Serum sperm antibodies are not elevated after mumps orchitis

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1 FERTILITY AND STERILITY VOL. 77, NO. 1, JANUARY 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Serum sperm antibodies are not elevated after mumps orchitis Svetoslav Kalaydjiev, M.D., a Dimitrina Dimitrova, M.D., a Marina Nenova, M.D., b Siika Peneva, M.D., c Ivan Dikov, M.D., c and Lyudmi Nakov, M.D. a Medical University of Sofia, Sofia, and Medical University of Varna, Varna, Bulgaria Objective: To assess the level of serum sperm antibodies after mumps orchitis. Design: Controlled descriptive study. Setting: Academic research environment. Patient(s): Seventy-four mumps orchitis patients. Intervention(s): Sampling of serum at different intervals after the onset of orchitis symptoms: 1 to 7 days, 31 to 60 days, and 61 to 431 days. Main Outcome Measure(s): Level of serum sperm antibodies, using Kibrick s gelatin agglutination test, Friberg s tray agglutination test, Isojima s sperm immobilization test, and ELISA. Result(s): Clinically relevant sperm antibody values were detected by the Friberg method among patients tested from 1 to 7 days (10.5%) and 61 to 431 days (10.5%) after the onset of disease. The Isojima test revealed a statistically insignificant higher incidence among patients at 61 to 431 days (31.6%) as compared with those sampled at 1 to 7 days (10.5%). None of the orchitis sera tested positive by the Kibrick and ELISA techniques. The established incidences did not differ significantly from the results for negative controls (blood donors) and were lower than the values acquired from positive controls (males with unexplained infertility). Conclusion(s): Mumps orchitis does not cause enhanced humoral immunity to spermatozoa. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Infertility, mumps orchitis, serum sperm antibodies Received April 5, 2001; revised and accepted June 26, Reprint requests: Svetoslav Kalaydjiev, M.D., Department of Biology, Medical University of Sofia, 2 Zdrave Str., 1431 Sofia, Bulgaria (FAX: ; skalay@ medfac.acad.bg). a Department of Biology, Medical University of Sofia. b Department of Infectious Diseases and Epidemiology, Higher Medical Institute, Varna, Bulgaria. c Department of Infectious Diseases, Epidemiology, Parasitology and Tropical Medicine, Medical University of Sofia /02/$22.00 PII S (01) Mumps is caused by a RNA virus belonging to the myxovirus group. One of its most notorious complications is mumps orchitis. Although it may cause infertility in some of the affected males, the exact mechanisms of fertility impairment are not entirely understood. Varying degrees of permanent damage to the seminiferous tubules usually occurred when pubertal or adult testes were involved (1). Testicular biopsies have shown that, in acute cases, edema, perivascular lymphocytic exudate, and diffuse infiltration of interstitial tissue with focal hemorrhage can be observed (2). Some investigators have speculated that male infertility can occur after mumps infection without mumps orchitis (3). It is generally accepted that one factor that can negatively affect fertility after mumps orchitis is the humoral immunity against spermatozoa the sperm antibodies (4 6). This assumption is based on several alternative hypothetical mechanisms. [1] Due to breaks in the blood testis barrier and leakage of sperm, the inflammation of the testis causes autoimmunization with sperm antigens. [2] Mumps viruses can attach to the sperm surface, and, serving as haptens, lead to the production of sperm antibodies. [3] The antigenic mimicry between the mumps virus and human spermatozoa causes the production of cross-reacting antibodies in the course of the disease. However, the limited experimental and clinical data have revealed a contradictory picture with regard to any of the above hypotheses, and the results of the few studies available have remained uncertain (7 10). None of these investigations have provided convincing data as to whether mumps orchitis can induce the production of systemic sperm antibodies. No data are available describing the sperm antibody activity at the beginning of the disease, when the patients were first diagnosed with orchitis. Such data may be important; it is well established that, although the incidence and serum 76

