Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

Altered circadian clock gene expression in the sperm of infertile men with asthenozoospermia.

PURPOSE: Male infertility is a complex multifactorial pathological condition, and asthenozoospermia (AZS) is one of the most common causes. Current evidence suggests the underlying role of the circadian clock on male fertility. This study aims to evaluate the expression levels of five principal clock genes in the sperm and their correlations with the sperm parameters in male infertility. METHODS: We determined the expression profiles of BMAL1, CLOCK, CRY1, PER1, and PER2 in the sperm of infertile men with AZS (n=38) and healthy fertile men (n=40) using quantitative real-time PCR. Then we performed comprehensive association analyses on the clock gene levels and the sperm parameters, including progressive and total motility, concentration, and normal morphology of the sperm. RESULTS: Our results showed that the expression levels of five clock genes (BMAL1, CLOCK, CRY1, PER1, and PER2) are significantly decreased in the sperm of the infertile men with AZS as compared with that of healthy fertile men (P< 0.01). All five clock gene levels are associated with the percentage of progressive/total sperm motility (r= 0.546/0.589~0.677/0.695, P< 0.01). We also discovered that a combination of BMAL1, CLOCK, CRY1, PER1, and PER2 could reach a high diagnostic performance (areas under the curves, 92%) for infertility with AZS. CONCLUSIONS: This study first reports that sperm BMAL1, CLOCK, CRY1, PER1, and PER2 levels are altered in AZS and may be molecular markers for male infertility with AZS. These findings indicate the possibility of stabilizing circadian rhythmicity through therapeutic intervention on clock genes to prevent and treat infertility.

A novel posttranslational modification of histone, H3 S-sulfhydration, is down-regulated in asthenozoospermic sperm.

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H2S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

Effect of Letrozole on sperm parameters, chromatin status and ROS level in idiopathic Oligo/Astheno/Teratozoospermia.

BACKGROUND: This study investigates the effect of letrozole on hormone profiles, semen parameters, body mass index (BMI), degree of oxidative stress and sperm chromatin integrity in men with idiopathic oligo/astheno/teratozoospermia (iOAT) and T:E2 ratio ≤ 10. MATERIALS AND METHODS: This study is a longitudinal, prospective, interventional and open-labelled clinical trial. Semen samples were collected from 20 iOAT men with low serum testosterone (T) to estradiol (E2) ratio (T:E2 ratio ≤ 10). The participants were treated with 2.5 mg letrozole orally per day for 3 months. Then, sperm parameters, hormone profiles, BMI, chromatin integrity and intracellular reactive oxygen species (ROS) level were assessed pre- and post- treatment. The chromatin integrity was evaluated by assessment of DNA fragmentation (with TUNEL assay) and protamine deficiency (with Chromomycin A3, CMA3). Also, the intracellular ROS levels were investigated by 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Finally, the differences between the parameters evaluated before and after letrozole treatment were analyzed with the t-test and the Wilcoxon signed-rank test. RESULTS: Sperm concentration, percentage of sperm motility and its normal morphology increased significantly after letrozole treatment. Moreover, serum testosterone level increased but estradiol level decreased significantly following treatment. The mean of T:E2 ratio improved 1600%. Also, letrozole treatment significantly reduced the percentage of sperm TUNEL positivity and sperm CMA3 positivity. While no significant difference was observed between intracellular ROS levels and BMI before and after treatment. Finally, as a notable result, four spontaneous pregnancies (20%) were achieved after treatment. CONCLUSIONS: Letrozole treatment can effectively increase spontaneous pregnancies by improving sperm parameters and sperm chromatin integrity in men with iOAT and T:E2 ratio ≤ 10. TRIAL REGISTRATION: Trial registration: IRCT, IRCT20191030045283N1. Registered 16 November 2019 - Retrospectively registered, https://fa.irct.ir/user/trial/43484/view.

DAAM1 and PREP are involved in human spermatogenesis.

