Fred K. Kirchner, M.D.t B. Jane Rogers, Ph.D.*:j:

Transcription

1 FERTILITY AND STERILITY Copyright c 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Establishment of TEST -yolk buffer enhanced sperm penetration assay limits for fertile males Randall M. Falk, M.D.* Kaylen M. Silverberg, M.D.* Peter M. Fetterolf, Ph.D.* Fred K. Kirchner, M.D.t B. Jane Rogers, Ph.D.*:j: Vanderbilt University Medical Center, Nashville, Tennessee TEST-yolk buffer has been shown to enhance sperm penetration of zona-free hamster eggs. Review of sperm penetration assay (SPA) data from a fertile population was undertaken to determine a normal range for SPA with TEST -yolk buffer enhancement. Thirtyeight intrauterine insemination patients and 4 artificial insemination donors who had successfully initiated a pregnancy within 18 months of SPA analysis were examined. All 42 enhanced SPAs demonstrated penetration of >10%, and 37 of these (88%) yielded SPA values of ~20%. Thirty-three percent (14/42) of these individuals achieved 0% penetration in the SPA without TEST-yolk buffer. The SPA performed with the TESTyolk modification has fewer false negatives than the assay done with the original methodology. Fertil SteriI54:121, 1990 Since the sperm penetration assay (SPA) was described by Yanagimachi et al. 1 and Rogers et al., 2 it has aroused great interest as an in vitro test of male fertility (see review by Rogers 3 ). Unlike the standard semen analysis, which evaluates the physical properties, count, motility, and morphology of the sperm, the SPA assesses the functional ability of the sperm in the fertilization process. The ability of the sperm to undergo capacitation, acrosome reaction, membrane fusion, and nuclear decondensation is measured by penetration of zonafree hamster eggs. This penetration potential correlates with fertility both in vitro 4-9 and in vivo. 3 Several modifications in the SPA over the past 10 years have been initiated in an attempt to improve sperm penetration. Sperm processing via a Received March 23, 1989; revised and accepted March 14, * Center for Fertility and Reproductive Research, Department of Obstetrics and Gynecology. t Department of Urology. * Reprint requests: B. Jane Rogers, Ph.D., Department of Obstetrics and Gynecology, C-FARR, D-3224, Medical Center North, Vanderbilt University Medical Center, Nashville, Tennessee swim-up,lo incubation of sperm with ionophore A23187,1l and preincubation in TEST-yolk buffer 3,12-15 cause more sperm to penetrate the zona-free eggs. The fertile range for SPA with buffer, i.e., a range of results that correlates with fertility in vivo, has not been established for our TESTyolk buffer methodology. The purpose of this study was to establish a fertile range for the SPA with TEST-yolk buffer enhancement, using the methodology established in this laboratory (Fig. 1). MATERIALS AND METHODS A fertile population of 42 men was identified from the intrauterine insemination (lui) (n = 38) and artificial insemination (AI) (n = 4) programs at Vanderbilt University. Each subject had successfully initiated a pregnancy within 18 months of the SPA utilized in this assessment. Known presence of antisperm antibodies precluded participation in this study. Of the select patient population, 25 pregnancies were achieved through lui and 13 pregnancies were achieved naturally. Semen samples were collected by masturbation after a period of at least 48 hours of sexual abstinence. The standard SPA with capacitation in Biggers, FaIk et ai. Enhanced SPA limits 121

