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Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality.

Stringent selection mechanisms, in both internal and external fertilisation systems, reject all but a significant minority of the spermatozoa released at ejaculation. Sperm competition theory provides circumstantial evidence that the selection process involves mechanisms by which the quality of the fertilising spermatozoon is controlled, thereby ensuring that females and their offspring receive high quality genetic material. In this review we examine some of these selection processes to see whether they could be exploited for the improvement of laboratory tests of sperm quality. Such tests are not only required for clinical and agricultural purposes, but are increasingly needed in fields such as reproductive and environmental toxicology where the species requirement is much broader. Despite many years of research, sperm quality assessment methods continue to provide imprecise data about fertility; here we suggest that this may be a consequence of using tests that focus on the spermatozoa that would normally be unable to fertilise under natural conditions. To achieve fertilisation a spermatozoon must be capable of responding appropriately to external signalling stimuli; those involving protein kinase-regulated flagellar function seem especially influential in governing effects ranging from non-Mendelian inheritance in mammals to sperm chemotaxis in sea urchins. Examination of the elicited responses reveals considerable heterogeneity in all species. Here we propose that this level of heterogeneity is meaningful both in terms of understanding how spermatozoa from some individuals possess fertility advantages over spermatozoa from their rivals in sperm competition, and in that the heterogeneity should be exploitable in the development of more accurate laboratory tests.

Animals↗

Quantification of bovine sperm separation by a swim-up method. Relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility.

The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter. In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure. In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97). Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination. Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated. Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06). On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06). In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained. This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

Acrosome↗

Mouse ICSI with frozen-thawed sperm: the impact of sperm freezing procedure and sperm donor strain.

Normal mouse offspring can be obtained from oocytes injected with frozen-thawed spermatozoa without cryoprotection, however, embryo development can be affected by sperm freezing procedure and sperm donor strain. In this study we observed that direct contact of mouse spermatozoa with liquid nitrogen did not affect their ability to activate injected oocytes but severely restricted subsequent in vitro embryo development to blastocyst stage. Tris-EDTA buffer and M2 were also shown to be better sperm freezing extenders than DPBS, allowing higher developmental potential. In addition, differences in embryo development obtained by intracytoplasmic sperm injection (ICSI) with frozen-thawed spermatozoa were observed between hybrid sperm donor strains. Frozen-thawed B6D2F1 spermatozoa provided higher embryo development than sperm cells from C57CBAF1.

Animals↗

Oviducal sperm storage in turkeys: spatial distribution of sperm within the uterovaginal junction sperm-storage tubules.

The spatial distribution of sperm within the sperm storage tubules (SST) found in the uterovaginal junction (UVJ) of the turkey is not known. In this study, we inseminated sperm stained with a fluorescent dye (Hoechst 33342) to determine their distribution in SST in the ventral, dorsal, and lateral regions of the proximal, middle, and distal regions of the UVJ. There was no preferential filling in the ventral-dorsal axis of the UVJ. In contrast, preferential filling of the SST was observed in the middle section of the UVJ. Here the individual SST were clearly longer and more pleomorphic than the SST in the more proximal and distal aspects of the UVJ. While no information on the temporal aspect of SST filling by sperm could be gleaned, it is evident that the more morphologically developed SST either accept sperm more readily or store sperm more efficiently than SST elsewhere in the UVJ.

Animals↗

Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection.