During differentiation of the male gamete, there is a massive remodelling in the shape and architecture of all the cells in the seminiferous epithelium. The cytoskeleton, as well as many associated proteins, plays a pivotal role in this process. To better characterise the factors involved, we analysed two proteins: the formin, dishevelled-associated activator of morphogenesis 1 (DAAM1), which participates in the regulation of actin polymerisation, and the protease, prolyl endopeptidase (PREP), engaged in microtubule-associated processes. In our previous studies we demonstrated their involvement in cytoskeletal dynamics necessary for correct postnatal development of the rat testis. Here, we used samples of testicular tissue obtained from infertile men by testicular sperm extraction and the spermatozoa of asthenoteratozoospermic patients. By western blot and immunofluorescent analysis, we found that DAAM1 and PREP expression and localisation were impaired in both the testis and spermatozoa, and in particular in the midpiece as well as in the principal and end-pieces of the flagella, as compared with spermatozoa of normospermic men. Our results provide new knowledge of the dynamics of spermatogenesis, raising the possibility of using DAAM1 and PREP as new markers of normal fertility.

Isoform-specific GSK3A activity is negatively correlated with human sperm motility.

In mouse and bovine sperm, GSK3 activity is inversely proportional to motility. Targeted disruption of the GSK3A gene in testis results in normal spermatogenesis, but mature sperm present a reduced motility, rendering male mice infertile. On the other hand, GSK3B testis-specific KO is fertile. Yet in human sperm, an isoform-specific correlation between GSK3A and sperm motility was never established. In order to analyze GSK3 function in human sperm motility, normospermic and asthenozoospermic samples from adult males were used to correlate GSK3 expression and activity levels with human sperm motility profiles. Moreover, testicular and sperm GSK3 interactomes were identified using a yeast two-hybrid screen and coimmunoprecipitation, respectively. An extensive in-silico analysis of the GSK3 interactome was performed. The results proved that inhibited GSK3A (serine phosphorylated) presents a significant strong positive correlation (r = 0.822, P = 0.023) with the percentage of progressive human sperm, whereas inhibited GSK3B is not significantly correlated with sperm motility (r = 0.577, P = 0.175). The importance of GSK3 in human sperm motility was further reinforced by in-silico analysis of the GSK3 interactome, which revealed a high level of involvement of GSK3 interactors in sperm motility-related functions. The limitation of techniques used for GSK3 interactome identification can be a drawback, since none completely mimics the physiological environment. Our findings prove that human sperm motility relies on isoform-specific functions of GSK3A within this cell. Given the reported relevance of GSK3 protein-protein interactions in sperm motility, we hypothesized that they stand as potential targets for male contraceptive strategies based on sperm motility modulation.

SCSA results correlated with rate of motility reduction after ejaculation in Asthenozoospermia.

Maintaining sperm motility after ejaculation is important for fertilisation. Apoptosis may play an important role to reduce sperm motility after ejaculation. The aim of this study was to perceive whether or not an increase in apoptosis reduces sperm motility in a higher degree after ejaculation and whether it can be predicted by laboratory tests, such as sperm chromatin structure assay (SCSA). Fifty-one Asthenozoospermia and 20 fertile subjects participated in this study. SCSA was applied using flow cytometry. Fluorescein-labelled inhibitors of Caspases (FLICA) method was used for assessment of active Caspase-3. Motility was assessed every 2 hr after ejaculation for 12 hr. Both SCSA and spermatozoa with active Caspase-3 were significantly correlated with the rate of motility reduction after ejaculation. In the subgroups who had SCSA <27% and active Caspase-3 <40%, the sperm motility reduction significantly occurred 6-8 hr after ejaculation compared to the fresh sample. In the cases of SCSA ≥27% and active Caspase-3 ≥ 40%, a significant decrease in motility was observed between 2 and 4 hr after ejaculation. The result demonstrated a significant trend in the rate of sperm motility reduction with SCSA increase, which suggests SCSA may indirectly show a good scheme of apoptosis status and may forecast the rate of motility reduction after ejaculation in Asthenozoospermia.

Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity.

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca2+ efflux pump, PMCA4. This is the major Ca2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca2+ ] in capacitated and Ca2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca2+ -dependent manner. In Pmca4-/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4-/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.

