Acrobeads test: a new diagnostic test for assessment of the fertilizing capacity of human spermatozoa*

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1 FERTILITY AND STERILITY Copyright 1995 American Society for Reproductive Medicine Vol. 63, No.3, March 1995 Printed on acid-free paper in U. S. A. Acrobeads test: a new diagnostic test for assessment of the fertilizing capacity of human spermatozoa* Kazutomo Ohashi, M,D,t Fumitaka Saji, M.D. Munehiro Kato, M.D. Tateki Tsutsui, M.D. Tatsuhiro Tomiyama, M.D. Osamu Tanizawa, M.D. Department of Obstetrics and Gynecology, Osaka University Medical School, Yamada-oka, Japan Objective: To determine the effectiveness of the Acrobeads test for predicting the outcome of IVF, Design: Human spermatozoa express the CD46 molecule (membrane cofactor protein) on their heads after the acrosome reaction. CD46-positive spermatozoa formed a sperm-bead complex with immunobeads coated with anti-cd46 monoclonal antibody. In the Acrobeads test, fertilizing capacity was determined by assessing sperm-bead agglutination. Setting: Department of Obstetrics and Gynecology, Osaka University Hospital. Participants: Thirty-seven donors of proven fertility and 88 male partners of infertile couples. Main Outcome Measures: We carried out the Acrobeads test and a sperm penetration assay (SPA) using zona-free hamster oocytes within 3 months before IVF and we then analyzed the results in relation to IVF outcome. Results: The sensitivity of the Acrobeads test and SPA was 100% and 88%, respectively, whereas the specificity was 43% and 52%, respectively. The negative predictive value of the Acrobeads test was 100%, whereas that of the SPA was 73%. These results indicate that there was no significant difference between these two tests in terms of predicting IVF outcome. Conclusion: We suggested that the Acrobeads test be used to evaluate the fertilizing capacity of human spermatozoa because we should avoid using the SPA to prevent cruelty to animals. Fertil Steril 1995;63: Key Words: Human spermatozoa, fertilizing capacity, in vitro fertilization, sperm penetration assay, Acrobeads test, CD46 The assessment of the fertilizing capacity of 00- cytes and spermatozoa before IVF is very important clinically. It is indispensable for the selection of appropriate treatment for infertility that we determine whether unsuccessful fertilization in IVF was due to the quality of the oocyte or that of the sper- Received April 24, 1994; revised and accepted September 21, * Supported in part by a Grant-in-Aid for Scientific Research (no , ) from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan. t Reprint requests to: Kazutomo Ohashi, M.D., Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2 Yamada-oka Suita, 565, Japan (FAX: ). matozoa or whether the lack of success was due to some technical aspect. The fertilizing capacity of spermatozoa depends on the function of the sperm head, including its capacity for the acrosome reaction and cell fusion to the oocyte, and on the sperm tail, e.g., its motility. We usually use a sperm penetration assay (SPA) using zona-free hamster 00- cytes (1, 2) to evaluate the fertilizing capacity of human spermatozoa; the usefulness of this assay in the treatment of infertility has been reported (3-5). The SPA is a bioassay that requires good quality hamster oocytes and a well-equipped laboratory and, therefore, it cannot be performed everywhere. Moreover, because hamsters are killed in every experiment, we should avoid using the SPA if possible. Vol. 63, No.3, March 1995 Ohashi et al. Assessment of sperm fertilizing capacity 625

2 We have developed a clinical diagnostic test for assessing the fertilizing capacity of human sperm a - tozoa; the test uses a MH61 monoclonal antibody (6) that recognizes the CD46 molecule on acrosome-reacted human spermatozoa (7) and is referred to as the Acrobeads test. The principles of the Acrobeads test are [1] motile spermatozoa make contact with immunobeads coated with the MH61 monoclonal antibody, referred to as MH61-beads; [2] when the MH61-bead meets the acrosomereacted spermatozoa, it binds to the sperm head; [3] the sperm-bead complex becomes larger as the acrosome reaction progresses; and [4] the spermbead complexes can be observed by agglutination, using an inverted phase-contrast microscope. The formation of the sperm-bead complex depends on acrosomal status and on sperm motility. The Acrobeads test can therefore be used to assess the function of both the sperm head and the tail at the same time. In the present study, we performed the Acrobeads test and the SPA within 3 