OBJECTIVE: To determine the prognostic value of sperm DNA fragmentation levels, as measured by the sperm chromatin dispersion (SCD) test, in predicting IVF and ICSI outcome. DESIGN: Double-blind prospective study. SETTING: University-affiliated private IVF setting. PATIENT(S): A total of 85 couples undergoing infertility treatment with IVF/ICSI. INTERVENTION(S): Analysis of DNA fragmentation by the SCD test in 170 aliquots obtained from the ejaculate and from the processed semen used for assisted reproductive technologies (ART). MAIN OUTCOME MEASURE(S): Percentage of spermatozoa with fragmented DNA was statistically correlated with embryo quality and reproductive success. RESULT(S): Fertilization rate was inversely correlated with DNA fragmentation (r = -0.245 P = .045). Higher DNA fragmentation rate gave an increased proportion of zygotes showing asynchrony between the nucleolar precursor bodies of zygote pronuclei (73.8% vs. 28.8% P < .001). In addition, the slower embryo development and worst morphology on day 6 was correlated with higher sperm DNA fragmentation (47.7% vs. 29.4% P = .044). We also observed a negative correlation between DNA fragmentation and the implantation rate (r = -0.250 P = .042). However, SCD test values were not statistically different in cycles that resulted in a pregnancy compared with those that did not (33.2 vs. 28.2 and 32.4 vs. 34.7). CONCLUSION(S): This is the first report that describes a correlation between sperm DNA integrity, as measured by the SCD test, and fertilization rate, embryo quality, and implantation rate in IVF/ICSI. The degree of DNA fragmentation was inversely correlated with fertilization rate, synchrony of the nucleolar precursor bodies' pattern in pronuclei, embryo ability to achieve blastocyst stage, and embryo morphological quality. Because SCD test values were correlated with embryo quality and blastocyst rate, the lack of correlation between sperm DNA fragmentation and pregnancy outcome in IVF might be due to embryo selection before transfer. The ability of the SCD test to predict the blastocyst rate after IVF/ICSI warrants further study.

Adult↗

Monoclonal antibody against a human sperm protein recognizes multiple epitopes on rabbit and human sperm and blocks sperm function.

A monoclonal antibody was raised against a human sperm protein of apparent molecular size of 40 kDa. Of the 6 hybridoma clones selected for study, one clone (B-12) was chosen for further investigations. The antibody secreted by the clone agglutinated human and rabbit spermatozoa in vitro. The antibody belonged to IgM class. In indirect immunofluorescence studies this antibody reacted to acrosome of living human and rabbit spermatozoa. On fixed sperm of the same species it recognized midpiece and parts of tail along with the acrosome. Mouse, hamster, guinea pig, monkey and rat sperm showed similar localization. Interaction between rabbit sperm and oocyte was inhibited in vitro by this antibody. On western blots of human and rabbit sperm extracts the antibody recognized more than one epitope.

Animals↗

Clinical adaptation of the sperm ubuquitin tag immunoassay (SUTI): relationship of sperm ubiquitylation with sperm quality in gradient-purified semen samples from 93 men from a general infertility clinic population.

BACKGROUND: The proteolytic chaperone peptide ubiquitin accumulates in defective human spermatozoa. Immunodetection of ubiquitin in human sperm samples correlates with semen quality and male fertility. METHODS: Semen samples from 93 men from couples seeking infertility treatment were separated on a PureSperm density gradient and screened by immunofluorescence microscopy with anti-ubiquitin antibodies. The percentage of spermatozoa with head ubiquitylation was recorded and compared with clinical semen evaluation and embryo development data after IVF or ICSI. Subjects were divided into the following four groups based on the initial clinical diagnosis of the couples; group 1, male factor; group 2, idiopathic infertility; group 3, female infertility with neither partner having children previously; and group 4, female infertility with male partners having children from previous relationships. RESULTS: The percentage of sperm with ubiquitylated heads remaining after PureSperm separation in the respective groups was 4.0% (male factor), 2.5% (idiopathic infertility), 0.7% (female infertility and presumed fertile male) and 0.9% (female infertility with established fertile male). Negative correlations between sperm ubiquitin and several parameters reflective of embryo development after assisted fertilization were found within the male factor group. CONCLUSIONS: Use of this simplified ubiquitin-based sperm quality assay is feasible in a clinical environment. Since the gradient separation does not completely deplete the defective spermatozoa, the modified light microscopic sperm ubiquitin tag immunoassay could add a new level of stringency to the selection of human spermatozoa for ICSI.

Antibodies, Monoclonal↗

Immunoprecipitation of sperm and somatic antigens with antibodies from sera of sperm-sensitized and anti-sperm antibody-free individuals.

