International Journal of Andrology, 1991, 14, pages 179-185
Detection of antisperm-antibodies in patients with primary testicular cancer R. S. FOSTER, L. R. RUBIN, A. MCNULTY, R. BIHRLEand J. P. DONOHUE Department of Urology and Department of Obstetrics and Gynaecology, Indiana University School of Medicine, U.S.A.
Summary Serum antisperm-antibodies have been noted to develop as a result of various types of testicular pathology and injury. Their presence sometimes correlates with impaired fertility. We examined 52 patients with non-seminomatous low-stage testicular cancer for the presence of serum antisperm-antibodies to determine whether or not auto-immunity is a factor in the depressed fertility known to exist in these patients. Twenty-one per cent were discovered to have serum antisperm-antibodies using an immunofluorescent technique. Keywords: antisperm-antibodies, immunofluorescence, testis cancer. Introduction Approximately 60% of men with newly diagnosed non-seminomatous testicular cancer are subfertile at diagnosis prior to any treatment (Fossa et al., 1982; Lange et al., 1987). The explanation for the lowered fertility has not been elucidated. Several factors, however, are thought to be contributory to this subfertile status. Semen analyses performed on men with primary testicular cancer, prior to any therapy, have revealed various abnormalities. Many patients have sperm counts below 20 x lo6 rnl-', many are severely oligospermic, and some have abnormalities of sperm motility and morphology (Berthelsen, 1983). After surgical treatment of testicular cancer approximately 50% of these individuals will exhibit an improvement in semen analysis parameters (Lange et d., 1987). Various hypotheses have been proposed to explain the subfertile status in this population of patients. Reports of abnormal chromatin patterns and high levels of antisperm antibodies in serum have been cited as factors contributing to subfertility (Evenson et al., 1984; Guazzien et al., 1985). The relevance of these findings is unclear as heterogeneous patient populations have been studied in these reports (Guazzien et af., 1985). Thus, a need to subclassify patients and clarify the reported observations exists. The development of antisperm antibodies, either locally or systemically, is felt to be related to disruption of the blood-testis barrier (Bronson et al., 1984). Carcinoma-in-situ (CIS) of the testis may be a precursor to subsequent invasive Correspondence: Dr R. S. Foster, Indiana University School of Medicine, Department of Urology, 926 W. Michigan Street, Rm. A112, Indianapolis, Indiana 46202-5250, U.S.A.
179
180 R. S. Foster et al. testicular carcinoma (Dauggard et al., 1987). The progression of disease from CIS to testicular cancer is associated potentially with disruption of the blood- testis barrier. Disruption of this barrier could then lead to the development of antispermantibodies in serum. Antisperm-antibodies have been associated with decreased sperm counts, poor sperm motility, and an increased number. of sperm with abnormal morphology (Mathur, 1985). These same abnormalities of semen analysis are associated frequently with testicular cancer prior to any therapy. Due to the availability of a large testicular cancer patient population at Indiana University, a study was undertaken to determine the prevalence of serum antisperm antibodies in men with primary testicular cancer. Because patients with various stages of testicular cancer were studied in previous work (Guazzien et al., 1985), this investigation was limited to a well defined group. The patients were clinical stage A or B1 (no or minimal retroperitoneal metastatic disease) and were studied after orchiectomy. Materials and methods
Patient details Patients with newly diagnosed primary testicular cancer were asked to participate in the study. A blood sample was drawn at the time of the initial clinic visit after obtaining informed consent. Patients accepted for study ( n = 52) were clinical stage A or B1, post-orchiectomy, but prior to further therapy. Clinical staging was performed using computed tomography of the chest and abdomen along with determination of serum alpha fetoprotein and human chorionic gonadotrophin. Blood samples from ‘control’ and ‘infertile’ patients were obtained as part of the routine diagnostics and quality control operation of the laboratory. Control serum samples (n = 24) were drawn from known fertile adult males. Samples (n= 118) classified as ‘infertile’ were drawn from the male partner of couples referred for an infertility work-up. The couples had been attempting to conceive for at least 1 year and, on average, had