Human antisperm monoclonal antibodies constructed postvasectomy

Sperm and spermatogenic cell antigens, escaping the blood-testis/blood-epididymal barrier, elicit an autoimmune response in patients following vasectomy. In this study, antisperm antibody-positive sera and peripheral blood lymphocytes were obtained 6-9 mo following vasectomy. Serum antisperm antibody levels were assessed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence. Lymphocyte-myeloma hybridomas were constructed by fusing peripheral blood lymphocytes, harvested from antisperm antibody-positive sera, with a hypoxanthine guanine-phosphoribosyltransferase (HGPRT)-negative mouse myeloma line. Immunoglobulin-secreting colonies surviving drug selection were detected by ELISA and screened for antisperm activity. Antisperm antibody-producing cultures were cloned and expanded for bulk antibody production both in culture and as ascites in athymic nude mice. Eight mouse-human fusions yielded 205 hybridomas secreting human monoclonal antibody, of which 11 demonstrated antisperm reactivity by ELISA. Two of these hybridomas are described in detail: HAS-1, which secretes human immunoglobulin M (IgM, kappa)-recognizing epitopes located on the sperm midpiece, and HAS-2 (IgM, lambda), which secretes monoclonal antibody-recognizing epitopes located on the entire sperm tail. The results indicate successful capture of human antisperm autoantibody from the postvasectomy autoimmune state using somatic cell hybridization techniques.     

The influence of vasovasostomy on antisperm antibodies in rats

Serum antisperm antibodies were studied in Sprague-Dawley rats after vasectomy and vasovasostomy. Animals received a bilateral vasectomy, a vasectomy followed 3 mo later by vasovasostomy, or sham operations. Blood samples were obtained at 1, 3, 4, and 7 mo, and antisperm antibodies were assayed by an enzyme-linked immunosorbent assay. After vasectomy reversal was performed at 3 mo, antisperm antibodies were significantly higher in rats in the vasovasostomy group at 4 mo than in animals that had a persisting vasectomy or sham operations. At 7 mo, the antisperm antibody level for the vasovasostomy group was approximately double that for the vasectomized rats. Spermatic granulomas occurred in 76% of rats after vasovasostomy. Antisperm antibody levels were higher in vasovasostomized animals with granulomas than in those lacking granulomas. The results suggest that vasovasostomy may stimulate an antibody response to sperm rather than lead to a reduced response, as was anticipated upon removal of the obstruction. Spermatic granulomas may serve as sires for continued antigenic challenge. The observed increase in antisperm antibodies after vasovasostomy in Sprague-Dawley rats may be related to their relatively low immunologic responsiveness to vasectomy, with vasovasostomy serving as a second major immunologic challenge, aided by the formation of an additional granuloma. In the more responsive Lewis strain, we previously observed a rise in antisperm antibodies after the initial vasectomy, with no further increase after vasovasostomy.     

Effects of antisperm antibodies on early cleavage of fertilized ova.

Early cleavage of zygotes was abnormal after oocytes were fertilized with sperm cells from one of 64 infertile couples studied in our human in vitro fertilization and embryo development procedure. The abnormal cleavage, which did not affect fertilization, was not due to polyspermy or parthenogenetic activation of oocytes, but to antisperm antibodies present on the sperm cells. These antibodies did not react with zona-intact or zona-free human oocytes, although they reacted with the acrosomal (strongly) and tail (weakly) regions of human sperm. The antibodies recognized a double band comprising two glycoproteins of 14 +/- 3 and 18 +/- 3 kDa and a protein band of 22 +/- 3 kDa on the Western blot of lithium diiodosalicylate-solubilized human sperm extract. Antisera were raised in rabbits against the double band (14 +/- 3- and 18 +/- 3-kDa antigens) and the 22 +/- 3-kDa protein. Passive transfer of affinity-purified human or rabbit immunoglobulins directed against the double band (14 +/- 3- and 18 +/- 3-kDa antigens), but not those directed against the 22 +/- 3-kDa protein, caused a significant inhibition of early cleavage of oocytes without affecting pronuclear formation in mice. These results suggest that the sperm surface antigens of 14 +/- 3 and 18 +/- 3 kDa may provide an extranuclear signal to oocytes to divide in mice and humans. These antigens may also find clinical applications in the management of immunoinfertility and in the development of an antisperm contraceptive vaccine for humans.     

