Patricia Haydee Chenlo

Asthenozoospermia: analysis of a large population.

Spermatozoa travel a long distance to meet and fertilize the oocyte, so sperm motility is a requisite for normal fertilization. Asthenozoospermia, or low sperm motility, is a common cause of human male infertility. This is a retrospective study (1992-1999) to document the prevalence of this pathology in infertile men and to clarify the probable factors associated to its etiology. The prevalence was 18.71% for asthenozoospermia and 63.13% for asthenozoospermia associated with oligo- and/or teratozoo-spermia; thus, 81.84% of the studied samples had altered motility. Leukocytospermia, the ratio of germ cells/sperm, anti-sperm antibodies, consistency, biochemical markers of accessory sex glands, and sperm response after swim-up were studied in normospermic (N), asthenozoospermic (A), and combined asthenozoospermic (C) samples. No significant difference was found in the frequency of leukocytospermia among groups. The rate of germ cells/(spermatozoa + germ cells) between C and N ( p <.01), and C and A ( p <.01) was statistically different, while no difference was found on comparing N and A. MAR-test over 40% was found in 6% of the A samples and 7.6% of the C, while no positive values were observed in the N group. The percentage of hyperviscous samples was higher in the low sperm motility samples than in the normal group. Data on concentration of the biochemical markers seem to be decreased in asthenozoospermia. Pure and combined asthenozoo-spermia showed different behavior in sperm recovery after swim-up. Two different asthenozoospermias could be defined: the pure one where sperm environment is involved (immunological factor, hyperviscosity, and secretory gland function) and the combined, where the testis is comprised.

Fragmentación del ADN espermático empleando el método de TUNEL

OBJECTIVES To establish the validity of the TUNEL assay in determining sperm DNA fragmentation, the relationship between the degree of fragmentation and the seminal parameters and the sample needed to conduct the test. MATERIAL AND METHODS We used semen samples from healthy fertile men (n=33), patients who consulted for infertility with a prescription for the TUNEL assay (n=77) and patients with intracytoplasmic sperm injection failure (n=20), analyzed according to the 2010 WHO. The TUNEL/propidium iodide test was performed by flow cytometry, on baseline and post-swim-up samples. RESULTS The cutoff value for the TUNEL assay (ROC curves) was 26%, with a sensitivity and specificity of 85% and 89%, respectively. The pre-swim-up and post-swim-up medians of the results from the TUNEL assay showed no significant differences (17.0% vs. 12.9%, respectively). However, 39.1% of the samples showed a difference greater than 15 in absolute value between the results of the baseline and post-swim-up TUNEL assays. The linear correlation study of the morphology, mobility and vitality using the post-swim-up TUNEL assay showed a greater correlation than preselection, with significant results (r: -0.394, P<.0001; r: -0.461, P<.0001; r: -0.526, P<.0001). CONCLUSIONS The TUNEL assay is a valid test for clinical use. DNA fragmentation is a factor independent from traditional semen tests. We found a greater susceptibility to damage generated in the laboratory procedures in the samples with lower quality. The sample of choice for evaluating DNA fragmentation will depend on whether the clinician is treating a natural or assisted fertilization.

Shedding of immature germ cells.

The immature germ cells (IGC) constitute the highest percentage (90%) of nonsperm cells (NSpC) in ejaculates from fertile or infertile men. The objective of this study was to evaluate IGC concentration and the IGC/(IGC + Sp) ratio, in normozoospermia and dispermia. Normozoospermia from men with proven fertility (NPF), nonproven fertility (NNPF), dispermia (D) and semen samples with excessive shedding of immature germ cells (G I : 1.7 2 10 6 to 5 2 10 6 IGC/mL and G II > 5.0 2 10 6 IGC/mL) were used in this study. The mean value + 2 SD for the NNPF (1.7 2 10 6 /mL) and the value proposed by WHO (5 2 10 6 /mL) were employed to define G I and G II groups. IGC concentration is statistically different in the studied groups. The IGC/Sp ratio showed a significant difference only between the NNPF and the D. When comparing semen parameters (Sp/ejaculate, grade (a) motility and morphology) there was a highly significant difference between NNPF and G I and G II; no difference was found between G I and G II. While studying 200 cases of dispermias 83% showed a high shedding of immature germ cells. The cytological study of nonsperm cells and the count and identification of the immature germ cells could be used to evaluate the dispermic disorders.

