FERTILITY AND STERILITY
Vol. 58, No.5, November 1992
Copyright v 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization*
Francisco J. Rojas, Ph.D. t:j: Anh-Tuan La, B.S.t Terri Ord, B.S. t Pasquale Patrizio, M.D. t
Jose P. Balmaceda, M.D.t Sherman J. Silber, M.D.§ Richardo H. Asch, M.D. t
University of California, Irvine Medical Center, Orange, California, and St. Luke's Hospital, St. Louis, Missouri
Objectives: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). Design: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. Patients, Participants: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. Results: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71 %. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. Conclusions: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm. Fertil Steril 1992;58:1000-5 Key Words: Human epididymal sperm, zona-free hamster oocytes, sperm penetration assay, human in vitro fertilization, sperm maturation, sperm fertilizing capacity, human epididymis
Numerous reports in several mammalian species indicate that transport of sperm through most of the epididymis is a mandatory condition for the
Received May 7, 1992; revised and accepted July 27, 1992. * Presented at the 47th Annual Meeting of The American Fer-
tility Society, Orlando, Florida, October 21 to 24, 1991. t Division of Reproductive Endocrinology and Gynecology, Department of Obstetrics and Gynecology, University of California, Irvine Medical Center. :j: Reprint requests: Francisco J. Rojas, Ph.D., Department of Obstetrics and Gynecology, University of California Irvine Medical Center, 101 The City Drive, B-41, Orange, California 92668. § Department of Urology, St. Luke's Hospital.
1000
Rojas et al.
SPA using human epididymal sperm
sperm to mature and acquire fertilizing capacity (see review by Robaire and Hermo [1]). In contrast to animal studies, information on the fertilizing capacity of sperm from the human testes and epididymis is very limited. Recently, men with azoospermia because of congenital absence of the vas deferens have been treated for infertility by direct microsurgical aspiration of sperm from the epididymis, combined with in vitro fertilization (IVF) and embryo transfer (2, 3). These studies indicate that epididymal sperm can fertilize the human oocyte in vitro and result in pregnancies with livebirths. The reported fertility of these patients suggests that epFertility and Sterility
ididymal sperm, at least in the human, may not be totally infertile. Thus, the role of the human epididymis in sperm maturation and fertilization remains to be defined. It becomes, however, clear that microsurgical aspiration of sperm directly from the testes combined with assisted reproductive techniques may be applied successfully to the treatment of azoospermia caused by congenital absence of the vas deferens or by idiopathic epididymal obstruction, and in men with failed reversals of vasectomy or other nonoperable inflammatory obstructive azoospermia (2-6). The use of the zona-free hamster egg sperm penetration assay (SPA) is a method that has gained increasing acceptance as an assay of functional activity of human spermatozoa (7-9). The assay measures some crucial steps in human spermoocyte interaction and fertilization, including sperm capacitation, acrosome reaction, and fusion of sperm with the plasma membrane of the oocyte. The advent of microsurgical techniques to obtain epididymal sperm provides the unique opportunity to use the SP A to directly evaluate the penetration potential of these sperm cells. The purpose of this study was to assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes as determined by SPA. Also, we used single sperm samples to directly compare the performance of epididymal sperm in SPA with that in zona-intact human oocytes as determined by IVF. MATERIALS AND METHODS Epididymal Sperm
Twenty-nine couples in whom the male partner had congenital absence of the vas deferens underwent direct microsurgical aspiration of epididymal spermatozoa for attempted IVF of their partner's oocytes. Two couples underwent the procedures two times. Results of both attempts were included. Aspirations were performed in the most proximal regions of the epididymis where usually sperm with progressive motility are found. The details of the surgical sperm retrieval technique have been described elsewhere (6). Epididymal aspirates were incubated with 3 mM pentoxifylline (Sigma, Holmdel, NJ) for 30 minutes at room temperature and then washed with Hepes-buffered human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, CA), supplemented with 0.5% human serum albumin (fraction V; Sigma, Holmdel, NJ). Pretreatment of sperm Vol. 58, No.5, November 1992
