- Email: [email protected]
Human sperm penetration assay as an indicator of sperm function in human in vitro fertilization
FERTILITY AND STERILITY
Vol. 48, No. 2, August 1987 Printed in U.S.A.
Copyright., 1987 The American Fertility Society
Human sperm penetration assay as an indicator of sperm function in human in vitro fertilization
Frank B. Kuzan, Ph.D.*t Charles H. Muller, Ph.D.*:j: Paul W. Zarutskie, M.D.*
L. Lynne Dixon, B.S.* Michael R. Soules, M.D.*
University of Washington, Seattle, Washington
Performance of spermatozoa in a hamster oocyte/human sperm penetration assay (SPA) was correlated with the results of in vitro fertilization (IVF). Forty-two patients underwent 50 IVF cycles. SPA scores were obtained before IVF cycles (screening SPA, n = 30) and, where practical, on the semen sample used for IVF (IVF SPA, n = 26). Screening SPA score did not correlate to IVF result, fertilization and cleavage rates were similar between normal (n = 17) and low (n = 13) SPA groups, pregnancy rates were 35 and 46%, respectively. In addition, SPA score at the time of IVF did not correlate with IVF result. Pregnancy rates were 33% for the normal group (n = 16) and 30% for the low SPA group (n = 10). Overall, the low SPA group (n = 16) exhibited a 78% fertilization rate and a 38% pregnancy rate, which was not different from the normal SPA group: 76 and 29%, respectively. The results of this study indicate that SPA score is a poor indicator of sperm function in IVF. Fertil Steril 48:282, 1987
I'
,,)i ~
I
The hamster oocyte/human sperm penetration assay (SPA) was introduced in 1976 as "a preliminary assessment of the fertilizing capacity of human spermatozoa. m Since then, the SPA has been found to correlate well with male fertility in vivo. 2- 4 With the advent of in vitro fertilization (IVF) as a therapy for human infertility, the correlation between SPA score and IVF result has been examined. 5_9 Wolf et al., 5 in a study of 24 patients, found SPA score correlated well with IVF success or failure, as did Margalioth et al., 8 •9 with 20 and 134 patients, respectively. Foreman et al. 6 reported that SPA scores in the low range correlated with an increased frequency of IVF failure; however, normal SPA scores were not reliable in predicting IVF success ..
'i Received November 18, 1986; revised and accepted April 6, 1987. * Department of Obstetrics and Gynecology. t Reprint requests: Frank B. Kuzan, Ph.D., Department of Obstetrics and Gynecology, RH-20, University of Washington, Seattle, Washington 98195. :J: Department of Biological Structure.
282
The results of Ausmanas et al. 7 agreed with those of Wolf et al., 5 but a higher false-negative (low SPA with successful IVF) rate of 23% was found. The approach to correlating SPA score with IVF result was different among these studies. In two studies,5 •6 SPA was performed on the same semen sample used for the IVF procedure, while three studies7- 9 obtained SPA on a semen sample before IVF. One study6 compared a select population of 15 men, who had failed IVF previously, with 15 randomly selected men. In summary, four studies5 •6 •8 •9 supported the hypothesis that SPA was a good predictor of the ability to fertilize human oocytes in vitro. In three of these studies, pregnancies were not reported. In a fifth study, 7 it was concluded that SPA was not helpful in predicting IVF performance. These reports are contradictory; therefore, in an attempt to clarify this apparent confusion, the present study was undertaken. Considering the discrepancy in SPA score within the male over time, 10 two types of SPA scores were obtained for analysis: (1) on a semen sample prior to the IVF cycle, screening-SPA; and (2) on the semen sample used for IVF, IVF-SPA.
Kuzan et al. Sperm penetration assay as an indicator for IVF
Fertility and Sterility
MATERIALS AND METHODS
dium. Only pregnancies confirmed by ultrasound at greater than 8 weeks' gestation are reported.