PROBLEM: Immune infertility may result from the presence of anti-sperm antibodies (ASA) in sera of sensitized individuals or in their genito-urinary millieu. According to some authors, sera of pre-pubertal boys with genital disorders may contain ASA, too. When identified and characterized, sperm antigenic components can be employed for diagnostic and/or for immunocontraceptive purposes. METHOD OF STUDY: Sperm auto- and isoimmune reactive surface antigens were identified by means of immunoprecipitation. Comparative immunoprecipitations of somatic antigens by using antibodies, contained in polyclonal sera of fertile and (immunologically) infertile adults as well as pre-pubertal boys with gonadal failures were performed. RESULTS AND CONCLUSIONS: Antibodies contained in sera of the all studied populations reacted with a number of sperm as well as somatic antigens. However, we distinguished a panel of antigens localized exclusively on the surface of sperm cells, reacting with ASA positive sera (in the range of molecular weight between 15 and 115 kDa), some of them seem to present novel entities as can be judged by the molecular weight.

Antibodies↗

Sperm retrieval in infertile males: comparison between testicular sperm extraction and testicular sperm aspiration techniques.

Improvements in using retrieved testicular spermatozoa in infertile males to achieve fertilization and pregnancy has enabled patients with clinical azoospermia and non-obstructive testicular failure to father children. In this review article, we compare the relative yields of the two major techniques for sperm retrieval in males with non-obstructive azoospermia; TESE (Testicular Sperm Extraction) and TESA (Testicular Sperm Aspiration). We also discuss the role of follicle-stimulating hormone (FSH), testicular volume, serum levels of inhibin B, testicular doppler flow pattern, chromosome Yq deletions, and presence of spermatids in azoospermic ejaculates as predictors of presence or absence of spermatozoa in biopsied testicular tissues. In conclusion, although most studies favor the more invasive TESE in terms of sperm retrieval success rate, the degree of certainty in this regard remains unsatisfactory, and future studies need to address the issues of standard error and differential misclassification attributable to needle gauge size in patients undergoing TESA.

Adult↗

Special applications of intracytoplasmic sperm injection: the influence of sperm count, motility, morphology, source and sperm antibody on the outcome of ICSI.

The relationship between the three basic parameters of ejaculated spermatozoa, i.e. concentration, motility and morphology, and the results of intracytoplasmic sperm injection (ICSI) were investigated in 838 microinjection cycles. A further 123 ICSI treatment cycles in which testicular spermatozoa were used for microinjection were also evaluated. The influence of anti-sperm antibodies (ASA) on the outcome of ICSI was investigated by analysing 55 cycles where the proportion of ASA-bound spermatozoa was > or =80%. After microinjection, oocyte intactness, fertilization, embryo cleavage, transfer and pregnancy rates were recorded and compared. The results showed that neither the type nor the extent of sperm impairment had an important influence on the outcome of ICSI when ejaculated spermatozoa were used. Only two very rare conditions had a strongly negative influence on the result of ICSI, i.e. where immotile (presumably dead) spermatozoa or where round-headed spermatozoa were injected into the oocyte. Neither the proportion of ASA-bound spermatozoa, the type of dominantly present ASA, nor the location of ASA on the spermatozoa had an important influence on fertilization, embryo development or pregnancy rates after ICSI. In most of the cycles combined with testicular biopsy (79%), there were enough motile spermatozoa present in the wet preparation for injection of all the oocytes. Injection of motile testicular spermatozoa led to a higher normal fertilization rate than did injection of non-motile spermatozoa (65 versus 21%). It can be concluded that injection of motile (living) spermatozoa into oocytes is the most important factor in determining good results with ICSI and that other sperm parameters do not have a strong influence on the outcome of ICSI.

Antibodies↗

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: evidence based on intracytoplasmic sperm injection experiments in the mouse.

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration.

Animals↗

Cryopreservation of ram semen facilitates sperm DNA damage: relationship between sperm andrological parameters and the sperm chromatin structure assay.