2 Add Equal Volume, ~~B ;~ension ~ Incubate THE SPERM PENETRATION ASSAY Sperm Preparation Semen Collection (Day 3) Two Washes with BWW Capacitation in TVB n R R U U ~ ~ Incubate 1 Hr 17 Hrs. at 4 C,+. at 37 C Two Washes with BWW. Adjust Sperm Concentration Capacitation in BWW ~ c==:1l ~ Add BWW to Adjust Sperm Concentration Incubate 18 Hrs. at 37 C Sperm and Eggs Incubated for 2 Hours Eggs Examined for Sperm Penetration Zona-Free Egg Preparation Hamster Injected with PMS (Day 1) and hcg (Day 3) Tlbes and Cumulus Removed (Day 4) Cumulus Dispersed with Hyaluronidase Zona Removed with Trypsin Figure 1 Methodology for performance of SPA with and without TEST -yolk buffer. Whitten, and Whittingham (BWW) medium was performed as described previously2 and is diagrammed in Figure 1. Sperm penetration assay methodology with capacitation in TEST-yolk buffer is also shown in Figure 1. After collection, the sample was allowed to liquefy for 30 minutes at room temperature before washing twice by centrifugation at 600 X g for 10 minutes in BWW (total volume 10 ml each time). The pelleted sperm was resuspended finally in 1 ml of BWW, and an equal volume of TEST-yolk buffer was added to the sperm suspension. The TEST -yolk buffer contains 21 mm TES (N-tris[hydroxymethyl]methyl-2- amino ethane sulfonic acid), 96 mm tris(hydroxymethyl)aminomethane, 11 mm dextrose, and 1 % penicillin-streptomycin, to which is added 20% fresh hen egg yolk. If the assay was to be performed both ways, the sample was divided before the TEST -yolk buffer addition. The sperm-buffer mixture, contained in a tube, was incubated overnight (17 hours) in a beaker of water in the refrigerator at 4 C. After the preincubation period, the warming step was done slowly by placing the beaker with the tube in the incubator at 3TC for 1 hour. Two washes with BWW were done by adding BWW up to 10 ml for the first spin at 600 X g for 5 minutes 122 Falk et al. Enhanced SPA limits and 2 ml for the second spin. The final pellet was resuspended and adjusted to a concentration of 10 X 10 6 sperm/ml. An aliquot (100 ttl) was placed in a Petri dish under oil before 30 zona-free hamster eggs were added. The results were expressed as penetration percentage (the number of eggs penetrated/number of eggs inseminated X 100) and as penetration index (the total number of swollen heads/number of eggs examined). RESULTS Sperm penetration assays were performed on 42 known fertile men, selected from the lui and AI programs, using the original BWW methodology and a TEST-yolk buffer protocol. Fourteen (33%) ofthese individuals achieved 0% penetration in the SPA without buffer enhancement (Fig. 2A). In contrast' all 42 TEST-yolk buffer SPAs demonstrated enhanced penetration values;;:: 10%, and 37 (88%) of these yielded SPA values of 20% (Fig. 2B). The mean penetration for this TEST-yolk buffer group was 48% versus 8% in the BWW group. An even greater enhancement was seen in the penetration index (Fig. 2C and D) by the use of TEST-yolk buffer (0.09 versus 1.14). Further evaluation of patient treatment cycles Fertility and Sterility

3 100 A 100 B Std.dav.! 50 --Mft',\ I -Sld.dey -Sid. de". - -Mean.I! C 1 '.0 D ~Sld.day Sid. de -'-Mean.;. 1.0 & i t Figure 2 Individual penetration rates and penetration indexes for fertile males. Assays were performed using the standard SPA methodology with BWW as buffer (A and C) and using a modified SPA methodology with TEST -yolk buffer (B and D). (Tables 1 and 2) revealed that 13 (34%) of this study population, though involved in an lui program, successfully initiated pregnancies by natural means (Table 2). Only 1 of these 13 men (8%) had a TEST-yolk buffer SPA value < 20%. Notably, 4 men (16%) of the assisted pregnancy group had <20% penetration with TEST -yolk buffer (Table 1). Limited sample size precluded the demonstration of statistical significance, though the trend was noted that fewer low penetrators «20%) achieved natural pregnancies than assisted pregnancies. All of these men represented couples reporting an inability to achieve pregnancy for at least 1 year of unprotected intercourse before entering the lui program. Of these natural pregnancies' the mean penetration in the SPA without TEST-yolk buffer enhancement was only 5%. In comparison, the mean percent penetration rose to 49% with TEST-yolk buffer. DISCUSSION During recent years, it has become apparent that male infertility is a dysfunction of a physiological process not entirely reflected in traditional semen analysis. Rogers et al. 2 reported the importance of zona-free hamster egg penetration as a means of assessing male fertility. TEST -yolk buffer enhancement has refined this assay, providing a means for synchronizing the acrosome reaction 3,9 and thereby evaluating penetrance of a higher percentage of the sperm in a given sample. Sperm motility has been demonstrated to be a critical parameter offertility Johnson et al. 15 suggested that Falk et al. Enhanced SPA limits 123

