High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age

Background

Sperm DNA damage is associated with male infertility, lower pregnancy rates and pregnancy loss.

Objective

The primary aim of our study was to evaluate the prevalence of sperm DNA damage in younger and older men with normozoospermia.

Design, Setting and Participants

We obtained semen from 277 consecutive non-azoospermic men presenting for sperm DNA testing.

Outcome Measurements and Statistical Analysis

The main outcome measures included sperm % DNA fragmentation index (%DFI, using sperm chromatin structure assay), sperm concentration, motility and morphology, and, paternal age.

Results and Limitations

Sperm % DFI was positively correlated with paternal age (r = 0.20, P < 0.001) and inversely correlated % progressive motility (r = −0.16, P = 0.01). Sperm %DFI was significantly higher in older (≥40 years) compared to younger (<40 years) normozoospermic men (17 ± 13 vs. 12 ± 8, respectively P = 0.008), whereas, sperm concentration, progressive motility and morphology were not significantly different in these two groups. Moreover, the prevalence of high levels of sperm DNA damage (>30 % DFI) was significantly higher in older compared to younger normozoospermic men (17 % vs. 3 %, respectively, P < 0.001).

Conclusion

The data indicate that a conventional semen analysis can often fail to detect a defect in spermatogenesis (high %DFI) in older men and suggest that infertile couples with advanced paternal age, including those with normal semen parameters, should consider sperm DNA testing as part of the couple evaluation.     

Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study

Background

There is good evidence to show that varicocele repair can improve conventional sperm parameters, as well as, sperm DNA integrity, in infertile men with a clinical varicocele.

Objective

To examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity and sperm mitochondrial DNA (mtDNA) copy number.

Design, Setting, and Participants

A prospective study done between March 2007 and January 2008. We evaluated a consecutive series of infertile men (n = 14) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and poor motility (<25 % rapid progressive and <50 % progressive).

Surgical Procedure

Microsurgical sub-inguinal varicocelectomy.

Outcome Measurements and Statistical Analysis

Conventional sperm parameters, sperm mtDNA copy number (by real time PCR) and sperm chromatin structure assay (SCSA) parameters (%DFI,% HDS) before and 4 months after microsurgical varicocelectomy.

Results and Limitations

Sperm concentration and SCSA parameters (%DFI and %HDS) improved significantly after surgery (P < 0.05). Sperm mitochondrial DNA copy number decreased significantly after surgery (27 ± 30 to 9 ± 6 copies per sperm, respectively, P = 0.032). There was a significant negative correlation between mitochondrial DNA copy number and sperm motility (r = − 0.71, P = 0.002).

Conclusion

These findings support the concept that correction of a varicocele can improve spermatogenesis and sperm function, as mitochondrial DNA copy number has been suggested to reflect the efficiency of spermatogenesis and has been inversely related to sperm motility.     

Sperm DNA and chromatin integrity in semen samples used for intrauterine insemination

Background

Sperm DNA damage is associated with male infertility but whether normozoospermic infertile men also have DNA damage is unknown.

Objective

To evaluate sperm DNA and chromatin integrity in men with mild male factor infertility.

Design, setting and participants

Prospective study of 102 consecutive men (78 normozoospermic, 15 asthenozoospermic, 9 oligozoospermic) enrolled for intrauterine insemination (IUI) and 15 fertile controls.

Outcome measurements and statistical analysis

Standard semen parameters and sperm chromatin and DNA integrity were assessed and compared between groups. Sperm chromatin quality was assessed by (1) aniline blue staining (AB is specific to histone lysines), (2) iodoacetamide fluorescein fluorescence (IAF targets free protamine sulfhydryl groups) and (3) sperm chromatin structure assay (SCSA) with the results expressed as % DNA fragmentation index (%DFI).