We hypothesized that cryopreservation and incubation in conditions that mimic the female genital tract following insemination increases the susceptibility of ram sperm DNA to denaturation. Ram sperm samples (n = 12) underwent the sperm chromatin structure assay (SCSA) and semen quality tests, including motility parameters, viability, and chlortetracycline fluorescence (CTC) patterns. We also assessed correlations between SCSA variables and semen quality parameters. Analyses were performed for both fresh and cryopreserved samples at 0, 3, and 20 hours of incubation in synthetic oviductal fluid (SOF; 39 degrees C, 5% CO(2)). The SCSA variables, mean alpha t (X alpha(t)) and standard deviation of alpha t (SD alpha(t)), were higher because of cryopreservation (P <.05, P <.001, respectively) after 20 hours in SOF. For both fresh and frozen spermatozoa, SCSA values (X alpha(t), SD alpha(t), and the percentage of cells outside the main population of alpha(t) [%COMP alpha(t)]) increased during incubation in SOF. Motility was negatively correlated with both SD alpha(t) and %COMP alpha(t), ranging from -0.39 (P <.01) to -0.59 (P <.001) for both fresh and cryopreserved semen; viability also was negatively correlated with X alpha(t), SD alpha(t), or %COMP alpha(t) (-0.36; P <.05, -.40 and -.46; P <.01, respectively) in fresh semen. The %COMP alpha(t) was positively correlated to the percentage of CTC pattern AR (P <.001) and negatively correlated to the percentages of patterns F and B (-0.33 to -0.60, P <.05 to P <.001). Variation among ejaculates within ram was observed (P <.01). Cryopreservation clearly facilitates DNA damage in physiological conditions. The low to moderate correlations between SCSA variables and classical semen quality parameters indicate that the SCSA provides additional information to standard tests for evaluating ram sperm quality.

Animals↗

Ultrasound-guided testicular sperm aspiration in azoospermic patients: a new sperm retrieval method for intracytoplasmic sperm injection.

PURPOSE: We investigated the technique of ultrasound-guided testicular sperm aspiration (USTSA) and compared it with "blind" testicular sperm aspiration (TSA) in patients with nonobstructive azoospermia. METHODS: Thirty-nine consecutive azoospermic men underwent TSA, 16 under sonographic guidance (USTSA group) and 23 with no imaging guidance (TSA group). Clinical and hormonal evaluation and sonography of the scrotum and testes were performed 1-2 days before the procedure. The aspiration was done using short-term general anesthesia. Follow-up consisted of sonographic reexamination of the scrotum and testes immediately and 1 month after the procedure. RESULTS: Intraoperative sonography with power Doppler imaging enabled good visualization of the testicular parenchyma, easy sampling, and avoidance of prominent vessels. Sufficient material was retrieved in 15 USTSA patients (94%) and 19 TSA patients (83%). No patients needed more than 4 hours' ambulatory hospitalization after the procedure. In the remaining 5 patients, aspiration failed to yield sperm, so open biopsy was performed. In those patients, postaspiration surgical exploration revealed subtunical bleeding in 3 patients after TSA but none after USTSA. Late minor complications occurred in 2 patients (13%) in the USTSA group and 7 (30%) in the TSA group. No difference was found between the 2 groups in pregnancy rate in the patients' female partners. CONCLUSIONS: USTSA is a safe and accurate method for sperm retrieval in azoospermic patients.

Adult↗

Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia.

The efficiency of testicular sperm retrieval by testicular fine needle aspiration (TEFNA) was compared with open biopsy and testicular sperm extraction (TESE), in 37 rigorously selected patients with non-obstructive azoospermia. All patients underwent TEFNA and TESE consecutively. Thus, each patient served as his own control. The case was regarded as successful if at least one testicular spermatozoon was found allowing intracytoplasmic sperm injection (ICSI) of at least one oocyte. The mean age of the male patients was 32.7 years (range 24-47). Whereas by TEFNA spermatozoa enabling performance of ICSI were found in only four patients out of 37 (11%), open biopsy and TESE yielded spermatozoa in 16 cases (43%). The negative predictive value of high serum follicle stimulating hormone (FSH) concentrations (> or =10 IU/l) (predicting failure to find spermatozoa for ICSI) was low (38.4%). The positive predictive value (predicting the chance to find spermatozoa for ICSI) of normal-sized testicle was not different from that of small-sized (<15 ml) testicle (50%). Complications included one case of testicular bleeding following fine needle aspiration, treated locally, and two cases of extratunical haematomata following TESE requiring no intervention. In patients with non-obstructive azoospermia, TEFNA has a significantly lower yield compared to TESE. Performance of ICSI with testicular sperm in these cases resulted in satisfactory fertilization and high embryo transfer rates. The implantation and pregnancy rates per embryo transfer were 13 and 29% respectively. Neither serum FSH values nor testicular size were predictive of the chances to find spermatozoa for ICSI. Some complications may occur even following TEFNA.