failed to conceive for 3 years or more. ‘Normal’ sperm profiles and true ‘male factor’ patients participated. Serum preparation Blood was allowed to clot for 20-30 min and centrifuged at 300 g for 10 min to separate serum from cells. The serum was then transferred to numerically-coded sterile-capped tubes. The code was recorded in a log opposite the patient’s name, date blood was drawn, and any relevant comments such as ‘haemolysis’was noted. All serum samples were heat-inactivated (56°C 2 30 min) and filtered (0.22 pm) prior to evaluation. In addition, all samples were initially frozen (-20OC) in order to permit batch testing. Antisperm-antibodies A micro-immunofluorescence antisperm-antibody assay protocol was used to screen the serum samples for the presence of IgG directed against sperm (Rubin et al., 1989). In the initial experiments pentavalent goat anti-human IgG monoclonal antibody conjugated to fluorescein (Atlantic Antibodies product #82917, lot
Antisperm-antibodies in primary testis cancer 181
#55083) was used. In subsequent work a monovalent goat anti-human IgG antibody (Fc fragment specific monoclonal #82907, lot #55316) was used. Each assay conducted, included a semen blank, a positive serum control, a negative serum control, and test samples. In addition, serum samples from 24 fertile men were run separately as controls. Pooled semen from known fertile, antibody-negative controls was used as an antigenic source. The same pool of donors was used to test all specimens throughout the study. Semen analyses were conducted on each sample submitted as a potential antigen source and was included in the pool only if the following criteria were met: sperm concentration >20 x lo6 m1-l; total sperm count >60 x lo6; sperm motility >75%; grade 3 activity or better (scale 1-4); morphology more than 50% normal forms; and < 1 x lo6 spermatid and/or white blood cells/ml. The initial whole semen samples were washed twice with PBS/BSA (phosphate buffered saline/bovine serum albumin (3% w:v) and resuspended to give a final concentration of 16 X lo6 ml-'. A 0.25 ml aliquot (4 X lo6 sperm) was placed in each of the labelled sterile test tubes. A 0.25 ml sample of PBS/BSA was added to the semen blank. The remaining tubes had the appropriate serum added (0.25 ml, final dilution 1:4). An incubation time of 30 min in a circulating water bath (37°C) was used. The sperm were washed three times with 1 ml PBS/BSA and centrifuged for 5 min at 500 g. The final pellet was resuspended in 0.25 ml PBS/BSA to which the fluorescein-conjugated antibody (1:20 dilution, 0.25 ml) was added (final antibody dilution 1:40). The tubes were incubated in the dark for 20 min in a circulating water bath (37°C). Samples were then washed a further three times with PBS/BSA and the final pellet resuspended in 0.25 ml PBS/BSA. A drop was immediately transferred to a clean slide, covered with a coverslip (22 x 50 mm) and read, using Nikon phase fluorescence optics and a B-1A Filter Cube (480/20 Excitation Filter, 520 Barrier filter). The test samples were required to exhibit 15% fluorescing cells over background fluorescence of the negative controls in order to be considered positive. In addition, the location of adherence of antibody on the sperm surface was noted. Later experiments were conducted as outlined above except the sperm antigen source used was sperm obtained by swim-up according to the method of Rogers (1985). These samples routinely had sperm motility >90% and grade 3-4 activity. Results
Antisperm-antibody status Of 61 serum samples collected and processed since January, 1987 and subsequently evaluated, only 52 had sufficient volume for more than one assessment. The following results represent data on serum which was evaluated on at least two separate occasions. Patients were considered to have a positive test result (serum positive for antisperm-antibodies) only if results from two or more experiments were concordant. Chi-square analysis of positive and negative serum samples for the pentavalent versus the monoclonal antibody was conducted. The two antibody population results were not significantly different (x2 = 0.00427, P > 0.975) and were combined subsequently into a single data pool. Discordant results (two
182 R. S. Foster et al. separate experiments showing conflicting results on the same specimen) were considered to be negative. Eleven patients out of 52 (21%) were positive for serum antisperm-antibodies. Six patients out of 52 (11%) showed discordant results and were therefore considered to be negative for antisperm-antibodies. Additionally, 38 patients showed negative results on two or more occasions.
