Spermatozoal characteristics from fresh and frozen donor semen and their correlation with fertility outcome after intrauterine insemination*

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1 FERTILITY AND STERILITY Copyright The American Fertility Society Printed on acid-free paper in U.S.A Spermatozoal characteristics from fresh and frozen donor semen and their correlation with fertility outcome after intrauterine insemination* Paul B. Marshburn, M.D. t+ Don McIntire, Ph.D. Bruce R. Carr, M.D.t William Byrd, Ph.D. t University of Texas Southwestern Medical Center at Dallas, Dallas, Texas Objective: To determine if conventional sperm parameters, specific characteristics of sperm motion determined by computer-aided semen analysis (CASA), sperm penetration assay (SPA), and/or spontaneous acrosome reaction assay could best predict fertility outcome after intrauterine insemination (lui) from frozen donor sperm. Design: A retrospective analysis of 2,245 cycles of therapeutic donor luis were initially studied; 1,147 cycles that met selection criteria were used in this report. Setting: A university-based assisted reproductive technology center. Patients, Participants: All luis were performed on women with documented patency of at least one fallopian tube, ovulatory cycles, and who did not receive human menopausal gonadotropins. Sperm donors had to be used for at least four different recipients (mean of 15) and at least 14 different cycles of insemination (mean of 41). Interventions: None. Main Outcome Measure: Pregnancy. Results: Statistical comparisons were made between donors of different relative fertility by using the Mann-Whitney test, Spearman's rank correlation, and multiple regression analysis. These analyses demonstrated that the most significant predictors of the fertility of frozen -thawed donor sperm were curvilinear velocity, straight line velocity, and the total number of motile sperm inseminated. The number of sperm with spontaneous acrosome reactions negatively correlated with fertility outcome, and SPA provided no predictive value. Conclusions: Our study supports the hypothesis that the study of sperm motion characteristics using CASA after thawing and washing of cryopreserved sperm is a better predictor of fertile outcome after lui than analysis of fresh semen. Fertil Steril 1992;58: Key Words: Donor sperm, sperm motility, computer-aided sperm analysis, intrauterine insemination Received November 20, 1991; revised and accepted February 26,1992. * Presented in part at the 38th Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20 to 23, t Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology. t Reprint requests: Paul B. Marshburn, M.D., Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas Division of Academic Computing Services. The poor correlation between spermatozoal characteristics and fertility stems, at least in part, from two major sources: (1) the subjective, imprecise, and poorly standardized methods to assess semen quality, and (2) the difficulty in defining factors that contribute to diminished fecundity in the infertile population. Recent advances in computerized semen analysis provide a more objective means of judging semen quality, especially with regards to sperm motion (1). The application of this technique to the Marshburn et al. Sperm motion and fecundity 179

2 study of semen quality in a program of therapeutic donor intrauterine insemination (lui) offers an excellent opportunity to examine spermatozoal characteristics and subsequent conception. Female patients with uncorrected infertility factors can be excluded, and the spermatozoal characteristics of donor semen can be determined for each insemination cycle. In addition, because these donors are used for multiple cycles of insemination with different recipients, the inherent variability of semen parameters between ejaculates in a given individual and occult infertility factors in recipients can be minimized. Donor semen is cryopreserved and quarantined for later use in lui. Because sperm survival, motility, and fecundity are diminished after thawing of frozen semen (2-4), the characteristics of the fresh ejaculate may correlate poorly with the quality of the inseminating specimen. Knowing the characteristics of a given inseminating specimen in its fresh, frozenthawed, and washed state would provide a direct comparison of sperm parameters at each stage of seminal preparation with conception outcome. The goal of this study was to determine whether conventional