Results and limitations

The mean (±SD) percentage of spermatozoa with positive IAF fluorescence was significantly higher in the IUI population compared to fertile controls (17 % ± 10 % vs. 8 % ± 6 %, P = 0.0011) and also in the normozoospermic subset (n = 78) compared to controls (16 % ± 9 % vs. 8 % ± 6 %, P < 0.0001, ANOVA). We also observed a trend toward lower %progressive motility, and higher %AB staining and %DFI in the IUI group compared to controls. We observed significant relationships between sperm %DFI and progressive motility (r = −0.40, P < 0.0001) and between positive AB staining and IAF fluorescence (r = 0.58, P < 0.0001).

Conclusions

The data indicate that sperm chromatin integrity may be abnormal in men enrolled in IUI treatment cycles, despite the fact that most of these men are normozoospermic.     

Scrotal heat stress causes sperm chromatin damage and cysteinyl aspartate-spicific proteinases 3 changes in fertile men

Purpose

To observe changes in semen parameters, sperm DNA integrity, chromatin condensation and cysteinyl aspartate-spicific proteinases (Caspase-3) in adult healthy men after scrotal heat stress (SHS).

Methods

The scrotums of 19 healthy male volunteers were exposed to the condition of 40–43 °C SHS belt warming 40 min each day for successive 2 days per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, eosin Y (EY) staining sperm HOS and chromatin dispersion (HOS/SCD) test, HOS and aniline blue (HOS/AB) staining test were carried out before, during and after SHS. The activated Caspase 3 levels of spermatozoa were determined with a microtiter plate reader.

Results

The mean parameters of sperm concentration, motility and normal morphological sperm were significantly decreased in groups with sperm being collected during SHS 1, 2 and 3 months when compared with those in groups of pre-SHS (P < 0.01). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane and vitality, and Caspase-3 activity were observed between the groups of before SHS and after SHS 3 months and the groups of during SHS 1, 2 and 3 months (P < 0.001). Three months the SHS stopped, various parameters recovered to the level before SHS. Abnormal sperm with HOS/AB and HOS/SCD showed a negatively significant correlation with normal sperm by HOS/EY test, and WBC in semen showed a positively significant correlation with Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS/SCD showed a positively significant correlation with that of HOS/AB.

Conclusions

The continuously constant SHS can impact the semen quality, sperm DNA integrity, chromatin condensation and Caspase-3, and the combination of HOS plus AB test may simultaneously determine the integrity of membrane and chromatin condensation at the same spermatozoon.     

Seminal superoxide dismutase activity and its relationship with semen quality and SOD gene polymorphism

Purpose

Superoxide dismutase (SOD) is an important component of antioxidative defense systems and plays an important role in protecting spermatozoa from oxidative damage. In this study, we assessed seminal SOD activity, its association with semen parameters, and also genetic and non-genetic factors contributing to the determination of SOD activity in infertile men.

Methods

Semen samples were obtained from 435 male infertility patients. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL) assay. Four single nucleotide polymorphisms (SNPs) in SOD2 and SOD3 genes were genotyped using OpenArray platform.

Results

We found that seminal SOD activity was positively associated with sperm concentration and overall motility, whereas inversely with sperm DNA fragmentation. In addition, infertile men with SOD2 rs4880 CC variants showed a low level of SOD activity when compared with TT carriers (Mean ± SD: 268.3 ± 102.3 and 342.8 ± 98.2, respectively, P = 0.005). Those who consumed vitamin C/E (≥3 times per week) had a significantly higher SOD activity level than those who did not (mean ± SD: 379.8 ± 93.3 and 332.2 ± 94.9, respectively, P = 0.001).

Conclusions

Seminal SOD activity and other factors influencing SOD activity play a role in determining sperm fertilization potential and male infertility.Keyword: SOD activity, Semen quality, Polymorphism, Vitamin C, Vitamin E, Male infertility     

Sperm processing by swim-up and density gradient is effective in elimination of sperm with DNA damage

Purpose

DNA damage may occur during sperm processing, thereby negatively influencing fertilizing ability of the sperm. The present study was designed to compare the effectiveness of gradient and swim-up, either alone or in combination, to eliminate sperm with DNA damage.