Relationship to disease status The correlation between serum antibody status and pathological stage is depicted in Table 1. Four out of 11 (36%) patients with serum antisperm-antibodies were found to have small volume metastatic disease in the retroperitoneum. Of the patients who were negative for serum antisperm antibodies 17/41 (41%) had retroperitoneal disease. This difference was not significant (P>0.25). Table 1. Pathologic stage of non-seminomatous testicular cancer Antibody status Disease stage A
B
Positive ( n = 11)
Negative (n = 41)
7 4
24 17
~~~~~~
Patients were classified according to stage of disease and outcome of immunofluorescence antibody assay. Chi square analysis for the two disease stages shown indicated no statistical difference (xz = 0.1222, P>0.25).
Relationship to sperm preparation method Twenty-nine samples were evaluated using both washed sperm and sperm obtained by Lswim-up’. Disagreement between the results using the two antigenic sources was noted in five instances. In four of the five samples, results were negative when washed sperm were used but positive under conditions utilizing sperm obtained by swim-up. Twenty one samples were tested using washed sperm only; two samples were evaluated using only sperm obtained by swim-up. Relationship to fertility status Of the 21% positive sera reported, eight out of 11 (73%) showed tail directed antisperm antibodies (Fig. 1). Twenty-four fertile males were tested using our immunofluorescent technique and were found to be negative for antisperm-antibodies. This value is compared with values obtained in our laboratory using the same immunofluorescence technique for men undergoing an infertility workup and for the present study in Fig. 2. There is no statistical difference between the antibody positive rate of infertile males or males with primary testicular cancer. However, the difference between testicular cancer patients (21%) and fertile controls ( 5 % ) was statistically significant (P
Detection of antisperm-antibodies in patients with primary testicular cancer R. S. FOSTER, L. R. RUBIN, A. MCNULTY, R. BIHRLEand J. P. DONOHUE Department of Urology and Department of Obstetrics and Gynaecology, Indiana University School of Medicine, U.S.A.
Summary Serum antisperm-antibodies have been noted to develop as a result of various types of testicular pathology and injury. Their presence sometimes correlates with impaired fertility. We examined 52 patients with non-seminomatous low-stage testicular cancer for the presence of serum antisperm-antibodies to determine whether or not auto-immunity is a factor in the depressed fertility known to exist in these patients. Twenty-one per cent were discovered to have serum antisperm-antibodies using an immunofluorescent technique. Keywords: antisperm-antibodies, immunofluorescence, testis cancer. Introduction Approximately 60% of men with newly diagnosed non-seminomatous testicular cancer are subfertile at diagnosis prior to any treatment (Fossa et al., 1982; Lange et al., 1987). The explanation for the lowered fertility has not been elucidated. Several factors, however, are thought to be contributory to this subfertile status. Semen analyses performed on men with primary testicular cancer, prior to any therapy, have revealed various abnormalities. Many patients have sperm counts below 20 x lo6 rnl-', many are severely oligospermic, and some have abnormalities of sperm motility and morphology (Berthelsen, 1983). After surgical treatment of testicular cancer approximately 50% of these individuals will exhibit an improvement in semen analysis parameters (Lange et d., 1987). Various hypotheses have been proposed to explain the subfertile status in this population of patients. Reports of abnormal chromatin patterns and high levels of antisperm antibodies in serum have been cited as factors contributing to subfertility (Evenson et al., 1984; Guazzien et al., 1985). The relevance of these findings is unclear as heterogeneous patient populations have been studied in these reports (Guazzien et af., 1985). Thus, a need to subclassify patients and clarify the reported observations exists. The development of antisperm antibodies, either locally or systemically, is felt to be related to disruption of the blood-testis barrier (Bronson et al., 1984). Carcinoma-in-situ (CIS) of the testis may be a precursor to subsequent invasive Correspondence: Dr R. S. Foster, Indiana University School of Medicine, Department of Urology, 926 W. Michigan Street, Rm. A112, Indianapolis, Indiana 46202-5250, U.S.A.