Disruption of Ssp411 causes impaired sperm head formation and male sterility in mice.

BACKGROUND: We previously cloned the Ssp411 gene. We found that the Ssp411 protein is predominantly expressed in elongated spermatids in the rat testis in a stage-dependent manner. Although our findings strongly suggested that Ssp411 might play an important role in mammalian spermatogenesis, this hypothesis has not been studied. METHODS: We first used real-time PCR, Western blotting and immunohistochemistry to confirm that the expression pattern of Ssp411 in several murine tissues is similar to its expression pattern in corresponding rat tissues. To better understand the roles of Ssp411 in male reproduction in vivo, we identified and characterized an Ssp411 expression-disrupted murine strain (Ssp411PB/PB) that was generated by piggyBac (PB) transposon insertion. We studied Ssp411-interacting proteins using proteome microarray, co-IP and GST pull-down assay. RESULTS: Both Ssp411 mRNA and protein were detected exclusively in spermatids after step 9 during spermiogenesis in testis. Phenotypic analysis suggested that only Ssp411PB/PB males are sterile. These males have smaller testes, reduced sperm counts, decreased sperm motility and deformed spermatozoa. Microscopy analysis indicated that the manchette, a structurally reshaped sperm head, is aberrant in Ssp411PB/PB spermatids. The results of proteome microarray analysis and GST pull-down assays suggested that Ssp411 participates the ubiquitin-proteasome system by interacting with PSMC3. This has been reported to be manchette-associated and important for the head shaping of spermatids. CONCLUSIONS: Our study suggested that Ssp411 is required for spermiogenesis. It seems to play a role in sperm head shaping. The lack of Ssp411 causes sperm deformation and results in male infertility. GENERAL SIGNIFICANCE: Ssp411PB/PB mouse strain is an animal model of idiopathic oligoasthenoteratozoospermia (iOAT), and the gene may represent a therapeutic target for iOAT patients.

Fluorescence in situ hybridisation sperm examination is significantly impaired in all categories of male infertility.

To study the outcome of FISH sperm examination in cases with sperm pathology and outline the potential correlation with certain chromosomal defects. A retrospective study of prospectively collected data was performed in IAKENTRO, Infertility Treatment Center. Rates of abnormal FISH semen examination were compared between male infertility patients and fertile controls. Detection of abnormal FISH semen examination as well as each chromosomal abnormality detected was correlated with each sperm deficiency (asthenozoospermia, oligozoospermia and teratozoospermia) in a univariate regression model. There were 72 male partners included, of which 52 male infertility patients and 20 controls. The rate of abnormal sperm FISH examination was significantly higher in patients' group (55.8% vs. 15.0% for controls, p = .002). Asthenozoospermia, oligozoospermia and teratozoospermia were significantly correlated with detection of abnormal FISH examination (p = .004, p = .01 and p < .001 respectively). Teratospermia was significantly correlated with increased aneuploidy rate for chromosome 17 (p = .005), chromosome X (p = .05) and Y (p = .03). FISH examination reveals pathology in a significant proportion of patients with sperm defects and should be recommended to achieve early detection of chromosomal defects that may postpone favourable reproductive outcome.

Effect of in vitro selenium supplementation on sperm quality in asthenoteratozoospermic men.

Sperm DNA damage, excessive oxidative stress and decrease in motility may lead to low fertilisation or poor assisted reproductive techniques outcomes in asthenoteratozoospermic men. Selenium was considered as essential element for male reproductive functions. Selenium has important role in enzymatic process for elimination of excessive reactive oxygen species and helps to maintain membrane integrity. The aim of this study was to determine the effect of selenium supplementation on sperm quality, DNA fragmentation, mitochondrial membrane potential and membrane lipid peroxidation during sperm sampling in vitro at different times. In this experimental study, semen samples were collected from 50 asthenoteratozoospermic men. Samples were divided into two groups as control group and test group (incubated with 2 µg/ml selenium at 37°C for 2, 4 and 6 hr). Motility and viability were assessed based on WHO 2010 criteria. Mitochondrial membrane potential, sperm DNA fragmentation and malondialdehyde levels were evaluated in each group. Results revealed that motility, viability and mitochondrial membrane potential were significantly higher in the test group (p < .05). Also malondialdehyde levels were significantly lower in the test group (p < .03). DNA fragmentation significantly decreased in the test group after 6 hr of incubation (p < .02). In conclusion, in vitro selenium supplementation may protect spermatozoa from maltreatment effect of reactive oxygen species (ROS) during sperm sampling via keeping enzymatic and antioxidant process in optimum condition.

