Circannual variation in human semen parameters.

The aim of the present study was to determine whether there were significant monthly variations in the semen parameters (i.e. volume, sperm count, total sperm count, motile and normal sperm count) of men living in a Mediterranean climate area. A total of 10 877 semen analysis results were included. Semen samples were obtained as a part of an initial screening of male partners from couples with infertility problems who were attending our laboratory from 1970 to 2000. Log transformation and cubic root transformation were used to test the sample distribution. Statistical significance was adjusted by year of examination, patient's age and sexual abstinence period by performing covariance analyses. Differences between months were assessed with the Bonferroni post-hoc test. There was an increase in March and a decrease in September in the adjusted mean sperm count (p < 0.0005), total sperm count (p < 0.0005), motile sperm count (p=0.01) and normal sperm count (p=0.002). There were no variations in semen volume in the study period. Monthly changes in semen quality are confirmed in this population.

Evolution of semen quality in North-eastern Spain: a study in 22,759 infertile men over a 36 year period.

A retrospective study was conducted in a large population to determine whether sperm quality has changed in Northeastern Spain between 1960 and 1996. From a total initial population of 22,759 men, two separate groups were studied: men with spermatozoa (n = 20,411) and those with azoospermia (n = 1364). After adjustment for age and sexual abstinence, multiple linear regression analyses were used to assess changes in semen parameters over time. A 0.2% decline was observed in semen volume in the spermatozoa group (P < 0.001). No significant increase (0.04%) in sperm count (x 10(6)/ml) was observed in the spermatozoa group. There was a 0.4% increase in motile spermatozoa in the spermatozoa group (P < 0.001). There was a statistically significant decline in normal spermatozoa (3.6%) in the spermatozoa group (P < 0.001). Of the total population, 1364 men had azoospermia (6.0%). The changes observed in the semen parameters analysed in this large population showed no evidence of a deteriorating sperm quality, although a statistically significant decline was observed in the percentage of normal spermatozoa.

Anti-endometrial autoantibodies in women with a diagnosis of infertility.

PROBLEM: The purpose of this study was to investigate the frequency of anti-endometrial antibodies (AEA) in infertile women. METHOD OF STUDY: Sera from fertile women (n = 6), and from patients with ovulatory dysfunction (n = 11), tubal obstruction (n = 9) and unexplained infertility (n = 5) were investigated for the presence of anti-endometrial membrane antibodies. We used two human endometrial cancer cell lines and human endometrial cells from gynecological biopsies as an antigenic source for analysis. The immunoenzymatic assay (ELISA) was performed with cultured endometrial cells in monolayers. Immunoblot analysis was performed with these two cell lines. RESULTS: A good correlation between the response with each cell line and with human endometrial cells was obtained, indicating that the antigens analyzed were probably similar. Endometrial antibodies were detectable in a high percentage of women with tubal obstruction (77.8 and 66.7%, respectively) and ovulatory dysfunction (54.5 and 45.5%, respectively). Unexplained infertility showed anti-endometrial immunological response (40 and 60%, respectively). Some endometrial antigens in infertile women are the target for autoimmune response. The serum from a patient with tubal obstruction and ovulatory dysfunction showed two antigens by immunoblot, with molecular weights of 97 and 50 kDa. CONCLUSION: The presence of anti-endometrial antibodies, detected by ELISA, is associated with infertility, mainly with ovulatory dysfunction and tubal obstruction. Some endometrial antigens may be involved in these two pathologies.

Human sperm coating antigen from seminal plasma origin.

Sperm surface glycoproteins may be involved in sperm-zona pellucida recognition. Some of these coating proteins are of seminal plasma origin and their expression may change in the process of capacitation and acrosome reaction. Sperm specific monoclonal antibodies (mAb) define the presence and role of sperm membrane associated proteins. We have isolated a monoclonal antibody (SEM-12) specific for human sperm that shows, by indirect immunofluorescence, a discontinuous distribution of the antigen on the head and tail surfaces of non capacitated sperm. This antigen is also present in human seminal plasma as detected by ELISA. The antigen is detectable in sperm of goat, ram, and mouse. Two proteins in the range of 80-84 kDa have been isolated by affinity chromatography with SEM-12 mAb. The same result is obtained by immunoprecipitation. This antibody inhibits sperm motility and acrosome reaction (spontaneous and A23187 ionophore induced.

