The correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele

This study aimed to assess the possible correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele. The study included sixty infertile males and fifty fertile males as controls. The infertile group was subdivided into the following subgroups: thirty males with varicocele and thirty males without varicocele. All subjects underwent medical history collection, clinical examination, semen analysis, sperm DNA integrity assessment, mTOR gene expression assessment and scrotal colour Doppler ultrasound. The mean mTOR gene expression in infertile patients with varicocele (23.52 ± 14.65) was significantly higher than that in infertile patients without varicocele (12.24 ± 12.44) and fertile control subjects (3.92 ± 3.26; p = 0.003 and p < 0.001 respectively). In the infertile varicocele‐positive group, mTOR gene expression showed a significant negative correlation with sperm count (p = 0.028, r = ?0.400) and progressive sperm motility (p = 0.038, r = ?0.381), as well as a significant positive correlation with the sperm DNA fragmentation index (DFI; p = 0.001, r = 0.578). In the infertile varicocele‐negative group, mTOR gene expression showed a significant negative correlation with progressive sperm motility (p = 0.018, r = ?0.429) and a significant positive correlation with sperm DFI (p < 0.001, r = 0.673). In conclusion, according to these results, there is a significant positive correlation between mTOR gene expression and sperm DFI among infertile patients with and without varicocele.     

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.     

Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD‐HaloSpermG2®) comparable?

Men affected with idiopathic infertility often display basic spermiogramme values similar to fertile individuals, questioning the diagnostic impact of the World Health Organization (WHO) thresholds used. This study explored sperm DNA fragmentation in single ejaculates from 14 fertile donors and 42 patients with idiopathic infertility providing semen for assisted reproductive techniques in a university fertility clinic. Each ejaculate was simultaneously studied for sperm DNA fragmentation by the flow cytometer‐based sperm chromatin structure analysis (SCSA) and the new light‐microscopy‐based sperm chromatin dispersion assay (SCD‐HaloSpermG2^®), before and after sperm selection for in vitro fertilisation with a colloid discontinuous gradient. The WHO semen variables did not differ between groups, but DNA fragmentation after SCSA (DFI) or SCD (SDF) was significantly (p < 0.05) higher in patients (DFI: 40.2% ± 3.0 vs. SDF: 40.3% ± 1.4) than in fertile donors (DFI: 17.1% ± 2.1 vs. SDF: 20.9% ± 2.5). Sperm selection led to lower proportions of DNA‐fragmented spermatozoa (DFI: 11.9 ± 1.7 vs. SCD: 10.0 ± 0.9, p < 0.05). The techniques output correlated highly and significantly (r² = 0.82). DNA fragmentation is confirmed as a relevant variable for scrutinising patients with idiopathic infertility, beyond the evidently insufficient WHO semen analyses. Since both techniques yielded similar results, the reduced necessity of complex equipment when running SCD ought to be considered for a clinical setting.     

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.     

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.     

Can leukocytospermia predict prostate cancer via its effects on mitochondrial DNA?

Leukocytospermia was previously reported to affect sperm quality by the production of reactive oxygen species (ROS) leading to oxidative stress (OS). In turn, OS decreases sperm functional integrity, increases sperm DNA damage and ultimately alters fertility status. To elucidate the impact of leukocytospermia on sperm nuclear DNA integrity and mitochondrial DNA (mtDNA) structure, we conducted a study including 67 samples from infertile patients with low level of leucocytes (Group 1: n = 20) and with leukocytospermia (Group 2: n = 47). In addition to standard sperm parameters’ assessment, we measured the levels of inflammation biomarkers [interleukin-6 (IL-6) and interleukin-8 (IL-8)] and evaluated the oxidative status [malondialdehyde (MDA) and enzymatic and non-enzymatic antioxidants]. In addition, we evaluated the level of sperm nuclear DNA fragmentation and analysed mitochondrial DNA (mtDNA) of sperm cells by sequencing of 5 genes [cytochrome oxidase I (COXI), cytochrome oxidase II (COXII), cytochrome oxidase III (COXIII), adenosine triphosphate synthase 6 (ATPase 6) and adenosine triphosphate synthase 8 (ATPase 8)]. As expected, patients with leukocytospermia had significantly higher MDA levels (32.56 ± 24.30 nmole/ml) than patients without leukocytospermia (17.59 ± 9.60 nmole/ml) (p < .018). Also, sperm DNA fragmentation index (DFI) was significantly higher in Group 2 (33.05 ± 18.14%) as compared to Group 1 (14.19 ± 9.50%) (p < .001). The sequencing of mtDNA revealed a high number of substitutions in Group 2 (n = 102) compared to Group 1 (n = 5). These substitutions were observed mainly in COXI. Among COXI substitutions found in Group 2, twelve changes were previously described in patients with prostate cancer and six of them were shown associated with this pathology. These findings suggest that leukocytospermia may predispose to the manifestation of prostate cancer through modification of mitochondrial DNA and this may be promoted by OS.     