2 levels of sperm antibodies may be considerably lower, they can be found in fertile individuals (11, 12). A statistically significant positive correlation is known to exist between the sperm antibody status and genital tract inflammation (13); peak sperm antibody incidence has been observed 1 month after the onset of the disease (12). Thus, a comparison of sperm antibody activity during the acute phase with the activity 1 month after the involvement of the testis in the inflammatory process would be efficient. With this data, it would be possible to evaluate whether mumps orchitis has any effect on the serum sperm antibody levels. The aim of our study was to measure the serum sperm antibody levels among mumps orchitis patients during the acute phase, when the disease was diagnosed for the first time, then to compare these levels with those found at least 1 month later, when the presence of sperm antibodies in the serum would be expected due to the inflammatory process in the testis. Because sperm antibodies differ in their effects (agglutination, immobilization, etc.), as well as in the antigens they recognize, we have applied the following detection techniques. To allow the uncovering of sperm agglutinins, we employed Kibrick s gelatin agglutination test (GAT) and Friberg s tray agglutination test (TAT). To measure sperm immobilizins, we used Isojima s sperm immobilization test (SIT). And to detect antibodies against antigens located beneath the sperm surface, we used the ELISA test with whole fixed sperm. MATERIALS AND METHODS Patients This investigation included mumps orchitis patients aged 12 to 50 years from the 1997 to 1998 mumps outbreak in Bulgaria (14, 15). Serum samples were obtained 1 to 7 days after the onset of clinical symptoms. The mumps orchitis (MO) group consisted of 38 patients in the acute phase of the disease; post mumps orchitis group 1 (PMO1) consisted of 7 patients examined 31 to 60 days after the onset of clinical symptoms; and post mumps orchitis group 2 (PMO2) consisted of 19 patients examined 61 to 431 days after the onset of clinical symptoms. Ten other patients were twice-sampled (TS), both in the acute phase (TS-MO) and after the disease (TS-PMO). Thirty-seven male blood donors (BD) who never had mumps served as the negative control group, and 22 male patients with unexplained infertility (INF) comprised the positive control group. The samples were heat inactivated at 56 C for 30 minutes and stored at 20 C until tested. All orchitis patients were hospitalized during the acute period of the disease and received corticosteroid medication ( mg/kg/24 hours of methylprednisolone for 2 to 18 days). Bed rest and suspensory bandage of the scrotum were also applied in the cases of profound swelling. This investigation was performed with institutional review board approval, following the ethical standards on human experimentation and the Helsinki declaration. Semen Semen samples to be used in the sperm antibody assays were collected from healthy donors according to World Health Organization criteria (16). Total ejaculates were used in the GAT test. Motile swim-up spermatozoa were employed in the TAT, SIT, and ELISA tests, acquired through the following method. Semen samples were centrifuged at 550 g for 10 minutes at room temperature, the supernatant was discarded, and the sedimented pellets were overlayered with 400 L of Baker s buffer (ph 8.1). The sample was kept for 30 to 60 minutes at 37 C. The motile spermatozoa that swam up into the medium were collected and used in the sperm antibody assays. Sperm Antibody Tests Serum sperm antibodies were detected by GAT, TAT, SIT, and ELISA according to standard laboratory protocols. GAT was performed in the following manner (17): A fresh semen sample was diluted in Baker s buffer to spermatozoa/ml, then 100 L of serially diluted sera in Baker s buffer were loaded in Kibrick test tubes. The same quantity of semen/gelatin suspension (1:1) was added and gently mixed, giving a final concentration of spermatozoa/ml in a 2.5% gelatin. The tubes were incubated at 37 C and read after 1 hour. The TAT (17) was performed in 60-well, low-profile stacking trays (Robbins Scientific, Sunnyvale, CA) that were loaded with serial dilution of sera in Baker s buffer (5 