with pentoxifylline was included in an attempt to improve fertilization rates (10). Sperm pellets were suspended in 0.3 mL of Hepes-buffered HTF medium and prepared for IVF using the mini-Percoll method (11), consisting of a discontinuous gradient formed by three layers of 0.3 mL each of 95%,70%, and 50% of isotonic Percoll (Pharmacia, Vppsala, Sweden). The gradient was centrifuged at 300 X g for 45 minutes. After centrifugation, the 95% Percoll layer was removed, and the recovered sperm were incubated with 3 mM 2' -deoxyadenosine (Sigma) for 30 minutes at 37°C (12). Sperm were then washed two times and suspended in 1 mL HTF medium plus 10% fetal cord serum and used for insemination in vitro. Controlled Ovarian Hyperstimulation (COH) and IVF
All the women were between 24 and 42 years of age. They had regular menstrual cycles and normal tubal patency on hysterosalpingography, laparoscopy, or both. Synchronization of the onset of their menses and COH were carried out as described in details elsewhere (6). The retrieval of oocytes was performed transvaginally with ultrasound guidance, 36 hours after the injection of human chorionic gonadotropin (hCG) (6). Oocytes were added to culture tubes containing sperm (0.4 to 3.3 X 106 sperm/mL) and incubated for 13 to 15 hours at 37°C in a humidified atmosphere of 5% CO 2 and air. Higher concentrations of sperm were added for some samples with poor motility in an attempt to achieve fertilization. After incubation, the oocytes were examined for signs of fertilization, indicated by the presence of two pronuclei, and were transferred to fresh growth medium for another 30 to 36 hours. Fertilization was considered confirmed only if cleavage occurred. Sperm Penetration Assay
Eggs were obtained from hamsters by inducing superovulation using the method described by Yanagimachi et al. (13). Briefly, adult female Golden Syrian Hamsters, 7 to 11 weeks old, were superovulated by intraperitoneal injection of 30 IV of pregnant mare serum gonadotropin (Sigma) on the postestrous morning during which a vaginal discharge was present. Fifty-five to 57 hours later, 30 IV of hCG (Sigma) was given to trigger ovulation. The animals were killed 15 to 17 hours after the hCG injection, and the oviducts were removed and placed in Biggers, Whitten, and Whittingham meRojas et al.
SPA using human epididymal sperm
1001
dium (BWW; Irvine Scientific) containing 0.3% bovine serum albumin (BSA). The cumulus masses were then retrieved and treated with 0.1% hyaluronidase (Sigma) in BWW medium to free the eggs. The eggs were collected, washed in BWW medium twice, and then transferred to a 0.1 % trypsin (Sigma) in BWW medium to remove the zona pellucida (ZP). Once the zona was dissolved, the eggs were washed three times in fresh BWW medium (14). In an effort to minimize differences between SPA and IVF in both sperm preparation and conditions for sperm capacitation, SPA was performed with the same sperm sample used for IVF. This included pretreatment with pentoxifylline/deoxyadenosine, mini-Percoll gradient, and incubation for 13 to 15 hours at 37°C in a humidified atmosphere of 5% CO 2 and air. Also, SPA was performed with a sperm concentration (0.5 to 1.0 X 106 sperm/mL) that was within the range of that used in IVF. Sperm were transferred in 100-IlL drops to the center of a 35 X lO-mm Petri dish and covered with warmed (37°C) paraffin oil (Fisher Scientific Company, Santa Clara, CA). Twenty to 30 zona-free hamster oocytes were added to each drop of sperm and incubated at 37°C for 3 hours. Then all eggs were collected and washed in BWW medium containing 0.3% BSA to remove the unattached sperm and were mounted under a coverslip supported by two solid lines of a 10:1 vaseline/paraffin mixture. The eggs were examined under a phase-contrast microscope at X400 for the presence of decondensing sperm heads and their visible tails in the cytoplasm, recording the percentage of penetrated eggs. Ejaculated sperm (fresh samples) from two fertile donors was prepared as described above and included for every run as an internal control. These internal controls were used at a sperm concentration equivalent to that used for patient samples. RESULTS Ability of Human Epididymal Sperm to Penetrate Zona-Free Hamster Oocytes
Penetration capacity of human epididymal sperm as determined by SPA is shown in Table 1. A wide range of penetration values was observed in the 31 cases analyzed. Penetration of at least one oocyte occurred in 22 of them. This represented 71 % of cases. However, penetration values were in general low and did not exceed 30% (Table 1). In contrast to epididymal sperm, internal controls using ejaculated, mature sperm from fertile donors 1002
Rojas et al.