semen parameters, specific sperm motion characteristics, or other tests of sperm function in fresh, thawed, and washed semen could best predict fertility outcome. To implement this goal, we employed the objective methods of computer-aided sperm analysis (CASA), the sperm penetration assay (SPA) using zona-free hamster oocytes, the spontaneous acrosome reaction determination, as well as the traditional semen analysis. Donor Selection MATERIALS AND METHODS Donors (n = 58) were selected from healthy medical and graduate student volunteers, 22 to 28 years of age. They were screened for heritable or sexually transmitted disease (STD) and had good prefreeze and post-thaw semen characteristics as described previously (4). Donors were tested before entry into the program for STDs and thereafter on a quarterly basis. All semen samples were quarantined for a minimum period of 6 months with a repeat negative STD screen before use. A donor was included for analysis only if his semen was used in at least four different recipients and in at least 14 lui cycles. The number of recipients required for donor inclusion was arbitrarily chosen as a minimum number of recipients to evaluate donor fertility after lui (5). Patient Selection Women (n = 279) entering into the program of lui at The University of Texas Southwestern Medical Center were considered for this study. Their average (±SD) age was 32.9 ± 4.5, and they had experienced 4.5 ± 2.8 years of primary or secondary infertility. Each patient had patency of at least one fallopian tube by hysterosalpingogram and/or laparoscopy. Ovulatory status was documented by midluteal phase serum progesterone (P) and/or endometrial biopsy. Women with ovulatory dysfunction as determined by a midluteal phase P of <4 ng/ml were treated with 50 to 100 mg of clomiphene citrate. Patients treated with human menopausal gonadotropin were excluded from the study. Serum P was measured on the 9th to 11th day after the luteinizing hormone (LH) surge to assess the probability of ovulation. Semen Analysis, Donor Cryopreservation, Thawing, and Preparation for lui Fresh ejaculates were allowed to liquefy for 30 minutes at 37 C before manual and CASA. All evaluations for sperm concentration were determined by hemocytometer counts, and percent sperm motility by manual determination using microcell chambers. Using the Cell Trak/S automated semen analysis system (Motion Analysis Corporation, Santa Rosa, CA), CASA was performed and determined on all fresh, thawed, and washed specimens. Aliquots of sperm (4 ttl) were placed into 12-ttmdeep disposable counting chambers (Fertility Technologies Inc., Natick, MA) on a 37 C microscope stage warmer. The CASA system used in this study has been described previously (4, 6). The computer image sampling was done at 30 Hz (30 frames/s). Minimum threshold velocity was set at 10 ttm/s and the maximum velocity at 400 ttm/s. A minimum of 100 sperm from at least four different fields was analyzed from each specimen. Variables of sperm motion that were analyzed included: (1) curvilinear velocity (VCL), the time-average velocity ofthe sperm head along its actual trajectory measured in ttm/s; (2) straight line velocity (VSL), the time average velocity of the sperm head along its straight line trajectory from its first to its last detected position measured in ttm/ s; (3) amplitude of lateral head displacement (ALH), the mean amplitude of variations ofthe actual sperm head trajectory measured in micrometers; and (4) linearity (LIN), the derived value calculated as the ratio VSL:VCL. After analysis, semen was suspended in a glycerolcryoprotectant medium in a final glycerol concen- 180 Marshburn et al. Sperm motion and fecundity Fertility and Sterility

3 tration of 7.5% (7). Sperm were frozen in a programmable rate freezer (Cryomed, Mt. Clemens, MI) as described previously (4). Samples were allowed to thaw at room temperature for 30 minutes before analysis and preparation for insemination. The thawed samples were re-suspended in five volumes of Hams F-I0 medium (GIBCO, Grand Island, NY) with 0.3% human serum albumin and centrifuged for 6 minutes at 200 X g. After centrifugation, the sperm pellet was resuspended in 0.5 ml of medium. There was no attempt to recover a swim-up fraction for insemination. Samples were then placed in a 37 C incubator until use. Timing and Methodology of lui Inseminations were timed by quantitative urinary LH determination in the endocrinology laboratory as published previously (4, 6). Washed samples