Patients Male partners of couples applying to the University of Washington IVF program from February to September 1984 were required to have a SPA before acceptance. Additionally, SPA tests were performed on most semen samples used for IVF from September 1984 to October 1985. Thus, two types of SPA scores were available: (1) screening-SPA (n = 30), those tests performed no more than 8 months prior to the IVF cycle; and (2) IVF -SPA (n = 26), those tests performed on the same semen sample used for IVF. No therapy of the male nor changes in IVF laboratory procedures, in regard to sperm preparation, were undertaken during the study interval based on SPA score. Couples who were included in this study had completed an IVF cycle, including oocyte retrieval, before November 1985. The patient population (n = 42) was composed of patients with tubal disease (n = 22), endometriosis (n = 2), male factor (n = 7), idiopathic source (n = 4), or a combination of these factors (n = 7) as the source of infertility. Superovulation for IVF cycles was accomplished by a combination regimen; 100 mg clomiphene citrate (CC) (Serophene, Serono Laboratories Inc., Randolph, MA) on cycle days 3 to 7 and 75 IU human menopausal gonadotropin (hMG) (Pergonal, Serono Laboratories Inc., Randolph, MA) starting day 3 until follicular maturity. Oocyte retrieval was accomplished laparoscopically on approximately day 12 (range, 11 to 14). After retrieval, oocytes were removed from follicular fluid within 2 minutes and placed into a culture tube (Falcon #3033, Becton Dickenson Co., Oxnard, CA) containing Ham's F-10 (HF-10, Gibco Laboratories, Santa Clara, CA) medium supplemented with 10% human fetal cord serum (FCS), 1 mM calcium lactate (Calbiochem-Behring Corp., LaJolla, CA), and 20 mM sodium bicarbonate (J. T. Baker Chemical Co., Phillipsburg, NY). Oocytes were incubated in organ-culture dishes (Falcon #3037, Becton Dickenson Co., Oxnard, CA) at 37°C in a 5% C02, 5% 0 2, and 90% N 2 gas mixture, 5 to 6 hours before the addition of spermatozoa. Seventeen hours after sperm addition, oocytes were examined for signs of fertilization, at which time they were placed into HF -10 containing 20% human FCS. The uterine placement of cleaving embryos, 40 to 42 hours after insemination, was accomplished with either a 3 or 4 French, side-port catheter (W. A. Cook Pty. Ltd., Melbourne, Australia) containing 20 to 30 ~1 meVol. 48, No.2, August 1987
Sperm Preparation for In Vitro Fertilization One-half milliliter of liquified (20 minutes at 37°C) neat semen was layered beneath 3 ml of HF-10 containing 0.5% human serum albumin (HSA; Sigma Chemical Co., St. Louis, MO), and incubated for 1 hour in a humidified atmosphere of 5% C02 in air at 37°C.U The upper 1.5 ml of medium, containing motile spermatozoa, was removed and centrifuged at 400 X g for 5 minutes. The supernatant was discarded, and spermatozoa were washed two additional times in HF-10 + 10% human FCS by centrifugation. The final pellet was drawn up in 0.5 ml HF-10, counted by hemocytometer, and 105 motile spermatozoa were added to each dish containing a single oocyte. The average time from semen collection to oocyte insemination was 3 hours. Semen samples ranged from 107 to 2 X 108 sperm cells/ml, with motilities of 20% to 70%, and abnormal forms ranging from 30% to 70%. No sample used for IVF had more than one abnormality detected by routine seminal fluid analysis (i.e., motility and sperm concentration). Oocyte Preparation for Sperm Penetration Assay Hamster oocytes and human spermatozoa were prepared by the method described by Karp et al. 3 Briefly, mature female hamsters were superovulated with pregnant mare's serum gonadotropin (PMSG; Organon, El Monte, CA) followed 56 hours later with human chorionic gonadotropin (hCG; Sigma Chemical Co., St. Louis, MO). Cumulus enclosed oocytes were collected into modified Biggers, Whitten, and Whittingham medium12 (BWW) 14 to 16 hours after hCG administration. Oocytes were treated with hyaluronidase (Sigma Chemical Co., St. Louis, MO) and trypsin (Sigma Chemical Co., St. Louis, MO) to remove the cumulus cells and the zona pellucida, respectively. Twenty-five zona pellucida-free oocytes were added to approximately 107 spermatozoa for each assay. Sperm Preparation for Sperm Penetration Assay After 20 minutes at 37°C to allow liquefaction, seminal components were separated from spermatozoa by three centrifugation steps (300 X g, 7 minutes, 5 minutes, 5 minutes). Spermatozoa were resuspended in BWW at a concentration of 1 to 2 X 107 and then incubated in a humidified gas atmo-
Kuzan et al.
Sperm penetration assay as an indicator for IVF
283
sphere of 1% C02 in air, at 37°C for 18 to 22 hours before hamster oocyte addition. The spermatozoa/ hamster oocyte mixture was incubated 2.0 to 2.5 hours, at which time the hamster oocytes were removed, lightly compressed between a glass slide and cover slip, and assessed for sperm penetrations (decondensed sperm head with associated tail). Results were reported as percent hamster oocytes penetrated.
cleavage rate, or pregnancy rate between the normal and low groups (Table 1). Of particular interest was the fact that mean number of embryos replaced after IVF was similar: 3. 7 for the normal group and 3.2 for the low group. Only 1 of 13 (7%) patients with a low SPA did not fertilize human oocytes in vitro and one pregnancy was initiated by a man who scored 0 on SPA. Twenty-six SPAs were performed on semen samples used for IVF; these data are presented in Table 1 (IVF -SPA). No difference was found in any IVF parameter between these groups. Two points of interest were: three patients scored zero on this SPA, yet initiated a pregnancy; and some men with a previous screening-SPA changed their SPA status. In particular, five patients changed from low (screening) to normal (IVF); one patient went from normal to low; and two patients who were low on the first IVF-SPA cycle were normal on the second IVF-SPA cycle. Only three men who had a SPA at screening and IVF remained in the same SPA category. Because men fluctuated between the low and normal groups during the course of this study, an additional effort was made to identify an infertile group of men whose spermatozoa would not perform well in IVF. Patients were separated into two groups based on low SPA score versus any normal SPA score achieved during the test period; these data are presented in Table 1 (all SPA scores). In 16 of the 50 cycles (32%), the male partner had never exhibited a normal SPA (Table 1). After arranging the data in this manner, no difference was found between the SPA groups in any IVF parameter. Additionally, two pregnancies were established by men with SPA scores of 0.
Data Analysis
Data concerning IVF were separated based on low (0% to 10%) and normal (~11%) SPA scores. Repeated measures analysis of variance was employed to detect differences between low and normal SPA groups relative to IVF and cleavage rates. Chi -square analysis was employed to determine whether IVF pregnancy rate differed between these SPA groups.
RESULTS
i.
!