Reproductive performance and fatty liver in Guernsey cows

The relationship between fatty liver and reproductive performance was investigated in a commercial herd of Guernsey dairy cattle. Forty-two cows were sampled by liver biopsy at 1 week after calving, and divided into two groups, a fatty liver and a non-fatty liver group, on the basis of the level of liver fat. The cows in the fatty liver group had a milk yield of 5267 kg and a calving interval of 428 days compared with 4407 kg and 369 days for the cows in the non-fatty liver group. The reduced fertility was associated with an elongation in the interval from calving to first service of 20 days and with a reduced conception rate. The occurrence of fatty liver significantly impaired the reproductive performance of the cows.     

Identification of bovine sperm specific polypeptides reactive with antisperm antibodies.

The present study was taken to characterize molecular weights of sperm specific polypeptides antigenic to rabbits and calf with the aim to assess their immunoreactivity with IgG antibodies in sera from immuno-infertile cows. Seropositivity for antisperm IgG antibodies in 75 repeat breeder and 15 pregnant control cattle was tested by cellular ELISA using washed spermatozoa antigen from 4 bulls. Molecular weights of bovine sperm polypeptides antigenic to rabbit and calf were determined by 10% SDS-PAGE and Western blotting. Molecular weights of sperm peptides reactive with sera from immuno-infertile cows were also determined. Seropositivity of antisperm IgG antibodies for bull I, II, III and IV was 23.6, 14.6, 26.6 and 20%, respectively. A total of 16 polypeptides were discernible on gel. Out of these, 7 polypeptides were immunoreactive with sera from hyperimmunized rabbits as compared to 3 poly-peptides which reacted with sera from hyper-immunized calf. Only two polypeptides were reactive with sera from immuno-infertile cows. Variable number of sperm polypeptides and their immunoreactivity have been reported in different species. Antigenicity of different polypeptides in sperm needs further investigations.     

Reproductive tract defense and disease in postpartum dairy cows

This paper briefly reviews recent data and concepts on the development and mitigation of infection and inflammation in the reproductive tract of dairy cows during the first 2 mo after calving. The incidence of metritis is typically between 10 and 20%, of clinical endometritis or purulent vaginal discharge (PVD) approximately 15%, and of subclinical or cytological endometritis a further 15%. Worse postpartum negative energy balance is associated with more severe or prolonged uterine inflammation. Changes in feed intake, expression of genes for pro-inflammatory cytokines, notably interleukin (IL) 1, IL6 and IL8, circulating concentrations of beta-hydroxybutyrate (BHBA) or nonesterified fatty acids (NEFA), and innate immune function precede both metritis and endometritis by several weeks. Infections with Escherichia coli and Arcanobacterium pyogenes are associated with both metritis and PVD. There are new data to suggest that specific virulence factors in E. coli associated with adherence may be important in metritis and PVD. Cytological endometritis and PVD are overlapping but largely distinct conditions, and there are emerging data that cervicitis exists both concurrent with and separate from endometritis. Much remains to be learned about what initiates and sustains harmful inflammation of the reproductive tract. Such information is necessary to develop effective treatments for the various forms of disease and, more importantly, to develop means to prevent endometritis and cervicitis. In particular, vaccination against specific uterine pathogens and interventions to modulate innate immune response appear to be important avenues for investigation. Presently, commonly recommended best management practices for cows in the transition period are likely to be helpful to mitigate the risk of reproductive disease.     