were aspirated into a 31 French Tomcat catheter (Richmond Veterinarian Supply, Richmond, VA) that was connected to a l-cc syringe (Becton Dickinson, Rutherford, NJ). The catheter was gently passed through the cervical canal into the uterine cavity, and the contents of the syringe were slowly injected. After resting 15 minutes in the supine position, the recipient resumed normal activities. More than one ejaculate from a given donor may have been used during an insemination cycle. A maximum of two luis were administered per cycle (approximately 20%), and in these cases semen parameters were averaged. A quantitative human chorionic gonadotropin beta subunit was performed approximately 2 weeks after insemination and repeated if positive. If both determinations demonstrated normal increases, ultrasound (US) confirmation of pregnancy was obtained 6 weeks after insemination. Positive fetal heart motion observed by US was considered an established pregnancy. Sperm Penetration Assay The SPA using human sperm and zona-free hamster oocytes was performed using the technique as described by Rogers et al. (8) except that washed sperm, either fresh or cryopreserved, were maintained in media for 6 rather than 18 hours before incubation with fresh zona-free hamster oocytes. Sperm penetration assay was performed on samples split from the same ejaculates used for lui. Detection of Acrosome-Reacted Sperm Analysis of the acrosomal status of sperm that had undergone capacitation in vitro was determined using a specific monoclonal antibody to the human acrosomal cap as described previously (9). Sperm for this assay were taken from the same population of cells used for incubation with zona-free hamster oocytes. Statistical Analysis Data analysis for comparison of sperm characteristics between the top and bottom quartile of donors ranked according to pregnancy success was by the Mann -Whitney U-test. The association of sperm characteristics with the proportion of pregnancy cycles for the 28 donors was determined by Spearman's rank correlation method. The proportion of successful attempts for each donor was transformed using an arcsine transformation. Then multiple regression was performed to test whether combinations of sperm characteristic variables would improve their association with pregnancy rate (PR). Response surface methodology was used to formulate a quadratic polynomial comprised of sperm variables found to provide independent positive correlation with cycle fecundity. This quadratic function was employed to calculate expected PRs from these variables for correlation with the observed PRs after lui. RESULTS A total of 279 women who underwent 2,245 cycles of lui with 58 donors comprised the original group under consideration for the study. A total of 1,147 lui cycles with 191 women were selected after imposing the selection criteria of at least 4 recipients per donor and at least 14 lui cycles per donor. Twenty-eight of 58 donors met the acceptance criteria; each donor was used to inseminate 15 different recipients on the average and underwent an average of 41 insemination cycles. The 28 donors were ranked according to their average fecundity per treatment cycle after lui (Fig. 1). Pregnancy resulted from lui with 25 of 28 donors (range of the percentage of cycles in which pregnancy occurred was 0% to 25%; average [±SDj 10.5 ± 6.0). Spermatozoal characteristics were compared between the top (most fertile) and bottom (least fertile) quartile of donors based on their overall fecundity (Table 1). We initially confirmed that there was no statistical difference between these two groups with regards to the number of lui cycles per donor or the number of ejaculates analyzed. Comparison with the Mann-Whitney U-test revealed that the only significant differences in the freshly ejaculated samples Marshburn et al. Sperm motion and fecundity 181

