Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men

This study was undertaken with the aim of investigating the cytogenetic constitution of normal as well as abnormal spermatozoa and immature germ cells found in semen of normal men and infertile patients. A specific protocol of double in-situ hybridization for chromosomes 1 and 17 based on colorimetric detection of the hybridization signals (ISH) and brightfield microscopy analysis of cellular morphology was applied. Also the influence of paternal age on sperm aneuploidy was investigated. We found that, at least in the age range analysed (28-54 years) and for semen of good quality (total normal motile counts above 10 x 10(6)) (n = 17), paternal age has no influence on baseline rates of sperm aneuploidy. However, with decreasing semen quality (total normal motile sperm counts below 5 x 10(6)) (n = 6) significantly higher rates of sperm aneuploidy for autosomes 1 and 17 were scored (0.8 versus 1.43%) (P < 0.001). Regardless of the type of semen analysed, a number of morphologically abnormal spermatozoa were found to be hyperhaploid or diploid in a high percentage of cases (20 and 10% respectively). The same was found for immature germ cells (aneuploidy rate of 18%). We conclude that in infertile men with poor quality semen a direct relationship may exist between the impairment of the spermatogenesis process (as reflected by an increased production of morphologically and cytogenetically abnormal germ cells) and rates of baseline aneuploidy occurring in normal spermatozoa. Infertile couples undergoing assisted reproduction treatment need to be counselled about the risk of using spermatozoa which may carry higher rates of non-disjunction for different chromosomes. While sperm hyper- or hypohaploidy for some chromosomes (X,Y) implies counselling couples about the risk of abnormal phenotype in their offspring, most autosomal sperm aneuploidies probably translate only into lower rates of embryo fertilization and survival.     

Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Rotational digital subtraction carotid angiography: technique and comparison with static digital subtraction angiography

To compare the efficiency of sperm preparation between the two-layer Percoll gradient and mini-Percoll methods, 50 normal and 33 abnormal semen samples from male partners of infertile couples were studied. The number of recovered spermatozoa, percentage of motility, percentage of normal morphology, and their survival at 24 and 48 hours were assessed. Both Percoll gradient techniques resulted in a significantly higher percentage of motility and percentage of normal morphology compared with the original semen samples (p < 0.0001). The two-layer Percoll gradient showed a higher sperm recovery than the mini-Percoll method (p < 0.001), but the latter resulted in a higher percentage of motility (p > 0.001) and a higher sperm survival rate at 24 hours (p < 0.05) than the former, regarding normal semen samples. These differences did not appear with abnormal semen samples when analyzed as a group. Considering each of the abnormal parameters separately, sperm recovery was significantly higher after the two-layer Percoll gradient in the case of astheno- and teratozoospermia (p < 0.05), but sperm survival at 48 hours was higher after the mini-Percoll gradient in the case of teratozoospermia (p < 0.05). It is concluded that both the two-layer Percoll gradient and mini-Percoll method can be used effectively for sperm preparation. The former yields a higher sperm recovery, but the latter should be considered regarding teratozoospermic samples and semen samples of very low volume.     

Status quo in "anesthesiology"? American Society of Anesthesiologists 1997 annual meeting--San Diego, October 18-22, 1997]

Percentage and types of morphological abnormalities found in canine spermatozoa were evaluated by three investigators using three stains (Giemsa-Wright stain [Diff-Quik], eosin Y/nigrosin [Hancock], and eosin B/nigrosin [Society for Theriogenology morphology stain] with conventional light microscopy, compared to phase contrast microscopy on unstained samples. The percentage of spermatozoa with abnormal heads, midpieces, and tails varied by technique and by investigator. Average percentages of morphologically normal spermatozoa were significantly higher in samples stained with Diff-Quik and samples examined by phase contrast microscopy than in samples stained with Hancock or Society for Theriogenology morphology stains. No effect of investigator on the percentage of morphologically normal spermatozoa was assessed. Results suggest that staining or preparation technique may alter the morphology of canine spermatozoa artifactually.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

Infraorbital nerve transection alters serotonin transporter expression in sensory pathways in early postnatal rat development

OBJECTIVE: To determine the relationship between sperm motility and sperm morphology parameters and IVF and pregnancy rates. DESIGN: Pre- and postpreparation analysis of semen samples from infertile couples undergoing IVF-ET. SETTING: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland. PATIENT(S): One hundred fifty couples undergoing IVF-ET treatment at the Regional Fertility Centre. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The ability of human sperm to achieve IVF and pregnancy was investigated in relation to motility parameters (assessed with computer-aided sperm analysis [Integrated Visual Optical System] and percent normal morphology (determined with the strict criteria). RESULT(S): Significant differences were observed in motility parameters and percent normal morphology in samples that achieved > or =50% fertilization compared with < or =50% fertilization and between samples that achieved a pregnancy compared with those that did not. Significant positive correlations were observed between percent progressive motility, the velocity of sperm movement, and morphology parameters and both IVF and pregnancy. CONCLUSION(S): Both sperm motility parameters and percent normal morphology are significant factors in predicting fertilization and pregnancy rates in IVF.     

