Kirk C. Lo

Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system.

Seminal plasma is a fluid that originates from the testis, epididymis,prostate, and seminal vesicles, and hence, proteomic studies may identify potential markers of infertility and other diseases of the genito-urinary tract. We profiled the proteomes of pooled seminal plasma from fertile Control and post-vasectomy (PV) men. PV seminal plasma samples are void of proteins originating from the testis and the epididymis due to ligation of the vas deferens, and hence, comparative analysis of Control and PV data sets allows for identification of proteins originating from these tissues. Utilizing offline MudPIT and high-resolution mass spectrometry, we were able to identify over 2000 proteins in Control and PV pools each and over 2300 proteins all together. With semiquantitative analysis using spectral counting, we catalogued 32 proteins unique to Control, 49 at lower abundance in PV, 3 unique to PV, and 25 at higher abundance in PV. We believe that proteins unique to Control or at lower abundance in PV have their origin in the testis and the epididymis. Public databases have confirmed that many of these proteins originate from the testis and epididymis and are linked to the reproductive tract. These proteins may serve as candidate biomarkers for future studies of infertility and urogenital diseases.

Isolation and Enrichment of Murine Spermatogonial Stem Cells Using Rhodamine 123 Mitochondrial Dye

Abstract Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rholow) or high (Rhohi) fluorescent intensity. Sterile recipients, W/Wv mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rholow, Rhohi, and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rholow cells showed a 17- and 20-fold enrichment over those of Rhohi and nonsorted cells, respectively.

The incidence and effect of bacteriospermia and elevated seminal leukocytes on semen parameters

OBJECTIVE To determine the incidence of bacteriospermia and elevated seminal leukocytes (ESL) in a subfertile male population and correlate these results with semen parameters. DESIGN Retrospective cohort study. SETTING Canadian tertiary-level male infertility clinic and university-affiliated andrology and microbiology laboratories. PATIENT(S) Four thousand nine hundred thirty-five nonazoospermic subfertile men. INTERVENTION(S) Analysis and concurrent culture of 7,852 semen samples. MAIN OUTCOME MEASURE(S) Incidence of bacteriospermia and ESL and comparison of semen parameters between these groups. RESULT(S) The rate of bacteriospermia was 15% (22 species), and the rate of ESL was 19%, with no statistical correlation between these groups. Bacteriospermic patients (without ESL) had a statistically significant deterioration in DNA fragmentation index (DFI) only, compared with patients without bacteriospermia and ESL (24.1 vs. 21.8%). ESL alone was associated with a statistically significant deterioration in sperm concentration (20.6 vs. 55.3 × 10(6)/mL), motility (21.8 vs. 26.9%), normal morphology (12.3 vs. 17.4%), and DFI (26.5 vs. 21.8%), with no additional deterioration identified with bacteriospermia. CONCLUSION(S) Bacteriospermia and ESL were prevalent, but not statistically associated, in subfertile men. Bacteriospermia alone was associated with an increase in DFI only, but the presence of ESL was the dominant factor associated with deterioration in semen parameters.

Testicular spermatozoa have statistically significantly lower DNA damage compared with ejaculated spermatozoa in patients with unsuccessful oral antioxidant treatment

OBJECTIVE To compare DNA damage in ejaculated and testicular spermatozoa in patients with previously unsuccessful oral antioxidant treatment. DESIGN Prospective clinical study. SETTING University-affiliated teaching hospital. PATIENT(S) Twelve men with persistently high sperm DNA damage. INTERVENTION(S) Evaluation of DNA damage of ejaculated and testicular spermatozoa by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. MAIN OUTCOME MEASURE(S) The DNA damage of ejaculated spermatozoa compared with that of testicular spermatozoa, both samples collected on the day of intracytoplasmic sperm injection. RESULT(S) Ejaculated spermatozoa showed a threefold higher DNA damage when compared with testicular samples (39.7% +/- 14.8 vs. 13.3% +/- 7.3). CONCLUSION(S) Our results indicated that in patients with previously unsuccessful oral antioxidant treatment the retrieved testicular spermatozoa had a lower degree of DNA damage compared with ejaculated sperm collected on the same day.

Analysis of seminal plasma from patients with non-obstructive azoospermia and identification of candidate biomarkers of male infertility.

Infertility affects approximately 15% of couples with equivalent male and female contribution. Absence of sperm in semen, referred to as azoospermia, accounts for 5-20% of male infertility cases and can result from pretesticular azoospermia, non-obstructive azoospermia (NOA), and obstructive azoospermia (OA). The current clinical methods of differentiating NOA cases from OA ones are indeterminate and often require surgical intervention for a conclusive diagnosis. We catalogued 2048 proteins in seminal plasma from men presented with NOA. Using spectral-counting, we compared the NOA proteome to our previously published proteomes of fertile control men and postvasectomy (PV) men and identified proteins at differential abundance levels among these clinical groups. To verify spectral counting ratios for candidate proteins, extracted ion current (XIC) intensities were also used to calculate abundance ratios. The Pearson correlation coefficient between spectral counting and XIC ratios for the Control-NOA and NOA-PV data sets is 0.83 and 0.80, respectively. Proteins that showed inconsistent spectral counting and XIC ratios were removed from analysis. There are 34 proteins elevated in Control relative to NOA, 18 decreased in Control relative to NOA, 59 elevated in NOA relative to PV, and 16 decreased in NOA relative to PV. Many of these proteins have expression in the testis and the epididymis and are linked to fertility. Some of these proteins may be useful as noninvasive biomarkers in discriminating NOA cases from OA.