4 > 30 () z c( z fb a: Q. c( 20 :c l- i en w...i () > () 10 II. o I- Z W () a: w Q. o DONOR Figure 1 The distribution of donor pregnancy success ranked according to the proportion of cycles in which conception occurred after lui. The 28 donors were selected from a group of 58 donors by the criteria stated in Materials and Methods. between these two groups of donors was LIN of sperm motion (P < 0.02) (Table 1). This difference, however, was not observed after examination of the thawed specimen. After preparation for insemination, there was no difference in the total number of motile cells inseminated. However, there appeared to be significant differences in two indices measured by CASA, i.e., VCL (P < 0.01) and VSL (P < 0.02). Comparison of zona-free hamster oocyte SPA scores for both fresh and frozen-thawed sperm indicated that there was no difference between the two different quartiles (Table 2). There was, however, a trend toward a lowered penetration score in the bottom quartile group. Furthermore, there was no significant difference in the motility of these sperm at 6 or 9 hours (data not shown) or the percent of sperm that had undergone a spontaneous acrosome reaction by 9 hours of incubation. The statistical comparison of the top and bottom quartiles of donor fertility limits the analysis of semen characteristics to a subpopulation of all donors. Therefore, we determined the correlation of PRs with sperm motility characteristics from fresh, frozen-thawed, and thawed-washed semen from all donors using Spearman's rank correlation method (Table 3). Standard semen analysis and CASA measurements of the fresh ejaculate demonstrated a positive correlation between conception outcome and total sperm number in the ejaculate, VCL, LIN, and ALH. When the motility characteristics of the thawed specimen and the washed specimen were determined, a decrease in the P value was noted for all motility characteristics. The association of conception with the motility characteristics of the donor spermatozoa from thawed and washed specimens revealed a highly significant correlation with VCL, VSL, and the total number of motile inseminating sperm. Spearman's rank correlation for conception after lui with SPA using zona-free hamster oocytes and measurement of the spontaneous acrosome reaction was determined. For SPA, the percent penetration, the mean number of sperm per oocyte and the motility at 6 hours were determined in both fresh and thawed semen specimens. Likewise, the percentage of sperm with a spontaneous acrosome reaction was evaluated in fresh and thawed specimens. None of these parameters were significantly correlated with PR in the fresh specimen. There was, however, a significant positive correlation between motility of the sample at 6 hours (P = 0.04) and a negative association with the percentage of sperm having a spontaneous acrosome reaction (P = 0.04) in the thawed samples. These assays represent at least three separate assays using fresh and frozen -sperm for each donor tested. Multiple hazard regression was applied to determine the interrelation between semen motility Table 1 Comparison of Semen Characteristics Between Donors in the Top Quartile and Bottom Quartile of Pregnancy Success' Top Bottom Variable quartile quartile Probability Fresh Motility (%) 66.7 ± ± Sperm/mL (10 6 ) ± ± Total sperm (10 6 ) ± ± VeL (I'm/s) 42.2 ± ± VSL (I'm/s) 21.7 ± ± LIN 49.6 ± ± t ALH(l'm) 10.1 ± ± Post-thaw Motility (%) 45.4 ± ± VeL (I'm/s) 37.5 ± ± VSL (I'm/s) 19.5 ± ± LIN 49.7 ± ± ALH(l'm) 5.8 ± ± Postwash Motility (%) 39.2 ± ± vel (I'm/s) 42.6 ± ± 2.9 O.D1t VSL (I'm/s) 21.1 ± ± t LIN 47.0 ± ± ALH(l'm) 7.4 ± ± Total motile sperm inseminated (10 6 ) 17.4 ± ± * Mann-Wbitney U-test; values are means ± SE. t p < Marshburn et al. Sperm motion and fecundity Fertility and Sterility

5 Table 2 Comparison of SPA Scores and Acrosomal Status of Sperm Between Donors in the Top Quartile and the Bottom Quartile* Variable Top quartile Bottom quartile Probability Fresh specimens Penetration (%) No. of sperm/oocyte Motility at 6 hours (%) Spontaneous acrosome reaction (%) Frozen samples Penetration (%) No. of sperm/oocyte Motility at 6 hours (%) Spontaneous acrosome reaction (%) 59.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± * Mann-Whitney U-test; values are means ± SE. characteristics for improving the association with PRs in the 28 donors (Table 4). In this model, the total number of motile sperm inseminated demonstrated the best association with fertile outcome (P < 0.003) and therefore was entered as the first independent variable in the stepwise multiple regression. Only the addition of VCL of the inseminating spermatozoa with the total number of motile sperm inseminated resulted in a decrease in the P value for predicting pregnancy. When the stepwise multiple regression analysis was performed on other binary and tertiary combinations of semen characteristics, no better predicted value for fertile outcome was found. Based on these observations, total motile sperm inseminated, VCL, and VSL were used as variables to formulate a quadratic equation for predicting the proportion of insemination cycles