L/well) and overlayered with liquid paraffin. Then 1 L of sperm suspension ( spermatozoa/ml) was added to each well. The plates were incubated for 1 hour at 37 C and observed for the degree and type of sperm agglutinates under an inverted Zeiss microscope ( 125 magnification). SIT (18), a semi-quantitative method, was applied for the detection of sperm immobilizing antibodies. Serum samples diluted 1:2 in Baker s buffer were loaded in a 60-well low-profile stacking tray plate, 8 wells/serum, 10 L/well, 1 L of swim-up sperm suspension ( spermatozoa/ ml), and 2 L of whole guinea pig complement (National Center for Infectious and Parasite Diseases, Sofia, Bulgaria; complement activity, CH units) were added. Sera were tested for sperm toxicity as in the above preparation, where, instead of complement, saline was added. The sperm toxicity of the complement was determined as inactivated complement was added to the controls. The plates were incubated for 1 hour at 37 C and then the percentage of sperm motility (T) was determined under an inverted Zeiss microscope (magnification 125), counting 100 spermatozoa per well. Normal human serum without sperm antibody activity was used as the negative control, and the percentage of sperm motility (C) was calculated as well. FERTILITY & STERILITY 77

3 For each serum, a sperm immobilization value (SIV) was calculated: SIV C/T, where C was the mean percentage of motile spermatozoa in the negative control, and T was the mean percentage of motile spermatozoa in the tested serum. ELISA was performed on flexible U-bottomed, 96-well assay plates (Falcon 3911 Microtest III, Becton Dickinson Labware, Oxnard, CA). To increase the adhesive capacity of the plates, they were pretreated with 50 g/ml poly-l-lysine hydrobromide (Sigma Chemical Company, St. Louis, MO) in distilled water for 1 hour at room temperature. Then the plates were rinsed with distilled water and the assay was performed as has been described elsewhere (19). Briefly, the plates were loaded with 50 L/well washed sperm suspension ( sperm/ml) and kept overnight at 37 C. Then the spermatozoa were methanol-fixed for 30 minutes at room temperature. The plates were rinsed thoroughly with phosphate-buffered saline (PBS), ph 7.2, containing 0.05% Tween 20 (PBS-Tween); nonspecific binding was reduced by incubating with 150 L/well bovine blocking buffer (1% bovine serum albumin [Sigma] in PBS) for 1 hour at room temperature. Serum dilutions of 1:20 were made in PBS, with 50 L/well added to the plates and incubated for 2 hours at 37 C. After extensive rinsing with PBS-Tween, anti-human immunoglobulin conjugated with horseradish peroxidase (National Center for Infectious and Parasite Diseases) diluted 1:1600, 50 L/well was added and incubated for 1 hour at 37 C. The plates were thoroughly rinsed with PBS-Tween and developed using an ex temporae prepared solution of 0.4 mg/ml orthophenylenediamine (Sigma) in 0.05 M citrate buffer (ph 5.0) and hydrogen peroxide. The reaction was stopped on the seventh minute by adding 50 L/well of 10% H 2 SO 4. The optical density was measured at 492 nm on a Uniscan 361 reader (Labsystems, Espoo, Finland). The ELISA assays were done in duplicate in three separate experiments. To correct for possible variation between plates, absorbance readings for all samples were normalized to the value for a serum from an infertile patient (strongly positive for sperm antibodies in GAT, TAT, SIT, and ELISA) on the same plate, which was considered to be 1. Control samples were included with each sperm antibody test: positive control samples from infertile patients with sperm antibodies, and negative control samples from fertile individuals. The following barrier levels were accepted, above which sperm antibodies were considered to be present in clinically relevant values: agglutinating titer 16 in GAT, and titer 32 in TAT (17); and SIV 2 in SIT (18). For the ELISA, from the readings of the negative controls (BD group), a cut-off level was established; sera were considered positive when their optical density was greater than x 2 SD (1.31), where x was the mean optical density of all sera from the BD group (19). The results were also evaluated as magnitude of positive response of all sera from a group, FIGURE 1 Sperm antibody incidence of mumps orchitis