Agnaldo Pereira Cedenho

Effect of varicocele on sperm function and semen oxidative stress

Study Type – Aetiology (case control)

Adolescent varicocele: improved sperm function after varicocelectomy

OBJECTIVE To assess the effect of varicocelectomy on sperm function (DNA integrity and mitochondrial activity) and levels of lipid peroxidation in seminal plasma of adolescents. DESIGN Prospective study. SETTING Patients recruited from a local public school. PATIENT(S) Adolescents (14-19 years old), Tanner stages IV or V with varicocele grades II or III, attending a local public school. INTERVENTION(S) Two semen collections with a one week interval between collections before bilateral varicocele repair using subinguinal microsurgical varicocelectomy, and two semen collections with a one week interval between collections three months after the surgery. MAIN OUTCOME MEASURE(S) Rate of sperm DNA fragmentation as assessed by the Comet assay and categorized as classes I (no DNA fragmentation) to IV (high DNA fragmentation). Rate of mitochondrial activity as assessed by the diaminobenzidine assay and categorized as grades I (all mitochondria active) to IV (all mitochondria inactive). Levels of lipid peroxidation in seminal plasma by a colorimetric method that quantifies a lipid peroxidation subproduct (malondialdehyde). RESULT(S) Concerning DNA integrity, the samples after varicocelectomy showed more spermatozoa with intact nuclear DNA (grade I) and less spermatozoa with Comet grades II, III, and IV. Regarding mitochondrial activity, the samples after varicocelectomy showed less cells with inactive mitochondria (class III). No differences were observed in classes I, II, and IV. Concerning lipid peroxidation, no significant differences were observed. CONCLUSION(S) This study was able to demonstrate that varicocelectomy in adolescents is associated with increased sperm DNA integrity and mitochondrial activity. However, levels of seminal products of lipid degradation (malondialdehyde) are not different.

Does varicocele grade determine extent of alteration to spermatogenesis in adolescents

OBJECTIVE To determine whether grade of varicocele determines extent of alterations to semen quality in adolescents. DESIGN Prospective study. SETTING Patients recruited from a local public school. PATIENT(S) Adolescents (14 to 18 y of age) attending a local public school. INTERVENTION(S) Scrotal palpation in a temperature-controlled room, testicular volume assessment with a Prader orchidometer, and semen analysis according to World Health Organization guidelines, with morphology by Krugers strict criteria. MAIN OUTCOME MEASURE(S) Presence, and grade, or absence of varicocele; testicular volume (assessed with a Prader orchidometer); semen analysis results; and prevalence of testicular asymmetry. RESULT(S) Among the adolescents, 27.8% (95% confidence interval [CI]: 23.2, 32.4) presented varicocele grades II and III, and 7.8% (95% CI: 5.0, 10.6) presented with a grade III varicocele. There was a high prevalence of testicular asymmetry in adolescents with left grade II (41.7%) and III varicocele (51.9%), whereas adolescents without varicocele showed very low testicular asymmetry (11.0%). Testicular asymetry was significantly less prevalent in adolescents without varicocele. Sperm progressive motility and concentration were lower in the two varicocele groups but were not different according to grade. However, the total number of progressively motile sperm in the ejaculate was lower in the varicocele grade II and III groups, and patients with varicocele grade III presented lower values than those with grade II. CONCLUSION(S) Grades II and III varicocele cause a decrease in testicular volume and in semen quality that is independent of grade, but when assessing the total number of progressively motile sperm in the ejaculate, grade III varicoceles place these adolescents very close to the World Health Organization cutoff rate, and thus, current guidelines for treating the adolescent varicocele may need to be revised.

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.

Male infertility in spinal cord trauma

Every year there are 10 thousand new cases of patients victimized by spinal cord trauma (SCT) in the United States and it is estimated that there are 7 thousand new cases in Brazil. Eighty percent of patients are fertile males. Infertility in this patient group is due to 3 main factors resulting from spinal cord lesions: erectile dysfunction, ejaculatory disorder and low sperm counts. Erectile dysfunction has been successfully treated with oral and injectable medications, use of vacuum devices and penile prosthesis implants. The technological improvement in penile vibratory stimulation devices (PVS) and rectal probe electro-ejaculation (RPE) has made such procedures safer and accessible to patients with ejaculatory dysfunction. Despite the normal number of spermatozoa found in semen of spinal cord-injured patients, their motility is abnormal. This change does not seem to be related to changes in scrotal thermal regulation, frequency of ejaculation or duration of spinal cord damage but to factors related to the seminal plasma. Despite the poor seminal quality, increasingly more men with SCT have become fathers through techniques ranging from simple homologous insemination to sophisticated assisted reproduction techniques such as intracytoplasmic sperm injection (ICSI).

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.

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.

SINGLE NUCLEOTIDE POLYMORPHISMS OF THE HEAT SHOCK PROTEIN 90 GENE IN VARICOCELE-ASSOCIATED INFERTILITY

PURPOSE Varicoceles are associated with impaired testicular function and male infertility, but the molecular mechanisms by which fertility is affected have not been satisfactorily explained. Spermatogenesis might be affected by increased scrotal temperature, such as that caused by varicocele. HSP90 is a molecular chaperone expressed in germ cells and is related to spermatogenesis, motility, and both heat and oxidative stress. Possible correlations between coding single region nucleotide polymorphisms (cSNPs) in the HSP90 gene in patients with varicocele associated with infertility were analyzed, and polymorphisms in these exons were characterized through DNA sequencing. MATERIALS AND METHODS PCR-SSCP and DNA sequencing were used to search for mutations in 18 infertile patients with varicocele, 11 patients with idiopathic infertility and 12 fertile men. DNA was extracted from leucocytes for PCR amplification and SSCP analysis. DNA from samples with an altered band pattern in the SSCP was then sequenced to search for polymorphisms. RESULTS Three silent polymorphisms that do not lead to amino acid substitutions were identified. CONCLUSION Mutations in the HSP90 gene do not appear to be a common cause of male factor infertility. The low incidence of gene variation, or SNPs, in infertile men demonstrates that this gene is highly conserved and thus confirms its key role in spermatogenesis and response to heat stress.