Production of reactive oxygen species by and hydrogen peroxide scavenging activity of spermatozoa in an IVF program

PURPOSE: Reactive oxygen species (ROS) including H2O2 produced by spermatozoa have been suggested, on one hand, to be associated with idiopathic male infertility and, on the other hand, to stimulate certain sperm function leading to fertilization. The influence of ROS on fertilization was investigated in 75 IVF patients by correlating fertilization rates with the production of ROS and the H2O2-scavenging activity of swim-up spermatozoa prepared in parallel with the IVF samples. RESULTS: Low rates of ROS production by the swim-up sperm was detected by the luminol-dependent chemiluminescence assay. They were not correlated with fertilization rates. The hydrogen peroxide scavenging capacity of these spermatozoa, measured as the removal of exogenous H2O2 assayed spectrophotometrically, decreased stepwise in groups of patients achieving higher fertilization rates, suggesting a positive effect of this ROS on fertilization. An alternative explanation of this correlation is plausible in view of the association of both high scavenging activities and poor fertilization rates with poor sperm morphology. CONCLUSIONS: ROS produced by spermatozoa selected by swim-up plays no negative, if not a positive, role in fertilization.     

Classification and quantification of abnormal sperm along the epididymal tract. Comparison between adult and aged hamsters

The types and averages of abnormal sperm were studied in the epididymis of adult and aged golden hamsters. Abnormal spermatozoa represent 14.6-19.6% of the total of spermatozoa in adults, 31.7-42.1% in middled-aged hamsters, and 39.3-50% in advanced-aged hamsters. Twelve abnormal shapes were found, with the lack of an acrosome, the lack of a head, and the coiling of the tail, being the most frequent in the three age groups. An important increase in the number of coiled spermatozoa was found in the corpus and proximal cauda of the epididymis, but a decrease was observed in the distal cauda. Our data suggest that the epididymis produces secondary defects in spermatozoa running from the proximal caput to the middle zone of the duct, but that many of these spermatozoa are eliminated in the distal cauda. Such a result is mainly found in aged animals, in which a higher percentage of abnormal sperm from secondary origin is found with respect to adults.     

Plasma membrane integrity of cryopreserved human sperm: an investigation of the results of the hypoosmotic swelling test, the water test, and eosin-Y staining

OBJECTIVE: [1] To examine the relationship between sperm membrane integrity and motion parameters before and after cryopreservation; [2] to determine the capacity of the membrane integrity tests to predict the outcome of cryopreservation in fertile and infertile men; and [3] to examine the degree of agreement between tail and head membrane integrity of testicular and ejaculated immotile sperm cryopreserved for intracytoplasmic sperm injection. DESIGN: Prospective study. SETTING: Academic tertiary care institution. PATIENT(S): Fertile donors and normozoospermic oligozoospermic, and asthenozoospermic subfertile men. INTERVENTION(S): Semen samples were cryopreserved and thawed for analysis. MAIN OUTCOME MEASURE(S): Sperm membrane integrity and computer-assisted motion parameters. RESULT(S): The hypoosmotic swelling test and water test had a significant and positive correlation in the fresh and cryopreserved ejaculates of all groups. The results of the hypoosmotic swelling test correlated positively with the percent motility in the fresh ejaculates of fertile and subfertile men. None of the membrane integrity tests correlated with the cryosurvival rate in any group. In the ejaculated and testicular samples with no postcryopreservation motility, the simultaneous assessment of hypoosmotic swelling test and eosin showed that of 33% sperm exhibiting coiling with the hypoosmotic swelling test, only 9% were eosin negative, whereas 24% were eosin positive. CONCLUSION(S): [1] The water test may be a simpler replacement for the hypoosmotic swelling test; [2] none of the membrane integrity tests predicted sperm motility after cryopreservation; and [3] there was a high degree of disagreement between the hypoosmotic swelling test and eosin in the samples with no postcryopreservation motility.     