Effect of oocyte-sperm co-incubation on acrosome reaction in the goat.

The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted spermatozoa was also determined. Ovulated oocytes were obtained from superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4% +/- 0.98 to 9% +/- 1.41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10.4% +/- 2.06 and 8.75% +/- 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction.

Which semen parameters have a predictive value for pregnancy in infertile couples?

A prospective study was carried out in 156 couples attending an infertility clinic. To assess the predictive value of semen parameters in relation to pregnancy, we defined a group of 16 couples (group II) in whom the female became pregnant by intra-uterine insemination (IUI), and therefore in whom a female factor could be ruled out. Studies of semen parameters before and after capacitation were carried out in the first trimester of pregnancy (< 12 weeks). The same studies were done in the remaining 140 men (group III) with primary infertility and then all results were compared with a control group of 27 healthy, fertile men (group I), with normal semen parameters. Our results showed that progressive motility and straight line velocity were significantly lower in group III compared with group II: 33.4 and 45.2% respectively (P < 0.001) for progressive motility, and 25.7 and 32.8% respectively (P < 0.005) for straight line velocity. Acrosome alterations, on the other hand, were significantly more frequent in group III compared with group II: 21.4 +/- 0.7 and 5.9 +/- 1.7 respectively (P < 0.003). After capacitation, the recovery in terms of numbers of motile spermatozoa, spermatozoa with normal morphology and acrosome-reacted spermatozoa could be a predictive parameter of fertilization, because all were significantly decreased in group III compared with group II (P < 0.01).

Characterization of monoclonal antibodies specific for human sperm: effect of CRL-10 on acrosome reaction.

Monoclonal antibodies to human sperm were obtained from hyperimmunized BALB/c mouse spleen cells fused with myeloma NS-1 cells. Each antibody recognized definite regions in fresh unfixed sperm: equatorial region, acrosome, postacrosome, midpiece, tail. All the antibodies were specific for sperm. We selected CRL-10 monoclonal antibody, specific for acrosome, for a detailed study. The expression of the CRL-10 antibody-bound antigen was detected in other mammalian species. When CRL-10 antibody was added prior to sperm incubation in a capacitating medium, promotion of the acrosome reaction was observed.

Characterization and regional binding of human sperm monoclonal antibodies.

Five monoclonal antibodies (MAb) with specific binding for tail, midpiece, equatorial region, and postacrosome of human sperm were selected. The reactivity of the MAbs was tested by three techniques: an ELISA with human sperm, an indirect immunostaining analyzed by a cell sorter, and immunofluorescence on the cell where the antigens were localized. The localization or exposure of the antigens changes throughout the in vitro capacitation. These antigens are distributed among other mammalian species: mouse, ram, and goat. Their presence was also analyzed in some human tissues: peripheral blood mononuclear cells, spleen, pancreas, thyroid, heart, skin, intestine, and lung. No cross-reactivity was detected. Some of the MAbs showing tail staining severely inhibited sperm motility after 5 hr of incubation with sperm, although no agglutination was present. This immobilizing activity of MAbs would prevent spermatozoa from reaching the oocyte in vivo.

Inhibition by anti-sperm monoclonal antibodies of the penetration of zona-free hamster oocytes by human spermatozoa.