patients sampled 1 to 7 days after the onset of clinical symptoms (MO group), 61 to 431 days after the onset of clinical symptoms (PMO2 group), and both in the acute phase (TS-MO group) and after the disease (TS-PMO group). Also sampled were blood donors (BD group) and males with unexplained infertility (INF group). GAT gelatin agglutination test; TAT tray agglutination test; SIT sperm immobilization test. None of the men tested positive by ELISA. where median sperm antibody levels (titer, SIV, or optical density) for each assay were compared among the groups. Statistical Analysis The data were processed with the software package SPSS 7.0 (SPSS, Inc., Chicago IL). Fisher s two-tailed exact test was applied to evaluate differences in the sperm antibody incidence among the tested groups of men. The Kruskal- Wallis nonparametric test was used for the analysis of variance in the median sperm antibody values among mumps orchitis patients and controls. The medians of the TS-MO and TS-PMO groups were compared by the Wilcoxon rank sum. P.01 was considered statistically significant. RESULTS The results from the sperm antibody tests did not demonstrate a statistically significant increased level of antibodies in the mumps orchitis patients after the disease, as compared with the patients who were sampled at the beginning, during the acute phase. Clinically relevant sperm antibodies could be established only by TAT and SIT, and none of the orchitis patients had positive levels as measured by GAT and ELISA (Fig. 1, Table 1). Four sera samples (10.5%) demonstrated clinically relevant titers at the beginning of the disease in TAT (MO group, titer 32, two sera; titer 64, one serum; titer 512, one serum). Another two sera (10.5%) reacted in a clinically relevant fashion after the disease (PMO2 group), demonstrating agglutination titers of 32. One 78 Kalaydjiev et al. Mumps orchitis and sperm antibodies Vol. 77, No. 1, January 2002

4 TABLE 1 Clinically relevant sperm antibody incidence among mumps orchitis patients and controls. Group No. of men tested GAT titer 16 TAT titer 32 SIT SIV 2 ELISA OD 1.31 MO a 4 0 PMO PMO a 6 0 TS-MO TS-PMO INF BD a 1 a 0 Abbreviations: GAT gelatin agglutination test; TAT tray agglutination test; SIT sperm immobilization test; MO patients 1 to 7 days after the onset of clinical symptoms; PMO1 patients 31 to 60 days after the onset of clinical symptoms; PMO2 patients 61 to 431 days after the onset of clinical symptoms; TS-MO patients in the acute phase of the disease, sampled twice; TS-PMO the same patients sampled after the disease; BD blood donors; INF males with unexplained infertility; OD optical density; SIV sperm immobilization value. a Fisher s exact test. Statistically significant differences with INF group (P.01). serum that demonstrated mixed agglutination during the acute phase at titer 8 became positive after the disease (TS-PMO group) titer 32, head-to-head agglutination. Mixed and head-to-head agglutinates were observed in all groups, whereas tail-to-tail agglutinates were demonstrated only in sera of patients in the acute phase of the disease. In SIT results, four sera (10.5%) were positive at the time the patient was diagnosed with orchitis (MO group), and six (31.6%) were positive after the disease (PMO2 group), but the established difference was not statistically significant (Fisher s exact test, P.01). One patient (10%) from the TS-MO group was also positive. None of the patients tested 31 to 60 days after the onset of clinical symptoms (PMO1 group) revealed clinically relevant sperm antibody values. The positive controls (INF group) yielded the highest sperm antibody incidence. One patient (4.5%) was positive by GAT, 10 (45.5%) in TAT, and 6 (27.3%) by SIT. None of the infertile patients was positive by the ELISA. These incidences were significantly higher as compared with the MO (Fisher s exact test, P.01), PMO2, and BD groups by TAT; and as compared with the BD group by SIT. The sperm antibody incidence among negative controls (BD group) was the lowest, and no statistically significant difference to the established values were found as compared with the orchitis patients groups. Only one man (2.7%) was positive for sperm antibodies by SIT. The rest of the assays registered no clinically relevant antibody