Gabriela Ruth Mendeluk

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.

Semen culture, leukocytospermia, and the presence of sperm antibodies in seminal hyperviscosity

Seminal hyperviscosity is generally thought to reveal genitourinary infection. The aim of the present work was to study this hypothesis. A total of 65 semen samples were obtained from males presenting for infertility screening. The samples were evaluated according to WHO criteria and microbiologically investigated, including culturing for Mycoplasma hominis and Ureaplasma urealyticum, and microscopic observation of Chlamydia trachomatis by a direct fluorescence assay. Determination of local antisperm antibodies was performed. Semen was categorized according to consistency: normal (n = 31) and high (n = 34). No difference was recorded either in the number of positive cultures, or in the number of species found in each sample. The number of white blood cells and the percentage of antibody-bound sperm showed no difference in the groups under study. There was no association between seminal hyperviscosity, positivity in semen cultures, number of species isolated in semen cultures, leukospermia, or presence of sperm antibodies. Further studies should be performed considering the evolution of the genital-infected patients to clarify the etiology of the hyperviscosity.

Viscosity of Human Seminal Fluid: Role of Lysozyme

The aim of this research was to evaluate the role of lysozyme in the phenomenon of seminal hyper-viscosity. The enzyme was determined in 142 samples of seminal plasma either leucospermic or not, with or without active macrophages classified according to their consistency (normal or high). The kinetic method with Micrococcus lysodeikticus as substrate was employed. No difference was found in enzymatic concentration expressed in nmol/L of enzymatic protein (mean +/- 2 SEM) on comparing normal and high seminal consistency groups, while differences proved highly significant in batches either leucospermic or not (n = 44, 197.2 +/- 51.3 vs. n = 98, 108.3 +/- 12.8; p < .0005). On subdividing the normal and high-consistency groups according to the count of polymorphonuclear leucocytes and the macrophagic responses, differences were also significant (p < .005 in both cases). Lysozyme concentration increases in presence of leucospermic reaction. In vitro lysozyme addition showed no significant effect on samples with high consistency. The results indicate that lysozyme plays no direct role in the phenomenon of seminal hyperviscosity, although its deficiency in cases of chronic infections may prove a factor aggravating the clinical picture.

Sperm Motility and ATP Content in Seminal Hyperviscosity

Objective spermatic motility (Hamilton Thorne Research), the rapid progressive spermatozoa (grade A) recovery after swim-up, and the spermatozoa ATP content (bioluminescence) were studied in normoviscous and hyperviscous asthenospermic samples. The amplitude of lateral head displacement (ALH) was significantly lower in hyperviscous semen (normal: 4.6 +/- 0.7 microns [n = 20], high: 3.5 +/- 1.2 microns [n = 16]; p < .05). The grade A recovery percentage after swim-up was significantly higher in semens with high consistency (normal: 71.0 +/- 38.0 [n = 14], high: 181.3 +/- 108.9 [n = 6]; p < .05). The ATP content per living spermatozoa was in the normal consistency group 449.4 +/- 65.1 pmol per million living spermatozoa (n = 29) and in the high consistency batch 605.1 +/- 242.8 (n = 9), p < .05. In asthenospermia, the spermatozoa from hyperviscous samples have minor ALH values, better response to swim-up, and high ATP content than those from normoviscous ejaculates.

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.

In vivo antigenotoxic activity of watercress juice (Nasturtium officinale) against induced DNA damage.

The present study was carried out to investigate the genotoxicity as well as possible protective activity against damage induced by cyclophosphamide (CP) of the aqueous juice of watercress (Nasturtium officinale, W.T. Aiton) in vivo. Male and female Swiss mice 7–8 weeks old (N = 48) were treated by gavage with 1 g kg−1 body weight and 0.5 g kg−1 body weight of watercress juice during 15 consecutive days. Genotoxicity and its possible protective effect were tested by the comet assay in peripheral blood cells and the micronucleus test in bone marrow. In addition, biopsies of the bladder, epididymis and testicles of mice were performed to extend the experimental design. Watercress juice per se did not induce genetic damage according to the comet assay and micronucleus study, exhibiting a protective activity against CP (P < 0.05 and P < 0.001, respectively). The comparative analysis of bladder histological changes obtained in the watercress plus CP group against those treated with CP alone suggests a probable protective effect. Further studies are needed in order to establish the protective role of watercress juice against DNA damage. Copyright

THE ONLY PRESENCE OF SPERM IN URINE DOES NOT IMPLY RETROGRADE EJACULATION

This is a retrospective study of clinical experience collected at the University Clinical Hospital over a 19-year period. Semen samples were analyzed according to WHO criteria. In the postmasturbatory urine, sperm count was performed. Data were expressed as total sperm number in urine (TSNU) and using a retroejaculation index. Patients were categorized into four groups according to the presence of sperm in the studied samples: a) in semen and urine; b) only in urine; c) only in semen; d) neither in semen nor in urine. A control group included nonretroejaculator patients. Retroejaculator patients are those whose TSNU is superior to 3.8 × 106 and the RI superior to 2.16%. While diagnosing retroejaculation, the only presence of sperm in the postmasturbatory urine is not adequate. The proposed index added to total sperm number in urine and semen volume may identify true retroejaculator patients.

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