Forty-two infertile couples and 50 IVF cycles were included in this study. A total of 228 human oocytes were recovered (4.6/cycle), 175 of these were fertilized (78%, 3.5/cycle), and 167 cleaved (73%), resulting in an average of 3.4 embryos per replacement. Sixteen cycles resulted in clinical pregnancies, ultrasound documented at 8 weeks' gestation, for a 32% pregnancy rate per laparoscopy. During the course of this study, only two failed fertilization cycles were completed. Twenty-six patients were evaluated by SPA prior to IVF (screening-SPA); results based on SPA score are presented in Table 1 (screening-SPA). No difference was detected in IVF fertilization rate,
Table 1 Relationship Between SPA Score and IVF Fertilization, Cleavage, and Pregnancy Rate Oocytes (low SPA score)
Oocytes (normal SPA score [""11%]) No. cycles
Recovered
Fertilized
Cleaved
No. pregnant
No. cycles
Recovered
Fertilized
Cleaved
No. pregnant
Screen SPA
17
5.3 ± 0.5" %
4.0 ± 0.4 75
3.7 ± 0.4 70
6b 35.2
13c
4.5 ± 0.5 %
3.2 ± 0.6 71
3.2 ± 0.6 71
6 46.2
IVF SPA
16
3.8 ± 0.3 %
3.1 ± 0.3 82
3.1 ± 0.3 82
5 31.2
lOd
4.8 ± 0.6 %
4.0 ± 0.8 83
3.9 ± 0.8 81
30
4.5 ± 0.3 %
3.5 ± 0.3 76
3.3 ± 0.3 73
lOb
16'
29.4
4.4 ± 0.5 %
3.3 ± 0.5 75
3.3 ± 0.5 75
6 37.5
All SPAs
34
• Mean ± standard error. b Includes one ectopic. c One 0 SPA score. 284
Kuzan et al.
3b
d Three 0 SPA scores. • Two 0 SPA scores.
Sperm penetration assay as an indicator for IVF
Fertility and Sterility
DISCUSSION
The findings of the present study do not support the concept that SPA can predict which semen sample possesses the ability to fertilize human oocytes in vitro. Therefore, these data do not support the findings of Wolf et al. 5 or Margalioth et al.8·9 because no correlation between negative IVF result and low SPA score was found. Two cases supported the findings of Foreman et al. 6 that semen samples that failed at IVF were associated with low SPA scores. The previous studies varied in the time relationship between SPA assay and IVF cycle. Wolf et al. 5 and Foreman et al. 6 performed SPA on the same semen sample used for IVF. The most meaningful scientific data would be obtained in this manner, since SPA score has been shown to fluctuate over time within individual men. 10 However, the data of Wolf et al. 5 were difficult to interpret because fertilization data often referred to a single oocyte per patient. Foreman et al. 6 compared the IVF results of 15 men who had failed a previous IVF cycle, "subfertile," with those of 15 randomly selected "normal" (by seminal fluid analysis) men. Although 6 "subfertile" men scored normal on SPA at IVF, none of the 15 semen samples fertilized human oocytes (n = 37) during that IVF treatment cycle. Only 1 of the 15 "normal" men scored in the low range on SPA and all semen samples were capable offertilizing human oocytes in vitro. Foreman et al. 6 concluded that low SPA scores were reliable in predicting IVF outcome. Margalioth et al.,S who did not indicate the time relationship between the SPA and IVF, found that a low SPA correlated well with poor IVF results. Spermatozoa from 7 patients with low SPA scores failed to fertilize human oocytes in vitro, while only 3 of 13 men with normal SPA scores failed IVF. Margalioth et al. 9 extended these observations to 134 couples in whom SPA was obtained sometime prior to the IVF cycle. Results showed that 94% of men with a normal SPA fertilized human oocytes in vitro, while 78% of men with low SPA failed to fertilize human oocytes. Data from this study do support the findings of Ausmanas et al. 7: that men from both the normal and low SPA groups can fertilize human oocytes and initiate IVF pregnancies. Ausmanas et al., 7 reported on 54 patients with SPA performed no more than 2 weeks before IVF. Two of 54 (4%) men with fertile SPA scores failed to fertilize oocytes at IVF. In addition, a 23% false-negative rate (men in the low SPA group and capable of fertilizing human Vol. 48, No. 2, August 1987
oocytes in vitro) was observed, compared with a 93% false-negative rate in the present study. Ausmanas et al. 7 concluded that no lower limit could be set on SPA score concerning IVF performance. Because there was no correlation between IVF result and SPA, what could the explanation be? Even though SPA and IVF examine sperm and oocytes in vitro, there are several differences between the test systems; specifically, the SPA measures the degree of acrosome reaction in a sperm population while IVF requires sperm motility in addition to the acrosome reaction. These differences may in part explain the incongruity of these tests. Obviously, the laboratory methods for IVF differ from those of SPA; this is true because SPA was unreliable when IVF methods were employed, i.e., hamster oocytes did not survive well in HF-10. Thus, the differences between the two test systems are mentioned in this section. Oocyte handling methods are considerably different. The hamster oocyte is treated enzymatically prior to the SPA assay, while the human oocyte is usually left intact during IVF. In the human IVF system, the zona pellucida and/or cumulus cells may aid the function of the spermatozoa, decreasing capacitation time and increasing the number of acrosomereacted spermatozoa. 13·14 Spermatozoa, at least in this study, were also handled much differently between the two systems. For SPA, both motile and immotile sperm cells were incubated for 18 hours before oocyte addition. For IVF, the spermatozoa were separated into a highly motile fraction and incubated for only 3 hours before oocyte addition. Spermatozoa for SPA were exposed to a lower protein concentration, a different protein source (bovine versus human), and much higher cell density during the test period. Although well characterized in laboratory animal IVF models, 15-18 little is known about these differences in human IVF and can only be pointed to as areas of future investigation. Because of technical difficulties, these differences were unavoidable in the present study. However, the specific phenomenon. of "fast" versus "slow" sperm capacitation has been implicated in some false-negative SPA results.19 Thus, problems with capacitation time would have been masked in the IVF system because spermatozoa and oocytes were mixed early in the assay period. As observed in this study, men fluctuate between the low and high SPA groups (IVF -SPA compared with screening-SPA). This fluctuation in SPA was not reflected by seminal fluid analysis or IVF per-