Adult↗

Studies on sperm chromatin structure alterations and cytogenetic damage of mouse sperm following in vitro incubation. Studies on in vitro-incubated mouse sperm.

Mouse epididymal sperm incubated in Tyrode's T6 fertilization media were analyzed over time for chromosome damage by two methods. First, cytogenetic analysis was done on paternal pronuclei metaphase chromosomes. After 6 hours incubation 11% of the cells demonstrated chromosome structural abnormalities. Secondly, sperm nuclei were measured by the sperm chromatin structure assay, which is a measure of the susceptibility of sperm DNA to the nuclei demonstrated an increased susceptibility to DNA denaturation, reaching near 100% by 48 hours. Changes in chromatin structure at the molecular level may lead to chromosome breaks seen in pronuclear chromosomes.

Animals↗

Microsurgical vasovasostomy versus microsurgical epididymal sperm aspiration/testicular extraction of sperm combined with intracytoplasmic sperm injection. A cost-benefit analysis.

PURPOSE: Vasovasostomy (VVS) represents the standard therapy of choice for the treatment of obstructive azoospermia following vasectomy. However, recently, intracytoplasmic sperm injection (ICSI) has been suggested by some to represent the solution for all cases of malefactor infertility regardless of its etiology based on its success rates. Therefore, we compared VVS to microsurgical epididymal sperm aspiration (MESA)/testicular extraction of sperm (TESE) and ICSI in terms of pregnancy, complications, and costs. PATIENTS AND METHODS: Between 1/93 and 6/98, 157 VVS were performed microsurgically using the double-layer technique. Between 9/94 and 9/97, 69 and 42 couples underwent MESA/ICSI and TESE/ICSI, respectively, for epididymal obstruction and azoospermia of testicular origin. RESULTS: The mean interval of vasal obstruction was 7.6 (0.5-18) years; patency after VVS was 77%, pregnancy rate was 52%. Local complication rate was 4.7%, no major complications were observed. Costs per life birth after VVS were 5,447 DM or 2,793 Euro. Pregnancy rates after MESA/TESE and ICSI were 22.5 and 19.5%, respectively, with 16 singletons, 3 twins and 3 abortions; local complications occurred in 3.9% of the men. Multiple births were noticed in 15.8% following ICSI, but in only 0.7% following VVS. 5.7 and 1.4% of the female partners experienced serious complications (mild or severe ovarian hyperstimulation syndrome, respectively). Costs per life birth after a MESA/TESE cycle amounted to 28,804 DM or 14,547 Euro. CONCLUSIONS: Even in the era of ICSI, microsurgical VVS represents the standard approach for obstructive azoospermia following vasectomy. Based on a cost-benefit analysis, VVS is more successful in terms of pregnancy rates (52 vs. 22.5%). VVS does not expose the female partners to complications following treatment of male infertility. In contrast to ICSI, multiple birth rates do not increase after VVS. We conclude that MESA/ICSI should be reversed for patients who are not amenable for microsurgical reconstruction.

Adult↗

Effect of human sperm exposure to progesterone on sperm-oocyte fusion and sperm-zona pellucida binding under various experimental conditions.

In this study, the effect of human sperm exposure to progesterone on sperm/oocyte fusion, using the hamster egg penetration test, and on sperm/zona pellucida (ZP) binding, using the hemizona assay, was investigated under various experimental conditions. A brief exposure of human spermatozoa to progesterone exerted a stimulatory effect on sperm/oocyte fusion which was dose-dependent, capacitation-dependent, influenced by the source of serum albumin in capacitating medium, and was higher than that produced by the exposure to progesterone from the onset of capacitation. The exposure of capacitated spermatozoa to progesterone during 20 min-spermatozoa/ZP-coincubation produced an enhancement of ZP-binding, which was not significantly influenced by the source of serum albumin in capacitating medium. A significantly lower ZP-binding was exhibited by spermatozoa exposed to progesterone from the beginning of capacitation. These results indicate that progesterone exerts a stimulatory effect on human sperm's fertilizing ability, which occurs mainly in post-capacitation events directly involved in sperm/oocyte fusion and in ZP-binding. Conditions optimizing these effects are provided. They should be taken into account in the standardization of experimental and clinical studies designed to evaluate the response of human spermatozoa to progesterone.

Animals↗