that resulted in pregnancy. Response surface methodology was employed to determine a more complete model because the quadratic expansion of the predictors would estimate a more complicated curved response (10). The strength of the quadratic function relies on the fact that the Taylor series expansion approximates many functions over a restricted domain, and often the approximation is quite appropriate at the quadratic expansion. The expression of this function in relation to the proportion of cycles resulting in pregnancy is presented in Figure 2A. The values for each variable are entered without units into the equation, and P is expressed as the percentage of cycles resulting in pregnancy. In this case, the quadratic expansion provides a fit to our data with the following statistics: df for error = 18; coefficient of determination (r2) = 0.7; adjusted r2 = The residuals range from -5.5% to 7.4 %. Residual analysis indicated no problems in the fit of the model to the observed values. Figure 2B presents a plot of the actual percentage pregnant by donor compared with the predicted proportion pregnant as determined by our model. DISCUSSION The association between conception and spermatozoal characteristics in men with normal semen quality was examined in a well-defined female population during ovulatory cycles with timed lui. We investigated two main issues: (1) whether objective measurements of spermatozoal motility by CASA or other tests of sperm function would correlate with pregnancy after lui and, if so, (2) would analysis of the fresh, thawed, or washed specimens used for insemination provide the best prediction for subsequent conception? Our data reveal that CASA of spermatozoal motility after thawing and washing provided the most significant information for predicting donor sperm fertility potential. The correlation of specific motility characteristics with pregnancy after lui demonstrated that VCL, VSL, and the total number of motile inseminating sperm of thawed specimens were positively associated with conception. The detection of spontaneously acrosome-reacted sperm in thawed specimens revealed a negative correlation of fertility outcome with an increased proportion of spontaneously reacted spermatozoa. In contrast, the results of the SPA test provided no predictive value. Several points in this study deserve emphasis. First, the analysis of sperm from our donors was performed on all inseminating samples so that pregnancy after lui could be correlated with spermatozoal characteristics. Second, the inherent variability of ejaculate quality within individual donors was minimized by the analysis of multiple specimens used in at least four different recipients. Third, we Marshburn et al. Sperm motion and fecundity 183

6 Table 3 The Correlation of PR With Motility Characteristics of Donor Spermatozoa From Fresh, Thawed, and Washed Specimens' Fresh Thawed Washed Characteristic Initial sperm concentration (l06/ml) Total sperm no. (10 6 ) Motility (%) VCL (/Lm/s) VSL (/Lm/s) LIN ALH (/Lm) Total motile inseminating sperm r P r P r P t 0.47 O.Olt ; :1: :1: 0.02t t t t, Values are Spearman's rank correlation data on the association of the averaged parameter for each donor over all cycles with PR. t P < :I: P < analyzed semen from donors with normal semen quality as judged by conventional semen parameters and not from men in the population of infertile coupies. Finally, the outcome variable of pregnancy after timed lui circumvents the difficulties of extrapolating data from in vitro testing to conception. Because we had follow-up on all of our cycles of lui, the data we present are a faithful reporting of pregnancies per cycle of timely pregnancy exposure. Difficulties arise in evaluating most studies that compare spermatozoal characteristics with cycle fecundity after insemination. Many reports base their conclusions on observations in a subfertile population that is comprised of couples with a heterogeneity of factors contributing to infertility. In these cases, the potential for confounding variables and selection bias is problematic. Dunphy et al. (11) studied the semen quality of the fresh ejaculate and conception outcome after intercourse III a large Table 4 Multiple Regression Analysis for the Significance of Semen Characteristics From Inseminations Specimens in Predicting Pregnancy Characteristic Sperm motility VSL VCL LIN ALH Total no. of motile sperm inseminated, Total motile. t Total motile and VCL. :I: P < P < Initially 0.007:1: :1: One variable