months before IVF and analyzed the results to predict the outcome ofivf. MATERIALS AND METHODS Sperm Preparation Semen was collected from donors of proven fertility and from patients in our IVF program. The semen sample was obtained by masturbation after ~3 days of abstinence from intercourse and was prepared within 2 hours of ejaculation. Motile spermatozoa were prepared by the swim-up method. The liquefied ejaculate, diluted with an equal volume of modified Biggers, Whitten, and Whittingham (BWW) medium (8, 9), was centrifuged for 5 minutes at 200 X g. The supernatant was discarded, after which 1.2 ml of modified BWW was layered over the sperm pellet. The test tube was kept at an angle of 5 for 60 minutes at 37 C. The upper 1 ml was removed and recentrifuged as described above. The pellet was washed twice in modified BWW supplemented with 0.3% human serum albumin (HSA), referred to as washing medium, ahd resuspended in modified BWW supplemented with 3.5% HSA, referred to as capacitation medium, at a concentration of 4 X 10 6 /ml. Acrobeads Test We performed the Acrobeads test as reported previously (10), with some slight modifications. The reaction of spermatozoa with MH61-beads was carried out in 4 wells of a 96-well fiat-bottomed tis- sue culture plate. In the first step, serial dilutions of sperm suspension in the wells were made as follows: [1] 100 JlL capacitation medium was added to the second, third, and fourth wells; [2] 100 JlL sperm suspension (4 X 10 6 /ml) was added to the first and second wells; [3] the medium in the second well was mixed by pipetting and then removed, in a volume of 100 JlL, and placed into the third well; and [4] the same procedure was carried out between the third and fourth wells, and 100 JlL of the medium was removed from the fourth well. In the next step, 10 JlL of MH61-beads (1.5 X 10 6 /ml) was added to each well, and the content of each well was mixed gently with the tip of a pipette. The 96-well tissue culture plate was incubated at 37 C in 5% CO 2 and agglutination of the bead-sperm complex was observed at 100X magnification with an inverted phase-contrast microscope on five fields of each well after 6 and 24 hours of incubation. Positive agglutination was defined as the absence of MH61- beads free from binding to spermatozoa in the microscopic field (Fig. 1A and B). Negative agglutination was defined as the presence of these beads (Fig. 1 C and D). If agglutination was considered positive for three or more of five fields, the well was assessed as positive, and the Acrobeads score was determined as shown in Table 1. A high Acrobeads score represents a high rate of acrosome reaction. Sperm Penetration Assay We used commercially available frozen hamster eggs (Nihon Nosan, Yokohama, Japan) for the hamster test. The frozen eggs were thawed at room temperature, washed three times in washing medium, and transferred to 0.1% trypsin. After the zona pellucida was removed, the eggs were removed from the trypsin and washed three times in the washing medium. Twenty to 30 eggs were placed in 200 JlL of sperm suspension (2 X 10 6 /ml) under mineral oil. Spermatozoa and eggs were incubated for 3 hours at 37 C in a 5% CO 2 atmosphere. When incubation was complete, the eggs were mounted on a slide glass and scored for sperm penetration by observing sperm head recondensation and/or male pronucleus formation, using a phase-contrast microscope at 400X magnification. Our lower limit for fertile spermatozoa was 10% penetration in the SPA. In Vitro Fertilization Patients received either hmg or buserelin acetate. Follicular growth was monitored by determining serum E 2 levels and by ultrasonography. Ovum recovery was performed 36 hours after the injection 626 Ohashi et al. Assessment of sperm fertilizing capacity Fertility and Sterility

3 Figure 1 Photographs ofpositive (A and B) and negative (C and D) agglutination as seen in the Acrobeads test at 24 hours of incubation (magnification, X200). Black arrows indicate MH61- beads free of human spermatozoa. of 10,000 IV hcg administrated when the leading follicle reached 18 mm in diameter. The quality of oocytes was assessed according to the Norfolk criteria (11), and the number of mature oocytes collected from any patient was at least four. Oocytes were washed with Ham's F-I0 medium containing 10% heat-inactivated serum obtained from the patient and were preincubated for 4 hours at 37 C in a humidified atmosphere of 5% CO 2 and air. Oocytes were then inseminated with 1 X 10 5 motile spermatozoa/oocyte in 1 ml of culture medium. Fertilization was defined by the presence of two or