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.     

In vitro effects of clomiphene citrate on sperm parameters and fertilisation rate in male NMRI mice

Clomiphene citrate (CC), as a medication in male infertility, improves the sperm parameters in oral consumption but various detrimental side effects have been reported including testicular tumours, gynaecomastia, skin allergic reactions and ocular symptoms. Therefore, this study was designed to evaluate the in vitro effects of CC on sperm parameters and fertilisation rate in IVF protocol. Sperm samples of NMRI adult mice were divided into six groups: group 1 received no treatment (control group), while groups of 2, 3, 4, 5 and 6 (experimental groups) were incubated with the doses of 0.001, 0.01, 0.1, 1 and 10 µg/ml of CC in culture medium respectively. Sperm parameters (viability, morphology and motility), DNA fragmentation levels and fertilisation rate in IVF were evaluated. The results demonstrated that the doses of 0.1 µg/ml (p = .000007 for viability and p = .00006 for fertilisation rate) and 1 µg/ml (p = .032 for viability and p = .005 for fertilisation rate) CC cause a significant improvements; also, the dose of 0.1 µg/ml CC found effective on sperm motility (p = .0003). In the field of IVF, the application of 0.1 and 1 µg/ml of CC in the culture medium may improve the sperm parameters in IVF protocol with no side effects.     

Impact of intra-operative doppler ultrasound assistance during microsurgical varicocelectomy on operative outcome and sperm parameters

The microsurgical varicocelectomy is the gold standard treatment with a low recurrence rate and less postoperative complications. We compared the surgical outcomes and difficulty in intra-operative vascular Doppler ultrasound-assisted microscopic varicocelectomy (IVDU-MV) with MV in primary and recurrent varicocele. A total of 228 infertile patients with clinically palpable varicocele were included in the study. One hundred fifteen patients were operated on with the standard MV approach, whereas the other 113 patients were operated on with IVDU-MV. Perioperative outcomes, sperm parameters and operative difficulty of the procedure were evaluated. The operative times were significantly shorter for the IVDU-MV group for primary and recurrent varicocele (p = .001). Mean number of veins ligated for primary and recurrent varicocele was significantly higher in the IVDU-MV group than in the MV group (6 ± 1.4 vs. 4.8 ± 1.8 and 3.7 ± 0.9 vs. 2.9 ± 1.2; p < .01). The increase in mean sperm motility was significantly higher in the IVDU-MV group for both primary and recurrent varicocelectomy patients (p < .05). A significant number of IVDU-MV procedures were described as easy in both primary and recurrent varicocelectomy procedures (p = .006). The use of Doppler ultrasound(US) revealed advantages in ligating veins, preserving arteries and improving sperm motility and facilitates the operation for the surgeon, especially during recurrent varicocele repair .     