Methods

A total of 51 subjects visiting the University infertility clinic with normozoospermic parameters, oligozoospermia and teratozoospermia were included. Semen characteristics were analysed by standard criteria; Terminal deoxy nucelotidyl transferase mediated dUTP nick end labeling assay was employed for DNA damage assessment.

Results

The percentage of TUNEL positive sperm after sperm processing was significantly lower in normozoospermic (P < 0.05), oligozoospermic (P < 0.001) and teratozoospermic samples (P < 0.01). No difference was observed in the incidence of TUNEL positive sperm between the various techniques, suggesting that they are comparable.

Conclusions

Sperm preparation has been found to result in enrichment of sperm with intact chromatin, which is likely to improve the chances of achieving a viable pregnancy.     

Supplementation of biotin to sperm preparation medium increases the motility and longevity in cryopreserved human spermatozoa

Purpose

To study the effect of supplementing biotin to sperm preparation medium on the motility of frozen-thawed spermatozoa.

Methods

Semen samples of men attending the University infertility clinic (n = 105) were cryopreserved using glycerol-egg yolk-citrate buffered cryoprotective medium in liquid nitrogen. After a period of two weeks, the semen samples were thawed and the motile spermatozoa were extracted by swim-up technique using Earle’s balanced salt solution (EBSS) medium supplemented with either biotin (10 nM) or pentoxifylline (1 mM). The post-wash motility was observed up to 4 h after incubation.

Results

Both biotin and pentoxifylline supplementation resulted in significant increase in total motility (p < 0.05), progressive motility (p < 0.001) and rapid progressive motility (p < 0.05 v/s biotin and p < 0.01 v/s pentoxifylline) compared to the control at 1 h post-incubation period. Significantly higher percentage of total (p < 0.01, p < 0.05 in biotin and pentoxifylline respectively), progressive (p < 0.001) and rapid progressive motilities (p < 0.01) were observed in these two groups even at 2 h compared to the control. In the control group at 4 h after incubation, ~11% decline in total motility and ~8% decline in progressive motility was observed. However, in both biotin and pentoxifylline group the motility was significantly higher than control (p < 0.001). No significant difference in the motility was observed between biotin and pentoxifylline groups at any of the time intervals studied.

Conclusions

Biotin can enhance the sperm motility and prolong the survival of frozen-thawed semen samples which may have potential benefit in assisted reproductive technology field.     

The prevalence of sperm with large nuclear vacuoles is a prognostic tool in the prediction of ICSI success

Purpose

To investigate if there is a correlation between the prevalence of sperm with large nuclear vacuoles (LNV) and intracytoplasmic sperm injection (ICSI) outcomes.

Methods

Two hundred male patients undergoing ICSI had their sperm morphology evaluated through motile sperm organelle morphology examination (MSOME) and the percentage of LNV sperm was recorded and correlated to the ICSI outcomes.

Results

The percentage of sperm with LNV negatively influenced the blastocyst formation (S: 16.9, R²: 20.5 %, p = 0.004) and implantation (S: 34.7, R²: 26.2 %, p = 0.001). There were significant differences in the percentage of sperm with LNV between patients in which pregnancy was achieved or not (22.2 % vs. 28.4 %, p < 0.001) and in patients with ongoing pregnancy or not (22.4 % vs. 28.5 %, p < 0.001). The incidence of sperm with LNV was determinant to the decreased odds of pregnancy (OR: 0.74, p < 0.001) and increased odds of miscarriage (OR: 1.46, p < 0.001). The area under the curve (AUC) was sufficient to distinguish between couples which did achieve pregnancy or not (AUC: 0.922, p < 0.001).

Conclusions

The MSOME is a prognostic tool in the prediction of ICSI success and could be used to select patients that should have their sperm selected by MSOME for ICSI.     

