Effects of human antisperm antibodies on development of preimplantation embryos.

The effects of antisperm antibodies (ASAs) present in sera of immunoinfertile patients and vasectomized men were investigated on preimplantation embryonic development in mice. Of the nine immunoinfertile sera tested, two were effective in inhibiting blastulation rates of in vitro cultured murine 2-cell embryos (p less than .05 to .002). Similarly, sera from two of the three vasectomized men were capable of affecting early embryonic development in mice (p less than .05 to .002). Specificities of the embryotoxic effects of ASAs were further confirmed by culturing embryos in the presence of affinity-purified monovalent Fab' antibodies isolated from these sera. Fab' antibodies from only one of the two immunoinfertile patients whose sera affected blastulation rates, and from one of the three vasectomized men were effective in influencing blastulation rates of in vitro cultured 2-cell murine embryos (p less than .05 to .001), mainly due to an arrest of development at 2 to 8-cell and morula stages. In the Western blot procedure, none of the immunoinfertile Fab' antibodies recognized any specific band on blots of extracts from murine ova or 2-cell embryos. However, all the immunoinfertile Fab', but not fertile control Fab', specifically recognized a protein band in the M(r) 25 +/- 2 kD region, on the Western blots of extract from murine blastocyst stage embryos. In addition, Fab' from one immunoinfertile serum, which inhibited embryonic development, reacted specifically with a protein band in the lower molecular range (approximate M(r) 12 kD) on Western blot involving exact from blastocysts. Fab' antibodies of sera from vasectomized men did not react with any specific protein band on blots of extracts from murine ova, 2-cell embryo, or blastocyst. These results suggest that ASAs from some immunoinfertile patients and vasectomized men, especially those reacting with 12-kD blastocyst protein, are capable of affecting preimplantation embryonic development in mice, and thus may contribute toward immunologically medicated infertility both at fertilization and postfertilization stages.     

Sperm antibodies after vasectomy with fulguration.

Whether antisperm antibodies develop after vasectomy probably depends on several variables, 1 of which may be the surgical technique. The levels of serum antisperm antibodies were compared in men vasectomized by 1 techniques: vasoligation and fulguration. No difference in the incidence of spermagglutinating antibody was found in the 2 groups. However, immobilizing antibodies were observed in 43 per cent of the men undergoing vasoligation but in only 29 per cent of the men vasectomized by fulguration.     

Occurrence of sperm antibodies in adult rats after prefertile traumatic vas lesions

The occurrence of sperm antibodies has been studied in adult rats, which at prefertile age had been subjected to unilateral vasectomy, unilateral or bilateral crush injury of the vas or bilateral resection of the ductal artery. By means of indirect immunofluorescence technique, circulating sperm antibodies could be demonstrated in the blood in all groups 13 weeks postoperatively: unilateral vasectomy 15/17 (88%), unilateral crush injury 14/20 (70%), bilateral crush injury 13/20 (65%) and vascular injury 14/20 (70%). Sperm granulomas were observed in the unilateral vasectomy group (14/17) and in the bilateral crush injury group (4/20), whereas no sperm granulomas were seen in the unilateral crush injury or the vascular injury group.     

Vasectomy: Study of Circulating Immune-Complexes and its Correlation with Antisperm Immunity in Man, with a Twelve-Month Follow-up Study

Circulating immune-complexes (CIC) have been detected in sera of vasectomized subjects using the Clq Binding Assay. Results seem to indicate that CIC are a feature of the early post-operative period and a consequence of acute immunization against sperm antigens. The progressive disappearance of CIC from the third month after vasectomy with the simultaneous increase in antisperm antibody percentage and titre suggests that CIC could be a temporary feature in vasectomized men and do not lead to a chronic disease, related to a Type III immune reaction.     