Changes of Sperm Parameters Along Time Among Groups of Different Qualities.

BACKGROUND: Male infertility is solely responsible for approximately 20% of all infertility in couples. Various factors have been proposed as having a negative effect on sperm quality; however, the reasons for the global decline in sperm parameters during the last few decades are still controversial. OBJECTIVES: To investigate the fluctuations of semen parameters (sperm concentration, motility, and morphology) in three sperm quality groups and to examine the trends of those parameters in the same men over time. RESULTS: Our data showed deterioration in all semen parameters assessed in the group of men originally considered as having normal semen values according to the 2010 criteria of the World Health Organization. In contrast, we found significant improvement over time in all semen parameters in the group of men with severe oligo-terato-asthenozoospermia. CONCLUSIONS: Our results suggest that, although there were changes in sperm quality over time in the groups assessed, the clinical significance is negligible and does not necessarily justify a change in the therapeutic approach to infertility or sperm cryopreservation.

Thiols of flagellar proteins are essential for progressive motility in human spermatozoa.

Male infertility is a disorder of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse. The presence of low-motile or immotile spermatozoa is one of many causes of infertility; however, this observation provides little or no information regarding the pathogenesis of the malfunction. Good sperm motility depends on correct assembly of the sperm tail in the testis and efficient maturation during epididymal transit. Thiols of flagellar proteins, such as outer dense fibre protein 1 (ODF1), are oxidised to form disulfides during epididymal transit and the spermatozoa become motile. This study was designed to determine how oxidative changes in protein thiol status affect progressive motility in human spermatozoa. Monobromobimane (mBBr) was used as a specific thiol marker and disruptor of sperm progressive motility. When mBBr was blocked by dithiothreitol it did not promote motility changes. The analysis of mBBr-treated spermatozoa revealed a reduction of progressive motility and an increased number of spermatozoa with non-progressive motility without affecting ATP production. Laser confocal microscopy and western blot analysis showed that one of the mBBr-positive proteins reacted with an antibody to ODF1. Monobromobimane fluorescence intensity of the sperm tail was lower in normozoospermic than asthenozoospermic men, suggesting that thiol oxidation in spermatozoa of asthenozoospermic men is incomplete. Our findings indicate that mBBr affects the thiol status of ODF1 in human spermatozoa and interferes with progressive motility.

Application of testosterone supplementation in semen to improve sperm motility in asthenozoospermic males.

PURPOSE: To evaluate the effect of different concentrations and durations of seminal testosterone supplementation upon the motility of sperm from asthenozoospermic males. METHODS: Semen was collected from 41 infertile men with asthenozoospermia. After liquefaction, 200 µL was extracted from each semen sample and divided equally into five groups for a negative control, a vehicle control, and three experimental portions mixed with 4.75, 7.75, and 17.75 nmol/L of testosterone, respectively. The sperm motility was evaluated at 5, 15, 30, and 45 min following the addition of testosterone. The supernatant from remaining samples were sent for testosterone assay. Sperm viability was also evaluated after 45 min. RESULTS: There was no difference in the number of samples in each group which showed a 20% improvement in sperm motility. Group 3 showed a significant retardation in the reduction of motility compared with Group 5 (P < 0.05). Semen samples with a final testosterone concentration of 4.51-10 nmol/L showed a significant improvement in sperm motility when measured 5 min after addition. In contrast, samples showing a rise in testosterone level above 10 nmol/L were associated with a reduction in both sperm motility and viability. CONCLUSION: Despite sperm motility decreasing over time, supplementation of semen samples with 4.75 nmol/L of testosterone could delay such reduction. A final seminal testosterone concentration of 4.51-10 nmol/L appears to be optimal for the best sperm motility.