Importance of sperm gluthatione treatment in ART

Purpose

The aim of this study was to investigate whether treatment of sperm from infertile patients would gluthatione could reduce sperm premature chromosome condensation (PCC). To reach the goals of this study, the frequency of sperm PCC formation in sperm of normal and sub-fertile men with/without glutathione treatment were compared and analyzed.

Methods

Hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. Following treatment with hyaluronidase, zonae was removed by trypsin digestion. Sperm were classified into 3 groups according to morphology, movement and counts, treated with glutathione(10 μg/ml) and then processed by swim up method. After capacitation, zona-free oocytes were incubated with sperm then transferred to fresh media containing colcemid. Cells were fixed and slides prepared using Tarkowskie’s standard air drying technique and after staining in 5% Giemsa, oocytes were analyzed at high magnification.

Results

Sperm penetration rate was higher and the rate of intact sperm head and PCC was lower in GSH treated samples compared to non treated groups. Sperm penetration rate was significantly higher in treated astheno sperm samples compared to non-treated ones (66.4% and 50. 97% respectively) (P < 0.001). We observed a significantly higher PCC frequency in infertile patients (P < 0.001). In addition, there was a significantly lower rate of intact sperm head in treated astheno sperm samples (17.49%) compared to non treated ones (26.79%) (P < 0.001). Finally, a significantly lower rate of PCC in treated astheno sperm samples comparing to non treated ones was seen (51.06% and 72.96% respectively) (P < 0. 001).

Conclusions

Our results show that sperm PCC formation could be one of the major causes of failed fertilization in individuals with sperm abnormalities. Also sperm PCC formation may be involved in the etiology of some cases of idiopathic infertility. Given that the susceptibility of sperm to oxidative stress is significantly greater in idiopathic infertile men, our results show that treatment with glutathione could significantly reduce these stress factors and increase ART outcome.     

Chromosomal abnormalities in patients with oligozoospermia and non-obstructive azoospermia

Purpose

To assess the frequency and types of chromosomal abnormalities in 204 Ukrainian patients with non-obstructive azoospermia and oligozoospermia and 87 men with normozoospermia.

Methods

Cytogenetic studies were performed on peripheral blood lymphocyte samples of 164 men with oligozoospermia, 40 men with non-obstructive azoospermia and 87 men with normozoospermia attending infertility clinic.

Results

Chromosomal abnormalities were detected in 17 % of patients with sperm disorders: in 35 % of men with azoospermia and in 12.7 % of men with oligozoospermia. The frequency of chromosomal abnormalities in patients with sperm disorders was significantly higher, than in patients with normozoospermia (P = 0.0001). An increase in the incidence of chromosomal abnormalities with the decrease of sperm count was observed. Chromosomal abnormalities were detected in 1.1 % of patients with normozoospermia, 6.5 % of patients with mild oligozoospermia (sperm count 5–15 × 10⁶/ml), 18.4 % of patients with severe oligozoospermia (sperm count <5 × 10⁶/ml) and 35 % of patients with azoospermia. A significant increase in the frequency of chromosomal abnormalities in patients with severe oligozoospermia was observed when compared to mild oligozoospermia (P = 0.01). A statistically significant association (P = 0.02) of chromosomal abnormalities and sex chromosome abnormalities (P = 0.0001) with azoospermia when compared to oligozoospermia was observed.

Conclusions

Our results highlight the importance of cytogenetic studies in patients with oligozoospermia (both mild and severe) and non-obstructive azoospermia. The presence of chromosomal abnormalities influences significantly the fertility treatment protocols, as well as provides a definite diagnosis to couples suffering from infertility.     

Intracytoplasmic morphologically selected sperm injection outcomes: the role of sperm preparation techniques

Purpose

To compare the results of intracytoplasmic morphologically selected sperm injection (IMSI) between cycles in which the swim-up (SUP) or the density gradient centrifugation (DGC) techniques were used for sperm preparation.

Methods

We evaluated 70 IMSI cycles performed in women with age ≤ 37 years, undergoing IMSI as result of male factor. The couples were divided into two groups: DGC group (n = 26) and SUP group (n = 44). The groups were compared with regard to IMSI outcomes.