179
180 R. S. Foster et al. testicular carcinoma (Dauggard et al., 1987). The progression of disease from CIS to testicular cancer is associated potentially with disruption of the blood- testis barrier. Disruption of this barrier could then lead to the development of antispermantibodies in serum. Antisperm-antibodies have been associated with decreased sperm counts, poor sperm motility, and an increased number. of sperm with abnormal morphology (Mathur, 1985). These same abnormalities of semen analysis are associated frequently with testicular cancer prior to any therapy. Due to the availability of a large testicular cancer patient population at Indiana University, a study was undertaken to determine the prevalence of serum antisperm antibodies in men with primary testicular cancer. Because patients with various stages of testicular cancer were studied in previous work (Guazzien et al., 1985), this investigation was limited to a well defined group. The patients were clinical stage A or B1 (no or minimal retroperitoneal metastatic disease) and were studied after orchiectomy. Materials and methods
Patient details Patients with newly diagnosed primary testicular cancer were asked to participate in the study. A blood sample was drawn at the time of the initial clinic visit after obtaining informed consent. Patients accepted for study ( n = 52) were clinical stage A or B1, post-orchiectomy, but prior to further therapy. Clinical staging was performed using computed tomography of the chest and abdomen along with determination of serum alpha fetoprotein and human chorionic gonadotrophin. Blood samples from ‘control’ and ‘infertile’ patients were obtained as part of the routine diagnostics and quality control operation of the laboratory. Control serum samples (n = 24) were drawn from known fertile adult males. Samples (n= 118) classified as ‘infertile’ were drawn from the male partner of couples referred for an infertility work-up. The couples had been attempting to conceive for at least 1 year and, on average, had failed to conceive for 3 years or more. ‘Normal’ sperm profiles and true ‘male factor’ patients participated. Serum preparation Blood was allowed to clot for 20-30 min and centrifuged at 300 g for 10 min to separate serum from cells. The serum was then transferred to numerically-coded sterile-capped tubes. The code was recorded in a log opposite the patient’s name, date blood was drawn, and any relevant comments such as ‘haemolysis’was noted. All serum samples were heat-inactivated (56°C 2 30 min) and filtered (0.22 pm) prior to evaluation. In addition, all samples were initially frozen (-20OC) in order to permit batch testing. Antisperm-antibodies A micro-immunofluorescence antisperm-antibody assay protocol was used to screen the serum samples for the presence of IgG directed against sperm (Rubin et al., 1989). In the initial experiments pentavalent goat anti-human IgG monoclonal antibody conjugated to fluorescein (Atlantic Antibodies product #82917, lot
Antisperm-antibodies in primary testis cancer 181
#55083) was used. In subsequent work a monovalent goat anti-human IgG antibody (Fc fragment specific monoclonal #82907, lot #55316) was used. Each assay conducted, included a semen blank, a positive serum control, a negative serum control, and test samples. In addition, serum samples from 24 fertile men were run separately as controls. Pooled semen from known fertile, antibody-negative controls was used as an antigenic source. The same pool of donors was used to test all specimens throughout the study. Semen analyses were conducted on each sample submitted as a potential antigen source and was included in the pool only if the following criteria were met: sperm concentration >20 x lo6 m1-l; total sperm count >60 x lo6; sperm motility >75%; grade 3 activity or better (scale 1-4); morphology more than 50% normal forms; and < 1 x lo6 spermatid and/or white blood cells/ml. The initial whole semen samples were washed twice with PBS/BSA (phosphate buffered saline/bovine serum albumin (3% w:v) and resuspended to give a final concentration of 16 X lo6 ml-'. A 0.25 ml aliquot (4 X lo6 sperm) was placed in each of the labelled sterile test tubes. A 0.25 ml sample of PBS/BSA was added to the semen blank. The remaining tubes had the appropriate serum added (0.25 ml, final dilution 