Data from men in greater Seattle area reveals no downward trend in semen quality: further evidence that deterioration of semen quality is not geographically uniform

OBJECTIVE: To determine whether there has been a decline in semen quality in a group of healthy men during the past 21 years. DESIGN: Retrospective analysis of the relationship between time and changes in semen parameters over 21 years using regression analysis. SETTING: A tertiary university center. PATIENTS: Five hundred ten healthy, normal men who donated multiple semen samples as participants in clinical studies between 1972 and 1993. MAIN OUTCOME MEASURES: Sperm concentration, semen volume, total numbers of sperm per ejaculate, and percent spermatozoa with normal morphology. RESULTS: There was no decrease in sperm concentration, semen volume, total number of sperm per ejaculate, and percent normal sperm morphology in 510 healthy men studied between 1972 and 1993. CONCLUSION: We conclude that in this population of healthy young men there has not been any decline in semen quality in the past 21 years.     

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep II filtration, swim-up and Percoll density gradient methods

The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim-up method, Percoll density gradient method, and SpermPrep II filtration method. Thirty-three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim-up method, whereas aliquot 2 was filtered via a SpermPrep II column. The Percoll density gradient method was compared with the SpermPrep II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim-up method. Furthermore, significant differences were found in most of sperm morphometric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swimup method.     

No evidence of deteriorating semen quality among men in infertile relationships during the last decade: a study of males from Southern Sweden

The aim of this study was to investigate whether the quality of semen has deteriorated during the last decade. Laboratory records containing semen analysis results were reviewed. The records, arranged according to date of birth, are kept in shelves. Every fifth record for analyses performed between 1985 and 1995, and only those of men in infertile relationships, were included. The data were abstracted in a data base, and time-related changes in semen characteristics were studied using linear regression analyses. During the study period, there was a slight, but significant increase in sperm concentration, percentage of motile spermatozoa, percentage of spermatozoa with normal morphology, and the base-value of the penetration test. The seminal volumes decreased slightly, but significantly. Sperm characteristics were not associated with age or date of birth of the men. In conclusion, these data show no evidence of deterioration in sperm quality during the last decade among men in infertile relationships.     

Discontinuous Percoll gradient preparation for donor insemination: determinants for success

The use of cryopreserved donor spermatozoa for insemination has become necessary to decrease the risk from sexually transmitted infectious diseases. Lower fecundity rates have been reported with this practice. Efforts have been applied to increase success, including identification of those sperm characteristics which correlate with increased fecundity. Data from in-vitro fertilization have revealed sperm morphology, motility and zona pellucida binding as important sperm parameters. Discontinuous Percoll gradient preparation yields a high concentration of motile spermatozoa. Using this preparation for thawed donor spermatozoa, we have identified post-preparation motility and progression as factors associated with increased fecundity. Consideration should be given to screening sperm donors with a freeze-thaw Percoll gradient preparation prior to acceptance into a donor bank.     

The Acridine Orange test: a clinically relevant screening method for sperm quality during infertility investigation?

To determine the clinical usefulness of Acridine Orange (AO) staining of spermatozoa as a screening test for the evaluation of semen quality during basic infertility investigation, semen smears from 103 randomly chosen males of subfertile couples were examined. The median duration of infertility was 4.5 years (range 1-15) and the median age was 33 years (range 21-43). The outcome of AO staining ranged from 5 to 81%, with a median of 24%, green fluorescent spermatozoa. Results were not significantly related to the parameters of semen analysis (sperm count, motility, standard morphology, viability, pH and volume, as well as fructose concentration and number of found cells) or to local sperm antibody testing and semen cultures. Fluorescence after AO staining was also not related to sperm functional capacity (evaluated using sperm-mucus interaction tests in vitro and in vivo), or the medical history of the patient. No significant differences in the AO test outcome were seen in patients with explained and unexplained infertility, or with regard to subsequent fertility [with a median value of 21% (range 5-46) green fluorescence in the fertile group, compared with a median value of 28% (range 9-81) green fluorescence in the other men]. The results of this prospective study indicate that under the usual conditions of conception, the AO test is not clinically useful as a screening procedure to determine semen quality during basic infertility investigation.     

Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility

During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.     

A radiomicroassay for cytotoxic antibody to human spermatozoa. Quantification by tritiated actinomycin d

A radiomicroassay for titration of spermocytotoxic antibody is described. The assay used [3H]AACTINOMYCIN D ([3H]Act D) to label damaged spermatozoa in a fashion analogous to penetration by vital dye. Optimal conditions for and some kinetics of the assay are presented. The assay is sensitive, reliable, simple to perform and uses only small amounts of serum and spermatozoa. Applied to sperm antibody positive human postvasectomy sera, the assay compared favourably in sensitivity eith vital dye microscopic observations and with parallel titration by the Isojima's immobilization tests.     