Detection of testicular endocrine abnormalities and their correlation with serum antisperm antibodies in men following vasectomy

We measured the serum gonadotropin response to gonadotropin-releasing hormone in 25 men who underwent vasectomy 2 to 64 months before the study. Ten age-matched fertile men were used as controls. Baseline serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were not significantly different between vasectomized men and controls. However, mean serum follicle-stimulating and luteinizing hormone responses to an intravenous bolus injection of 100 mcg. gonadotropin-releasing hormone were significantly greater in the vasectomy group (p equals 0.008 and 0.003, respectively). There was no correlation between these responses and the interval after vasectomy. Serum antisperm antibodies were present in 13 vasectomized men (52 per cent) using enzyme-linked immunosorbent assay and microagglutination techniques. A significant correlation (p equals 0.003) was found between the presence of serum antisperm antibodies and a normal follicle-stimulating hormone response to gonadotropin-releasing hormone stimulation. Of 13 patients with demonstrable antisperm antibody titers 9 (69 per cent) had normal follicle-stimulating hormone responses, compared to only 1 of 12 (8 per cent) without identifiable antisperm antibody titers. Our data suggest that certain men following vasectomy have abnormalities in seminiferous tubule and Leydig cell functions of the testes. These abnormalities are unrelated to the interval after vasectomy and are not identifiable with routine static hormonal measurements. In addition, serum antisperm antibodies are most likely to be present in men who demonstrate normal seminiferous tubular activity after vasectomy.     

Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies

The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies‐containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.     

A new method for isolation of human antisperm antibodies

Summary. A study was undertaken to isolate pure human antisperm antibodies from the sera of infertile couples. One hundred infertile couples attending the Infertility and IVF Unit (Beilinson Medical Center) because of unexplained infertility were tested (both partners) for antisperm antibodies. Sixty-eight experiments were performed with positive sera containing antisperm antibodies and normal donor sperm. These experiments were followed by experiments in order to elute pure human antisperm antibodies from the sperm surface. Three experiments were performed with human sperm which were found to be coated by antisperm antibodies, in order to directly elute these antibodies from the sperm surface. In all experiments we eluted antisperm antibodies of the IgG and IgA isotypes from the sperm surface. These antibodies were demonstrated in the eluate, in each case by either the indirect immunobead test, the radial immune diffusion assay, or the electrophoresis method. Control experiments were performed as follows: (i) normal donor sperm incubated with normal serum; (ii) normal donor sperm without serum incubation; (iii) normal donor lymphocytes incubated with serum containing antisperm antibodies; (iv) normal donor lymphocytes without serum incubation. No antisperm antibodies were obtained in any of these control experiments. Absorption and elution experiments can be used for the isolation of pure human antisperm antibodies, which may then be used for the production of anti-idiotypic antibodies to antisperm antibodies. The anti-idiotypic antibodies could be further utilized as antigen substitutes for the production of a contraceptive vaccine and/or for application in the treatment of spontaneous abortion and infertility.     

Vasectomy in rhesus monkeys II. Failure to demonstrate humoral and cellular immune responses specific for sperm

A study designed to determine whether humoral antibodies and a cell-mediated immune response to sperm antigen(s) in rhesus monkeys after unilateral and bilateral vas ligations has revealed negative findings. Kibrick agglutination, indirect hemagglutination, and sperm immobilization tests on pre- and postvasectomy sera obtained at timed intervals of one to one hundred and two weeks indicated that significant sperm agglutinating, hemagglutinating, and immobilizing antibodies did not develop in these animals. Also, lymphocyte transformation studies showed that a significant cellular antibody response to sperm antigen(s) did not develop in vasectomized animals. Furthermore, sera from rhesus monkeys injected with autologous and homologous washed sperm in Freund's adjuvant failed to demonstrate a positive reaction in agglutination and immobilization tests. On the other hand, immunization of a female rhesus monkey with washed sperm in Freund's adjuvant produced high titer sperm agglutinating and immobilizing antibodies. Therefore, it has not been possible to repeat or contribute supporting data to literature reports concerning the presence of antibodies to sperm in vasectomized rhesus monkeys. It is further suggested that a number of antigens unrelated to sperm might react with sera obtained from vasectomized animals and lead to positive findings in sperm agglutination and immobilization tests. As a result, erroneous conclusions may be drawn about the development of antibodies specific for sperm in vasectomized rhesus monkeys. Definitive conclusions concerning the development of specific antibodies to sperm antigens after vasectomy will be possible only when sperm antigens have been isolated, purified, and chemically and immunologically characterized for use in serologic tests.     

