Determination of testicular function in adolescents with varicocoele - a proteomics approach.

The goal of this study was to determine seminal plasma biomarkers of testicular function in adolescents with varicocoele and to verify enriched gene ontology terms associated to these differential proteomes. An observational study was carried out in an academic research environment. A total of 77 adolescent patients were recruited from a local public school, of which 23 were without varicocoele and with normal semen analysis (control group), 37 were with varicocoele and normal semen (VNS) parameters, and 17 were with varicocoele and altered semen (VAS) parameters. Two semen collections were provided with a 1-week interval, after 2-5 days of ejaculatory abstinence. Seminal plasma proteins were identified and quantified utilizing a label-free shotgun proteomics approach, generating (i) proteins differentially expressed in each group (control, VNS, and VAS) and putative biomarkers using multivariate statistics followed by discriminant analysis. Confirmatory analysis was performed for two proteins by western blotting. Enriched biological processes and molecular functions were determined using gene ontology analysis. In total, 541 proteins were identified and quantified: 108 exclusive or overexpressed in controls, 26 in the VNS group, and 13 in the VAS group. The suggested biomarkers are Cab45/SDF4 (Q9BRK5), protein lefty-1 (O75610), DNase I (P24855), PAP2-alpha (O14494), IBP-7 (Q16270), HDC (P01860), and CRISP-3 (P54108). Western blotting results showed that Cab45 was significantly underexpressed in both varicocoele groups, and CRISP-3 was significantly overexpressed in seminal plasma of adolescents with VAS. In conclusion, specific biomarkers of spermatogenesis and homeostasis are observed in adolescents without varicocoele, and the presence of a palpable varicocoele progressively shifts these adolescents toward initially an immune response, and finally toward a chronic inflammatory profile. This shift is accompanied by decreased semen quality.

Reproductive function of the male obese Zucker rats: alteration in sperm production and sperm DNA damage.

Obesity has been considered a public health issue in many countries and is of increasing concern for authorities over the past 6 years. The Zucker rat is a good experimental model for obesity and diabetes studies due to its metabolic characteristics that are similar to those developed by humans. A total of 12 obese Zucker rats and their lean littermates were killed in pubertal and young adult phases for assessing organ weights (testis and epididymis), testicular histomorphometric and stereological analyses, daily sperm production, and transit time in the epididymis. Sperm integrity was also investigated in the adult animals using the Comet assay. Alterations in organ weights, seminiferous epithelium architecture, sperm production, and transit time were noticed in the pubertal fatty rats. The volume density of the lymphatic space was decreased in both the ages. Adult animals had a significant increase in the extent of damage found in sperm DNA. Our results show for the first time that leptin receptor deficiency compromises sperm production during puberty and that genetic obese Zucker rats have increased sperm DNA fragmentation.

Microdissection testicular sperm extraction causes spermatogenic alterations in the contralateral testis.

Testicular sperm extraction (TESE) associated with intracytoplasmic sperm injection has allowed many men presenting non-obstructive azoospermia to achieve fatherhood. Microdissection TESE (microTESE) was proposed as a method to improve sperm retrieval rates in these patients; however, there have been failures. Little is known about whether microTESE leads to spermatogenic alterations in the contralateral testis. We assessed histological outcomes of experimental microTESE in the contralateral testis of adult male rabbits. Nine adult male rabbits were divided into three groups: control (testicular biopsy to observe normal histological and morphometric values), sham (incision of the tunica vaginalis, and a contralateral testicular biopsy to observe histological and morphometric patterns, 45 days later), and study (left testicular microTESE, and a right testicular biopsy to observe histological and morphometric patterns, 45 days later). Sections were assessed by calculating Johnsen-like scores, and measuring total tubule diameter, lumen diameter and epithelial height. The results were compared using ANOVA and Bonferroni's statistical analysis. Morphometric evaluation of the seminiferous tubules did not demonstrate differences between the three groups. However, microTESE caused spermatogenic alterations, leading to maturation arrest in the contralateral testis.

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.

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 percent)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3 percent)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2 percent)) 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 percent); 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.

Oligozoospermia and heat-shock protein expression in ejaculated spermatozoa.