more pronuclei 16 to 20 hours after insemination. Statistical Analysis Data in Tables 2 and 3 were analyzed by the Student's t-test, and differences of P < 0.05 were con- Table 1 Serial dilution of sperm Xl X2 X4 X Judgment of Acrobeads Test Acrobeads score Judgement Negative agglutination at all wells a well of Xl dilution sperm wells of Xl and X2 dilutions of sperm wells Xl, X2, and X4 dilutions of sperm all wells side red to be significant. In Table 4, the significance of differences was assessed by the x 2 test. RESULTS Of 37 males of proven fertility, 13 (35%) had an Acrobeads score of 0 at 6 hours of incubation, whereas none of the 37 had this score at 24 hours of incubation (Table 2). On the other hand, 58 of 88 infertile subjects (66%) had an Acrobeads score of 0 at 6 hours of incubation, and 13 (15%) retained a score of 0 at 24 hours of incubation (Table 2). The mean ± SD of Acrobeads scores at 6 and 24 hours of incubation in fertile donors was 0.95 ± 0.88 and 2.62 ± 0.83, respectively, whereas these scores in infer- Table 2 Acrobeads Scores at 6 and 24 Hours of Incubation Males of proven fertility (n = 37) at 24 h h D D D D D D 0 2 Male partners of infertile couples (n = 8) at 24 h h D D D D D D 1 1 Vol. 63, No.3, March 1995 Ohashi et al. Assessment of sperm fertilizing capacity 627

4 Table 3 Comparison of Acrobeads Test and SPA DISCUSSION Acrobeads score 6h 24 h SPA penetrated ± 1.8 (0 to 6)* ± 5.5 (0 to 75) 0 ~ ± 25.0 (0 to 80) 1 ~ ± 18.5 (0 to 67) ~2 ~ ± 32.0 (8 to 94) * Values are means ± SE, with ranges in parentheses. No. of samples tile patients were 0.43 ± 0.69 and 1.75 ± The Acrobeads scores in fertile donors were significantly higher (P < 0.01) than those in infertile subjects at both 6 and 24 hours of incubation. In the next examination, we compared the results in the Acrobeads test with those in the SPA, using the same semen samples obtained from 79 males of infertile couples. As shown in the first line of Table 3, the spermatozoa of 10 males whose Acrobeads scores were 0 at both 6 and 24 hours of incubation showed significantly poor (P < 0.01) potential to penetrate the zona-free hamster oocytes in comparison with the other four groups shown in Table' 3, who had equal levels of penetrating capacity. Consequently, we determined an abnormal score in the Acrobeads test as 0 at 24 hours of incubation. To determine the effectiveness of the Acrobeads test for assessing the fertilizing capacity of human spermatozoa before IVF, we performed this test and the SPA within 3 months before IVF and analyzed the results in relation to IVF outcome (Table 4). The spermatozoa of 34 of 46 patients (74%) who scored normal values in the Acrobeads test ferti Ii zed oocytes in IVF, whereas the spermatozoa of the 9 patients who had abnormal scores in the Acrobeads test failed to fertilize oocytes in IVF. In 30 of 40 patients (75%) who showed positive fertilization in the SPA, there was subsequent successful fertilization of human oocytes in IVF, whereas 4 of 15 patients (27%) successfully fertilized in IVF despite their negative fertilization in the SPA. The sensitivity of the Acrobeads test and the SPA as screening tests for IVF was 100% and 88% and their specificities were 43% and 52%, respectively. A negative predictive value indicates the likelihood that the spermatozoa of a man with an abnormal result will fail in IVF and is the most important diagnostic test parameter before IVF-ET. The negative predictive value of the Acrobeads test was 100%, whereas that of the SPA was 73%. Assisted reproductive technology has been developing in recent years: worldwide, 26,411 clinical pregnancies and 19,319 live births due to such technology were reported in 1991 (12). Data concerning IVF in a 1993 world collaborative report on assisted reproduction showed 119,992 retrieval cycles and 99,314 transfer cycles (12), suggesting that there were 20,678 cases (17.2%) in which no embryos for replacement were obtained. We often experience such unsuccessful fertilization in cases of male indication. The world collaborative report indicates that the percentage of male indication in assisted reproduction increased from 15.1% in 1989 to 21.5% in 1991 (12). We possibly will experience more cases of unsuccessful fertilization in the future. When spermatozoa fail to fertilize oocytes in vitro, we cannot determine whether the unsuccessful fertilization is due to the quality of the spermatozoa, to the quality of the oocytes, to conditions of fertilization, or to some technical aspect. Determination of the cause of unsuccessful fertilization is very important for the further