1:4). An incubation time of 30 min in a circulating water bath (37°C) was used. The sperm were washed three times with 1 ml PBS/BSA and centrifuged for 5 min at 500 g. The final pellet was resuspended in 0.25 ml PBS/BSA to which the fluorescein-conjugated antibody (1:20 dilution, 0.25 ml) was added (final antibody dilution 1:40). The tubes were incubated in the dark for 20 min in a circulating water bath (37°C). Samples were then washed a further three times with PBS/BSA and the final pellet resuspended in 0.25 ml PBS/BSA. A drop was immediately transferred to a clean slide, covered with a coverslip (22 x 50 mm) and read, using Nikon phase fluorescence optics and a B-1A Filter Cube (480/20 Excitation Filter, 520 Barrier filter). The test samples were required to exhibit 15% fluorescing cells over background fluorescence of the negative controls in order to be considered positive. In addition, the location of adherence of antibody on the sperm surface was noted. Later experiments were conducted as outlined above except the sperm antigen source used was sperm obtained by swim-up according to the method of Rogers (1985). These samples routinely had sperm motility >90% and grade 3-4 activity. Results
Antisperm-antibody status Of 61 serum samples collected and processed since January, 1987 and subsequently evaluated, only 52 had sufficient volume for more than one assessment. The following results represent data on serum which was evaluated on at least two separate occasions. Patients were considered to have a positive test result (serum positive for antisperm-antibodies) only if results from two or more experiments were concordant. Chi-square analysis of positive and negative serum samples for the pentavalent versus the monoclonal antibody was conducted. The two antibody population results were not significantly different (x2 = 0.00427, P > 0.975) and were combined subsequently into a single data pool. Discordant results (two
182 R. S. Foster et al. separate experiments showing conflicting results on the same specimen) were considered to be negative. Eleven patients out of 52 (21%) were positive for serum antisperm-antibodies. Six patients out of 52 (11%) showed discordant results and were therefore considered to be negative for antisperm-antibodies. Additionally, 38 patients showed negative results on two or more occasions.
Relationship to disease status The correlation between serum antibody status and pathological stage is depicted in Table 1. Four out of 11 (36%) patients with serum antisperm-antibodies were found to have small volume metastatic disease in the retroperitoneum. Of the patients who were negative for serum antisperm antibodies 17/41 (41%) had retroperitoneal disease. This difference was not significant (P>0.25). Table 1. Pathologic stage of non-seminomatous testicular cancer Antibody status Disease stage A
B
Positive ( n = 11)
Negative (n = 41)
7 4
24 17
~~~~~~
Patients were classified according to stage of disease and outcome of immunofluorescence antibody assay. Chi square analysis for the two disease stages shown indicated no statistical difference (xz = 0.1222, P>0.25).
Relationship to sperm preparation method Twenty-nine samples were evaluated using both washed sperm and sperm obtained by Lswim-up’. Disagreement between the results using the two antigenic sources was noted in five instances. In four of the five samples, results were negative when washed sperm were used but positive under conditions utilizing sperm obtained by swim-up. Twenty one samples were tested using washed sperm only; two samples were evaluated using only sperm obtained by swim-up. Relationship to fertility status Of the 21% positive sera reported, eight out of 11 (73%) showed tail directed antisperm antibodies (Fig. 1). Twenty-four fertile males were tested using our immunofluorescent technique and were found to be negative for antisperm-antibodies. This value is compared with values obtained in our laboratory using the same immunofluorescence technique for men undergoing an infertility workup and for the present study in Fig. 2. There is no statistical difference between the antibody positive rate of infertile males or males with primary testicular cancer. However, the difference between testicular cancer patients (21%) and fertile controls ( 5 % ) was statistically significant (P