The changes in semen quality,arginase activity and nitric oxide level in dexamethasone-treated rams

This study was made to investigate the effects of intramuscular administrations of dexamethasone on seminal plasma nitric oxide levels and arginase activity, and some spermatological parameters in rams. Ten Akkaraman rams weighing 50–60 kg and 2 years old were used as material in this study. The study was performed during the breeding season (September–November) for rams. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th, 72nd and 96th hours for control group before dexamethasone administration. For treatment group, 0.25 mg/kg dexamethasone was administered and semen was collected at the time points described for control group. Spermatological characteristics of semen samples (semen volume, pH, sperm motility, density and abnormal sperm rate), seminal plasma arginase enzyme activities and nitric oxide levels were determined. It was determined that the administration of dexamethasone was detected to decrease seminal plasma arginase activity (p < .05 and .01) and nitric oxide level (p < .05), semen volume (p < .05 and .01), mass activity (p < .05 and .01), sperm density (p < .05) and sperm motility (p < .05 and .01), and to increase abnormal sperm rate (p < .05 and .01). In conclusion, dexamethasone is not recommended to be used during the breeding season as it damages the sperm quality of the rams.     

Assessment of seminal mast cells in infertile men with varicocele after surgical repair

This study aimed to assess seminal mast cells in infertile men associated with varicocele (Vx) pre‐ and post‐surgical repair. Forty‐five infertile men associated with Vx were subjected to history taking and clinical examination. In addition, semen parameters and seminal mast cells stained with 1% toluidine blue were estimated pre‐varicocelectomy and three months post‐varicocelectomy. Vx surgical repair revealed a significant improvement in the mean sperm concentration, progressive sperm motility, total sperm motility and sperm abnormal morphology and a significant decrement in seminal mast cells (mean ± SD, 3.56 ± 2.23 cells per high‐power field (HPF) vs. 2.22 ± 1.06 cells per HPF, p = .01). The pre‐operative mean mast cell count demonstrated significant increases in cases with Vx grade III compared with other Vx grades and in cases with bilateral Vx compared with unilateral Vx cases. Seminal mast cells demonstrated a significant correlation with sperm concentration, progressive sperm motility and total sperm motility and a nonsignificant correlation with age and sperm abnormal morphology. It is concluded that seminal mast cells decrease significantly in infertile men with Vx after surgical repair showing a significant negative correlation with sperm concentration, progressive sperm motility and total sperm motility.     

Sperm binding to hyaluronan is an excellent predictor of Nili-Ravi buffalo bull fertility

This study reports the first evaluation of sperm hyaluronan binding assay (HBA) for predicting the fertility of Nili-Ravi buffalo bulls in relation to standard parameters of sperm quality. Cryopreserved semen doses of low (n = 6), medium (n = 3) and high fertility (n = 8) bulls based on their respective return rates were used. Significantly, more spermatozoa bound to hyaluronan from the most fertile bulls (57.15% ± 1.44) compared with medium (42.46% ± 1.08) and low fertility bulls (29.70% ± 0.78). A strongly positive correlation (r = .824, p < .01) was found between HBA and fertility that predicts a 67.9% variability (r² = .679, p < .01) in fertility. HBA was also strongly positively correlated with sperm viability (r = .679, p < .01) followed by their live/dead ratio (r = .637, p < .01), uncapacitated spermatozoa (r = .631, p < .01), normal apical ridge (r = .459, p < .01), motility (r = .434, p < .01), mature spermatozoa with low residual histones (r = .364, p < .01), high plasma membrane integrity (r = .316, p < .01) and nonfragmented DNA levels (r = .236, p < .05). It was negatively correlated with spermatozoa having reacted acrosome (r = −.654, p < .01). A fertility model built using a combination of sperm HBA and either sperm livability or viability predicts, respectively, 86.1% (r² = .861, p < .01) and 85.9% (r² = .859, p < .01) variability in buffalo bull fertility. In conclusion, sperm HBA may prove to be a single robust predictor of Nili-Ravi buffalo bull fertility.     