Reproductive tract defense and disease in postpartum dairy cows

This paper briefly reviews recent data and concepts on the development and mitigation of infection and inflammation in the reproductive tract of dairy cows during the first 2 mo after calving. The incidence of metritis is typically between 10 and 20%, of clinical endometritis or purulent vaginal discharge (PVD) approximately 15%, and of subclinical or cytological endometritis a further 15%. Worse postpartum negative energy balance is associated with more severe or prolonged uterine inflammation. Changes in feed intake, expression of genes for pro-inflammatory cytokines, notably interleukin (IL) 1, IL6 and IL8, circulating concentrations of beta-hydroxybutyrate (BHBA) or nonesterified fatty acids (NEFA), and innate immune function precede both metritis and endometritis by several weeks. Infections with Escherichia coli and Arcanobacterium pyogenes are associated with both metritis and PVD. There are new data to suggest that specific virulence factors in E. coli associated with adherence may be important in metritis and PVD. Cytological endometritis and PVD are overlapping but largely distinct conditions, and there are emerging data that cervicitis exists both concurrent with and separate from endometritis. Much remains to be learned about what initiates and sustains harmful inflammation of the reproductive tract. Such information is necessary to develop effective treatments for the various forms of disease and, more importantly, to develop means to prevent endometritis and cervicitis. In particular, vaccination against specific uterine pathogens and interventions to modulate innate immune response appear to be important avenues for investigation. Presently, commonly recommended best management practices for cows in the transition period are likely to be helpful to mitigate the risk of reproductive disease.     

Effects of experimentally generated bull antisperm antibodies on in vitro fertilization.

To test the hypothesis that bull antisperm antibodies have the capacity to interfere with fertilization, antisperm antibodies were generated in three 13-mo-old Holstein bulls by auto-immunizing each bull with sperm three times. All bulls produced serum antisperm IgG1 and IgG2 antibodies. No serum antisperm IgA nor seminal plasma antisperm antibodies of any isotype could be detected by ELISA. Western blots were performed with immunopurified IgG1 and IgG2 from pre- and post-immunization sera from one test bull. Both post-immunization IgG1 and IgG2 recognized a 45-kDa sperm antigen. Serum samples from a normal bull stud population tested by ELISA had significantly higher levels of antisperm antibodies than did heifers. The bull stud serum samples giving the highest ELISA values differed from those of the immunized bulls in that their antisperm antibodies were of the IgM isotype only. Bull sperm were incubated with serum from the immunized and control bulls, then added to bovine oocytes in vitro. Incubation of sperm with post-immunization serum reduced in vitro fertilization rates (p < 0.01). This study demonstrated that antisperm IgG1 and IgG2 generated by sperm auto-immunizations reduced fertility in vitro, and therefore naturally occurring antisperm antibodies may affect fertility in bulls.     

Standardization of a method to detect bovine sperm-bound antisperm antibodies by flow cytometry

The objectives were to standardize some methodological and analytical aspects of a direct technique to detect sperm-bound antisperm antibodies (ASAs) in bovine semen using flow cytometry, including the effects of prefixation of sperm membranes with formalin buffer solution and inclusion of dead cells in the analysis. Fourteen Angus bulls, including ASA-positive (experimentally induced ASAs) and 10 reproductively normal ASA-negative bulls, were used. Fixation of sperm membranes had no significant effect on the percentage of IgG- or IgA-bound spermatozoa detected by flow cytometry. However, including dead cells in the analysis increased the percentage of IgG-bound spermatozoa in fixed (live and dead 18.6 ± 9.7% and live 1.3 ± 0.5%; median ± SEM) and nonfixed samples (live and dead 18.8 ± 9.2%, live 1.5 ± 0.6%; P = 0.0029), as well as IgA-bound spermatozoa in fixed (live and dead 16.3 ± 6.4%, live 0.3 ± 0.5%) and nonfixed samples (live and dead 21.4 ± 4.6%, live 1.0 ± 0.5%; P = 0.0041) in semen from ASA-negative bulls. Intrasample, intra-assay, and interassay coefficients of variation (CV) were 0.8%, 4.6%, and 5.3%, respectively, for determination of sperm-bound IgG, and were 2.8%, 8.4%, and 40.3% for determination of sperm-bound IgA. Despite the high interassay CV for IgA determination, all ASA-positive bulls consistently had high percentages of IgA-bound spermatozoa. Flow cytometry correctly identified ASA-positive bulls. Confocal laser microscopy confirmed binding of ASAs to sperm heads and cytoplasmic droplets, and less frequently to midpieces and principal piece. In conclusion, although fixation was not necessary, dead cells should be excluded from the analysis, because ejaculates with a large proportion of dead cells can yield false-positive results. Flow cytometry was accurate and reliable for detection of sperm-bound IgG and IgA and discrimination between ASA-positive and ASA-negative bulls.     