Experimental Escherichia coli epididymitis in rabbits

We describe an experimental model of bacterial epididymitis in New Zealand white rabbits. Inoculation of 10(7) colony-forming units of Escherichia coli in a retrograde fashion into the vas deferens reliably produced clinical, bacteriologic, and pathologic epididymitis. Inflammation was maximum at two weeks and subsided by one month without treatment. E. coli could be reisolated from the epididymides for up to two weeks post inoculation. We detected loss of spermatogenesis in both the ipsilateral and contralateral testes and the appearance of antisperm antibodies subsequent to the infection in some animals. There were 2 cases (11%) of histologic bilateral epididymitis after unilateral inoculation; one of these had bilateral clinical epididymitis with E. coli recovered from both epididymides at two weeks.     

The relationship of circulating antisperm antibodies to sperm surface antibodies in infertile men

The correlation between the amount and location of antisperm antibody binding to the sperm surface and the level measured in the serum has not been previously reported. Hence, the value and limitations of screening blood sera from men with suspected immunologic infertility are not currently known. In this study 70 paired sera and semen samples were assayed by the immunobead test (IBT). A screening protocol for blood sera was constructed to be 100% sensitive for detecting semen specimens with 20% or more of sperm binding IgG or IgA immunobeads. The specificity of this screening protocol was determined to be 79%. Serum IgA was not a good predictor of IgA on the sperm surface. The true positive predictive rate for antisperm antibodies on the sperm surface using circulating antisperm antibodies as a screening assay was estimated to be as low as 35%. There was little correlation between the site of immunobead binding following passive antibody transfer from patients' sera to donor sperm and the site of naturally occurring antibodies on the patients' sperm surface. Although direct assessment of antibodies on the sperm surface is preferred, these data suggest that serum IgG alone can be used as a sensitive screening assay for antisperm antibodies in men. A positive screen dictates that a direct assay on semen should be performed.     

Vasectomy in rhesus monkeys II. Failure to demonstrate dumoral and cellular immune responses specific for sperm.

A study designed to determine whether humoral antibodies and a cell-mediated immune response to sperm antigen(s) in rhesus monkeys after unilateral and bilateral vas ligations has revealed negative findings. Kibrick agglutination, indirect hemagglutination, and sperm immobilization tests on pre- and postvasectomy sera obtained at timed intervals of one to one hundred and two weeks indicated that significant sperm agglutinating, hemagglutinating, and immobilizing antibodies did not develop in these animals. Also, lymphocyte transformation studies showed that a significant cellular antibody response to sperm antigen(s) did not develop in vasectomized animals. Furthermore, sera from rhesus monkeys injected with autologous and homologous washed sperm in Freund's adjuvant failed to demonstrate a positive reaction in agglutination and immobilization tests. On the other hand, immunization of a female rhesus monkey with washed sperm in Freund's adjuvant produced high titer sperm agglutinating and immobilizing antibodies. Therefore, it has not been possible to repeat or contribute supporting data to literature reports concerning the presence of antibodies to sperm in vasectomized rhesus monkeys. It is further suggested that a number of antigens unrelated to sperm might react with sera obtained from vasectomized animals and lead to positive findings in sperm agglutination and immobilization tests. As a result, erroneous conclusions may be drawn about the development of antibodies specific for sperm in vasectomized rhesus monkeys. Definitive conclusions concerning the development of specific antibodies to sperm antigens after vasectomy will be possible only when sperm antigens have been isolated, purified, and chemically and immunologically characterized for use in serologic tests.     