Clinical significance of human sperm-zona pellucida binding

OBJECTIVE: To assess the relationship between sperm morphology and motion parameters and sperm-zona pellucida (ZP) binding capacity under hemizona assay (HZA) conditions and to determine the discriminatory power of the HZA for the prediction of in vitro sperm fertilizing ability. DESIGN: Prospectively designed study. SETTING: Academic tertiary centers. PATIENT(S): One hundred ninety-six couples undergoing IVF therapy participated in this study. INTERVENTION(S): Hemizona assay and IVF results were determined for each couple. MAIN OUTCOME MEASURE(S): Computerized sperm motion analysis, sperm morphology (strict) criteria), and HZA results were correlated with fertilization outcome. RESULT(S): Among sperm parameters from the original ejaculates, morphology was the best predictor of sperm-ZP binding ability; hyperactivated motility was the best predictor of HZA results after swim-up separation of the motile sperm fractions. The HZA index provided the highest discriminatory power for fertilization success/failure, with an overall accuracy of 86%. CONCLUSION(S): Sperm morphology and hyperactivated motility showed a high correlation with the capacity of sperm to achieve tight binding to the ZP. The excellent positive and negative predictive values of the HZA for fertilization outcome provide additional support for the use of this functional bioassay in the decision-making process within the assisted reproduction setting.     

Apolipoprotein A-I promotes cholesterol release and apolipoprotein E recruitment from THP-1 macrophage-like foam cells

Nitric oxide (NO) is a free radical involved in the regulation of several functions of the male genitourinary system. It is produced by neurons and the endothelium and epithelia of reproductive system; it mediates penile erection and regulates sperm motility, viability, and metabolism. Here we show that human spermatozoa exhibit a detectable NO synthase (NOS) activity, measured both as ability of the intact sperm and cell lysate to convert L-[3H]arginine into L-[3H]citrulline and as 24 h accumulation of extracellular nitrite in intact sperm suspensions. NOS activity (identified as an endothelial isoform) was inhibited by L-canavanine and N(G)-monomethyl-L-arginine, and nitrite accumulation was inhibited by the NO scavenger hemoglobin; both enzyme activity and nitrite production were increased by a 24 h incubation of spermatozoa with protein-enriched extracts of human follicular fluid (PFF); a significant increase of citrulline synthesis was observed only after a 4 h incubation with 40% PFF, a time period during which acrosomal reactivity was significantly increased. PFF-induced acrosomal reaction was inhibited by L-canavanine and hemoglobin, and the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP), and DETA NONOate were able to increase the percentage of reacted spermatozoa. Our results suggest that NO synthesized by human sperm may play a role in follicular fluid-induced acrosomal reaction.     

Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue

To maximize the total number of spermatozoa which can be retrieved from a testicular biopsy, four mechanical methods for preparation were compared. In all, 17 biopsies were divided into four equal fractions, which were weighed individually and prepared by rough shredding (control), fine mincing, vortexing and crushing (electrical Potter). After resuspension in an erythrocyte-lysing buffer, removal of the remaining tissue pieces, washing and centrifugation, the following parameters were evaluated: number of spermatozoa per 100 mg tissue, percentage motility, percentage vitality and percentage normal morphology (strict Kruger criteria). After further purification by discontinuous Percoll centrifugation, the same parameters were assessed again. This procedure was efficient in obtaining only spermatozoa in the final fraction. Before Percoll centrifugation, only minor differences between methods were observed. Percoll centrifugation generally resulted in a pronounced loss of sperm cells, partly because of the abnormal dimensions of the eliminated cells. After Percoll centrifugation, treatment of the testicular tissue by fine mincing was the most effective method in terms of the total number of motile spermatozoa and percentage normal morphology.     

Binding of 125I-prolactin to spermatozoa from normospermic and asthenospermic men

Scatchard analysis of prolactin binding sites (PRL-BS) from ejaculated spermatozoa showed a single population of binding sites (apparent association constant: 2.51+/-0.186 nmol/l[-1]) with 0.317+/-0.0743 fmol/10(6) sperm binding sites. Different pools of spermatozoa were incubated with increasing concentrations of several hormones. There was a decrease in [125I]-oPRL binding with purified ovine prolactin (oPRL) and human growth hormone (hGH) which was not observed in the presence of synthetic ACTH and recombinant FSH, suggesting that binding was hormone specific. When the patient's samples were analyzed using the single point assay at saturation concentration, asthenospermic patients showed a significantly higher concentration of binding sites compared to normospermic ones. Both groups of patients displayed similar PRL levels in seminal plasma measured by DELFIA. Moreover, individual values of PRL levels in seminal plasma did not correlate with PRL-BS concentrations. We thus conclude that [125I]-oPRL binding to ejaculated spermatozoa was hormone specific and with similar parameters as seen in other target tissues. PRL-BS concentration in asthenospermic patients was significantly higher than in normospermic but this was not due to different levels of PRL in seminal plasma.