LABORATORY METHODS FOR THE DIAGNOSIS OF ASTHENOZOOSPERMIA

Human sperm samples from 46 men were evaluated for standard semen parameters by two trained technicians, according to WHO criteria. A computer-aided semen analyzer (CASA, HTM-IVOS) was employed. The overall mean coefficient of variation for the 2 participants and the 46 samples studied was 17% for the percentage of progressive motile spermatozoa ( r = 0.77). Twenty-nine (63%) samples were classified as asthenozoospermia by both methods. From the studied samples, 12 (26%) were classified as asthenozoospermia by the subjective method and 2 (4.3%) by CASA ( p =. 01). The two methods do not provide directly comparable data.

Flow cytometry TUNEL standardization for assaying sperm DNA fragmentation.

We read with great interest Muratori’s paper (Muratoriet al, 2010), because we believe that terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling(TUNEL) assay standardization is a matter of greatconcern and that there are certain steps that must befollowed to achieve accurate results.We agree with the authors of the paper but feel theneed to stress several important considerations. As theauthors mention in their introduction, the labeling ofDNA breaks should be carried out by nuclear stainingwith propidium iodide (PI; Muratori et al, 2008). Wehave noted in oligozoospermia samples a clear increasein the percentage of PI-negative events with forwardscatter/side scatter similar to sperm, yielding lowerresults. The upper sperm count limit that could beconsidered a threshold for this phenomenon is difficultto establish; thus, TUNEL/PI should be applied to allsamples, and reference values should be recalculated forfuture clinical use.The authors propose 2 methods to quantify the DNAfragmentation percentage within a sperm sample: athreshold-setting method (Muratori et al, 2000) and ablank subtraction method. The results differed substan-tially between these different methods. The authorschose the first one, stating that it correlated well withsemen quality; in our hands, however, no associationwas found between DNA fragmentation and motility,morphology, and sperm count (Pearson correlation; n 5210). We believe that our data do not invalidate the useof this method because new assays might give differentinformation than previously determined parameters.As the authors report, fixed samples should not bestored at 4uCorat220uC because it affects reproduc-ibility. In our experience, the results obtained using freshcompared with stored samples varied unpredictably by,50% (from 247% to +52%). Other authors suggeststoring the samples (4uC, 220uC) until testing (Stronatiet al, 2006; Ramos et al, 2008), but we do not agree withthem. This point is essential for assay standardization.When the procedure is followed rigorously, the resultsare precise: our intra-assay coefficient of variation is 3%(aliquot processed immediately after fixation), which isin agreement with data reported by Muratori et al(2010). Finally, we congratulate Muratori’s teambecause their comprehensive studies have helped usimprove the analysis of sperm DNA fragmentation.Susana M. Curi, Patricia H. Chenlo, Luis A. Billordo,Pla´cida Baz, Melba L. Sardi, Julia I. Ariagno,Herberto Repetto, Gabriela R. Mendeluk,and Mercedes N. PuglieseLaboratorio de Fertilidad MasculinaDepartamento de Bioqui´mica Cli´nicaINFIBIOC, Facultad de Farmacia y Bioqui´micaUniversidad de Buenos Aires, Argentina

Computer-aided sperm analysis: a useful tool to evaluate patient's response to varicocelectomy.

Preoperative and postoperative sperm parameter values from infertile men with varicocele were analyzed by computer-aided sperm analysis (CASA) to assess if sperm characteristics improved after varicocelectomy. Semen samples of men with proven fertility (n = 38) and men with varicocele-related infertility (n = 61) were also analyzed. Conventional semen analysis was performed according to WHO (2010) criteria and a CASA system was employed to assess kinetic parameters and sperm concentration. Seminal parameters values in the fertile group were very far above from those of the patients, either before or after surgery. No significant improvement in the percentage normal sperm morphology (P = 0.10), sperm concentration (P = 0.52), total sperm count (P = 0.76), subjective motility (%) (P = 0.97) nor kinematics (P = 0.30) was observed after varicocelectomy when all groups were compared. Neither was significant improvement found in percentage normal sperm morphology (P = 0.91), sperm concentration (P = 0.10), total sperm count (P = 0.89) or percentage motility (P = 0.77) after varicocelectomy in paired comparisons of preoperative and postoperative data. Analysis of paired samples revealed that the total sperm count (P = 0.01) and most sperm kinetic parameters: curvilinear velocity (P = 0.002), straight-line velocity (P = 0.0004), average path velocity (P = 0.0005), linearity (P = 0.02), and wobble (P = 0.006) improved after surgery. CASA offers the potential for accurate quantitative assessment of each patient′s response to varicocelectomy.