David L. Fulgham

Antisperm antibodies to sperm surface antigens in women with genital tract infection

Antisperm antibodies to sperm surface antigens in nulligravid women with primary upper genital tract infections were measured by the sperm mixed agglutination reaction assay. As many as 56% of women with a primary episode of pelvic inflammatory disease had antisperm antibodies. In addition, 69% of those women with no history of genital tract infection but with laparoscopic evidence of past pelvic infection had significant levels of circulating antisperm antibodies. Electroimmunoblots of sperm preparations probed with the sera of women who had either known or presumed upper genital tract infection revealed a uniformly recognized 69 kd antigen. In contrast, women with circulating antisperm antibodies before primary upper genital tract infection recognized up to five distinct sperm antigen determinants of 27, 54, 131, 146, and 174 kd. It is a distinct possibility that genital tract infections may lead to immunopotentiation of antisperm antibodies that could affect fertility.

Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody

A monoclonal antibody, T-6, useful for detecting acrosome-reacted sperm based on an immunofluorescent assay, was employed to evaluate acrosomal status of human sperm that were tightly bound to hemisected human zonae pellucidae (hemizona assay). Over 90% of the bound sperm evaluated exhibited immunofluorescent patterns indicative of acrosome reaction. This staining method for evaluating the acrosomal status of sperm bound to the zona pellucida may enable definition of a group of male infertility patients heretofore not recognized.

Gender after artificial induction of ovulation and artificial insemination

Several studies on artificial insemination by donor (AID) semen have suggested that the gender of infants can be influenced by treatment of the women with clomiphene citrate (CC) and by the type of semen used (fresh versus cryopreserved). We conducted a 3-year prospective clinical trial to test these hypotheses. Two groups of pregnant women were evaluated. Group I (n = 130) comprised women whose ovulation was induced by CC; group II (n = 190) comprised those who conceived during spontaneous ovulatory cycles. In a total of 320 pregnancies, 55 spontaneous abortions occurred, 23.1% in group I and 13.2% in group II (P less than or equal to 0.05). Two tubal ectopic pregnancies occurred in group I. Of the 100 and 165 pregnancies carried to term in the treated and control groups, respectively, 11% and 1.8% involved twins (P less than or equal to 0.005). When only single births were considered, group I had 46.1% males in 89 term pregnancies, and group II had 60.5% males in 162 term pregnancies. Significantly more female offspring occurred in the group treated with CC (P less than or equal to 0.05). Because it is possible that a portion of the effects observed in this study were a function of cryopreservation of the AID semen, we compared data on frozen sperm with data on fresh sperm in terms of abortion, gender, and incidence of multiple births; there were no significant differences. Fertil Steril 40:481, 1983.

Circulating Immune Complexes and Antisperm Antibodies in Yasectomized and Vasovasostomized Rhesus Macaques

Sperm contain many antigens that can elicit autoantibody production. After a vasectomy, spermatozoa are confined to the epididymis and vas deferens, where they degenerate and release antigens that enter the circulation directly or are phagocytosed by macrophages. A humoral immune response is initiated and circulating antibodies to sperm develop in both men and animals. We measured the levels of circulating antibodies to spermatozoa in 51 vasectomized rhesus monkeys (Mucaca mulatta), some of whom had been vasectomized for as long as 14 years, and compared these values to data on control monkeys. Antibodies to sperm, measured by immobilization’ and sperm agglutination,2 developed in 43% of the vasectomized monkeys but in none of the controls (FIGURE I ) . We gave I5 monkeys vasectomy reversals six months after their initial operation. One year later, 53% (8/15) remained positive for circulating antibodies to sperm. Spermatozoa produced after a vasectomy are a constant source of antigen that results in antibody production. The presence of antigen and persistent antibody may result in immune-complex disease. We have postulated that the increase in atherosclerosis found in vasectomized monkeys is due to injury of the arterial wall by immune-complex dep~si t ion.~.~ To test this hypothesis, we evaluated the levels of circulating immune complexes in the same 51 vasectomized monkeys. We measured the circulating immune complexes either by a Staphylococcus aureus binding assay5 or a Clq solid-phase assay.6 Circulating immune complexes were present in 57% of the vasectomized monkeys but in only 15% of the control group 0, < 0.005 by x 2 ) (FIGURE 1). There was no association between the presence of antibodies and circulating immune complexes. Before reversal, circulating immune complexes were present in 10 of the 15 monkeys; 6 had antibodies. One year

Studies on the in Vitro Spermicidal Activity of Synthetic Magainins

Two synthetic magainins A and G are shown to have spermicidal activity. Transmission electron microscopic micrographs show that both magainins alter the plasma membranes of sperm and that these actions are rapid. Further studies will better delineate the contraceptive potential of synthetic magainins.

Serum hormone levels affect sperm function**Supported by a grant of the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, under a Cooperative Agreement (DPE-2644-A-00-6063-00) with the United States Agency for International Development (AID). The views expressed by the authors do not necessarily reflect the views of AID.

If hormone levels affect sperm motility during capacitation, then the serum added to samples prepared for artificial insemination could affect sperm fertilizability. We investigated various sperm functional movement characteristics (percent motile, progressive velocity, linearity, beat cross frequency, lateral head displacement, longevity, and hyperactivation) in specimens incubated with womens sera, as well as exogenous hormone preparations of estradiol (E2) and progesterone (P). Early follicular phase serum (low E2 and low P) maintained motility and longevity. Sperm in E2-treated medium exhibited higher progressive velocity, linear motility, and longevity. In contrast, the percent motility decreased as sperm exhibited hyperactivated motility with P.