The relation between antisperm antibodies and testicular alterations after vasectomy and vasovasostomy in Lewis rats

The relationship between antisperm antibodies as determined by enzyme-linked immunosorbent assay (ELISA) and the occurrence of alterations in testicular weight and histology was studied following vasectomy in Lewis rats. The effects of vasovasostomy on antisperm antibody levels were also examined. At 1, 3, and 4 months after vasectomy, the mean absorbance values in an ELISA for sera from animals with altered testes was significantly greater than that from animals lacking testicular alterations. However, animals showing positive antisperm antibody responses were represented both in the group with testicular alterations and among those that lacked testicular damage. Levels of antisperm antibody in both vasectomy and vasovasostomy groups significantly exceeded that for sham-operated animals, but the level of antisperm antibodies in vasovasostomized animals with positive responses was similar to vasectomized animals one and four months after reanastomosis. It is suggested that persistence of antisperm antibodies or testicular alterations, or both, may play roles in limiting the restoration of fertility after vasovasostomy.     

Anti-leucocyte antibodies and embryonic mortality in embryo transferred cows

Cows carrying unrelated transferred embryos (ET) produced anti-leucocyte serum antibodies (aLA) more often than cows carrying their own embryos. Cows carrying the ET showed a higher frequency of cytotoxic reactions against leucocytes from 40-60 randomly chosen cows than individuals carrying their own embryos. The percentage of animals with aLA was higher in cows carrying their second or third transferred embryo than in those with their first transferred embryo. There was no change in the frequency of cytotoxic reactions with repeated pregnancies from transferred embryos. There was no difference in the toxicity of aLA in normal pregnant cows and those carrying transferred embryos. Embryonic mortality (EM) of 35, 73 and 88% was noted during pregnancies from the first, second and third successful ET, respectively. Mortality of 48% occurred in the first pregnancy following an unsuccessful ET. Embryonic mortality of 31% occurred in cows simultaneously carrying their own and a transferred embryo. A direct relationship between the presence of aLA and EM in recipients was not proved. Other fertility problems may lead to EM in cows subjected to repeated transfer of foreign embryos.     

Livestock production in central Mali: Reproductive aspects of sedentary cows

The results of a 6-year study of cow reproductive traits in an agro-pastoral system in semi-arid central Mali are reported. Age at first calving was 1505 days (49.5 months). Calving intervals averaged 665 days with a standard deviation of 202 days, but these showed a bimodal distribution centred on 13–16 and 23–26 months. Least squares analysis indicated that parturition number had a significant effect on the calving interval. Some 56% of calves were born in the period April to June, there being a very highly significant correlation between these births and rainfall 9 and 10 months previously. The average number of calves produced per cow in the herd was 2.9. Nutritional stress appears to be the main cause of low reproductive rates and of the seasonal pattern of births and mitigation of this stress would lead to higher calving percentages.     

The use of flow cytofluorometry for determining antisperm antibodies by sperm immobilization and sperm toxicity methods]

During antisperm antibody detection by conventional methods of immobilization and cytotoxicity investigators used flow cytometry to detect dead and/or immovable sperm cells. It was shown that after incubation of sperm cells with low antiserum dilution immovable spermatozoa are still living. Evidently, cytotoxicity test in this case brings about false-negative results. Therefore, flow cytometry will be a success for ASAB detection through immobilization test.     