Fourteen mAb specific for human sperm membrane antigens were selected to investigate their inhibitory effect on fertilization. The antigens were not expressed in other human somatic tissues but were present in sperm from other species. The antibodies were purified from ascites fluid produced in mice. The zona-free hamster egg penetration assay was used for the evaluation of the blocking ability of these antibodies. The inhibition rate was generally related to a decrease in the number of adherent sperm. Three groups of antibodies were distinguished: (i) four mAb that have high inhibition at any concentration; (ii) four mAb with an intermediate inhibitory effect, that is more dependent on the antibody concentration tested; and (iii) six mAb with little or no effect at any concentration. The presence of antibodies leads to a lower penetration index, or number of penetrating sperm per oocyte. MAb specific for head antigens promote high inhibition of fertilization; these antibodies show a patchy staining on the sperm head. The antibodies localized in the midpiece have an intermediate inhibitory effect. No inhibition is detected with the equatorial region binding pattern. Sperm agglutination does not play any role in the inhibition caused by the antibodies described here.

Effect of anti-human sperm monoclonal antibodies on mouse in vitro fertilization.

We employed anti-human sperm monoclonal antibodies to investigate how sperm membrane antigens are involved in gamete interactions. We have produced seven monoclonal antibodies specific for human sperm antigens, that showed reaction with mouse sperm by ELISA and by immunofluorescence. These antibodies did not react with zona pellucida or any other somatic human tissue. Some degree of toxicity was detected for oocytes at high antibody concentration and this was correlated with their inhibitory effect on fertilization. Unrelated to the degree of antigen expression or localization on sperm membrane, the antibodies showed several degrees of inhibition. The participation in sperm-zona pellucida interaction for every antigen could be evidenced by the impaired penetration of sperm caused by the presence of several concentrations of antibody. Thus, DAN-2, MOU-8 and VAC-4 inhibit mouse in vitro fertilization.

Detection of induced anti-sperm antibodies by an improved enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) has been adapted for the detection of human anti-sperm antibodies (ASA). The use of peroxidase in the enzyme-anti-immunoglobulin conjugate, together with a nontoxic chromogenic substrate (MBTH-DMAB), renders the assay highly sensitive for detection of sperm surface antigens. This ELISA is highly reproducible, not only intra-assay (94% to 97%) but also interassay (80% to 96%). A great advantage of this ELISA is that microplates can be kept at -20 degrees C for months without any decrease in the response. New Zealand white rabbits showed a significant response to human sperm and tail fractions obtained by sonication. The sperm were extremely immunogenic, reaching titers of antisera of 1/10,000 or more. The results show that this ELISA is a good tool for the objective measurement of the anti-sperm response, and is significantly more sensitive than other techniques for anti-sperm antibody analysis.

Detection of anti-sperm antibodies in serum, seminal plasma and cervical mucus by the immunobead test.

Anti-sperm antibodies in serum and seminal plasma were detected by means of an indirect immunobead test (IBT). Immunobeads with separate specificites for each immunoglobulin class (IBT-IgG, IBT-IgM, and IBT-IgA) were used. Semen parameters were controlled in all sperm donors and Biggers-Whitten-Whittingham (BWW) medium supplemented with human serum albumin (HSA) was used to increase sperm motility. This technique was tested with high titre anti-human sperm sera induced in rabbits. Sperm tails showed a good response by IBT. We included in this study 178 men and 35 women evaluated for infertility and the sera were also tested by the Tray Agglutination Test (TAT). Although the presence of semen markers such as agglutination or trembling of spermatozoa is meaningful even by itself, the percentage of anti-sperm antibodies was increased in the patients with markers, both using IBT (21.4%) and using TAT (35.7%). At high titres of specific immunoglobulins (rabbit antisera and vasectomized men), the correlation between IBT and TAT techniques was better than in sera with very low titres, in which more positive TAT's were detected.

Improvement of sperm quality in abnormal semen samples using a modified swim-up procedure.

A modified swim-up procedure for the selection of motile and morphologically normal spermatozoa is described. Applied to abnormal (astheno and asthenoterato) semen samples, the recovery figures are comparable to those obtained by other authors in normal samples. The elimination of seminal plasma from the beginning of the procedure avoids contact of spermatozoa with decapacitating factors.