Kuzan et al. Sperm penetration assay as an indicator for IVF
285
formance. Fifty percent (8) of the men with low screening-SPAs scored high on an IVF -SPA at least once during the 11-month test period. Associated with fluctuation of SPA score is the difference between a score of 10 and 11%. In this study, only 4 of 42 patients scored 0 in any SPA, 3 at the time of IVF; all other patients exhibited some positive SPA score. Thus, it is possible that any positive SPA is predictive of IVF fertilization success. The data from the present study support the conclusion that SPA was unreliable in predicting IVF outcome. From a practical standpoint, nine pregnancies would have been lost if IVF had been denied patients based on a low or no screening SPA. Additionally, four pregnancies were initiated by men with 0 SPA scores before or during IVF therapy. Two of these men had scored in the normal range on other SPA tests. Thus, it is not advisable to eliminate patients from an IVF program based on single SPA scores. In conclusion, even though the SPA correlates well with male fertility in vivo, no correlation was found between SPA and IVF in this study. The lack of correlation is most likely due to inherent differences between the assays, which test different physiologic functions of the spermatozoa. Because SPA is a partial assessment of sperm function, 1 it should not be used exclusively to determine the fertility of semen samples, as observed by Stenchever and Muller. 2° Finally, the results of this study support the conclusion of Mahadevan et al. 21 and Trounson et al. 22 that diagnostic IVF is the most sensitive assay of male fertility. However, because of the cost of the procedure, diagnostic IVF is impractical for the routine analysis of male factor infertility. 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, 1976 2. Martin RH, Taylor PJ: Reliability and accuracy of the zona-free hamster ova assay in assessment of male fertility. Br J Obstet Gynaecol 89:951, 1982 3. Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620, 1981 4. Stenchever MA, Spadoni LR, Smith WD, Karp LE, Shy KK, Moore DE, Berger R: Benefits of the sperm (hamster ova) penetration assay in the evaluation ofthe infertile couple. Am J Obstet Gynecol143:91, 1982 5. Wolf DP, Sokoloski JE, Quigley MM: Correlation of human
286
in vitro fertilization with the hamster egg bioassay. Fertil Steril 40:53, 1983 6. Foreman R, Cohen J, Fehilly CB, 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, 1984 7. Ausmanas M, Tureck RW, Blasco L, Kopf GS, Ribas J, Mastroianni L Jr: The zona-free hamster egg penetration assay as a prognostic indicator in a human in vitro fertilization program. Fertil Steril 43:433, 1985 8. Margalioth EJ, Navot D, Laufer N, Yosef SM, Rabinowitz R, Yarkoni S, Schenker JG: Zona-free hamster ovum penetration assay as a screening procedure for in vitro fertilization. Fertil Steril 40:386, 1983 9. Margalioth EJ, Navot D, Laufer N, Lewin A, Rabinowitz R, Schenker JG: Correlation between the zona-free hamster egg sperm penetration assay and human in vitro fertilization. Fertil Steril 45:665, 1986 10. Pavlok A, Flechon JE: Some factors influencing the interaction of ram spermatozoa with zona-free hamster eggs. J Reprod Fertil 74:597, 1985 11. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW: Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534, 1980 12. Biggers JD, Whitten WK, Whittingham DF: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, Freeman and Co, 1971, p 86 13. Bavister BD: Evidence for a role of post-ovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J Androl 3:365, 1982 14. Tsarik J: Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid, and ionophore A23817 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 74:383, 1985 15. Bavister BD: Fertilization of hamster eggs in vitro at sperm:egg ratios close to unity. J Exp Zoo! 210:259, 1979 16. Niwa K, Chang MC: Optimal sperm concentration and minimal number of spermatozoa for fertilization in vitro of rat eggs. J Reprod Fertil40:471, 1974 17. Nishimura H, Niwa K, Miyabe M, Iritani A: The effect of sperm concentrations during preincubation and insemination on fertilization of rat eggs in vitro. J Exp Zoo! 223:75, 1982 18. Martin RH, Taylor PJ: Effect of sperm concentration in the zona-free hamster ova penetration assay. Fertil Steril 39:379, 1983 19. Perreault SD, Rogers BJ: Capacitation pattern of human spermatozoa. Fertil Steril 38:258, 1982 20. Stenchever MA, Muller CH: Hamster penetration test and sperm fertilizing ability. In Foundations of In Vitro Fertilization, Edited by C Fredricks. Washington, DC, Hemisphere Publishing, 1986, p 41 21. Mahadevan MM, Trounson AO, Leeton JF: The relationship of tubal blockage, infertility of unknown cause, suspected male infertility, and endometriosis to success of in vitro fertilization and embryo transfer. Fertil Steril 40:755, 1983 22. Trounson AO, Leeton JF, Wood C, Webb J, Kovacs G: The investigation of idiopathic infertility by in vitro fertilization. Fertil Steril 34:431, 1980
Kuzan et al. Sperm penetration assay as an indicator for IVF
Fertility and Sterility
Vol. 48, No. 2, August 1987 Printed in U.S.A.