Results

There were no significant differences between SUP and DGC groups regarding the number of follicles, oocytes, mature oocytes, oocyte yield and mature oocyte rate. Fertilization rate and high-quality embryos rate on day 5 of development were similar between SUP and DGC groups. Implantation, pregnancy and miscarriage rates were not statistically different between SUP and DGC groups (28.8 vs 33.3 %, 46.2 vs 57.1 % and 8.3 vs 4.2 %, respectively).

Conclusions

Both the SUP and the DGC techniques recover improved sperm fractions and result in similar IMSI outcomes. Further randomized trials analyzing both the quality of sperm through MSOME and the IMSI outcomes are needed to elucidate the role of sperm preparation techniques and morphology on IMSI outcomes.     

Coenzyme Q10 and male infertility: a meta-analysis

Objective

To evaluate the effect of coenzyme Q10 treatments in male infertility, specifically in these parameters: live birth and pregnancy rates, CoQ10 seminal concentration, sperm concentration, and sperm motility.

Materials and methods

Systematic review and meta-analysis in male infertility patients with CoQ10 oral treatments. Three trials were included: 149 males in CoQ10 group and 147 males in placebo group.

Results

None of the included trials provided any data regarding live births. The results of this meta-analysis show that supplementing infertile men with CoQ10 does not increase pregnancy rates. The analysis showed, among patients receiving CoQ10 treatment, a statistically significant increase in: CoQ10 seminal concentration (RR 49.55, 95 % CI 46.44 to 52.66, I² = 17 %), sperm concentration (RR 5.33, 95 % CI 4.18 to 6.47, I² = 58 %), and sperm motility (RR 4.50, 95 % CI 3.92 to 5.08, I² = 0 %)

Conclusion

There is no evidence in the literature that CoQ10 increases either live birth or pregnancy rates, but there is a global improvement in sperm parameters. Adequately powered, robust trials of individual and combination antioxidant therapies are required to guide clinical practice.     

A proteomic analysis on human sperm tail: comparison between normozoospermia and asthenozoospermia

Purpose

Asthenozoospermia is a common cause of human male infertility characterized by reduced sperm motility. The molecular mechanism that impairs sperm motility is not fully understood. This study proposed to identify novel biomarkers by focusing on sperm tail proteomic analysis of asthenozoospermic patients.

Methods

Sperm were isolated from normozoospermic and asthenozoospermic semen samples. Tail fractions were obtained by sonication followed by Percoll gradient. The proteins were extracted by solubilization and subjected to two-dimensional gel electrophoresis (2-DE); then, the spots were analyzed using Image Master 2D Platinum software. The significantly increased/decreased amounts of proteins in the two groups were exploited by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry.

Results

Three hundred ninety protein spots were detected in both groups. Twenty-one protein spots that had significantly altered amounts (p < 0.05) were excised and exploited using MALDI-TOF-TOF mass spectrometry. They led to the identification of the following 14 unique proteins: Tubulin beta 2B; glutathione S-transferase Mu 3; keratin, type II cytoskeletal 1; outer dense fiber protein 2; voltage-dependent anion-selective channel protein 2; A-kinase anchor protein 4; cytochrome c oxidase subunit 6B; sperm protein associated with the nucleus on the X chromosome B; phospholipid hydroperoxide glutathione peroxidase-mitochondrial; isoaspartyl peptidase/L-asparaginase; heat shock-related 70 kDa protein 2; stress-70 protein, mitochondrial; glyceraldehyde-3-phosphate dehydrogenase, testis-specific and clusterin.

Conclusion

Fourteen proteins present in different amounts in asthenozoospermic sperm tail samples were identified, four of which are reported here for the first time. These proteins might be used as markers for the better diagnosis of sperm dysfunctions, targets for male contraceptive development, and to predict embryo quality.     