4 Table 1 Semen Parameters of Men Initiating Assisted Pregnancies Penetration Penetration percentage Penetration index Patient Normal percentage Penetration index with TEST- with TEST-yolk no. Concentration Motility morphology with Bwwa with BWW b yolk buffera buffer b Xl(f/mL % % c c lo c lo c 0.10 Average a Penetration percentage = (no. eggs penetrated/no. eggs inseminated) X 100. b Penetration index = (no. swollen sperm heads/no. eggs inseminated). TEST -yolk buffer preserves sperm motility during the capacitation phase in vitro. If, in fact, dysfunctional penetration potential is a key to male infer- c Four of the five patients with SPA results <20% achieved pregnancy by assisted reproductive technology (lui). tility, this method of evaluating a larger number of motile sperm as they enter the acrosome reaction phase is valuable. Table 2 Semen Parameters of Men Initiating Natural Pregnancies Penetration Penetration percentage Penetration index Patient Normal percentage Penetration index with TEST- with TEST-yolk no. Concentration Motility morphology withbww withbww yolk buffer buffer XJ(f/mL % % a 0.36 Average a Only one of the five patients with SPA results < 20% achieved pregnancy naturally. 124 Falk et al. Enhanced SPA limits Fertility and Sterility

5 The mechanism of action of TEST -yolk buffer in enhancing penetration ofthe zona-free hamster egg is still unclear. Our current hypothesis is that the sperm are allowed to capacitate in this cold cholesterol-rich milieu provided by the buffer, but are blocked from acrosome reacting. When the buffer is removed after the overnight preincubation, a large number of sperm are capable of acrosome reaction (25% to 35%19). In contrast, in the BWW situation with no cholesterol and a high temperature (37 C), when a sperm becomes capacitated, it undergoes an acrosome reaction, thus entering a very finite phase of existence. When the preincubation period in BWW is over, many of these acrosome-reacted sperm lack viability. Thus, the live acrosome reaction percentage in this group is much lower. The lipid components in the yolk appear to prevent acrosome reaction sufficiently to allow a synchronization of reaction at the time of removal of buffer by washing. In an effort to establish a normal range for the TEST-yolk buffer-enhanced SPA, this study included subjects who initiated a pregnancy within 18 months of their SPA. Pregnancies included in this study were achieved by both natural means and AI. Thirty-three percent of the patients in this study population tested 0% penetration with the standard SPA and would have been expected to be infertile based on this result but, in fact, were fertile. All of our patients demonstrated> 10% penetration when TEST -yolk buffer enhancement was employed, and 88% demonstrated >20% penetration when TEST -yolk buffer was utilized. In this study, the enhancement methodology has thus eliminated the occurrence of 0% penetrations in the fertile population. Two points are elucidated by these data. First, TEST-yolk buffer enhancement more clearly delineates the fertile male population. Men demonstratingpenetration < 20% with TEST-yolk buffer are outside one standard deviation and may be considered to have reduced fertility potential. Those scoring 0% with TEST -yolk buffer may be the subset oftruly infertile males. In our in vitro fertilization (IVF) program, we have had five patients with 0% TEST-yolk buffer SPA attempt IVF, and all failed to achieve fertilization~ This is somewhat anecdotal information but is suggestive that the subset of 0% penetrators is a small group, and they are truly impaired. This information should be used to counsel couples before they pursue long, expensive programs aimed at achieving pregnancy. Second, because 33 % of this fertile study population achieved pregnancy despite 0% standard SPA re- sults, evaluation of men using the SPA should employ TEST -yolk buffer methodology. It is the lower end of the penetration spectrum that TEST -yolk buffer delineates more clearly. Furthermore, when TEST -yolk buffer-enhanced SPAs are positive, participation in an lui program might be considered. Those men with enhanced SPA penetrations between 0% and 10% may have limited success with IVF, but the outlook for pregnancy in these patients utilizing lui or natural means is less favorable. In light of the finding that 34 % of the study population achieved pregnancy by natural means during a nontreatment cycle, the value of patient and couple education cannot be overemphasized. In comparing the penetration percentage and penetration index for these two groups, the penetration percentage is not statistically different (43% for assisted versus 49% for natural pregnancies). The penetration index, however, reaches a low level of significance (0.66 versus 1.45, P < 0.05). This may indicate that the natural pregnancy group had a higher fertility potential than the assisted group. Each of these couples in the study population had been unsuccessful in achieving pregnancy after at least 1 year of unprotected intercourse. The fact that so many pregnancies were achieved during nontreatment cycles after entering the lui pro.gram reinforces the supposition that higher pregnancy rates in the fertility center population are a function of education as well as technology. Education should not become sidelined as the thrust toward new technology continues. REFERENCES 1. 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 15:471, Rogers BJ, Van Campen H, Veno M, Lambert H, Bronson R, Hale R: Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil Steril32:664, Rogers BJ: The sperm penetration assay: its usefulness reevaluated. Fertil SteriI43:821, WolfDP, Sokoloski JE, Quigley MM: Correlation of human in vitro fertilization with the hamster egg bioassay. Fertil Steril40:53, Margalioth EJ, Navot D, Laufer N, Yosef SM, Rabinowitz R, Yarkoni S, Schenker J G: Zona-free hamster ovum penetration assay as a screening procedure for in vitro fertilization. Fertil Steril 40:386, Foreman R, Cohen J, Fehilly CV, Fishel SB, Edwards RG: The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization. J In Vitro Fert Embryo Transfer 1:166, Ausmanas M, Tureck RW, Mastroianni L, Jr, Kopf G, Ri- Falk et al. Enhanced SPA limits 125