[In vitro culture medium for sparse spermatozoa improves human sperm motility].

OBJECTIVE: To investigate whether in vitro culture medium (IVCM) for sparse spermatozoa can improve human sperm motility for the purpose of helping clinicians, laboratorians and patients choose a better strategy of assisted reproduction. METHODS: Semen samples were obtained from 178 males for routine semen examination from March to August 2016, including 151 cases of asthenozoospermia and 27 cases of normal sperm motility. A total of 200 µl was collected from each sample and divided into two equal portions and equal volumes of IVCM (experimental group) and F10 (1×) (control group) were added to the two portions, respectively, followed by 30-minute incubation at 37℃ in an incubator with 5% CO2. Sperm concentration, motility and viability and the percentages of progressively motile, non-progressively motile and immotile sperm were recorded before and after incubation. RESULTS: After activated with IVCM, neither the samples with asthenozoospermia nor those with normal sperm motility showed any statistically significant difference in sperm viability from the baseline or the control group (P>0.05). The rates of progressively and non-progressively motile sperm from the asthenozoospermia males were increased by 14.02% and 4.86% respectively, while that of immotile sperm decreased by 19.01% in the experimental group (P >0.01), and similar results were observed in the semen samples from the men with normal sperm motility. The percentage of reduced immotile viable sperm was positively correlated with that of immotile viable sperm in both the asthenozoospermia patients (r = 0.260, P <0.01) and the men with normal sperm motility (r = 0.679, P <0.01). CONCLUSIONS: IVCM can increase sperm motility without affecting sperm viability in men with either asthenozoospermia or normal sperm motility. The larger the proportion of immotile viable sperm, the higher the percentages of progressively and non-progressively motile sperm in the semen after IVCM activation, and this correlation is more significant in men with normal sperm motility than in asthenozoospermia patients.

[Expressions of ODF2 mRNA and protein are down-regulated in the sperm of asthenospermia patients].

OBJECTIVE: To investigate the mRNA and protein expressions of outer dense fiber 2 (ODF2) in the sperm of the asthenospermia patient and their differences from those in normal healthy men. METHODS: According to the WHO criteria, we collected semen samples from 45 asthenozoospermia patients and 15 normal healthy volunteers. Using computer-assisted sperm analysis (CASA), we divided the semen samples from the asthenospermia patients into a mild, a moderate and a severe group, and determined the mRNA and protein expressions of ODF2 in different groups by RT-PCR and Western blot. RESULTS: Compared with the normal healthy men, the expression of the ODF2 gene showed no statistically significant difference in the mild asthenospermia group (1.112 0 ± 0.525 5 vs 0.688 0 ± 0.372 0, P >0.05) but remarkably decreased in the moderate (0.483 3 ± 0.186 3, P <0.05) and severe asthenospermia patients (0.448 3 ± 0.340 8, P <0.01). The OD value (ODF2/ß-actin) of the ODF2 protein in the normal men exhibited no statistically significant difference from that in the mild asthenospermia group (0.458 7 ± 0.052 1 vs 0.326 1 ± 0.071 4, P >0.05), but markedly lower than in the moderate (0.145 4 ± 0.053 6, P <0.05) and severe asthenospermia patients (0.122 7 ± 0.045 7, P <0.01), which was consistent with the results of RT-PCR. CONCLUSIONS: Decreased mRNA and protein expressions of ODF2 in the sperm are positively correlated with declined sperm motility of the asthenospermia patient, which is suggestive of the involvement of the ODF2 gene in the regulation of sperm motility.

TAT-peroxiredoxin 2 Fusion Protein Supplementation Improves Sperm Motility and DNA Integrity in Sperm Samples from Asthenozoospermic Men.