Temporal appearance of antisperm autoantibodies in Lewis rats following vasectomy

An indirect enzyme-linked immunosorbent assay (ELISA) was employed to monitor antisperm autoantibodies in 16 Lewis rats for up to 36 weeks following vasectomy. This assay was capable of discriminating all prevasectomy from postvasectomy sera at a 1:16 dilution. Weekly serum samples were obtained for the first 13 weeks and bimonthly samples thereafter. Half of the animals developed a positive antisperm autoantibody response by the end of the first postoperative week. By the end of the second week, 81% of the animals had positive responses. The greatest proportion (88%) of animals having a positive response over the course of the study was found at the end of the seventh postoperative week and the highest mean absorbance value for all 16 animals was observed at this time. Only 25% of the animals had positive responses for antisperm autoantibody at the end of the 35th week of the study. These findings indicate that circulating antisperm autoantibodies arise in the Lewis rat earlier than has been generally appreciated. The time course is similar to that of antibody titers to infectious agents or arising from inoculation of rats with spermatozoa. These findings on autoantibody levels in the Lewis rat are compared with the dynamics of antisperm autoantibody formation in man.     

Do men with prostate abnormalities (prostatitis/benign prostatic hyperplasia/prostate cancer) develop immunity to spermatozoa or seminal plasma?

Prostate is an immunocompetent and not an immunoprivileged organ. It has an active immunologic armamentarium. There are three major prostate abnormalities namely, prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer. In all these abnormalities, infection/inflammation has been implicated. As infection/inflammation of the male genital tract can also be involved in induction of antisperm antibodies (ASA), this study was conducted to examine if these prostate abnormalities lead to the formation of ASA. Sera were obtained from normal healthy men (n = 20), men with chronic prostatitis (n = 20), men with BPH (n = 25), men with prostate cancer (n = 25) and immunoinfertile men (n = 10). The presence of antisperm antibodies against lithium diiodosalicylate (LIS)-solubilized human sperm extract (HSE), seminal plasma and synthetic peptides based upon sperm-specific antigens namely fertilization antigen (FA-1) and YLP(12), were analysed using the sperm immobilization technique (SIT), tray agglutination technique (TAT), enzyme-linked immunosorbent assay (ELISA) and indirect immunobead binding technique (IBT). All the sera from normal men and men with prostate abnormalities (chronic prostatitis/BPH/prostate cancer) were found to be negative in SIT and TAT. In ELISA, a few sera from men having prostate abnormalities (4-24%) showed a weak positive immunoreactivity (2-3 SD units) with some of the spermatozoa/seminal plasma antigens. Majority of the samples did not show any immunoreactivity (<2 SD units) in ELISA. Even the samples that showed a weak positive immunoreactivity in ELISA did not bind to live human sperm in IBT, indicating lack of sperm binding antibodies in these sera. In all these assays, the sera from immunoinfertile men were positive. Our findings indicate that chronic prostatitis, BPH and prostate cancer do not induce antibodies to spermatozoa, sperm-specific antigens and seminal plasma components. Although prostate is an immunologically competent organ, and its abnormalities cause a rise in circulating prostate-specific antigen (PSA), it appears that there is no concomitant induction of immunity to spermatozoa/seminal components including sperm-specific fertility-related antigens, thus not causing ASA-induced immunoinfertlity. This is the first study to our knowledge reporting the absence of ASA in men with BPH and prostate cancer.     