Copyright., 1987 The American Fertility Society
Human sperm penetration assay as an indicator of sperm function in human in vitro fertilization
Frank B. Kuzan, Ph.D.*t Charles H. Muller, Ph.D.*:j: Paul W. Zarutskie, M.D.*
L. Lynne Dixon, B.S.* Michael R. Soules, M.D.*
University of Washington, Seattle, Washington
Performance of spermatozoa in a hamster oocyte/human sperm penetration assay (SPA) was correlated with the results of in vitro fertilization (IVF). Forty-two patients underwent 50 IVF cycles. SPA scores were obtained before IVF cycles (screening SPA, n = 30) and, where practical, on the semen sample used for IVF (IVF SPA, n = 26). Screening SPA score did not correlate to IVF result, fertilization and cleavage rates were similar between normal (n = 17) and low (n = 13) SPA groups, pregnancy rates were 35 and 46%, respectively. In addition, SPA score at the time of IVF did not correlate with IVF result. Pregnancy rates were 33% for the normal group (n = 16) and 30% for the low SPA group (n = 10). Overall, the low SPA group (n = 16) exhibited a 78% fertilization rate and a 38% pregnancy rate, which was not different from the normal SPA group: 76 and 29%, respectively. The results of this study indicate that SPA score is a poor indicator of sperm function in IVF. Fertil Steril 48:282, 1987
I'
,,)i ~
I
The hamster oocyte/human sperm penetration assay (SPA) was introduced in 1976 as "a preliminary assessment of the fertilizing capacity of human spermatozoa. m Since then, the SPA has been found to correlate well with male fertility in vivo. 2- 4 With the advent of in vitro fertilization (IVF) as a therapy for human infertility, the correlation between SPA score and IVF result has been examined. 5_9 Wolf et al., 5 in a study of 24 patients, found SPA score correlated well with IVF success or failure, as did Margalioth et al., 8 •9 with 20 and 134 patients, respectively. Foreman et al. 6 reported that SPA scores in the low range correlated with an increased frequency of IVF failure; however, normal SPA scores were not reliable in predicting IVF success ..
'i Received November 18, 1986; revised and accepted April 6, 1987. * Department of Obstetrics and Gynecology. t Reprint requests: Frank B. Kuzan, Ph.D., Department of Obstetrics and Gynecology, RH-20, University of Washington, Seattle, Washington 98195. :J: Department of Biological Structure.
282
The results of Ausmanas et al. 7 agreed with those of Wolf et al., 5 but a higher false-negative (low SPA with successful IVF) rate of 23% was found. The approach to correlating SPA score with IVF result was different among these studies. In two studies,5 •6 SPA was performed on the same semen sample used for the IVF procedure, while three studies7- 9 obtained SPA on a semen sample before IVF. One study6 compared a select population of 15 men, who had failed IVF previously, with 15 randomly selected men. In summary, four studies5 •6 •8 •9 supported the hypothesis that SPA was a good predictor of the ability to fertilize human oocytes in vitro. In three of these studies, pregnancies were not reported. In a fifth study, 7 it was concluded that SPA was not helpful in predicting IVF performance. These reports are contradictory; therefore, in an attempt to clarify this apparent confusion, the present study was undertaken. Considering the discrepancy in SPA score within the male over time, 10 two types of SPA scores were obtained for analysis: (1) on a semen sample prior to the IVF cycle, screening-SPA; and (2) on the semen sample used for IVF, IVF-SPA.
Kuzan et al. Sperm penetration assay as an indicator for IVF
Fertility and Sterility
MATERIALS AND METHODS
dium. Only pregnancies confirmed by ultrasound at greater than 8 weeks' gestation are reported.
Patients Male partners of couples applying to the University of Washington IVF program from February to September 1984 were required to have a SPA before acceptance. Additionally, SPA tests were performed on most semen samples used for IVF from September 1984 to October 1985. Thus, two types of SPA scores were available: (1) screening-SPA (n = 30), those tests performed no more than 8 months prior to the IVF cycle; and (2) IVF -SPA (n = 26), those tests performed on the same semen sample used for IVF. No therapy of the male nor changes in IVF laboratory procedures, in regard to sperm preparation, were undertaken during the study interval based on SPA score. Couples who were included in this study had completed an IVF cycle, including oocyte retrieval, before November 1985. The patient population (n = 42) was composed of patients with tubal disease (n = 22), endometriosis (n = 2), male factor (n = 7), idiopathic source (n = 4), or a combination of these factors (n = 7) as the source of infertility. Superovulation for IVF cycles was accomplished by a combination regimen; 100 mg clomiphene citrate (CC) (Serophene, Serono Laboratories Inc., Randolph, MA) on cycle days 3 to 7 and 75 IU human menopausal gonadotropin (hMG) (Pergonal, Serono Laboratories Inc., Randolph, MA) starting day 3 until follicular maturity. Oocyte retrieval was accomplished laparoscopically on approximately day 12 (range, 11 to 14). After retrieval, oocytes were removed from follicular fluid within 2 minutes and placed into a culture tube (Falcon #3033, Becton Dickenson Co., Oxnard, CA) containing Ham's F-10 (HF-10, Gibco Laboratories, Santa Clara, CA) medium supplemented with 10% human fetal cord serum (FCS), 1 mM calcium lactate (Calbiochem-Behring Corp., LaJolla, CA), and 20 mM sodium bicarbonate (J. T. Baker Chemical Co., Phillipsburg, NY). Oocytes were incubated in organ-culture dishes (Falcon #3037, Becton Dickenson Co., Oxnard, CA) at 37°C in a 5% C02, 5% 0 2, and 90% N 2 gas mixture, 5 to 6 hours before the addition of spermatozoa. Seventeen hours after sperm addition, oocytes were examined for signs of fertilization, at which time they were placed into HF -10 containing 20% human FCS. The uterine placement of cleaving embryos, 40 to 42 hours after insemination, was accomplished with either a 3 or 4 French, side-port catheter (W. A. Cook Pty. Ltd., Melbourne, Australia) containing 20 to 30 ~1 meVol. 48, No.2, August 1987