PURPOSE: We compared levels of peroxiredoxin 2 in semen samples from normozoospermic and asthenozoospermic men. The potential effects of TAT-peroxiredoxin 2 fusion protein on sperm motility and DNA integrity were also evaluated. MATERIALS AND METHODS: Semen samples were obtained from 50 normozoospermic and 50 asthenozoospermic men. Lipid peroxidation of semen was determined using a commercial malondialdehyde kit. Sperm DNA fragmentation was evaluated by TUNEL assay. Western blot and immunofluorescence were performed to detect the amount of peroxiredoxin 2 protein in seminal plasma and spermatozoa. Sperm motility, DNA damage and levels of reactive oxygen species were evaluated after TAT-peroxiredoxin 2 fusion protein supplementation to the sperm suspension for 2 and 12 hours of incubation. RESULTS: In asthenozoospermic semen samples a significantly higher level of malondialdehyde and DNA damage was discovered. However, the expression of peroxiredoxin 2 was significantly lower in seminal plasma and spermatozoa compared with that of normozoospermic men. TAT-peroxiredoxin 2 fusion protein was successfully prepared and delivered to the spermatozoa. Interestingly adding TAT-peroxiredoxin 2 in asthenozoospermic sperm suspension effectively defended against the decrease in progressive motility and the increase in DNA damage. CONCLUSIONS: This study shows that supplementation of TAT-peroxiredoxin 2 fusion protein in the sperm suspension from asthenozoospermic men effectively improved sperm motility and DNA integrity by reducing levels of reactive oxygen species. Therefore, we speculate that peroxiredoxin 2 may have an important role as an antioxidant defense in semen and would provide new prevention and therapy alternatives for asthenozoospermia.

Protective effects of l-carnitine on astheno- and normozoospermic human semen samples during cryopreservation.

This study was conducted to determine the effects of l-carnitine (LC), as an antioxidant, in preventing spermatozoa damage during the freezing-thawing process in both astheno- and normozoospermic human semen samples. Seventy semen samples (37 asthenozoospermic and 33 normozoospermic) were involved in this study. Cryopreservation medium supplemented with 1.0 g/l LC was mixed with semen at a ratio of 1:1 (v/v). Controls were cryopreserved with freezing medium only. Assessment of motility, viability (VIA), mitochondrial membrane potential (MMP) and DNA fragmentation index (DFI) were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed LC treated samples. Supplementation of the cryopreservation medium with LC induced a significant improvement in post-thaw sperm parameters in both the asthenozoospermic and normozoospermic semen samples, compared with those of the control, regarding sperm fast forward motility, forward motility, total motility and VIA. LC showed better protective effects towards asthenozoospermia for DFI (F = 115.85, P < 0.01) and VIA (F = 67.14, P < 0.01) than did normozoospermic semen samples. We conclude that supplementation with LC prior to the cryopreservation process reduced spermatozoa cryodamage in both asthenozoospermic and normozoospermic semen samples. LC had better protective effects for asthenozoospermic human semen samples. Future research should focus on the molecular mechanism for and the different protective effects of LC between asthenozoospermic and normozoospermic semen samples during cryopreservation.

Comparison of the outcome of conventional in vitro fertilization and intracytoplasmic sperm injection in moderate male infertility from ejaculate.

OBJECTIVE: To evaluate whether couples with moderate male infertility should be treated with conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). PATIENTS AND METHODS: A total of 249 couples with moderate male infertility undergoing their first IVF/ICSI cycle were enrolled in the study. The couples were divided into two groups according to the results of semen analysis: moderate oligozoospermia (O group) and moderate oligoasthenozoospermia (OA group). Sibling oocytes were randomized into groups to be inseminated either by conventional IVF or ICSI. Fertilization rate, embryo quality, implantation rate, and clinical pregnancy rate were examined. RESULTS: There was no difference in the fertilization, implantation, and pregnancy rates between conventional IVF and ICSI in either the O group or OA group (p > 0.05). Additionally, in the OA group, the good quality embryo rate was similar after IVF or ICSI (p > 0.05). However, in the O group, the good quality embryo rate was significantly higher after ICSI than after IVF (p < 0.05). CONCLUSIONS: Couples with moderate oligozoospermia or moderate oligoasthenozoospermia did not influence the major indices of IVF. Because of the uncertainties concerning the safety of ICSI, couples with moderate oligozoospermia or moderate oligoasthenozoospermia need not be subjected to this procedure.