Development of a predictive model for optimal zona pellucida binding using insemination volume and sperm concentration*

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1 FERTILITY AND STERILITY Copyright c 1994 The American Fertility Society Vol. 62, No. 4, October 1994 Printed on acid-free paper in U. S. A. Development of a predictive model for optimal zona pellucida binding using insemination volume and sperm concentration* Kemal Ozgiir*, M.D.t Daniel R. Franken, Ph.D.:j: Khalied Kaskar, B.Sc. Honn.:j: Carl J. Lombard, Ph.D. II Thinus F. Kruger, M.D.:j: Akdeniz University Medical Faculty, Antalya, Turkey; Tygerberg Hospital, Tygerberg; and Institute for Biostatistics, South African Medical Research Council, Tygerberg, South Africa Objective: To develop a predictive model under hemizona assay (HZA) conditions for human spermatozoa concentrations and insemination volume for optimum zona pellucida (ZP) binding. Design: Analysis of 20 different insemination volumes for zona binding and sperm morphology under HZA conditions. Setting: Reproductive biology unit, tertiary medical center. Patients: Four proven fertile sperm donors. Main Outcome Measures: 5-, 20-, 50-,80-, and 100-JLL droplets were analyzed with four different concentrations of0.5 X 10 6, 1.0 X 10 6, 2.0 X 10 6, and 4.0 X 10 6 cells/ml to determine the number of sperm bound to each hemizona. Fifteen hemizonae were used for each insemination volume or microdroplet. Response surface regression model with volume and concentration as the regressor variables has been used. Results: The response surface of binding for the factors concentration and volume showed nonlinear association. A formula, indicating the optimal sperm insemination volume for maximum sperm binding to the ZP, V max= -(b 1 + b 5c)/2b 6c, is described. The transformed data indicated 60 11L containing 4 X 10 6 sperm/ml to be optimal. Although morphology of zona spermatozoa is superior compared with seminal and postswim-up samples, no difference among the percentage of the normal morphology in different microdroplets could be demonstrated. Conclusion: Optimal volume for the obtained concentration of spermatozoa from a patient can be calculated and therapeutically used for cases of severe oligozoospermic patients by microvolume inseminations in IVF practice. Fertil Steril 1994;62:845-9 Key Words: Zona binding, microvolume insemination, male factor infertility Clinicians and scientists who work in reproductive endocrinology and infertility are faced with Received November 22, 1993; revised and accepted May 12, *Part of the study was supported by the South African Medical Research Council. t Department of Obstetrics and Gynaecology, Akdeniz University Medical Faculty. :j: Department of Obstetrics and Gynaecology, Tygerberg Hospital. Reprint requests: D. R. Franken, Ph.D., Department of Obstetrics and Gynaecology, Third Floor, Tygerberg Hospital, Tygerberg, 7505, South Africa ( ). II Institute for Biostatistics, South African Medical Research Council. very important and relevant questions in terms of identifying and treating a male factor. An immediate question is what therapeutic modality is available or will be the most feasible one to apply to a specific patient. Apart from the standard IVF, GIFT, and microfertilization techniques, the clinician has little to present as alternative approaches to the problem. During the 15 years of IVF practice, the challenge of male infertility problems has not been resolved completely, especially for the more severe cases. Fertilization failures reported during IVF procedures are significantly higher among male factor cases compared with female etiologies. However, Ozgur et al. ZP binding in microdroplets 845

2 once fertilization is achieved in male factors, the cleavage rate, implantation rates, and pregnancy rates do not differ from the other causes (1, 2). Therefore, fertilization failure among the male factor patients attending an assisted reproductive treatment program can be regarded as the prominent and remaining cause for their infertility. According to Ahlgren (3), the number of spermatozoa that eventually reach the ampulla is limited to a few hundred cells. However, the fertilization rates among male factor cases in an assisted reproductive program is known to be impaired using spermatozoa concentrations ranging from 50,000 to 200,000 motile spermatozoa per oocyte (4, 5). The basic limitation, therefore, in the treatment of severe male factor infertility is, apart from severe teratozoospermia, inadequate numbers of spermatozoa in ejaculate (6). The literature reports two methods for the treatment of severe male factor cases, namely, micromanipulation techniques that are very sophisticated, expensive, and show varying success rates (1, 7). Second, the use of microvolume insemination techniques has not been very popular and is less sophisticated, although it is inexpensive, convenient, and with satisfying success rates (2, 8). The spermatozoa of male factor men often need to be concentrated to optimize the insemination volume required during IVF or GIFT. The culture of early mammalian embryos is generally performed in