Sperm Preparation for In Vitro Fertilization One-half milliliter of liquified (20 minutes at 37°C) neat semen was layered beneath 3 ml of HF-10 containing 0.5% human serum albumin (HSA; Sigma Chemical Co., St. Louis, MO), and incubated for 1 hour in a humidified atmosphere of 5% C02 in air at 37°C.U The upper 1.5 ml of medium, containing motile spermatozoa, was removed and centrifuged at 400 X g for 5 minutes. The supernatant was discarded, and spermatozoa were washed two additional times in HF-10 + 10% human FCS by centrifugation. The final pellet was drawn up in 0.5 ml HF-10, counted by hemocytometer, and 105 motile spermatozoa were added to each dish containing a single oocyte. The average time from semen collection to oocyte insemination was 3 hours. Semen samples ranged from 107 to 2 X 108 sperm cells/ml, with motilities of 20% to 70%, and abnormal forms ranging from 30% to 70%. No sample used for IVF had more than one abnormality detected by routine seminal fluid analysis (i.e., motility and sperm concentration). Oocyte Preparation for Sperm Penetration Assay Hamster oocytes and human spermatozoa were prepared by the method described by Karp et al. 3 Briefly, mature female hamsters were superovulated with pregnant mare's serum gonadotropin (PMSG; Organon, El Monte, CA) followed 56 hours later with human chorionic gonadotropin (hCG; Sigma Chemical Co., St. Louis, MO). Cumulus enclosed oocytes were collected into modified Biggers, Whitten, and Whittingham medium12 (BWW) 14 to 16 hours after hCG administration. Oocytes were treated with hyaluronidase (Sigma Chemical Co., St. Louis, MO) and trypsin (Sigma Chemical Co., St. Louis, MO) to remove the cumulus cells and the zona pellucida, respectively. Twenty-five zona pellucida-free oocytes were added to approximately 107 spermatozoa for each assay. Sperm Preparation for Sperm Penetration Assay After 20 minutes at 37°C to allow liquefaction, seminal components were separated from spermatozoa by three centrifugation steps (300 X g, 7 minutes, 5 minutes, 5 minutes). Spermatozoa were resuspended in BWW at a concentration of 1 to 2 X 107 and then incubated in a humidified gas atmo-
Kuzan et al.
Sperm penetration assay as an indicator for IVF
283
sphere of 1% C02 in air, at 37°C for 18 to 22 hours before hamster oocyte addition. The spermatozoa/ hamster oocyte mixture was incubated 2.0 to 2.5 hours, at which time the hamster oocytes were removed, lightly compressed between a glass slide and cover slip, and assessed for sperm penetrations (decondensed sperm head with associated tail). Results were reported as percent hamster oocytes penetrated.
cleavage rate, or pregnancy rate between the normal and low groups (Table 1). Of particular interest was the fact that mean number of embryos replaced after IVF was similar: 3. 7 for the normal group and 3.2 for the low group. Only 1 of 13 (7%) patients with a low SPA did not fertilize human oocytes in vitro and one pregnancy was initiated by a man who scored 0 on SPA. Twenty-six SPAs were performed on semen samples used for IVF; these data are presented in Table 1 (IVF -SPA). No difference was found in any IVF parameter between these groups. Two points of interest were: three patients scored zero on this SPA, yet initiated a pregnancy; and some men with a previous screening-SPA changed their SPA status. In particular, five patients changed from low (screening) to normal (IVF); one patient went from normal to low; and two patients who were low on the first IVF-SPA cycle were normal on the second IVF-SPA cycle. Only three men who had a SPA at screening and IVF remained in the same SPA category. Because men fluctuated between the low and normal groups during the course of this study, an additional effort was made to identify an infertile group of men whose spermatozoa would not perform well in IVF. Patients were separated into two groups based on low SPA score versus any normal SPA score achieved during the test period; these data are presented in Table 1 (all SPA scores). In 16 of the 50 cycles (32%), the male partner had never exhibited a normal SPA (Table 1). After arranging the data in this manner, no difference was found between the SPA groups in any IVF parameter. Additionally, two pregnancies were established by men with SPA scores of 0.
Data Analysis
Data concerning IVF were separated based on low (0% to 10%) and normal (~11%) SPA scores. Repeated measures analysis of variance was employed to detect differences between low and normal SPA groups relative to IVF and cleavage rates. Chi -square analysis was employed to determine whether IVF pregnancy rate differed between these SPA groups.
RESULTS
i.
!