Determination of Sertoli cell numbers in the developing rat testis by stereological methods

Stereological studies were performed to determine the number of Sertoli cells present during the postnatal development of the rat testes. Sprague-Dawley rats aged from 1 to 70 days were used in two experiments, and in each were fixed by vascular perfusion and embedded in Epon-Araldite, subsequent to which 1 micron sections stained with Toluidine blue were prepared. In the first experiment, rats aged from 1 to 20 days were used in groups of three, and number estimates were made using a direct counting method. In the second, which used groups of four rats aged from 20 to 70 days, a point sampled intercept was used to estimate nuclear volume and thence number. The results of the experiments indicate that the newborn rat testis contains 1.3 +/- 0.2 x 10(6) Sertoli cells and that this number increases to 38.4 +/- 2.7 x 10(6) at day 15. No further increase in Sertoli cell number occurred thereafter up to day 70 of age.     

Recovery of water maze performance in aged versus young rats after brain injury with the impact acceleration model

Clinically, elderly patients have a higher cognitive morbidity from head trauma than young patients. We have modeled injury in aged rats in an effort to elucidate the pathophysiology of this enhanced sensitivity and, in particular, to determine if there are susceptibility differences in forebrain cholinergic innervation in young versus aged rats. Aged (20-23 months) and young (2-3 months) rats were subjected to injury under halothane anesthesia using the Marmarou impact acceleration model. Injury parameters required adjustment downward for the aged rats (323 g at 1.61 m versus 494 g at 2.06 m) to provide equivalent mortality (30% versus 20%) and loss of righting-reflex times (10-12 min average). At 1 week following injury, the aged animals were markedly more impaired in water maze performance than were young rats, and this difference persisted at least up to 5 weeks following injury. The extent of improvement in performance from 1 to 5 weeks was markedly worse for aged animals compared to young animals. Forebrain synaptosomal choline uptake was decreased in aged injured rats by 8-14% at 1, 3, and 5 weeks postinjury, but not decreased in young injured rats. No differences were noted in entorhinal cortex or hippocampal choline uptake. This model effectively demonstrates the markedly increased susceptibility of older animals to head injury and their decreased capacity for recovery. The neurophysiological basis for this difference is presently unknown, but the differences in cognitive dysfunction between young and aged rats appears to be much greater than would seem to be explained by the small differences in forebrain cholinergic innervation.     

The incidence of spermatic granulomas and their relation to testis weight after vasectomy and vasovasostomy in Lewis rats

The occurrence of spermatic granulomas of the vas deferens was studied in Lewis rats at intervals up to 7 months after vasectomy or vasectomy followed 3 months later by vasovasostomy. The incidence of granuloma progressed with time to involve one or both tracts in 100% of vasectomized rats. In addition, the majority of animals developed new granulomas after vasovasostomy, even though fluid flow through the reconnected vas deferens was demonstrated in vitro. When individual tracts were analyzed, the weight of the testis was related to ipsilateral spermatic granuloma formation in both vasectomy and vasovasostomy groups at 3 and 4 months after initial operation. Testes were small in the absence of a granuloma but similar to those of sham-operated rats if a granuloma was present. The possible protective effect of spermatic granuloma formation on the testis is discussed.     

Complement-fixing islet cell antibodies in the spontaneously diabetic BB rat

Fourteen rats of the spontaneously diabetic BB line were bled from the retroorbital sinus approximately every 10 days. Sera taken from an early age up to 20 days after the onset of overt diabetes were assayed for complement-fixing antibodies against antigens of the surface of islet cells (CFA). Dispersed islet cells from normal Wistar rats prelabeled with 3H-leucine were used as targets. Target cells in suspension were incubated with heat-inactivated rat sera and then, after washing, exposed to guinea pig complement. Cytolytic "injury" was measured by the percentage of labeled cellular proteins released into the medium. Sera from sequential bleedings from eight normal Wistar rats and three rats from a nondiabetic BB subline were assayed to establish basal control cytolytic activity. The mean response +/- SD obtained with all control sera was 7.7 +/- 1.7%. A response exceeding the mean + 3 SD (12.8%) was considered significantly different from the basal value. Thirteen of the fourteen BB rats developed strongly positive sera. The cytolytic activity preceded the onset of overt diabetes. In several rats CFA appeared 4-8 wk preceding diabetes while in other rats CFA appeared 1-2 wk preceding the manifestation of the disease. These results indicate that CFA may contribute to the destruction of pancreatic islets directly or by attracting mononuclear cells.     