microdrops under oil (9). The use of capillary tubes or cryopreservation straws as opposed to droplets under mineral oil has been suggested for microvolume insemination (2, 8, 10). In this study, we used 306 human hemizonae within 20 different microdroplets under mineral oil to evaluate the zona binding. These results are used in a response surface regression model to create a predictive formula for the insemination volume needed for a given sperm concentration to achieve optimal zona binding. MATERIALS AND METHODS Patients and Semen Preparation Four known fertile donors were used. Semen samples were collected by masturbation and evaluated immediately after liquefaction. Sperm concentration, motility, and forward progression were microscopically assessed following World Health Organization standards (11). Sperm morphology was evaluated by strict criteria following established guidelines (6). Separation of the motile sperm fraction was accomplished after double wash and centrifugation with Ham's F-10 medium (GIBCO, Grand Island, NY) supplemented with 0.3% bovine serum albumin. The final sperm pellet was overlaid with 0.5 to 1.0 ml of medium and incubated for 1 hour at 37 C in 5% C0 2 in air to allow the motile sperm to swim up. Sperm concentration was evaluated at X100 magnification using the outer 16 white blood cell blocks of the Neubauer hemocytometer. The slides were air dried for morphology assessment before and after swim-up procedure. The supernatant of the swim-up was diluted into four tubes that consisted of0.5 X 10 6, 1.0 X 10 6,2.0 X 10 6, and 4.0 X 10 6 spermatozoa/ml. Hemizona Assay (HZA) Oocytes for the HZA were obtained from postmortem material. Ovarian tissue was collected and processed within 24 to 36 hours after death. Great care was taken by all persons involved to adhere to ethical and legal guidelines surrounding the handling and collection of the postmortem tissue. Zona-intact prophase I oocytes were denuded of granulosa cells and placed in plastic vials containing 1.5 MgCl 2 (Mallinkrodt Chemical Work, St Louis, MO), 0.01% polyvinylpyrrolidone (MW 40,000 PVP-40; Sigma Chemical Co., St. Louis, MO), and 40 mm of HEPES buffer and stored at 4 C for up to 30 days under controlled conditions (12). For each sperm donor, 40 oocytes were removed from storage and desalted by thorough rinsing with Ham's F-10 culture medium. Oocytes were bisected, resulting in two identical hemizonae using previously reported micromanipulation techniques (13). Microdroplets Five microdroplets, 5-, 20-, 50-, 80-, and L, were prepared from four different concentrations (0.5 X 10 6, 1 X 10 6, 2 X 10 6, 4 X 10 6 cells/ml) achieved by serial dilution. Four hemizonae from different oocytes were placed in each droplet, whereas the hemizonae were randomly selected. The droplets were covered with mineral oil (M- 3516; Sigma Chemical Co.) and incubated for 4 hours at 37 C. Each hemizona from the same droplet was removed and rinsed in medium five times with a finely drawn pipette to remove loosely bound spermatozoa. Spermatozoa that were tightly bound 846 Ozgiir et al. ZP binding in microdroplets Fertility and Sterility

3 Table 1 Six Regressors as Calculated From Second- and Third-Order Products of the Sperm Concentration and Insemination Volumes Parameter b 0 (Intercept) b, b2 ba h. hs b6 *Values are means± SE. Estimate* ± ± ± ± ± ± ± Pvalue to hemizonae were counted in each droplet. The air-dried samples of the hemizonae from each droplet were stained by Papanicolaou technique, and sperm morphology was evaluated according to strict criteria previously published (6). Twenty microdroplets ( 4 concentrations in 5 different volumes) were evaluated for each spermatozoa donor, and 15 hemizonae were counted for each droplet. Statistical Analysis and Development of a Regression Model The data consisted of 306 measurements of sperm binding under varying conditions of sperm volumes (5, 20, 50, 80, 100) (v) and sperm concentration (0.5 X 106, 1 X 106, 2 X 106, 4 X 106 cells/ml) (c). Because the outcome variable sperm binding is a count of the absolute number of sperm binding to a unit surface on the oocyte, these counts can be considered to have a Poisson distribution. The natural logarithm of the count is the transformed outcome variable modeled in a response surface regression model with volume and concentration as the regressor variables (14). The regression model derived is: ln (count) = b 0 + b 1v + b 2c + b 3c 2 + b 4c 3 + b5vc + b6vc 2 + error The model consists of six regressors (b 0 to b6) that are made up of second- and third-order products of sperm concentration (c) and microdroplet insemination volume (v). All regression coefficients are given in Table 1. The lack of fit test was used to establish the adequacy of the model (14). The final model represents the most parsimonious model in terms of the number of regressors and goodness of fit. The lack of fit test for the model showed F(14, 285) = 1.346, P > 0.05 indicating that the model is adequate. The R 2 for the model is 0.37, and the overall mean value is with the root SE and coefficient of variation RESULTS The mean (±SD) for the semen parameters of