D.M. Spaine

Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity

PurposeTo assess the effect of leukocytospermia and semen processing on sperm DNA and mitochondria.MethodsTwenty-two patients with and 41 without leukocytospermia were included. Sperm DNA fragmentation was assessed by the Comet assay, and mitochondrial activity by a colorimetric method for active mitochondria. Semen was processed using Percoll, and motility, DNA fragmentation, and mitochondrial activity were analyzed pre- and post-processing.ResultsNo differences were observed in age, abstinence, volume, sperm morphology, progressive motility, concentration, and vitality (p > 0.10). Variables were grouped according to time (pre- vs post-processing) and group (leukocytospermia vs non-leukocytospermia) because no interactions could be observed. Leukocytospermia was associated to increased DNA fragmentation, while semen processing led to a decrease in DNA fragmentation and to increased mitochondrial activity.ConclusionWhile semen processing selects sperm with higher rates of DNA integrity independent of the presence or absence of leukocytes in semen, samples without leukocytospermia present more sperm without DNA fragmentation. Semen processing also selects sperm with higher mitochondrial activity.

Microbiologic aerobic studies on normal male urethra

OBJECTIVES To carefully collect samples from the external urethral orifice, navicular fossa, and penile urethra and perform a semiquantitative evaluation and identification of gram-positive and gram-negative bacteria present in the normal male urethra. METHODS Thirty uncircumcised male patients 18 to 40 years old without any inflammatory and/or infectious urethral processes were enrolled in this study. Samples were collected from the external urethral orifice, navicular fossa, and penile urethra with sterile alginate swabs that were immediately transferred to tubes containing buffered phosphate solution. Inoculation was done by spreading 0.01 mL of the buffered solutions on sheep blood agar plates and MacConkey agar plates; the plates were then incubated at 36.5 degrees C for 24 hours. After this period, the quantification and identification of each type of colony was performed. RESULTS Among the 30 patients studied, 12 (40%) had bacteria isolated from the three segments, 10 (33.3%) had bacterial colonization in two segments, and 8 (26.7%) had colonization in only one segment (external urethral orifice). Staphylococcus coagulase-negative species, group viridans alpha-hemolytic streptococci, Corynebacterium species, and Enterococcus species were the bacteria more frequently isolated from these three segments. CONCLUSIONS From the findings in this study, it was clear that the bacterial urethral flora was abundant, not evenly distributed, concentrated in the external urethral orifice and navicular fossa, and basically consisted of gram-positive aerobic bacteria.

Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.

Proteomic analysis of seminal plasma in adolescents with and without varicocele

OBJECTIVE To compare proteomic profiles of seminal plasma from adolescents with varicocele and changes in semen quality with the plasma from adolescents with varicocele without seminal changes and from adolescents without varicocele. DESIGN Observational study. SETTING Patients in an academic research environment. PATIENT(S) Adolescents without varicocele (control group), adolescents with varicocele and normal semen quality (VNS group), adolescents with varicocele and abnormal semen quality (VAS group). INTERVENTION(S) Two semen collections at 1-week interval. Protein separation by two-dimensional protein electrophoresis, analysis by gel densitometry, and identification by mass spectrometry. MAIN OUTCOME MEASURE(S) Overexpressed proteins in each group, observed by increased densitometric signal in gels, and exclusively identified proteins in each group. RESULT(S) No differences were observed among the three groups regarding clinical parameters. In semen analysis, the VAS group presented lower sperm concentration, motility, and morphology compared with the VNS and control groups. Forty-seven protein spots of interest were submitted to mass spectrometry identification. Apoptosis regulation proteins were overexpressed in the VAS group, whereas spermatogenesis proteins were overexpressed in the VNS group. Controls presented proteins related to homeostasis. CONCLUSION(S) Changes in the proteomic profile of adolescents with varicocele and normal semen parameters (VNS group) indicate that normal semen analysis may not reflect alterations in proteins in seminal plasma. Implementation of proteomics will help characterize proteins identified in seminal plasma and will facilitate detection of new proteins associated with spermatogenesis and sperm function.

Quality and functional aspects of sperm retrieved through assisted ejaculation in men with spinal cord injury

