreed to participate, four of whom fulfilled the Amsterdam II criteria. Loss of expression of MLH1 was found in 20 patients, and loss of expression of MSH2 in ten patients. Four of the MLH1-deficient patients had a germline MLH1 point mutation (positive predictive value (PPV) 20 (95 per cent confidence interval (c.i.) 2 to 38 per cent) and 11 had promoter methylation. Seven of the MSH2-deficient patients had a germline MSH2 point mutation (PPV 70 (95 per cent c.i. 54 to 96 per cent), and none showed promoter methylation. CONCLUSION: MLH1-deficient patients who are young or have a positive family history of cancer should be referred for genetic testing and counselling, whereas MSH2-deficient patients should be counselled in the same way as patients with HNPCC.     

Relationship of semen hyperviscosity with IL‐6, TNF‐α, IL‐10 and ROS production in seminal plasma of infertile patients with prostatitis and prostato‐vesiculitis

Changes in levels of oxidative damage products in semen and their relationship to seminal fluid viscosity (SFV) have recently received increasing research interest. We analysed whether SFV was associated with ROS generation, levels of cytokines TNF‐alpha (TNF‐α), IL‐6 and IL‐10 and seminal leucocyte concentration, and whether ROS production was related to the extent of infections/inflammations at one (prostatitis) or two (prostato‐vesiculitis) male accessory glands. We studied 169 infertile patients, with chronic bacterial prostatitis (PR, n = 74) and/or bilateral prostato‐vesiculitis (PV, n = 95), as diagnosed by the ultrasound (US) criteria. Healthy fertile men (n = 42) served as controls. In the PV patient group, SFV, semen characteristics and ROS production had median values that were significantly higher than those found in PR patients and controls, although other sperm variables had values significantly lower than those found in PR patients or controls. In PV infertile patients, ROS generation and pro‐inflammatory cytokines levels were higher than those found in PR infertile patients and controls, although seminal IL‐10 levels in PV and PR patients were lower than those found in the controls. In PR patients, the levels of SFV were positively related to TNF‐α (r = 0.67; P < 0.01), fMLP‐stimulated ROS production in the 45% Percoll fraction (r = 0.687, P < 0.01) and the 90% Percoll fraction in basal condition (r = 0.695, P < 0.01), and after fMLP‐stimulation (r = 0.688, P < 0.01). Thus, our data indicated that seminal hyperviscosity is associated with increased oxidative stress in infertile men and increased pro‐inflammatory interleukins in patients with male accessory gland infection, more when the infection was extended to the seminal vesicles.     

Does Lepidium meyenii (Maca) improve seminal quality?

Maca (Lepidium meyenii) is a herbaceous plant grown at over 4,000 m in Peru. It has been studied worldwide for its properties on fertility. Previous studies have assessed maca effects on semen quality, but there is need of randomised, double-blind trials in order to make clinical decisions. The objective of this study was to assess the effect of maca on seminal parameters in infertile adult men. This is a double-blind, randomised, placebo-controlled pilot trial in which sixty-nine patients diagnosed with mild asthenozoospermia and/or mild oligozoospermia were supplied by maca (n = 35) or placebo (n = 34) (2 g/day) for a period of 12 weeks. When compared patients treated with maca and patients treated with placebo, there were no significant differences in semen volume (2.95 ± 0.52 vs. 2.90 ± 0.52; p = .392), sperm motility (22.34 ± 2.22 vs. 23.05 ± 2.22; p = .462) and normal sperm morphology (7.89 ± 1.89 vs. 7.04 ± 2.28; p = .801), but there was a significant difference in sperm concentration (15.04 ± 5.61 vs. 10.16 ± 3.59, respectively; p = .011). In conclusion, patients treated with 2 g of maca for a period of 12 weeks showed a significant improvement in seminal concentration compared with patients treated with placebo. There were no significant differences in semen volume, sperm mobility and morphology when compared both groups.