the four sperm donors were: sperm concentration, 79.5 ± 25 X 106 cells/ml; percentage motile, 57.5% ± 9%; percentage normal sperm morphology, 14.7% ± 2%. The percentage normal sperm morphology showed an increase from 15% to 23% to 35% among semen, swim-up, and hemizona-bound populations, respectively. The mean number of bound sperm for the different insemination concentrations used for each of the microdroplets is shown in Figure 1. There was a tendency toward increased binding with an increase in volume and concentration. However, for the optimal binding, a nonlinear relation between volume and concentration has been calculated. Using a regression model, the optimum microdroplet volume (V max) for each sperm concentration could be calculated (Fig. 2). From the significance of the cross-products, between microdroplet volume (v) and sperm concentration (c), we calculated a significant interaction between these contributing factors. For given sperm concentration (c), therefore, the calculated optimum volume of the microdroplet for obtaining maximum ZP binding under HZA conditions (vmax) is given by: V max= -(bl + b5c)/2b6c Optimum zona binding for a specific patient can therefore be calculated on condition that the postswim-up sperm concentration is known. For example, the calculated optimum insemination volume for postswim-up sperm concentrations of 4 X 106 sperm/ml ( c = 4) where optimum zona binding was 0 z ::::> ~ 150 ::; a: ~ 100 t'5 a: w 50 Ill ::; ::::> z 200~ , Figure 1 The effect of sperm insemination volume and concentration (, 0.5 X 10 6 ; ~. 1 X 10 6 ; D, 2 X 10 6 ; -, 4 X 10 6 sperm/ml) on the number of sperm bound to human ZP under HZA conditions. Ozgiir et al. ZP binding in microdroplets 847

4 ~~~ :::r "'300 ~ 250 " ~ ol,-,..,,..,-rr~-.~~~ Concentration (milllon/ml) o Optimal binding Figure 2 The relationship between insemination concentration and volume to yield optimal sperm-zp binding. recorded is 60 ~tl and in cases in which c = 1, the calculated V max is 89 ~tl. Transforming back to the original scale, the predicted mean values are compared with the geometric means of the counts at each combination of the concentration and volume. DISCUSSION The initial interaction between human gametes that eventually leads to fertilization seems to be predominantly defined by the zona binding potential of the spermatozoa. Despite the fact that zona binding does not reflect any other sperm function, the ability of sperm to bind to the ZP has been shown to be predictive of IVF rates (13, 15). Although zona binding per se does not guarantee fertilization, poor binding under HZA conditions has been correlated with IVF failure. Despite the reported success of expensive techniques such as microfertilization (7), namely, intracytoplasmic sperm injections that are not readily available, clinicians still need a simple and in expensive method to treat male factor couples. Sci entists are therefore urged to find methods to assist in the diagnosis and treatment of male factor men. Specific sperm-oocyte defects among couples attending an assisted reproductive program should be diagnosed using a sequential analytic approach (16), thereby improving the understanding of the couples infertility problem. For example, in cases in which low to normal sperm binding (13, 17) are reported in the presence of a severe male factor, the use of alternative gamete manipulating techniques should be investigated. In this study, ZP binding was performed during different volumes and sperm concentrations in mi- crodroplets overlaid with mineral oil. Generally, one should expect greater binding in small volumes because 5 and 20 ~tl cause an increase in collision probability of spermatozoa and hemizonae. However, sperm concentration and volume show nonlinear associations; volumes greater than 50 ~tl revealed better binding results than the droplets of 5 and 20 ~tl. Microdroplets under mineral oil have been described to absorb physiological amounts of Pin the microdroplets (18), which might have an effect on zona binding. Progesterone, ultimately interferes with the capacitational status of the sperm population, thereby affecting zona binding capacity of the spermatozoa. Sueldo et al. (19) demonstrated an elevated number of sperm bound to hemizonae after 1 hour incubation with 1.0 ~tg/ml of P. According to the regression model used in this study, the relationship between sperm concentration and microdroplet volume to obtain optimal zona binding is nonlinear and exponential (Fig. 2). At low sperm concentrations ( <0.5 X 10 6 sperm/ ml), insemination volume plays a critical role in obtaining optimal zona binding. However, at concentrations > 1 X 10 6 sperm/ml, the volume of the microdroplet becomes less critical. Between sperm concentrations 100,000 to 700,000 sperm/ml, the relation (Fig. 2) does, however, show linear associations; thus an increase in concentration can bemanipulated by an increase in volume. This range is important for gamete manipulation because it includes sperm concentration ranges often found among male factor men that the consulting clinician is confronted with in the final diagnosis. Its usefulness