BACKGROUND: Heat-shock protein A2 (HspA2) is correlated with sperm maturity, function and fertility, and a dysfunctional expression of such a gene results in abnormal spermatogenesis. The purpose of this study was to compare HspA2 gene expression in spermatozoa from oligozoospermic men and normozoospermic controls. METHODS: Semen was obtained and analysed according to World Health Organization (World Health Organization, 1999) guidelines, morphology by Kruger's strict criteria. Seventeen patients with oligozoospermia and 21 fertile controls were studied. Total RNA was extracted from ejaculated and Percoll density-gradient-separated spermatozoa followed by semiquantitative RT-PCR analysis. The relative expression level of HspA2 was analysed according to the expression level of the housekeeping beta-actin gene. Serum hormonal profiles (FSH, LH and testosterone) and a peripheral karyotype were also performed. RESULTS: All patients possessed normal karyotype, and no significant hormonal differences were found between the two groups. The study group had significantly lower sperm concentration and normal morphology than the controls. Semiquantitative RT-PCR analysis of HspA2 showed significantly lower expression levels in the oligoteratozoospermic men when compared to controls (P=0.0021). CONCLUSIONS: The HspA2 gene was down-regulated in sperm from infertile men with idiopathic oligoteratozoospermia, suggesting that such anomalies of gene expression might be associated with pathogenesis in some subtypes of male infertility.

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.

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 percent) 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

Correlation between endometrial dating of luteal phase days 6 and 10 of the same menstrual cycle.

CONTEXT: Endometrial maturation, important in the diagnosis of infertile couples, has been evaluated since 1950 using the Noyes criteria. Nevertheless, there is no consensus regarding the most suitable period of the luteal phase for performing the biopsy. OBJECTIVE: This study evaluated the correlation between the histological dating of two endometrial biopsies performed in the same menstrual cycle, on luteal phase days six and ten. DESIGN: Prospective study. SETTING: Human Reproduction Division of the Federal University of São Paulo, referral center. PATIENTS: Twenty-five women complaining of infertility had their menstrual cycles monitored by ultrasound and LH plasma levels, to obtain evidence of ovulation. PROCEDURES: Endometrial biopsies were performed on luteal phase days LH + 6 and LH + 10 (luteal phase day 1 = LH + 1 = the day that follows LH peak). Dating was done according to morphometric criteria, in which an endometrium sample is considered out of phase if the minimum maturation delay is one day. On day LH + 6, blood was drawn for plasma progesterone level determination. RESULTS: All patients had an ovulatory cycle (mean LH peak: 47.4 U/L; mean follicular diameter on LH peak day: 18.9 mm; mean endometrial thickness on LH peak day: 10.3 mm; mean plasma progesterone level on day LH + 6: 14.4 ng/ml). 14 patients had both biopsies in phase; 5 patients had out of phase biopsies only on day LH + 6; 3 had out of phase biopsies only on day LH + 10 and 3 patients had out of phase biopsies on both days. McNemar's test showed no statistical difference between these data (p > 33.36%). CONCLUSIONS: The correlation found between the endometrial datings suggests that biopsies performed on either of these two days are suitable for evaluation of endometrial maturation.

Testicular alterations in hansen's disease

Estudaram-se 58 fragmentos testiculares e o espermograma de 10 indivíduos normais e 24 hansenianos. Verificaram-se as alteraçöes gerais e precoces na histologia, a freqüência de espermátides jovens no lumem tubular e a medida dos diâmetros de 1.450 túbulos seminíferos. De acordo com os resultados obtidos, näo foi possível estabelecer uma correlaçäo qualitativa entre as descriçöes histológicas e ou quantitativa entre a morfometria, e um grupo clínico específico de hansenianos. Assim, também, a simultaneidade e a variabilidade dos achados observados em um mesmo testículo, se opöe às fases evolutivas, descritas por H. Grabstald & L.L. Swan e à classificaçäo proposta por B. Kumar et al. Os autores sugerem que as alteraçöes testiculares sejam conseqüência de uma auto-agressäo à glândula

Centrifuged and noncentrifuged first-cath urine screening for male urethritis

In this investigation a comparison was made between the method for dounting leucocytes in first and final voided centrifuged urine samples and that for noncentrifuged urine samples. Among 417 patients, with a presumed diagnosis of urethritis, 216 had their diagnosis confirmed by the centrifuged urine method and 221 by that which did not use centrifugation. This difference was considered signficant and showed the superiority of the method which did not use centrifugation

Microsurgical anastomosis of vas deferens by a telescopic technique.

A telescopic microsurgical anastomosis of the vas deferens was performed on 24 rats. Sequential histological examination demonstrated mucosal healing in 7 days. Healing was complete in 21 days, and tubal patency was confirmed by histology in 100% of the cases.

Fertility of the monorchid rat after partial resection of testes.