values. When the magnitude of positive response was analyzed (Table 2), we found no statistically significant difference (Kruskal-Wallis test, P.01) between the median sperm antibody levels at the beginning (MO group) and after the disease (PMO1 and PMO2 groups) by GAT, TAT, and SIT. Interestingly, the median optical density via ELISA was significantly higher for the MO group (P.01). The differ- TABLE 2 Sperm antibody levels among mumps orchitis patients and controls. Group No. of men tested GAT Median titer (range) TAT Median titer (range) SIT Median SIV (range) ELISA Median OD (range) MO 38 0 (0 8) 8 (0 512) 1.27 ( ) 0.86 a ( ) PMO1 7 0 (0) 4 (2 16) 1.33 ( ) 0.62 ( ) PMO (0 2) 8 (0 32) 1.42 ( ) 0.50 ( ) TS-MO 10 0 (0 2) 4 (0 8) 1.57 ( ) 0.79 ( ) TS-PMO 10 0 (0) 4 (0 32) 1.30 ( ) 0.73 ( ) INF 22 0 (0 32) 12 (0 16,384) 1.62 ( ) 0.55 ( ) BD 37 0 (0 4) 0 (0 8) 1.16 ( ) 0.72 ( ) Abbreviations: See Table 1. a Kruskal-Wallis test. Statistically significantly higher value (P.01). FERTILITY & STERILITY 79

5 FIGURE 2 Results from the Kibrick gelatin agglutination test (GAT), the Friberg tray agglutination test (TAT), the Isojima sperm immobilization test (SIT), and ELISA for sera sampled on different intervals (X-axis) after the onset of orchitis symptoms. The sperm antibody levels (Y-axis) are presented as log 2 titer for GAT and TAT, sperm immobilization value (SIV) for SIT, and optical density (OD) measured at 492 nm in ELISA. The dashed line indicates the cut-off level above which sera are considered positive. ences between the TS-MO and TS-PMO groups were not found to be statistically significant by any of the four assays (Wilcoxon rank sum, P.01). The median titer (TAT) and SIV of the positive controls were the highest, but the established variation was not statistically significant (Kruskal-Wallis test, P.01). However, their median optical density was lower than most of the other groups. The median titer (TAT) and the median SIV of the BD group were lower than found among the groups of orchitis patients. Figure 2 plots the results from the four sperm antibody tests (Y-axis) against the day the serum was sampled (Xaxis, orchitis diagnosis is day 0). Obviously, sperm antibodies did not tend to persist in clinically relevant values after the disease. Only sperm immobilizing antibodies, but not in very high values, were detected during the whole period of the investigation; this was in contrast to sperm agglutinins, which were found in clinically relevant values mainly in the acute period of the disease. DISCUSSION The causal link between mumps orchitis and sperm antibodies has been rather unclear. Although these antibodies were suspected to impair fertility after mumps orchitis, their increased levels and involvement have never been convincingly established. In one study, the application of the indirect immunofluorescent test to 1340 patients with fertility problems revealed sperm antibodies in seven patients who had suffered mumps orchitis after puberty. In two of these men, sperm antibodies were found on the third week after the onset of mumps, but no such activity could be detected during the first week of the disease (8). In another report, the analysis of the presence of immunity against the antigens of sperm and testis in 75 patients with mumps orchitis (complement fixation, hemagglutination, double immunodiffusion, indirect immunofluorescent test, gelatin agglutination, sperm immobilization, and delayed hypersensitivity skin tests) showed controversial data 80 Kalaydjiev et al. Mumps orchitis and sperm antibodies Vol. 77, No. 1, January 2002

6 about the significance of cellular immunity and circulating antibodies in this disease. Although a considerable number of patients exhibited positive delayed hypersensitivity reactions to homologous spermatozoa, only some of the sera were positive for sperm antibodies: head-to-head agglutination at titer 32 were found in sera from five (14%) mumps orchitis patients (two acute and three chronic cases), as well as in eight control sera (20%). No clinically relevant sperm immobilization activity could be demonstrated (9). In a study of 19 soldiers who had suffered from mumps orchitis, approximately 1 year from their original diagnosis, GAT, TAT, and ELISA were applied on sera samples. A low incidence of sperm