Could androgen receptor gene CAG tract polymorphism affect spermatogenesis in men with idiopathic infertility?

Purpose

This study examined whether the AR-CAG repeat length might affect clinical characteristics (testis volume) seminal parameters (sperm count and its mobility) along with hormonal serum profile [FSH, LH, Testosterone (T) and Inhibin B (InhB)] both in idiopathic male infertility (IM) and in infertility due to a previous condition of cryptorchidism (CryM) or to Y chromosome long arm microdeletions (YM).

Design

Observational study without intervention(s).

Patients

One hundred and ten IM patients [90 idiopathic olizoospermic males (IOM) and 20 idiopathic azoospermic males (IAM)], 19 CryM male and 10 YM patients were included. Sixty-one age-matched healthy men who had fathered within 3 years were involved representing the control group (FM).

Results

AR-CAG repeats stretch was significantly longer in IOM (p < 0.05), CryM (p < 0.05) and YM (p < 0.001) than FM. When the AR-CAG repeat tracts were subdivided in three subgroups according to the length of CAG repeats tract assessed in fertile subjects (the one with the middle (n 19–21) belonging to the 25 and 75 % inter-quartile, the ends belonging to the <25 % inter-quartile and >75 % inter-quartile, respectively), there was a statistically significant difference of distribution of AR-CAG tract length among fertile and different groups of infertile men (p = < 0.0005; chi-square test). Moreover, the subgroup of AR-CAG repeat stretch with 22–28 triplets was associated with lower levels of InhB both in idiopathic oligozoospermic (Scheffe, Bonferroni and Dunett tests p = < 0.01) and azoospermic men (Scheffe, Bonferroni and Dunett test p = < 0.05), while, when FM and men with idiopathic infertility were gathered in a single group, both the subgroup of AR- CAG tract with 15–18 repeats and the one with 22–28 repeats are associated with lower testis volume, reduced sperm count and serum InhB levels.

Conclusions

Our study showed that the outliers of AR-CAG repeat length seem to influence the function of AR, affecting testis volume and Sertoli cell function and consequently sperm production in both fertile and idiopathic infertile men.     

Analysis of telomere length in couples experiencing idiopathic recurrent pregnancy loss

Purpose

Telomere length plays a significant role in various disorders; however, its role in idiopathic recurrent pregnancy loss (iRPL) is not known. The objective of this study was to assess telomere length in peripheral blood leukocytes in couples experiencing unexplained recurrent pregnancy loss (iRPL).

Methods

The study included 25 couples experiencing iRPL and 20 controls. The mean relative telomere length was measured by quantitative Real Time PCR (Q-PCR) based assay, which measures the average ratio of telomere repeat copy number to a single copy gene (36B4) copy number (T/S ratio) in each sample.

Results

The relative leukocyte mean telomere length (T/S) in both men and women from iRPL group was significantly lower (p < 0.05) when compared to controls. A significant (P < 0.05) negative correlation was found between age and leukocyte telomere length (T/S ratio). Among the sperm parameters seminal volume was found to be negatively (r = −0.4679) associated with the telomere T/S ratio. The DNA fragmentation index of sperm showed positive correlation (r = 0.4744) with telomere length. In this preliminary study, we found that shorter telomere length in both men and women may be associated with early pregnancy loss.

Conclusion

In conclusion, shorter telomere length in both male and female partners appears to play a role in the idiopathic recurrent pregnancy loss. Loss of telomeric DNA due to oxidative stress needs further analysis. Analysis of telomere length in germ cells are needed to further substantiate the findings of this study.     

Y chromosome AZFc microdeletion may not affect the outcomes of ICSI for infertile males with fresh ejaculated sperm

Purpose

To explore whether the presence of a Y chromosome AZFc microdeletion confers any adverse effect on the outcomes of intracytoplasmic sperm injection (ICSI) with fresh ejaculated sperm.