SPA using human epididymal sperm
Table 1 Penetration of Zona-Free Hamster Oocytes by Sperm Aspirated From the Human Epididymis Penetration rate*
No. of cases
%
o 1 to 6 to 11 to 16 to 21 to
5 10 15 20 30
9 (29) t 2 (6)
5 (16) 6 (19) 7 (23) 2 (6)
* Percentage of ova penetrated; results are divided according to categories of penetration rates. t Values in parentheses are percentages of the total number of 31 cases.
showed penetration rates in SPA that ranged from 30% to 71 %, with a mean (±SD) value of 58% ± 12% for 15 determinations (data not shown). These data indicate that ejaculated sperm from fertile men penetrated zona-free hamster eggs to a greater degree compared with epididymal sperm. Penetration of Zona-Free Hamster Oocytes Using Human Epididymal Sperm Compared With HumanIVF
Table 2 shows the rate of fertilization as determined by IVF and the penetration scores as determined by SPA for each of the cases using epididymal sperm. Cases in which no fertilization in IVF was observed are presented in first place. In general, no fertilization was associated with either no penetration or low penetration values in SPA. As fertilization rate increased, penetration values also increased. A good association was more evident at higher fertilization rates. Thus, the highest penetration values were observed in cases showing the highest fertilization values (Table 2). Penetration rates in SPA expressed as percentage of ova penetrated ranged from 0% to 30%. On the other hand, the range of fertilization rates in IVF was wider, with the lowest value being 0% and the highest 94% (Table 2). The association between percentage of oocytes penetrated and the percentage of oocytes cleaved in IVF is illustrated in Figure 1. Linear regression analysis indicated a positive correlation that was highly significative (P < 0.001). The equation line intercepted the y-axis (0% fertilization rate) at 6% penetration rate in SPA (Fig. 1). Assuming that ~10% penetration rate in SPA represents potential fertility, a cutoff value used by other investigators (8, 15), a positive SPA was associated with a positive Fertility and Sterility
Table 2 Penetration of Zona-Free Hamster Oocytes Using Human Epididymal Sperm Compared With the Results of Human IVF
Case no.
IVF -oocytes cleaved
SPA-oocytes penetrated
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0/6 (O)* 0/22 {OJ 1/16 {OJ 0/27 {OJ 0/10 {OJ 0/11 {OJ 0/50 (OJ 0/16 (OJ 0/34 (OJ 0/9 (OJ 0/18 (OJ 1/27 (4) 1/26 (4) 1/20 (5) 2/21 (9) 2/18 (11) 4/38 (11) 2/13 (15) 4/27 (15) 6/13 (16) 3/16 (19) 5/21 (24) 3/12 (25) 8/20 (40) 3/7 (43) 17/36 (47) 7/12 (58) 29/38 (76) 10/11 (91) 10/11 (91) 15/16 (94)
0/22 {OJ 0/25 {OJ 0/22 {OJ 0/25 {OJ 0/25 {OJ 0/25 {OJ 1/23 (4) 1/20 (5) 2/24 (8) 2/23 (9) 4/25 (16) 0/24 (OJ 2/20 (10) 0/20 (OJ 0/21 (OJ 3/24 (13) 4/20 (20) 4/29 (14) 2/25 (8) 5/24 (21) 2/22 (9) 5/27 (19) 4/24 (17) 5/26 (19) 3/22 (14) 4/33 (12) 3/25 (12) 3/22 (14) 5/29 (17) 5/25 (20) 6/20 (30)
* Values in parentheses are percents.