Total antioxidant status and lipid peroxidation with and without in vitro zinc supplementation in infertile men

The aim of this study was to assess the total antioxidant capacity (TAC) and malondialdehyde (MDA) level in infertile men with asthenozoospermia and asthenoteratozoospermia compared to fertile donors, and to examine the effect of zinc on sperm lipid peroxidation and antioxidant status in infertile and fertile men. Semen samples provided by infertile men (n = 38) and fertile donors (controls; n = 12) were exposed to 6 mmol/L of zinc for 2 hr at 37°C. After semen analysis, lipid peroxidation was detected by MDA assay and seminal TAC was assessed by colorimetric method using TAS (total antioxidant status) Kit. TAC was significantly lower in infertile group compared to controls (p = .037). However, lipid peroxidation did not alter in infertile patients compared to controls (p > .05). After in vitro incubation of samples with zinc, a significant increase in TAC level was found only in infertile men (p < .001). Meanwhile, zinc had no effect on sperm lipid peroxidation in both fertile and infertile men (p > .05). Our data indicate that antioxidant treatment based on zinc in vitro supplementation may be helpful to enhance the rate of seminal antioxidant status in infertile men; however, it does not prevent sperm lipid peroxidation.     

Evaluation of the role of L-arginine on spermatological parameters,seminal plasma nitric oxide levels and arginase enzyme activities in rams

The purpose of this study is to investigate the effects of L-arginine on spermatological parameters, seminal plasma nitric oxide levels and arginase enzyme activities. Fertile rams that are 2–3 years old and weighing 50–60 kg were used as material. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th, 72nd, 96th and 120th hours for the control group before L-arginine administration. For treatment groups, L-arginine was administered intraperitoneally at a dose of 5 mg kg⁻¹ bw⁻¹ and semen was collected at the time point described for the control group. Spermatological characteristics of semen samples (semen volume, pH, sperm motility, concentration and abnormal sperm rate), seminal plasma nitric oxide levels and arginase enzyme activities were determined. Increased seminal plasma nitric oxide level (p < .01), seminal plasma arginase activity (p < .01), semen volume (p < .05), semen mass activity (p < .05), sperm motility (p < .05) and concentration (p < .01) and decreased abnormal sperm rate (p < .05 and p < .01) were observed by L-arginine administration. In conclusion, it may be concluded that L-arginine application in rams during the breeding season may have positive effects on rams' reproductive performance.     

Elevated sperm DNA fragmentation does not predict recurrent implantation failure

In this study, we sought to determine whether sperm DNA fragmentation (DFI%) and high DNA stainability (HDS%) evaluated by sperm chromatin structure assay (SCSA) predict recurrent implantation failure (RIF) or pregnancy rate. A retrospective study was performed of consecutive cycles of ICSI treatment from 2009 to 2018. A total of 386 couples that underwent 1,216 frozen embryo transfer (FET) cycles were analysed. Mean female and male age was 34 ± 3.6 years and 37.3 ± 6.6 years, respectively, and a median total motile sperm count (TMSC) was 43.5 [9.9–105.5] million. Overall median DFI% and HDS% was 12 [7.1–18.9] and 9.6 [6.5–14.4] respectively. On multivariable analysis, DFI% and HDS% were not associated with RIF (DFI%: OR = 1.01, 95% CI: 0.98–1.04, p = .414; HDS%: OR = 0.97, 95% CI: 0.94–1.01, p = .107) or IVF success, defined as clinical pregnancy (DFI%: OR = 1.00, 95% CI: 0.99–1.01, p = .641; HDS%: OR = 1.01, 95% CI: 0.99–1.02, p = .565). We found that neither DFI% or HDS%, as assessed by SCSA, were predictive of RIF or pregnancy rate. This finding suggests that sperm DNA fragmentation does not predict RIF or pregnancy rate.     

Oxidative stress-related miRNAs in spermatozoa may reveal the severity of damage in grade III varicocele

Varicocele is associated with excessive production of reactive oxygen species (ROS). Although the harmful effects of ROS on sperm DNA, proteins and lipids are well documented, its impact on the expression of miRNAs in spermatozoa has not been fully understood. In this study, the expression patterns of microRNAs (miRNAs), miR-21, miR-34a and miR-122a as well as the level of ROS in the fertile control (FC; proven fertility without varicocele, n = 15) and grade III varicocele patients with normal (VN; n = 15) and abnormal (VA; n = 15) spermogram were investigated. The real-time PCR was performed to analyse the expression of the miRNAs, while oxidative stress was evaluated by measuring the concentrations of MDA. Our results showed that the expression levels of miR-21 (p = .001), miR-34a, (p = .007) and miR-122a (p < .001) were significantly decreased in spermatozoa of VN and VA patients in comparison with the fertile group. Also, increased levels of oxidative stress were detected in semen samples of varicocele patients compared with the fertile control (p < .0001). Overall, these findings demonstrate oxidative stress changes the expression pattern of some miRNAs, and these alterations could be a valuable diagnostic marker for the diagnosis and prognosis of varicocele-induced oxidative stress to retain the male fertility during the spermatogenesis process.     