treatment of infertility. The quality of semen is generally assessed by the parameters of sperm concentration, motility, forward progression, and morphology, determined according to World Health Organization criteria (13). Kruger et al. (14) reported that the assessment of sperm morphology, determined according to their original criteria, yielded good information in regard to IVF. These parameters, however, cannot directly indicate the fertilizing capacity of human spermatozoa. Techniques to evaluate this capacity include SPAs using zona-free hamster oocytes (1, 2), nonliving intact human oocytes (15), or bisected human zona pellucida (hemizona assay) (16). The SPA using zona-free hamster oocytes is the most Table 4 Acrobeads test Relation of In Vitro Assay Results to IVF Outcome Acrobeads test IVF + + SPA SPA IVF Sensitivity (%) Specificity (%) Negative predictive value (%) Ohashi et al. Assessment of sperm fertilizing capacity Fertility and Sterility

5 common examination for evaluating the fertilizing capacity of human spermatozoa before IVF, but its value for prediction of the results of IVF remains controversial (17) because there is no standardized method for this test and because its value has been determined only in small studies. Recently, Inoue (18), in a report of 4,050 cases, found that the SPA was of value in predicting the results ofivf. Accordingly, we consider that the SPA using zona-free hamster oocytes does yield information useful for predicting the outcome ofivf, ifit is performed in a well-equipped laboratory. However, there are many ambiguous aspects of the SPA because of its complicated procedure. Against this background, we developed the Acrobeads test as a new nonbioassay to evaluate the fertilizing capacity of human spermatozoa (10); for this test, fresh eggs and a wellequipped laboratory are not necessary. In developing the Acrobeads test, we focused on the CD46 antigen expressed on acrosome-reacted spermatozoa (19-22) because anti-cd46 monoclonal antibodies have been reported to inhibit fertilization (6), suggesting that CD46-positive spermatozoa have fertilizing capacity. In our preclinical study (10), we reported significant (P < 0.01) correlations between the percentage acrosome-reacted viable spermatozoa determined by the fluorescein isothicyanate-conjugated Pisium sativum agglutinin staining and the Acrobeads score, and good reproducibility ofthe results, when we used samples from six donors at two or three different times within 2 weeks. The results correlated positively with the outcome of IVF when we performed the Acrobeads test at the time of IVF. In the present study, we used the Acrobeads test and the SPA as screening tests before attempting IVF, and we analyzed the results in relation to IVF outcome. The results shown in Table 4 were analyzed by the x 2 test, and there was no significant difference between the Acrobeads test and the SPAin predicting IVF outcome. False negatives have been reported frequently in the SPA (16); indeed, in this study, 4 of 15 males with an abnormal result in the SPA succeeded in IVF. In contrast to the SPA, all nine males with abnormal results in the Acrobeads test failed in IVF. These results indicate that the Acrobeads test is a useful predictor of IVF outcome. Based on these findings, we recommend that, for infertile males with abnormal results in the Acrobeads test, microfertilization with an adequate acrosome induction should be used rather than conventional IVF. The reproducibility of the SPA has not been established and frequent or multiple exam- inations are recommended (23), but the SPA is very expensive ($150 to $325) (24). The Acrobeads test, however, is inexpensive, and frequent tests can be performed in infertile males who have received urologic treatment. In conclusion, the advantages of the Acrobeads test are [1] simple procedure, [2] nonanimal experiment, [3] cost benefit, [4] good reproducibility, and [5] good predictor of IVF failure. We consider that the Acrobeads test will be an important method for evaluating the fertilizing capacity of human spermatozoa in the future. Acknowledgment. The authors gratefully acknowledge the laboratory assistance of Mr. Teruki Nakazawa at Fuso Pharmaceutical Co. Ltd., Osaka, Japan. REFERENCES 1. Yanagimachi R. Penetration of guinea-pig spermatozoa into hamster eggs in vitro. J Reprod FertiI1972;28: Yanagimachi R, Yanagimachi H, Rogers BJ. The use of zona-free animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa. BioI Reprod 1976;15: Wolf DP, Sokoloski JE, Quigley MM. 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