antibodies and no difference between patients and controls were encountered. By the means of GAT, the investigators found sperm agglutinins in the serum of only one soldier (5%); TAT was negative in all sera; ELISA was positive in one orchitis patient (5%) and two controls (11%) who had suffered only mumps (10). Another study examined 13 men who had contracted mumps orchitis after puberty, 1 to 12 years before the study; a GAT investigation revealed no sperm agglutinins in their sera (7). An examination of the interactions between germ cells and viruses observed that members of the myxovirus group agglutinated the spermatozoa of rooster, ram, bull, and human, especially the Newcastle disease virus and the influenza virus (20). Another study on a rabbit model of acute orchitis caused by the myxoma virus revealed that serum sperm antibodies were detectable on day 5 after infection, along with degeneration of the seminiferous epithelium and impaired steroidogenesis (21). Our study tested mumps orchitis patients both in the acute phase of the disease and at least 1 month later, when the presence of sperm antibodies in the serum could be expected due to the inflammatory process in the testis. We applied four sperm antibody techniques, allowing the detection of antibodies with different effects on spermatozoa and against different antigens. This approach had never been used in previous studies. It permits a precise evaluation of the impact of mumps orchitis on the production of serum sperm antibodies, especially of the changes in their levels after the disease. Our results have demonstrated no statistically significant elevation in the sperm antibody incidence among patients with mumps orchitis after the acute period. Although other investigators have found a peak incidence of sperm antibodies 1 month after the acute inflammation (12), we could not establish any similar pattern among our patients. The antibody incidence did not differ significantly from the incidence among the male BDs in our control group, and it was lower than the incidence among the control patients with unexplained infertility. Here, we should note that some investigators failed to establish correlation between sperm antibodies and certain markers of genital tract infection such as leucocytospermia and seminal hyperviscosity (22), or between sperm antibodies and genital infection caused by Chlamydia (23, 24). It is interesting that the median ELISA optical density of patients tested during the first week of the disease (MO group) was significantly higher than in patients tested after their diagnosis (PMO1 and PMO2 groups). One possible explanation is that the anti-mumps virus antibodies, whose level is known to be relatively high in patients with orchitis (25), nonspecifically bind Fc-receptors on the sperm surface, resulting in higher optical density values. Or it can be supposed that some elevated antisperm antibody activity was detected by this sensitive test at the time the patient s mumps orchitis was diagnosed, as sperm antibodies detected by ELISA in epididymitis patients increase rapidly and reach maximum levels on the seventh day (12). However, the sperm antibody levels decreased and did not tend to reach clinically relevant values with time after the disease. The results from this investigation should be evaluated in the context of the applied treatment. All of the patents who were tested received a corticosteroid medication during the acute period as part of an accepted treatment scheme for mumps orchitis. It is difficult to judge whether this therapy influenced the levels of serum sperm antibodies, because such medication does not seem to alter significantly the circulating sperm antibodies (26 29). In conclusion, both the incidence and the median levels of serum sperm antibodies among mumps orchitis patients were low and did not increase significantly after the disease. These results do not support the hypothesis for an enhanced humoral immunity against spermatozoa after mumps orchitis. Acknowledgments: The authors thank Dr. E. Alexandrova and Dr. I. Radeva from the Department of Infectious Diseases, Epidemiology, Parasitology and Tropical Medicine, Medical University of Sofia, for data collection and participation in this study. References 1. Diehl K, Hondl H. Mumps