Methods

A total of 143 oligozoospermia patients with Y chromosome AZFc microdeletion in ICSI cycles in a five-year period were studied. Infertile men with normal Y chromosome in ICSI at the same time-frame were used as controls matched to the study group for age of female, female’s body mass index, male’s age, infertility duration and number of oocytes retrieved. Retrospective case–control study was used.

Results

There were no significant differences between groups in clinical outcomes of endometrial thickness, transferred embryos, good embryo rates, implantation rates, biochemical pregnancy rates, clinical pregnancy rates, ectopic pregnancy rates, miscarriage rates, preterm birth rates, the ratio of male and female babies, newborn body height, newborn weight, low birth weight and birth defects (P > 0.05). Patients with Y chromosome AZFc microdeletion had a lower fertilization rate (61.8 % vs. 67.8 %, P < 0.05) and higher cleaved embryo rate (94.0 % vs. 88.1 %, P < 0.05).

Conclusions

ICSI clinical outcomes for oligozoospermic patients with Y chromosome AZFc microdeletion are basically comparable to that of infertile patients with normal Y chromosomes. The results of ICSI were not affected by the AZFc deletion. Preimplantation genetic diagnosis (PGD) before ICSI for Y chromosome AZFc microdeletion may not be a justifiable regular procedure if the couples didn’t care the vertical transmission of Y chromosome deletion.     

Calcium- and integrin-binding protein-1 is down-regulated in the sperm of patients with oligoasthenozoospermia

Purpose

The aim of this study was to determine whether altered expression and distribution of calcium- and integrin-binding protein-1 (CIB1) is involved in the pathogenesis of patients with oligoasthenozoospermia.

Methods

Sperm samples were obtained from 25 infertile Chinese men who had failed to achieve conception after a period of 1–2 y and had been referred to the Reproductive Laboratory of the second hospital affiliated to the Shandong University of Traditional Chinese Medicine. Participants were divided into two groups: oligoasthenozoospermia (n = 13) and asthenozoospermia (n = 12); as a third group, fertile men (n = 19) were included as controls. The expression levels of mRNA and protein levels of CIB1 and cyclin-dependent kinase 1 (CDK1) were measured using qRT-PCR and western blotting.

Results

mRNA and protein expression levels of CIB1 were decreased in the oligoasthenozoospermia patients. Interestingly mRNA and protein expression levels of CDK1 were increased in the oligoasthenozoospermia patients.

Conclusion

The results of the present study indicate that that CIB1 may be involved in the pathogenesis of oligoasthenozoospermia by the CDK1 signaling pathway.     

Assessment of density gradient centrifugation (DGC) and sperm chromatin dispersion (SCD) measurements in couples with male factor infertility undergoing ICSI

Purpose

To investigate how effectively density gradient centrifugation (DGC) improves sperm nuclear integrity and to determine whether the sperm chromatin dispersion (SCD) test of sperm nuclear integrity in native or DGC-treated semen can predict the outcome of assisted reproductive technology (ART) in couples undergoing intracytoplasmic sperm injection (ICSI).

Methods

The DNA integrity of spermatozoa from 63 male factor infertility patients undergoing ICSI was analyzed by the SCD test before and after DGC. The predictive value of the sperm DNA fragmentation index (DFI) for ART outcomes was assessed in a cohort of 45 patients who were undergoing fresh embryo transfer. For the analysis, they were divided into pregnant and non-pregnant groups and, independently, into high sperm DFI (DFI > 30 %) and low sperm DFI (DFI ≤ 30 %) groups. Both raw and DGC semen parameters were examined.

Results

In the asthenospermia and oligozoospermia groups, DGC decreased the sperm DFI from 31.5 ± 19.7 and 28.5 ± 10.3 to 19.2 ± 18.3 and 16.0 ± 12.8, respectively (P < 0.01). DGC decreased the sperm DFI in the severe oligozoospermia group from 41.4 ± 19.0 to 36.3 ± 20.6 (P > 0.01). The pregnant and non-pregnant groups did not differ in their fertilization rate and sperm DFI in native or DGC semen (P > 0.05). There was also no significant difference between the high sperm DFI (DFI > 30 %) and low sperm DFI (DFI ≤ 30 %) groups with regard to fertilization rate, implantation rate, and clinical pregnancy rate for both native and DGC semen (P > 0.05). The patients undergoing ICSI with a high sperm DFI had a higher pregnancy loss rate (defined as spontaneous miscarriage or biochemical pregnancy) compared with patients with a low sperm DFI in both the native and DGC semen groups.