Socioeconomic status and incidence of sudden cardiac arrest

Background:

Low socioeconomic status is associated with poor cardiovascular health. We evaluated the association between socioeconomic status and the incidence of sudden cardiac arrest, a condition that accounts for a substantial proportion of cardiovascular-related deaths, in seven large North American urban populations.

Methods:

Using a population-based registry, we collected data on out-of-hospital sudden cardiac arrests occurring at home or at a residential institution from Apr. 1, 2006, to Mar. 31, 2007. We limited the analysis to cardiac arrests in seven metropolitan areas in the United States (Dallas, Texas; Pittsburgh, Pennsylvania; Portland, Oregon; and Seattle–King County, Washington) and Canada (Ottawa and Toronto, Ontario; and Vancouver, British Columbia). Each incident was linked to a census tract; tracts were classified into quartiles of median household income.

Results:

A total of 9235 sudden cardiac arrests were included in the analysis. For all sites combined, the incidence of sudden cardiac arrestin the lowest socioeconomic quartile was nearly double that in the highest quartile (incidence rate ratio [IRR] 1.9, 95% confidence interval [CI] 1.8–2.0). This disparity was greater among people less than 65 years old (IRR 2.7, 95% CI 2.5–3.0) than among those 65 or older (IRR 1.3, 95% CI 1.2–1.4). After adjustment for study site and for population age structure of each census tract, the disparity across socioeconomic quartiles for all ages combined was greater in the United States (IRR 2.0, 95% CI 1.9–2.2) than in Canada (IRR 1.8, 95% CI 1.6–2.0) (p < 0.001 for interaction).

Interpretation:

The incidence of sudden cardiac arrest at home or at a residential institution was higher in poorer neighbourhoods of the US and Canadian sites studied, although the association was attenuated in Canada. The disparity across socioeconomic quartiles was greatest among people younger than 65. The association between socioeconomic status and incidence of sudden cardiac arrest merits consideration in the development of strategies to improve survival from sudden cardiac arrest, and possibly to identify opportunities for prevention.An estimated 250 000–300 000 sudden cardiac arrests occur each year in the United States,¹ accounting for up to 63% of cardiac-related deaths annually.² Despite advances in resuscitation, more than 95% of people who experience sudden cardiac arrest die,³ and up to 50% of sudden cardiac arrests occur in people who do not have a history of coronary artery disease.⁴Socioeconomic status has been shown to predict many health outcomes, including all-cause mortality,⁵ prevalence of risk factors for cardiovascular disease⁶ and incidence of cardiovascular disease.^7–9 Despite this substantial literature, we found only three studies that examined the potential association between socioeconomic status and sudden cardiac arrest. Although the studies were small and conducted in single communities, each showed that the incidence of sudden cardiac arrest was significantly higher in lower socioeconomic areas.^10–12 The Oregon Sudden Unexplained Death Study (Ore-SUDS) reported a 30%–80% higher incidence of sudden cardiac arrest in poorer neighbourhoods. A stronger association was observed among people less than 65 years old, a group for whom basic health care funding is not guaranteed in the United States.¹¹Low socioeconomic status may be linked to an increased risk of sudden cardiac arrest by a variety of mechanisms related to individual risk factors or health-promoting behaviours or neighbourhood characteristics. Individuals of lower socioeconomic status have been found to have a greater burden of risk factors for cardiovascular disease,¹³ poorer control of established cardiovascular risk factors¹⁴ and longer delays in seeking hospital care for acute myocardial infarction.¹⁵ Numerous studies have also shown that disparities in health outcomes are apparent across the spectrum of socioeconomic status.¹⁶A better understanding of community-level patterns in the distribution of sudden cardiac arrest may identify opportunities for improving survival, such as effective targeting of community training for cardiopulmonary resuscitation and placement of automated external defibrillators in lower-income communities. We tested the hypothesis that disparities in the incidence of sudden cardiac arrest by level of socioeconomic status would be evident in a variety of urban communities in the United States and Canada, and that this association would be most prominent among people less than 65 years old residing in US communities.     