Regeneration of the seminiferous tubules was tested in monorchid rats whose testes were resected 25%, 50%, and 75% of the wet weight of testis. After a 50-day recovery the animals were paired with primiparous rats to verify the male fertility. All the rats with 25% of testicular resection sired young, while those with 75% of resection were sterile. Among rats that suffered 50% of resection, only 17% were fertile. A comparative histomorphometry of the control testes and the resected testes demonstrated that parenchyma of the adult rat testis was not able to regenerate. However, the fertilizing capacity of rats depended on the amount of preserved testicular parenchyma.

The mechanism of spontaneous recanalization of human vasectomized ductus deferens.

The longitudinal and serial histologic sections of spontaneously recanalized ductus deferens and those of the contralateral ductus deferens displayed many tortuous epithelial tubules growing from the mucosal epithelium of distal stumps intruding into the fibrous scar tissue toward proximal stumps. One of the growing gland-like tubules might perform the spontaneous recanalization.

PIP: Many cases of spontaneous ductus deferens recanalizations have been recorded in the last few years, since vasectomy became an effective and safe male contraceptive method. The exact mechanism of spontaneous recanalization is unknown. The patient, a 46-year old man, declared that his wife became pregnant 3 years following his vasectomy. Examination of the patient revealed a nodule that was 1 cm in length on the left ductus deferens 2 cm from the superior pole of the left testis. In the right ductus deferens a lesser nodule was seen--0.4 cm long 2 cm from the superior pole of the right testis. A spermiogram showed a sperm count of 39 million/ml with 70% motility at the 1st hour and normal forms. Following surgical exploration, a bilateral vasectomy with recession of 3 cm long segments, just at the sites of former vasoligations, was performed. The right and left segments were fixed in Bouin's fluid for 24 hours and embedded in paraffin wax. A dense fibrous scar tissue filled the space of 4 mm between the distal and proximal stumps. The lumen of the proximal cut end was slightly dilated and had a blind end. The lining epithelium was pseudostratified and columnar and involved some spermatozoal debris and other cells. The lumen of the distal stumps lined by some epithelia was irregular. Many tortuous blind-ended tubules lined by ciliated cubic epithelium that arose from this cut end invaded the scar tissue. The lamina propria and muscular wall were morphologically normal and ended in sudden constriction at the rim of scar tissue. A dense fibrous scar tissue filled the space of 2 mm between the distal and proximal cut ends and covered the ductus deferens on both sides. The extension was 8 mm long. The distal and proximal stumps were connected by a tortuous canal lined by pseudostratified low columnar epithelium. Close to the scar tissue, the muscular walls of distal and proximal stumps ended abruptly, and only a tortuous epithelial canal passed through the scar tissue. In sum, the longitudinal and serial histologic sections of spontaneously recanalized ductus deferens and those of the contralateral ductus deferens displayed many tortuous epithelial tubules growing from the mucosal epithelium of distal stumps intruding into the fibrous scar tissue toward proximal stumps. 1 of the growing gland-like tubules might perform the spontaneous recanalization.

Fertilizing capacity of early pubertal cryptorchid rat after orchiopexy.

Early pubertal albino rats aged 20 days were made bilaterally cryptorchid by surgery, and groups with 5, 15, 25, and 35 days of cryptorchidism were submitted to bilateral orchiopexy. Fertility of all rats 110 days of age was tested by mating with female rats. Despite histological changes in the germinal epithelium of 35-day cryptorchid testes, orchiopexy restored fertility.

Histology of artificial cryptorchid testes followed by orchiopexy in pubertal rats.

Artificial bilateral cryptorchidism was induced in 4 groups of pubertal male albino rats for different periods. Left orchiopexy and right orchiodectomy were performed. The left testes were orchidectomized after 70 days in the scrotum, the necessary period for possible recovery of spermatogenesis. The comparative histology of both testes showed that the longer the period of cryptorchidism, the more histological degeneration could be observed. However, after 70 days of recovery, only the 4 days cryptorchid testes succeeded in recovery and reorganization of spermatogenesis in the majority of the seminiferous tubules.

Fertilizing capacity of the cryptorchid rat.

Rats were surgically made bilaterally cryptorchid and after 4-8 days the testes were returned to the scrotum. After 70 days fertility was tested by pairing with females. Fertility was restored in 5/6 rats with testes cryptorchid for 4 or 5 days, but only 2/9 were fertile when the duration of cryptorchidism was 6-8 days. The sterility was due to irreversible degeneration of the spermatogonial stem cells. The testes of infertile males were smaller and lighter than those of fertile males and the seminiferous tubule diameters were reduced.