Distribution of T cell subsets in follicular fluid**Supported by the Contraceptive Research and Development Project, Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (A.I.D.) grant DPE-2044-A-00-6063-00. The views expressed by the authors do not necessarily reflect the views of A.I.D.

Examination of follicular fluid (FF) from in vitro fertilization patients revealed a significant difference in concentrations of lymphocytes and T cell subpopulations with increased oocyte maturation. A total of 111 follicles containing 82 oocytes were aspirated from 10 patients undergoing laparoscopic oocyte retrieval. FF from 61.3% of the follicles was classified as clear and 38.7% as bloody, based on gross and microscopic appearance. A mean of 1.78 X 10(6) lymphocytes/ml was obtained from peripheral blood (PB) as compared to 2.14 X 10(5) and 2.79 X 10(5) lymphocytes/ml for clear and bloody FF, respectively. There were 6.3 X 10(5) T4 and 3.7 X 10(5) T8 lymphocytes in PB, resulting in a T4/T8 ratio of 1.72, which is not significantly different from that of the general population. The mean concentration of FF T4 and T8 lymphocytes decreased with increased oocyte maturation; the T8 reduction was statistically significant (P less than 0.05). The proportion of T4 to T8 lymphocytes in FF remained unchanged and was unaffected by maturity of the oocyte. Although estradiol (E2) did not vary with oocyte maturity, progesterone (P) increased and E2/P decreased. There was no correlation between E2 or P levels and distribution of T cells. Fertilization rates were higher in more mature oocytes, but there was no correlation between fertilization and E2, P, E2/P, or T cell subpopulations. It remains to be determined what factors result in the decrease in lymphocytes with increased oocyte maturity and the observed difference in FF T4/T8 compared to PB.

In vitro immune absorption of antisperm antibodies with immunobead-rise, immunomagnetic, and immunocolumn separation techniques

Fourteen men with a mean duration of infertility greater than 3 years who had significant sperm immobilizing or sperm-agglutinating antibodies were studied. All patients had greater than 20% IgG or IgA immunobinding to sperm in their seminal plasma and 7 had immunobinding levels of greater than 50%. Sperm from these men were less able to penetrate an overlaying buffer layer than sperm from a fertile control. Addition of immunobeads to the specimen was of little use, because few motile sperm could swim into the overlaying buffer; retained immunobeads were noted in the buffer layer of 18-hour capacitated specimens. Magnetic isolation of antibody-coated sperm from antibody-free sperm avoids potential damage to fragile sperm through centrifugation. Viable spermatozoa were isolated from magnetite-complexed spermatozoa, but the motility of the isolated spermatozoa deteriorated rapidly during the subsequent capacitation period. Passage of diluted ejaculate through a column of dextran beads for antisperm antibody processing (ASAP) was associated with superior sperm quality and fertilizing potential. The use of ASAP resulted in good sperm velocity and linearity and improved sperm function, as measured with the hamster egg penetration test. Sperm from men with immunologically mediated infertility can be processed through the ASAP and used for artificial insemination of their partners or in an in vitro fertilization program.

Antibodies to Spermatozoa in Male Monkeys: Mode of Action**Publication No. 990 from the Oregon Regional Primate Research Center. Supported by Northwestern University/PARFR Subcontract 70N under Subcontract AID/csd-3608, National Institutes of Health Grant RR000163 and Contract NO-1-4-2866.††Presented in part at the Thirty-Fourth Annual Meeting of The American Fertility Society, March 29 to April 1, 1978, New Orleans, La.

Studies were conducted to determine how antisperm antibodies affect male fertility. First we determined that all serum components revealing activity by sperm immobilization, spermagglutination, and indirect immunofluorescence were immunoglobulins by serum fractionation. Antisperm activity was limited to the immunoglobulin components; this activity was sperm-specific and could be removed by adsorption with homologous epididymal or ejaculated sperm but not with liver powder. Next, we studied whether circulating antisperm antibodies could affect spermatozoa. The blood-testis barrier was found to be intact even in animals with high circulating antisperm antibodies. Furthermore, epididymal spermatozoa from these animals exhibited no adsorbed antibodies. We incubated semen samples from rhesus monkeys (Macaca mulatta) that had high levels of circulating antisperm antibodies in order to assess sperm motility over time. We checked whether sperm from infertile animals that had received a vasovasostomy had a shorter life-span or reduced motility with time, phenomena one might expect if antisperm antibodies were contained in seminal plasma. We employed a similar assay to determine whether normal monkey spermatozoa incubated in seminal plasma from vasectomized or vasovasostomized animals with circulating antisperm antibodies would exhibit reduced motility with time. Some seminal plasma samples appeared to impede motility, but these were not necessarily the same samples that exhibited high immunoglobulin levels. However, normal spermatozoa incubated in serum containing antisperm activity were less able to penetrate and migrate through cervical mucus in vitro. We conclude that high circulating antisperm antibody levels are not always correlated with seminal plasma antibody levels and are not necessarily indicative of poor sperm motility or longevity.