Forty-two infertile couples and 50 IVF cycles were included in this study. A total of 228 human oocytes were recovered (4.6/cycle), 175 of these were fertilized (78%, 3.5/cycle), and 167 cleaved (73%), resulting in an average of 3.4 embryos per replacement. Sixteen cycles resulted in clinical pregnancies, ultrasound documented at 8 weeks' gestation, for a 32% pregnancy rate per laparoscopy. During the course of this study, only two failed fertilization cycles were completed. Twenty-six patients were evaluated by SPA prior to IVF (screening-SPA); results based on SPA score are presented in Table 1 (screening-SPA). No difference was detected in IVF fertilization rate,
Table 1 Relationship Between SPA Score and IVF Fertilization, Cleavage, and Pregnancy Rate Oocytes (low SPA score)
Oocytes (normal SPA score [""11%]) No. cycles
Recovered
Fertilized
Cleaved
No. pregnant
No. cycles
Recovered
Fertilized
Cleaved
No. pregnant
Screen SPA
17
5.3 ± 0.5" %
4.0 ± 0.4 75
3.7 ± 0.4 70
6b 35.2
13c
4.5 ± 0.5 %
3.2 ± 0.6 71
3.2 ± 0.6 71
6 46.2
IVF SPA
16
3.8 ± 0.3 %
3.1 ± 0.3 82
3.1 ± 0.3 82
5 31.2
lOd
4.8 ± 0.6 %
4.0 ± 0.8 83
3.9 ± 0.8 81
30
4.5 ± 0.3 %
3.5 ± 0.3 76
3.3 ± 0.3 73
lOb
16'
29.4
4.4 ± 0.5 %
3.3 ± 0.5 75
3.3 ± 0.5 75
6 37.5
All SPAs
34
• Mean ± standard error. b Includes one ectopic. c One 0 SPA score. 284
Kuzan et al.
3b
d Three 0 SPA scores. • Two 0 SPA scores.
Sperm penetration assay as an indicator for IVF
Fertility and Sterility
DISCUSSION
The findings of the present study do not support the concept that SPA can predict which semen sample possesses the ability to fertilize human oocytes in vitro. Therefore, these data do not support the findings of Wolf et al. 5 or Margalioth et al.8·9 because no correlation between negative IVF result and low SPA score was found. Two cases supported the findings of Foreman et al. 6 that semen samples that failed at IVF were associated with low SPA scores. The previous studies varied in the time relationship between SPA assay and IVF cycle. Wolf et al. 5 and Foreman et al. 6 performed SPA on the same semen sample used for IVF. The most meaningful scientific data would be obtained in this manner, since SPA score has been shown to fluctuate over time within individual men. 10 However, the data of Wolf et al. 5 were difficult to interpret because fertilization data often referred to a single oocyte per patient. Foreman et al. 6 compared the IVF results of 15 men who had failed a previous IVF cycle, "subfertile," with those of 15 randomly selected "normal" (by seminal fluid analysis) men. Although 6 "subfertile" men scored normal on SPA at IVF, none of the 15 semen samples fertilized human oocytes (n = 37) during that IVF treatment cycle. Only 1 of the 15 "normal" men scored in the low range on SPA and all semen samples were capable offertilizing human oocytes in vitro. Foreman et al. 6 concluded that low SPA scores were reliable in predicting IVF outcome. Margalioth et al.,S who did not indicate the time relationship between the SPA and IVF, found that a low SPA correlated well with poor IVF results. Spermatozoa from 7 patients with low SPA scores failed to fertilize human oocytes in vitro, while only 3 of 13 men with normal SPA scores failed IVF. Margalioth et al. 9 extended these observations to 134 couples in whom SPA was obtained sometime prior to the IVF cycle. Results showed that 94% of men with a normal SPA fertilized human oocytes in vitro, while 78% of men with low SPA failed to fertilize human oocytes. Data from this study do support the findings of Ausmanas et al. 7: that men from both the normal and low SPA groups can fertilize human oocytes and initiate IVF pregnancies. Ausmanas et al., 7 reported on 54 patients with SPA performed no more than 2 weeks before IVF. Two of 54 (4%) men with fertile SPA scores failed to fertilize oocytes at IVF. In addition, a 23% false-negative rate (men in the low SPA group and capable of fertilizing human Vol. 48, No. 2, August 1987
oocytes in vitro) was observed, compared with a 93% false-negative rate in the present study. Ausmanas et al. 7 concluded that no lower limit could be set on SPA score concerning IVF performance. Because there was no correlation between IVF result and SPA, what could the explanation be? Even though SPA and IVF examine sperm and oocytes in vitro, there are several differences between the test systems; specifically, the SPA measures the degree of acrosome reaction in a sperm population while IVF requires sperm motility in addition to the acrosome reaction. These differences may in part explain the incongruity of these tests. Obviously, the laboratory methods for IVF differ from those of SPA; this is true because SPA was unreliable when IVF methods were employed, i.e., hamster oocytes did not survive well in HF-10. Thus, the differences between the two test systems are mentioned in this section. Oocyte handling methods are considerably different. The hamster oocyte is treated enzymatically prior to the SPA assay, while the human oocyte is usually left intact during IVF. In the human IVF system, the zona pellucida and/or cumulus cells may aid the function of the spermatozoa, decreasing capacitation time and increasing the number of acrosomereacted spermatozoa. 13·14 Spermatozoa, at least in this study, were also handled much differently between the two systems. For SPA, both motile and immotile sperm cells were incubated for 18 hours before oocyte addition. For IVF, the spermatozoa were separated into a highly motile fraction and incubated for only 3 hours before oocyte addition. Spermatozoa for SPA were exposed to a lower protein concentration, a different protein source (bovine versus human), and much higher cell density during the test period. Although well characterized in laboratory animal IVF models, 15-18 little is known about these differences in human IVF and can only be pointed to as areas of future investigation. Because of technical difficulties, these differences were unavoidable in the present study. However, the specific phenomenon. of "fast" versus "slow" sperm capacitation has been implicated in some false-negative SPA results.19 Thus, problems with capacitation time would have been masked in the IVF system because spermatozoa and oocytes were mixed early in the assay period. As observed in this study, men fluctuate between the low and high SPA groups (IVF -SPA compared with screening-SPA). This fluctuation in SPA was not reflected by seminal fluid analysis or IVF per-