Spermotoxic properties of IgG fractions from normal rabbit sera.

The antisperm activities of IgG fractions prepared from normal rabbit sera by three different techniques (ammonium sulphate precipitation, gel filtration, and DEAE-Sephadex absorption) were tested using a cytotoxic assay, and antisperm activity detected in fractions prepared by each of these techniques. The nature of the interactions between sperm and IgG molecules from normal sera, and possible functions of "natural" antisperm antibodies, are discussed.     

Analysis of spermatozoa from the proximal vas deferens of vasectomized men

This study assessed the condition of spermatozoa from the proximal vas deferens of men after vasectomy. The fluids of both proximal vas deferens were collected from 67 vasectomized men by cannulating the vas deferens at the time of vasectomy reversal. Selected sperm parameters were analysed after incubation of the spermatozoa for 30 min at 37°C. Spera concentration in the proximal vas from vasectomized men (16 312 ± 21 496 million per ml, geometric mean: 7948 ± 398 million per ml) was significantly higher than that of fertile men and was maintained at a constant level independent of the duration of vas obstruction. The means of sperm motility (36.2 ± 26.2%), spermatozoa with normal morphology (50.7 ± 21.7%), sperm viability (53.0 ± 25.3%) and hypo-osmotic swelling test (HOS-test, 53.9 ± 21.7%) were statistically lower than the respective values for normal fertile men. There was no significant correlation between the duration of vas obstruction and the above semen parameters. In 46.4% of vas fluids all spermatozoa were immotile and this condition was more common after 3 years of vasectomy. Immotile spermatozoa in the proximal vas fluids at the time of vasectomy reversal may be an important factor for predicting semen quality and fertilizing ability after vasovasostomy. There were no significant differences in the results of sperm-cervical mucus penetration test (CMPT) between spermatozoa fiom vasectomized and fertile men. Antisperm antibodies on the surface of spermatozoa from the vas of vasectomized men were determined by the immunobead test (IBT; 78.6% for IgG, 32.1% for IgA) and sperm cervical mucus contact test (SCMC, 36.4%). The presence of antisperm antibodies on the spermatozoa from the vas of vasectomized men may explain, in part, the lower pregnancy rate after vasovasostomy. These parameters of spermatozoa from the proximal vas of vasectomized men may closely reflect those in the cauda epididymis after vasectomy.     

Age-dependence of the dose-response curve of vecuronium in pediatric patients during balanced anesthesia

The effect of age on the log-based cumulative dose-response curve of vecuronium was determined in ten age groups of 80 pediatric patients ranging from neonates to adolescents during thiopental-fentanyl-N2O/O2 anesthesia. Neuromuscular block was recorded as the evoked thenar electromyographic response to train-of-four stimulation of the ulnar nerve (2 Hz at 20-second intervals). The dose-response curves were parallel to each other in all ten age groups studied. In neonates and infants, the ED95 of vecuronium was 47 +/- 11 (SD) micrograms/kg. This was significantly lower than the ED95 of 81 +/- 12 micrograms/kg in children between 3 and 10 years of age (P less than 0.01). In patients aged 13 years or older, the ED95 was 55 +/- 12 micrograms/kg, which did not differ from the neonatal and infant values but was significantly lower than the ED95 of children between 3 and 10 years of age. The results indicate that the dose of vecuronium necessary for tracheal intubation is age-dependent. The individual ED95 values varied between 22 and 103 micrograms/kg. This suggests that an individually optimal dose of vecuronium can be administered to pediatric patients only if neuromuscular block is adequately monitored.