Production of foetally stimulated lymphocytotoxic antibodies by primiparous cows

Serum samples from groups of heifers at different pregnancy-related stages (preconception, month of pregnancy, and postpartum) were examined for the presence of lymphocytotoxic antibodies. Only a few weak and transitory antibodies were observed before or during the first pregnancy. These did not appear to be foetally stimulated. Strong or medium strength antibodies were detected in first or second month postpartum samples from 8 of 27 heifers (30%). The reactive sera were in every case reactive with the offspring of the cows from which the respective sera were collected.     

Production of foetally stimulated lymphocytotoxic antibodies by multiparous cows

Serum samples were collected monthly from second gestation cows and examined for the presence of lymphocytotoxic antibodies. Of 25 cows studied 16 (64 %) raised antibodies during or immediately after second gestation. Ten of these cows (40 %) raised antibodies during gestation, some as early as 5 months before parturition. Reactors which had had first-gestation reactivity responded earlier, but had peak antibody titers no higher than cows without observable first gestation reactions. Cows with antibodies of similar specificity in the first two gestations responded earlier than those with different antibody specificities. Regardless of the time of first antibody detrection, peak titers were usually achieved during the first month postpartum. Antibody persistence increased with parity. Among cows of all ages sampled at random times, there was a linear relationship of serum reactivity to cow age.     

Production of foetally stimulated lymphocytotoxic antibodies by primiparous cows

Serum samples from groups of heifers at different pregnancy-related stages (preconception, month of pregnancy, and postpartum) were examined for the presence of lymphocytotoxic antibodies. Only a few weak and transitory antibodies were observed before or during the first pregnancy. These did not appear to be foetaliy stimulated. Strong or medium strength antibodies were detected in first or second month postpartum samples from 8 of 27 heifers (30%). The reactive sera were in every case reactive with the offspring of the cows from which the respective sera were collected.     

Reproductive function in beef cows calving in the spring or fall

Plasma progesterone was determined twice weekly for approximately 100 days postpartum in suckled purebred (Hereford) and crossbred (beef x dairy) cows that calved in the spring or late summer/fall season. The progestèrone profiles and occurrence of estrus were used to determine ovulation times and to monitor ovarian function. Postpartum ovulations occurred significantly earlier in crossbred than in purebred cows (38.1 ± 18.5 vs. 58.1 ± 21.8, P<0.01) and in fall compared to spring calving cows (32 ± 13.9 vs. 59.1 ± 20.3, P<0.001). Rations providing either 70 or 100% of requirements for metabolizable energy were fed from 30 days prepartum until the end of the subsequent rebreeding period. Cows receiving the 70% energy ration ovulated slightly earlier but there was no effect of ration on days to pregnancy. The minimal effect of energy ration was not unexpected in this trial since many of the cows were overconditioned during late gestation.The correlation between calendar date of calving and interval to first ovulation was significant for spring (r = ?0.38, P<0.01) but not for fall calving animals. Since cows were confined and received a balanced ration of stored feeds throughout the year, photoperiod and/or temperature rather than nutritional factors would be the probable cause of delayed ovarian activity in spring calvers.Reproductive performance was assessed during the period when samples were collected and the response for purebred (n=105) and crossbreds (n=142), respectively were: ovulated by day 60 postpartum, 57 vs. 87%; mated by day 100 without conception, 17 vs. 26%; ovulated before day 100 without detection and mating, 10 vs. 4%; anovulatory to day 100, 7 vs. 0%; pregnant by day 100, 67 vs. 70%. Similar comparisons for spring (n=133) and summer/fall (n=144) calvers, respectively, were: ovulated by 60 days postpartum, 56 vs. 96%; mated by day 100 without conception, 27 vs. 17%; ovulated by day 100 without detection and mating, 8 vs. 5%; anovulatory to day 100, 5 vs. 0%; pregnant by day 100, 60 vs. 78%. The difference with the greatest practical significance is that a higher proportion of the late summer/fall calving animals were pregnant by day 100 postpartum (P<0.01) which indicates that reproductive performance is superior in fall calving beef cows.