Kuzan et al. Sperm penetration assay as an indicator for IVF
285
formance. Fifty percent (8) of the men with low screening-SPAs scored high on an IVF -SPA at least once during the 11-month test period. Associated with fluctuation of SPA score is the difference between a score of 10 and 11%. In this study, only 4 of 42 patients scored 0 in any SPA, 3 at the time of IVF; all other patients exhibited some positive SPA score. Thus, it is possible that any positive SPA is predictive of IVF fertilization success. The data from the present study support the conclusion that SPA was unreliable in predicting IVF outcome. From a practical standpoint, nine pregnancies would have been lost if IVF had been denied patients based on a low or no screening SPA. Additionally, four pregnancies were initiated by men with 0 SPA scores before or during IVF therapy. Two of these men had scored in the normal range on other SPA tests. Thus, it is not advisable to eliminate patients from an IVF program based on single SPA scores. In conclusion, even though the SPA correlates well with male fertility in vivo, no correlation was found between SPA and IVF in this study. The lack of correlation is most likely due to inherent differences between the assays, which test different physiologic functions of the spermatozoa. Because SPA is a partial assessment of sperm function, 1 it should not be used exclusively to determine the fertility of semen samples, as observed by Stenchever and Muller. 2° Finally, the results of this study support the conclusion of Mahadevan et al. 21 and Trounson et al. 22 that diagnostic IVF is the most sensitive assay of male fertility. However, because of the cost of the procedure, diagnostic IVF is impractical for the routine analysis of male factor infertility. 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, 1976 2. Martin RH, Taylor PJ: Reliability and accuracy of the zona-free hamster ova assay in assessment of male fertility. Br J Obstet Gynaecol 89:951, 1982 3. Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620, 1981 4. Stenchever MA, Spadoni LR, Smith WD, Karp LE, Shy KK, Moore DE, Berger R: Benefits of the sperm (hamster ova) penetration assay in the evaluation ofthe infertile couple. Am J Obstet Gynecol143:91, 1982 5. Wolf DP, Sokoloski JE, Quigley MM: Correlation of human
286
in vitro fertilization with the hamster egg bioassay. Fertil Steril 40:53, 1983 6. Foreman R, Cohen J, Fehilly CB, 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, 1984 7. Ausmanas M, Tureck RW, Blasco L, Kopf GS, Ribas J, Mastroianni L Jr: The zona-free hamster egg penetration assay as a prognostic indicator in a human in vitro fertilization program. Fertil Steril 43:433, 1985 8. Margalioth EJ, Navot D, Laufer N, Yosef SM, Rabinowitz R, Yarkoni S, Schenker JG: Zona-free hamster ovum penetration assay as a screening procedure for in vitro fertilization. Fertil Steril 40:386, 1983 9. Margalioth EJ, Navot D, Laufer N, Lewin A, Rabinowitz R, Schenker JG: Correlation between the zona-free hamster egg sperm penetration assay and human in vitro fertilization. Fertil Steril 45:665, 1986 10. Pavlok A, Flechon JE: Some factors influencing the interaction of ram spermatozoa with zona-free hamster eggs. J Reprod Fertil 74:597, 1985 11. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW: Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534, 1980 12. Biggers JD, Whitten WK, Whittingham DF: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, Freeman and Co, 1971, p 86 13. Bavister BD: Evidence for a role of post-ovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J Androl 3:365, 1982 14. Tsarik J: Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid, and ionophore A23817 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 74:383, 1985 15. Bavister BD: Fertilization of hamster eggs in vitro at sperm:egg ratios close to unity. J Exp Zoo! 210:259, 1979 16. Niwa K, Chang MC: Optimal sperm concentration and minimal number of spermatozoa for fertilization in vitro of rat eggs. J Reprod Fertil40:471, 1974 17. Nishimura H, Niwa K, Miyabe M, Iritani A: The effect of sperm concentrations during preincubation and insemination on fertilization of rat eggs in vitro. J Exp Zoo! 223:75, 1982 18. Martin RH, Taylor PJ: Effect of sperm concentration in the zona-free hamster ova penetration assay. Fertil Steril 39:379, 1983 19. Perreault SD, Rogers BJ: Capacitation pattern of human spermatozoa. Fertil Steril 38:258, 1982 20. Stenchever MA, Muller CH: Hamster penetration test and sperm fertilizing ability. In Foundations of In Vitro Fertilization, Edited by C Fredricks. Washington, DC, Hemisphere Publishing, 1986, p 41 21. Mahadevan MM, Trounson AO, Leeton JF: The relationship of tubal blockage, infertility of unknown cause, suspected male infertility, and endometriosis to success of in vitro fertilization and embryo transfer. Fertil Steril 40:755, 1983 22. Trounson AO, Leeton JF, Wood C, Webb J, Kovacs G: The investigation of idiopathic infertility by in vitro fertilization. Fertil Steril 34:431, 1980
Kuzan et al. Sperm penetration assay as an indicator for IVF
Fertility and Sterility