Conclusions

DGC highly significantly reduces sperm DNA fragmentation in the semen of ICSI patients, with the exception of those with severe oligozoospermia. The results of the SCD test of sperm DNA fragmentation in native or DGC semen do not correlate with the fertilization rate, implantation rate, or clinical pregnancy rate in patients undergoing ICSI.     

Sperm head vacuolization affects clinical outcome in ICSI cycle. A proposal of a cut-off value

Objective

To evaluate the relationship between sperm nuclear vacuoles and sperm morphology and to investigate the influence of the rate of spermatozoa with head vacuolization (SVR) in a seminal sample on the clinical outcomes in couples undergoing intracytoplasmic sperm injection.

Materials

26 patients undergoing infertility investigations were included and were divided in two groups according to an SVR ≤ 20,28 % (Group A) or > 20,28 % (Group B), and were investigated to verify the influence of SVR on the fertilization rate, embryo quality, pregnancy and implantation rates.

Results

Abnormal spermatozoa with nuclear vacuoles were significantly higher (p < 0.001) than the percentage of normal spermatozoa with nuclear vacuoles. Patients in group A had a percentage of abnormal sperm with nuclear vacuole significantly lower compared to group B (p < 0,001), but there was no difference in the percentage of normal sperm with nuclear vacuoles. Fertilization rates and the number of top quality embryos did not differ between the two groups. The pregnancy and implantation rates were significantly higher in Group A compared to Group B (respectively p < 0,05 and p < 0.001).

Conclusions

For the first time, we propose a cut off value in the proportion of sperms with nuclear vacuolization on the total of sperm in seminal samples, and demonstrate a relationship between SNV and clinical outcomes after ICSI. The SNV rate could be introduced as an easy diagnostic evaluation prior to perform an ICSI cycle.     

Next day determination of ejaculatory sperm motility after overnight shipment of semen to remote locations

PurposeTo develop a method for delayed assessment of sperm motility, after shipment of semen to a remote laboratory. Sperm in semen were labeled with the MitoTracker® Red CM-H₂XRos reagent, and fixed with 3.7 % formaldehyde by the laboratory technicians at the origin of the semen. This treatment reflected well sperm mitochondrial activity, and the MitoTracker® signal was related to sperm motility and velocity for 2–3 days following ejaculation.MethodsSperm motility and velocity were evaluated manually and by computer assisted semen analysis (CASA), respectively. Fluorescence assessment of individual sperm was carried out with the computer assisted Metamorph v4.6.9 program. Emission levels of MitoTracker® spermatozoa were studied in room temperature and cooled semen, or in the respective room temperature swim-up sperm fractions following ejaculation, and on the second day (N = 103 samples, 89 men) and third day (N = 10 samples, 8 men).ResultsSperm with optical density (O.D.) ≥0.7 showed close correlations with ejaculatory sperm motility and velocity even after second day (r = 0.92, p < 0.001, N = 103 samples). Further, the multiple of sperm motility and velocity was also related to the proportion of high MitoTracker® reagent emission sperm (r = 0.83, p < 0.001, N = 103 samples). MitoTracker® dye fluorescence on the second day accurately reflected the ejaculatory sperm motility (r = 0.90, p < 0.001). Thus, a shipping delay would not adversely affect the results.ConclusionsThe delayed assessment of sperm motility in samples treated with MitoTracker® Red CM-H₂XRos reagent and shipped to remote laboratory truly reflects the level of sperm motility at the time of the ejaculation.