selected' Two variables selected t number of couples without identifiable female factors. They found that only in a subgroup of couples with a duration of infertility that exceeded 4 years did the single semen variable of motile sperm density significantly correlate with cumulative PRo In other studies, however, no differences in standard semen analysis parameters were found between conception and nonconception cycles after intercourse or inseminatiqn (12-14). Although CASA provides an objective characterization of sperm motility, the superiority of CASA A. B. Let P = 'proportion of pregnancy cycles Then x = total motile inseminated spenm x 106 Y = curvilinear velocity (VC) in ~M/sec z = straight line velocity (VSL) in ~M!sec P = x+ 7.88y-B.62z x xy- 0.23xz y yz- 0.75z2 " 30 g, 0 ~ ~ 20 c L ~ Il. c: ~ 10,.. Il. " Cii ~ Predicted Percent Pregnant per Cycle Figure 2 (A), Mathematical relationship between proportion of pregnancy cycles (P) and CAS A parameters (see text for description). (B), The correlation of the actual PR rate per treatment cycle with the predicted PR per treatment cycle based on total motile sperm inseminated, VCL, and VSL derived by response surface methodology. 184 Marshburn et al. Sperm motion and fecundity Fertility and Sterility

7 over conventional measures of semen quality in correlating with fertilization of oocytes in vitro, however, has not been consistently demonstrated. Milligan et al. (15) observed that spermatozoa from fertile and infertile men could be distinguished on the basis of sperm velocity measurements. In two studies, sperm speed measurements correlated with penetration of zona-free hamster eggs (16) and human oocytes in vitro (17). In a different report, however, conventional semen parameters were more predictive of the potential for sperm to fertilize 00- cytes in vitro than movement characteristics obtained by CASA (18). The biological significance of the fertilization of oocytes in vitro in predicting pregnancy after insemination is unclear because factors necessary for in vitro oocyte fertilization may not be identical to that required for sperm transport and fertilization in vivo. In this report, we show that even though certain semen characteristics of the fresh ejaculate correlate with conception after lui, a much stronger association is found between cycle fecundity and CASA measurements of sperm motion in the thawed and washed specimen. The frozen-thawed semen compared with fresh semen has a diminished capacity to achieve pregnancy after donor insemination (2-4). It is reasonable that the spermatozoal characteristics of the thawed and washed specimen that is inseminated correlates better with conception outcome than would the quality of fresh semen before freezing. Indeed, Paraskevaides et al. (19) found that the pre freeze: postfreeze sperm ratio of the percentage of motile sperm positively reflected fertility after donor insemination, despite the insensitivity of the conventional semen analysis parameters to do so. The quantitative analysis of sperm motion characteristics of thawed and washed specimens provided independent information toward helping to predict conception after lui. The multiple regression analysis of thawed and washed specimens indicated that when the variable total number of motile sperm was combined with sperm VCL, the association with fertile outcome was strengthened. Other investigators have demonstrated the positive correlation of cycle fecundity after insemination with the total number of sperm inseminated (3, 4, 20). We confirm and extend these observations by providing data that sperm VCL of the thawed and washed specimen also provides independent predictive value for conception outcome. The percentage of motile sperm, however, gave no independent information with multiple regression analysis that was not provided by the variables total motile sperm inseminated and the VCL. Thus, these data support the hypothesis that the detection of sperm motion alone is less predictive of fertility potential than the specific characteristics of that motion. Other indices of spermatozoal function in fresh and thawed sperm such as the hamster egg SPA and the number of spontaneously acrosome-reacted sperm were tested in our donors. Only the percent spontaneously acrosome-reacted sperm after thawing was found to have negative association with cycle fecundity. Our data support that of others who have found limited usefulness of the hamster egg penetration assessment of fertility outcome (21). Rogers et al. (8) and Wiltback et al. (22), however, suggested that SPA testing is effective in estimating fertility potential, and others found that sperm speed measurements correlate with SPA (16, 17). Our data do not confirm, however, that SPA predicts the fertility potential of frozen donor sperm after lui. A significant correlation with conception rate was found with donors who had a lower proportion of sperm with a spontaneous acrosome reaction. One might reason that the higher number of sperm with a spontaneous acrosome reaction would lessen the proportion of capacitated sperm capable of participating in binding to and penetrating the zona pellucida. Another interpretation of this finding is that the higher proportion of spontaneous acrosome-reacted sperm in less fertile donors reflects a higher susceptibility to membrane damage after cryopreservation in this group. In this regard, it is possible that sperm viability, rather than or in addition to, acrosome reactions is an important parameter. A previous study with frozen semen showed that fertile donors had a greater proportion of spermatozoa that could become capacitated and undergo a physiologically induced acrosome reaction (5). Therefore the proportion of capacitated and nonacrosome-reacted sperm may indicate semen with greater fertility potential. Our study supports the hypothesis that the analysis of sperm motility after thawing and washing cryopreserved semen is a better predictor of fertile outcome after lui than examination of the fresh semen parameters. We advocate the practice of performing CASA measurements and other tests of sperm function on thawed and washed specimens for evaluating the fertile potential of donor sperm for several reasons. The capability of spermatozoa to survive the physiological insult of cryopreservation may provide an independent test of sperm quality (19). Other reasons relate to the appropriate Marshburn et al. Sperm motion and fecundity 185

8 r, specimen preparation for accurate and reliable CASA measurements. The variability of semen viscosity is known to affect characteristics of sperm motion (23), and washing of spermatozoa will remove debris in semen that may interfere with accurate CASA measurements (24). Expression of the variables, total motile sperm inseminated, VCL, and VSL, in a quadratic function provided good correlation between the PRs predicted by this model and the actual PRs observed (r2 = 0.7). Thus CASA appears to be a powerful tool to aid in the discrimination of fertile and sub fertile donors, offering the possibility that mathematical indices can be derived that can prospectively assess semen donor fertility potential. We emphasize that the model derived for calculation of the proportion of pregnancy cycles from the variables total motile inseminated, VCL, and VSL was obtained by application of certain selection criteria of donors and recipients. Therefore, the extension of this equation to other programs that use different selection criteria for donors, recipients, or CASA parameters may not be valid. Thus, more work is necessary to specify sperm motion criteria that can be universally applied to the screening of men for fertility potential in artificial insemination by donor programs. REFERENCES 1. Boyers SP, Davis RO, Katz DF. Automated semen analysis. In: Barbieri RL, editor. Current problems in obstetrics and gynecology and fertility. Chicago: Year Book Medical Publishers, 1989;12: Hammond MG, Jordan S, Sloan CS. Factors affecting pregnancy rates in a donor insemination program using frozen semen. Am J Obstet Gynecol 1986;155: Brown CA, Boone WR, Shapiro SS. Improved cyropreserved semen fecundability in an alternating fresh-frozen artificial insemination program. Fertil Steril 1988;50: Byrd W, Bradshaw K, Carr B, Edman C, Odom J, Ackerman G. 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The sperm penetration assay: can it discriminate between fertile and infertile men? Am J Obstet Gynecol 1988;159: Wiltbank MC, Kosasa S, Rogers B. Treatment of infertile patients by intrauterine insemination of washed spermatozoa. Andrologia 1985;17: Amann RP, Hammerstedt RH. Validation of a system for computerized measurements of spermatozoal velocity and percentage of motile sperm. BioI Reprod 1980;23: Calamera JC, Brugo S, Quiros MC, Nicholson RF. Computer assisted measurement in normal and pathological human semen, fresh and post swim-up technique. Andrologia 1989;21: Marshburn et al. Sperm motion and fecundity Fertility and Sterility