OBJECTIVE To assess semen quality, sperm DNA fragmentation, and mitochondrial activity in fertile men as well as in men with spinal cord injury who were collecting semen through different methods. DESIGN Prospective controlled study. SETTING Academic research environment. PATIENT(S) Men with spinal cord injury who achieved ejaculation through electroejaculation (n = 12) and penile vibratory stimulation (n = 10); 30 fertile control men without spinal cord injury. INTERVENTION(S) Electroejaculation or penile vibratory stimulation, semen analysis according to World Health Organization guidelines, morphology by Krugers strict criteria. MAIN OUTCOME MEASURE(S) Semen was analyzed according to World Health Organization guidelines; morphology was analyzed according to Krugers strict criteria. Sperm DNA fragmentation, as assessed by the TUNEL technique, was classified as percentage positive. Mitochondrial activity was assessed by incorporation of diaminobenzidine by mitochondria. Cells were classified as I (all active) to IV (all inactive). RESULT(S) The control group presented a statistically significantly higher percentage of sperm with active mitochondria and a statistically significantly lower percentage of sperm with inactive mitochondria. Although sperm DNA fragmentation was not significantly different when considering collection method (electroejaculation: 30; 8.4; penile vibratory stimulation: 31.2; 8), both groups presented statistically significantly higher DNA fragmentation than did controls (11.8; 4.5). A strong inverse correlation was observed between sperm DNA fragmentation (assessed by in situ DNA nick end labeling) and mitochondrial activity in the case of electroejaculation (r = -0.714), but not in the case of penile vibratory stimulation (r = 0.060). CONCLUSION(S) Spinal cord injury led to a decrease in sperm mitochondrial activity and an increase in sperm DNA fragmentation, and the latter is a sign of testicular alterations. Studies should focus on improving the testicular environment in these men.

Prevalence of Y chromosome deletions in a Brazilian population of nonobstructive azoospermic and severely oligozoospermic men

We determined the prevalence of Y chromosome deletions in a population of 60 Brazilian nonobstructive azoospermic and severely oligozoospermic men. PCR-based screening of microdeletions was performed on lymphocyte DNA for the presence of 14 sequence-tagged sites (STS) located in the azoospermic factor (AZF) on the Yq chromosome. All STS were amplified efficiently in samples from 12 fertile men tested, but failed to be amplified in samples from fertile women, indicating the specificity of PCR conditions for Yq screening. Overall, 4 of the 60 infertile patients tested (6.7%) exhibited deletion of the Y chromosome, 2 of them being severely oligozoospermic patients (P10 and P32) and 2 azoospermic men (patients P47 and P57). Patients P47 and P57 presented larger deletions in the AZFa, AZFb and AZFc subregions, with apparent loss of Yq material evidenced by karyotype analysis. Patients P10 and P32 presented deletions confined to the AZFc region, involving the DAZ locus. Male relatives of patients P10 and P32 had no Y chromosome deletions and presented a normal karyotype, suggesting a de novo status of the deletions found. Our data add to the growing literature showing that microdeletions of the Y chromosome can be the cause of male idiopathic infertility.

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

PurposeSperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation.MethodsSperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MSE). Differentially expressed proteins were used for a functional enrichment study.ResultsTwo hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells.ConclusionsSperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

DIFFERENTIAL SEMINAL PLASMA PROTEOME ACCORDING TO SEMEN RETRIEVAL IN MEN WITH SPINAL CORD INJURY

OBJECTIVE To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI. DESIGN Experimental study. SETTING University hospital. PATIENT(S) Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration. INTERVENTION(S) EEJ and PVS. MAIN OUTCOME MEASURE(S) The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). RESULT(S) A total of 638 different proteins were identified by MS(E) and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group. CONCLUSION(S) This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility.

Changes in the seminal plasma proteome of adolescents before and after varicocelectomy

OBJECTIVE To compare seminal plasma protein profiles before and after varicocele correction to assess if surgical intervention alters the protein profile. DESIGN Prospective study. SETTING Academic research environment. PATIENT(S) Nineteen adolescent boys with varicocele grades II or III. INTERVENTION(S) Two semen samples were collected before bilateral subinguinal microsurgical varicocelectomy, and two semen samples were collected 3 months after surgery. Seminal plasma protein profiles were determined with the use of two-dimensional gel electrophoresis. Proteins were separated in 18-cm 3-10 pH strips and 10%-17.5% gradient gels. Gels were stained, scanned, and compared with the use of Imagemaster 2D platinum 7.0. Spots of interest were removed from gels, and protein digestion was performed with the use of trypsin. Digests were identified with the use of electrospray ionization-quadrupole/time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS), and spectra were analyzed with the use of the Mascot software. MAIN OUTCOME MEASURE(S) Proteins uniquely or overexpressed in each period (before or after varicocelectomy). RESULT(S) Nineteen spots were differentially expressed between pre- and postsurgery samples. Identified proteins were albumin, proteasome subunit alpha type 6, alpha-1-antitrypsin, fibronectin, CD177, prostatic acid phosphatase, specific prostatic antigen, alpha-2-antiplasmin, vitamin D-binding protein, gastricsin, clusterin, semenogelin-1, semenogelin-2, superoxide dismutase, protein-glutamine gamma glutamyltransferase-4, and prolactin-inducing protein. CONCLUSION(S) Varicocelectomy is associated with changes in the seminal plasma protein profile. Understanding specific pathways leading to male infertility may further assist physicians in demonstrating deviation from homeostasis in male infertility. In addition, it may be possible to observe if surgical intervention does indeed revert altered pathways toward a homeostatic state.

Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post‐genomic pathways associated with sperm DNA fragmentation.