is demonstrated in assisted reproductive technologies for patients with low swim-up sperm concentrations. The suggested model, therefore, can serve as a clinical guideline to indicate the optimum insemination volume that should be used for a given sperm concentration. Microvolume insemination of human oocytes has been successfully employed using capillary tubes of 5 to 10 ~tl and straws with 100-~tL volume (2, 8). Previous studies reported that an increase in sperm insemination concentration resulted in an increase in the number of morphologically abnormal sperm bound to murine ZP (20). This, however, is not consistent with our findings. Our results showed no significant difference in sperm morphology between the different microdroplets. This may be due to the selectivity of human zonae for normal forms that was previously demonstrated (21). Results indicate no increase in the percentage abnor- 848 Ozgiir et al. ZP binding in microdroplets Fertility and Sterility

5 mal bound spermatozoa with an increase in insemination concentration. The development of the formula in the present study will enable clinicians to calculate the optimal insemination volume needed for a specific sperm concentration to achieve maximum zona binding. These results demonstrate that zona binding depends on the insemination volume and spermatozoa concentration, which show nonlinear associations. Our results indicate the practical use of the statistical formula that can be used for specific assessments of male fertilizing capacity in conjunction with infertility treatment programs. Acknowledgments. The authors thank Miss Helga Nel for her technical assistance, Roelof Menkveld, Ph.D., and Mr. Leon Gabriel, M.Sc., for their scientific advice during the project. REFERENCES 1. Cohen J, Edwards R, Fehilly C, Fishel S, Hewitt J, Durdy J, et al. In vitro fertilization: a treatment for male infertility. Fertil Steril 1985;43: Vander Ven HH, Hoebbel K, Al-Hasani A, Diedrich K, Krebs D. Fertilization of human oocytes in capillary tubes with very small numbers of spermatozoa. Hum Reprod 1989;4: Ahlgren M. Sperm transport to and survival in the human fallopian tube. Gynecol Invest 1975;6: Diamond MP, Rogers BJ, Vaughn WK, Wenz AC. Effect of the number of inseminating sperm and the follicular stimulation protocol on in vitro fertilization of human oocytes in male factor and non-male factor couples. Fertil Steril 1985;44: Oehninger SC, Acosta AA, Morsedi MM, Veeck LL, Swanson RJ, Simmons K, et al. Corrective measurements and pregnancy outcome in in vitro fertilization in patients with severe morphology abnormalities. Fertil Steril1988;50: Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, Van Zyl JA, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril1986;46: van Stierteghem AC, Liu J, Joris H, Nagy Z, Jansenswillen C, Tournaye H, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8: Hammitt DG, Walker DL, Syrop CH, Miller TM, Bennett MR. Treatment of severe male factor infertility with high concentrations of motile sperm by microinseminations in embryo cryopreservations straws. J in Vitro Fertil Embryo Transf 1991;8: Brinster RL. A method for in vitro cultivation of mouse ova from two cell to blastocyst. Exp Cell Res 1963;32: Renoux C, Seibel MM. New techniques in fertilization: intravaginal culture and microvolume straw. J in Vitro Fert Embryo Transf 1990;7: World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction, 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987: Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck L, Kruger TF, et al. The hemizona assay using salt stored human oocytes: evaluation of zona pellucida capacity for binding spermatozoa. Gamete Res 21;1989;22: Franken DR, Kruger TF, Menkveld R, Oehninger S, Coddington CC, Hodgen GD. Hemizona assay and teratozoospermia: increasing sperm insemination concentrations to enhance zona pellucida binding. Fertil Steril 1990;54: Box GEP, Draper NR. Empirical model-building andresponse surfaces. New York: John Wiley, Franken DR, Acosta AA, Kruger TF, Lombard CJ, Oehninger SC, Hodgen GD. The hemizona assay: its role in identifying male factor infertility in assisted reproduction. Fertil Steril1993;59: Oehninger S, Coddington CC, Scott R, Franken DR, Burkman LJ, Acosta AA, et al. Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil Steril1989;51: Franken DR, Kruger TF, Oehninger S, Coddington CC, Lombard C, Smith K, et al. The ability of the hemizona assay to predict human fertilization in different and consecutive in-vitro fertilization cycles. Hum Reprod 1993;8: Miller KF, Pursel VG. Absorption of compounds in medium by the oil covering microdrop cultures. Gamete Res 1987;17: Sueldo CE, Oehninger S, Subias E, Mahony M, Alexander NJ, Burkman LJ, et al. Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity. Fertil Steril 1993;60: Krzanowska H, Lorenc E. Influence of egg investments on in vitro penetration of mouse eggs by misshapen spermatozoa. J Reprod Fertil1983;68: Menkveld R, Franken DR, Kruger TF, Oehninger S, Hodgen GD. Sperm selection capacity of the human zona pellucida. Mol Reprod Dev 1991;30: Ozgiir et al. ZP binding in microdroplets 849