orchitis: symptoms and treatment possibilities. Z Urol Nephrol 1990;83: Gall E. The histopathology of acute mumps orchitis. Am J Pathol 1947;23: Niermann H. Male infertility after mumps infection without mumps orchitis? Med Welt 1980;31: Hafez ESE, ed. Human semen and fertility regulation in men. Saint Louis: C.V. Mosby, Tung KSK, Menge AC. Sperm and testicular autoimmunity. In: Rose NR, Mackay IR, eds. The autoimmune diseases. San Diego: Academic Press, 1985: Shulman S. Immunological reactions and infertility. In: Kurpisz M, Fernandez N, eds. Immunology of human reproduction. Oxford: BIOS Scientific, 1995: Rumke P. Autospermagglutinins: a cause of infertility in men. Ann NY Acad Sci 1965;124: Haensch R. Fluorescenzimmunologische Spermienautoantikörper-Befunde bei männlichen Fertilitätsstörungen. Arch Gynakol 1969;208: Andrada JA, von der Walde F, Hoschoian JC, Comini E, Mancini E. FERTILITY & STERILITY 81

7 Immunological studies in patients with mumps orchitis. Andrologia 1977;9: Shulman A, Shohat B, Gillis D. Mumps orchitis among soldiers: frequency, effect on sperm quality and sperm antibodies. Fertil Steril 1992;57: Eggert-Kruse W, Huber K, Rohr G, Runnebaum B. Determination of antisperm antibodies in serum samples by means of enzyme-linked immunosorbent assay a procedure to be recommended during infertility investigation? Hum Reprod 1993;8: Heidenreich A, Bonfig R, Wilbert DM, Strohmaier WL, Engelmann UH. Risk factors for antisperm antibodies in infertile men. Am J Reprod Immunol 1994;31: Gubin DA, Dmochowski R, Kutteh WH. Multivariant analysis of men from infertile couples with and without sperm antibodies. Am J Reprod Immunol 1998;39: Gospodinova M, Gancheva T, Nenova M. Epidemic parotitis-characteristics of epidemic process and clinical course in the town of Varna and region Infectology 1999;36: Komitova R, Popivanova N, Madzharova L, Todorov G, Bozhilova M, Kostadinova M, et al. Mumps orchitis in postvaccinal era. Infectology 1999;36: World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, Rose NR, Hjort T, Rumke P, Harper MJK, Vyazov O. Techniques for detection of iso- and auto-antibodies to spermatozoa. Clin Exp Immunol 1976;23: Isojima S, Koyama K. Microtechnique of sperm immobilization test. In: Bratanov K, Vulchanov VD, Georgieva T, Somlev B, eds. Immunology of reproduction. Sofia: BAS Press, 1978: Fichorova R, Nakov L. The use of ELISA to evaluate human antibody binding to epididymal sperm from different species. Am J Reprod Immunol 1993;29: Peleg BA, Ianconescu M. Spermagglutination and spermadsorption due to myxoviruses. Nature 1966;211: Fountain S, Holland MK, Hinds LA, Janssens PA, Kerr PJ. Interstitial orchitis with impaired steroidogenesis and spermatogenesis in the testes of rabbits infected with an attenuated strain of myxoma virus. J Reprod Fertil 1997;110: Munuce MJ, Bregni C, Carizza C, Mendeluk G. Semen culture, leucocytospermia, and the presence of sperm antibodies in seminal hyperviscosity. Arch Androl 1999;42: Habermann B, Krause W. Altered sperm function or sperm antibodies are not associated with chlamydial antibodies in infertile men with leucocytospermia. J Eur Acad Dermatol Venerol 1999;12: Eggert-Kruse W, Rohr G, Demirakca T, Rusu R, Naher H, Petzoldt D, et al. Chlamydial serology in 1303 asymptomatic subfertile couples. Hum Reprod 1997;12: Ukkonen P, Granstrom ML, Penttinen K. Mumps-specific immunoglobulin M and G antibodies in natural mumps infection as measured by the enzyme-linked immunosorbent assay. J Med Virol 1981;8: Butler WT, Rossen RD. Effects of corticosteroids on immunity in man. I. Decreased serum IgG concentration caused by the 3 or 5 days of high doses of methylprednisolone. J Clin Invest 1973;52: Hendry WF, Stedronska J, Parslow J, Hughes L. The results of intermittent high dose steroid therapy for male infertility due to antisperm antibodies. Fertil Steril 1981;36: De Almeida M, Feneux D, Rigaud C, Jouannet P. Steroid therapy for male infertility associated with antisperm antibodies. Results of a small randomized clinical trial. Int J Androl 1985;8: Fredricsson B. Infertility caused by antispermatozoal antibodies in the male experience from an intermittent high dose cortisone regimen. Andrologia 1988;20: Kalaydjiev et al. Mumps orchitis and sperm antibodies Vol. 77, No. 1, January 2002