Microencapsulation of human spermatozoa increases membrane stability and DNA longevity

The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation on human sperm membrane integrity (viability) and sperm DNA fragmentation (SDF) following storage for 24 hr at 37°C. The cumulative sperm viability (Log-rank, Mantel–Cox; Chi-square = 114.95, p = .000) and cumulative sperm DNA fragmentation (Log-rank, Mantel–Cox; Chi-square = 187.86, p = .000) of encapsulated spermatozoa were substantially improved when compared to control spermatozoa. Significant differences in the dynamic behaviour of different individuals were only apparent for sperm viability in microencapsulated samples (p = .021) while no significant differences were observed in control spermatozoa (p = .245); the equivalent comparison for SDF showed no differences (control p = .320; microencapsulated p = .432). We present potential scenarios for the use of microencapsulated human spermatozoa in reproductive medicine.     

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients’ samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.     

High levels of oxidation–reduction potential in frozen-thawed human semen are significantly correlated with poor post-thaw sperm quality

This study examined the relationship between oxidation–reduction potential (ORP) in frozen-thawed semen and the post-thaw sperm parameters. Levels of ORP were measured in 25 samples from men presenting for routine infertility work-up and were expressed as millivolt (mV)/10⁶ sperm/ml. Frozen-thawed samples were examined for post-thaw total motility (TM%), progressive motility (PM%), total sperm count (TSC) and ORP. The cryo-survival rate (CSR) was calculated as post-thaw TM/pre-freeze TM × 100. Data are provided as median and interquartile range (25th and 75th percentiles). The post-thaw TM% (10.0% [4.00%, 15.1%]), PM% (5.88% [2.97%, 9.33%]) and TSC (12.5 [10.0, 17.5] × 10⁶ sperm) were significantly lower than the pre-freeze TM% (45.9% [32.9%, 59.1%], PM% (31.5% [24.4%, 40.0%] and TSC (120 [90, 250] ×10⁶ sperm) (p < .001). Post-thaw ORP (2.62 [2.52, 3.13] mV/10⁶ sperm/ml) was significantly higher than pre-freeze ORP (0.73 [0.54, 1.21] mV/10⁶ sperm/ml; p < .001). The CSR was 21.7% (11.3%, 31.9%). The post-thaw seminal ORP was negatively correlated with post-thaw TM% (r = −.5; p = .02), PM% (r = −.41; p = .03), TSC (r = −.60; p = .03) and CSR (r = −.52; p = .01). Increased levels of ORP are significantly correlated with poor post-thaw sperm quality and CSR.     

Seminal cell-free DNA and sperm characteristic’s: An added biomarker for male infertility investigation

Cell-free DNA (Cf-DNA) fragments may constitute an easy-to-measure molecular tool for guiding the choice of care provided to infertile couples who benefit assisted reproductive technology (ART) programmes. Data on Cf-DNA levels in the seminal plasma of men with sperm alterations are scarce. The objective of the present study was to quantify the presence of Cf-DNA in semen by using a quantitative real-time PCR. We compared men with abnormal sperm characteristics (n = 21) with normospermic controls (n = 21). The PCR assay evidenced significantly higher mean Cf-DNA levels in patients with sperm abnormalities than in controls (2.09 versus 1.18 µg/ml, respectively; p = .0003). The Cf-DNA levels were notably higher in men with azoospermia (3.65 µg/ml, versus 1.34 µg/ml in matched controls; p = .03) and men with teratozoospermia (1.80 µg/ml, versus 1.29 µg/ml in matched controls; p = .008). Our data report a significant association between elevated Cf-DNA levels and sperm abnormalities. These results may open up new diagnostic and prognostic perspectives in male infertility.