6 bas J, Blasco L: The zona-free hamster penetration assay as a prognostic indicator in an in vitro fertilization program. (Abstr. 254) Fertil Steril41:106S, Rogers BJ, Herbert CM, Wentz AC: Comparison of human egg and hamster zona-free egg penetration. Presented at the Third World Congress ofin Vitro Fertilization and Embryo Transfer, Helsinki, Finland, May 14 to 17, Bastias C, Wentz AC, Rogers BJ: Predictive potential of the modified SPA for IVF success. (Abstr. pp-90) Presented at the Fifth World Congress on In Vitro Fertilization and Embryo Transfer, Norfolk, Virginia, April 5 to 10, Published by The American Fertility Society, in the program supplement, 1987, p Russell LD, Rogers BJ: Improvement in the quality and fertilization potential of a human sperm population using the rise technique. J AndroI8:25, Aitken RJ, Ross A, Hargreave T, Richardson D, Best F: Analysis of human sperm function following exposure to ionophore A J AndroI5:321, Smith RG, Johnson A, Lamb D, Lipshultz LI: Functional tests of spermatozoa. Urol Clin North Am 14:451, Johnson AR, Lipshultz LI, Smith RG: Thermal shock (37 C) to spermatozoa stored at 4 C optimizes capacitation. J UroI133:74, Chan SYW, Li SQ, Wang C: TEST-egg yolk buffer storage increases the capacity of human sperm to penetrate hamster eggs in vitro. Int J Androl10:517, Johnson AR, Syms AJ, Lipshultz LI, Smith RG: Conditions influencing human sperm capacitation and penetration of zona-free hamster ova. Fertil SteriI41:603, Aitken RJ, Best FSM, Richardson DW, Djahanbakhch 0, Lees MM: The correlates of fertilizing capacity in normal fertile men. Fertil Steril 38:68, Aitken RJ, Best FSM, Richardson DW, Djahanbakhch 0, Mortimer D, Templeton AA, Lees MM: An analysis of sperm function in cases of unexplained infertility: conventional criteria, movement characteristics, and fertilizing capacity. Fertil SteriI38:212, Aitken RJ, Best FSM, Richardson DW, Djahanbakhch 0, Templeton A, Lees MM: An analysis of semen quality and sperm function in cases of oligozoospermia. Fertil Steril 38: 705, Rogers BJ, Bastias C: Unpublished data 126 Falk et al. Enhanced SPA limits Fertility and Sterility