Cause-Specific Treatment in Patients with High Sperm DNA Damage Resulted in Significant DNA Improvement

Assessment of sperm DNA damage has been suggested as a negative predictor of fertility potential. Multiple pathological factors acting at both the intra-testicular and post-testicular levels may contribute to sperm DNA damage. The relative contribution of each of these factors in an individual with high DNA damage (>30%) is unclear. The management of patients with elevated DNA damage is also challenging. The purpose of our retrospective study was to evaluate the clinical course of patients with sperm DNA damage over 30% and to assess the effect of non-specific (oral antioxidant) and cause-specific treatments on the quality of their sperm DNA. Results of our retrospective study suggest that the evaluated group with high DNA damage was diagnostically heterogeneous and comprised patients with varicoceles, bacteriospermia and idiopathic infertility. A three month course of antioxidant therapy reduced sperm DNA damage in only 30/61 (49%) patients with significant improvement between the initial and post-treatment DNA Fragmentation Index (DFI) results (46.8%±14.1 vs. 36.7%±16.6, p <. 001). The positive effect of antioxidants could be age-dependent, as patients older that 40 years of age showed no improvement after such treatment. The cause-specific treatments showed superior results compared to antioxidants alone. Improvement was observed in 7/9 (78%) of patients after surgical varicocele repair between the initial and post-treatment DFI results (44.7%±12.8 vs. 28.4%±9.5, p <. 03). The majority of the patients 13/14 (93%) with bacteriospermia had improvement in sperm DFI results after antibiotic treatment (50.4%±19.1 vs. 38.6%±18.7, p <. 001).

Use of the aromatase inhibitor letrozole to treat male infertility

OBJECTIVE Report the case of induction of spermatogenesis with the aromatase inhibitor letrozole. DESIGN Case report. SETTING University Infertility center. PATIENT(S) A 31-year-old man with primary infertility, normal volume azoospermia, normal follicle stimulating hormone (FSH) levels and pattern of nonobstructive azoospermia (NOA) on a testicular biopsy. INTERVENTION(S) The patient was given the aromatase inhibitor letrozole for 4 months and had repeated FSH, testosterone, LH levels, semen analyses, and finally a testicular biopsy. MAIN OUTCOME MEASURE(S) Results of a testis biopsy. RESULT(S) Testis biopsy showed normal spermatogenesis following 4 months of letrozole therapy. CONCLUSION(S) This is the first case report on the use of letrozole to treat male infertility and the first case report on the induction of spermatogenesis in a man with NOA using any aromatase inhibitor.

CUA Guideline: The workup of azoospermic males.

A committee was established at the request of the CUA to determine guidelines for the investigation and management of azoospermia. Members of the committee, all of whom have special expertise in the investigation and management of male infertility, were chosen from different communities across Canada. The members represent different practices in different communities.

Finasteride use in the male infertility population: effects on semen and hormone parameters.

OBJECTIVE To determine the degree of improvement in semen parameters after finasteride discontinuation. DESIGN A prospective database of men presenting for a fertility evaluation was analyzed for semen and hormone parameters before and after discontinuation of finasteride. SETTING A male infertility specialty clinic. PATIENT(S) Men presenting for fertility evaluation from 2008-2012 on finasteride. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Semen and hormone parameters before and after discontinuation of finasteride. RESULT(S) At presentation, 27 (0.6%) of 4,400 men on finasteride. The mean duration of treatment with finasteride was 57.4 months, and mean dose was 1.04 mg/day. There was an average 11.6-fold increase in sperm counts after finasteride discontinuation. Of the men with severe oligospermia (<5 M/mL), 57% had counts increase to >15 M/mL after finasteride cessation. No man had a decrease in sperm count. There was no change in hormone parameters, sperm motility, or sperm morphology. CONCLUSION(S) Finasteride, even at low doses, may cause reduced sperm counts in some men. In this population, counts improved dramatically for the majority of men after finasteride discontinuation. The hormone parameters, sperm motility, and sperm morphology were unchanged after cessation. Finasteride should be discontinued in subfertile men with oligospermia, and used with caution in men who desire fertility.

A comparison of ejaculated and testicular spermatozoa aneuploidy rates in patients with high sperm DNA damage

Testicular spermatozoa are utilized to achieve pregnancy in couples with severe male factor infertility. Several studies suggest that aneuploidy rates in spermatozoa are elevated at the testicular level in infertile patients compared to ejaculates of normal controls. However, essential data regarding aneuploidy rates between ejaculated and testicular spermatozoa in the same individuals is lacking. The purpose of our study was to compare aneuploidy rates at the testicular and post-testicular level from the same patients with persistently high sperm DNA damage. Ejaculates and testicular biopsies were obtained from eight patients with persistently high DNA damage (>30%). Both ejaculated and testicular samples were analyzed for sperm DNA damage and sperm aneuploidy for chromosomes 13, 18, 21, X, and Y. In addition, semen samples from ten normozoospermic men presenting for fertility evaluation served as a control group. A strong correlation between the alteration of spermatogenesis and chromatin deterioration was observed in our study. In the same individuals, testicular samples showed a significantly lower DNA damage compared to ejaculated spermatozoa (14.9% ± 5.0 vs. 40.6% ± 14.8, P < 0.05), but significantly higher aneuploidy rates for the five analyzed chromosomes (12.41% ± 3.7 vs. 5.77% ± 1.2, P < 0.05). While testicular spermatozoa appear favourable for ICSI in terms of lower DNA damage, this potential advantage could be offset by the higher aneuploidy rates in testicular spermatozoa.