IVF in 15 of 20 cases (75%). On the other hand, a negative «10% penetration rate) SPA was associated with a negative IVF in 10 of 11 cases (91%). Table 3 summarizes the SPA results using a cutoff point of 10% penetration rate, compared with the ability of human sperm aspirated from the epididymis to fertilize the human oocytes in vitro. DISCUSSION
In this study, we report the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes determined by SPA. Demonstration of penetration capacity of these cells is consistent with previous clinical studies that indicated the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies (2, 3). Also, we report that human epididymal sperm penetrate zona-free hamster oocytes with a wide range of penVol. 58, No.5, November 1992
l-30
•
R -0.87 P < 0.001 N -31
..--~~--~~--.--.--.--.~
o~
OW.
30
•
•
•
~
•
•
~
IVF - Oocytes cleaved (%) Figure 1 Correlation between the zona-free hamster egg SPA and human IVF using sperm aspirated from the epididymis. The line equation was y = 0.18x - 5.89.
etration scores. At least one oocyte was penetrated in most (71%) cases. However, penetration rate, as expressed as percentage of ova penetrated, was low compared with ejaculated sperm and failed to exceed 30%. Fertilization rates in IVF, on the other hand, showed values ranging from 0% to 94%. Incidence of high fertilization rates, nevertheless, was low; table 2 indicates that only 5 of the 31 cases had a fertilization rate in IVF higher than 50%. Similar data have been reported in previous studies (6). Thus, our observations using SPA agree with data on IVF showing that epididymal sperm present low fertilization rates per oocyte compared with rates observed with ejaculated sperm (16). Diminished penetration rate of zona-free hamster oocytes suggests that human epididymal sperm has reduced functional competence. For a sperm to pen-
Table 3 Correlation Between the Results of SPA and the Outcome of IVF Using Human Epididymal Sperm Results *
No. of cases
+SPA/+IVF +SPA/-IVF -SPAj+IVF -SPA/-IVF
15 (75)t 1 (9)t 5 (25)t 10 {9I)t
* A cutoff of 10% penetration rate in SPA was considered to represent potential fertility. t Percentage of positive IVF (20 cases). t Percentage of negative IVF (11 cases). Rojas et al.
SPA using human epididymal sperm
1003
etrate the oocyte, it must complete capacitation, undergo an acrosome reaction, develop a fusogenic region in its plasma membrane, collide with and bind to the oocyte surface, complete membrane fusion, and begin decondensation of the nucleus (15). Low penetration rate, therefore, indicates limited capacity of epididymal sperm to undergo these reactions. Altogether, our data support the concept that the fertilizing capacity of human sperm retrieved from an obstructed epididymis is lower than that of ejaculated sperm. In addition, although a population of sperm retrieved directly from the epididymis has fertilizing potential and can produce successful pregnancy, our results demonstrating reduced penetration rate in SPA suggest that sperm must pass through a certain length of the human epididymis to become fully mature. We have also compared the performance of epididymal sperm in the SPA with the results of IVF. Because performance of SPA has been shown to significantly depend on the conditions selected for sperm preparations and capacitation (17), we carried out SPA on the same sperm sample used for the IVF procedure. This minimized variations in sperm preparations and conditions for sperm capacitation. We found a good association between penetration scores of zona-free hamster oocytes in SPA and fertilization of zona-intact human oocytes in IVF. Linear regression analysis indicated that at zero fertilization rate in IVF, the penetration rate in SPA was 6% (Fig. 1, y-intercept). Interestingly, assuming that ~10% penetration rate in SPA may represent potential fertility, we found that the overall sensitivity of SPA, i.e., its true-positive rate (18) was 75%, and the overall specificity of the assay, i.e., its true-negative rate (18) was 91 %. These observations indicate that a positive SPA (~1O% penetration) was a good indicator of successful fertilization by human epididymal sperm and that the false-positive rate was low. Similarly, a negative SPA correlated remarkably well with failure of human epididymal sperm to fertilize human oocytes in vitro. This would be compatible with studies in ejaculated sperm, indicating that SPA score is a good indicator of sperm function in IVF and of chances to achieve pregnancy (7-9). Accordingly, in one of largest clinical studies, a positive SPA has been reported to predict subsequent in vitro cleavage in 91 (85%) of 107 cases, whereas a negative SP A predicted an overall fertilization failure in 78% of the cases (7). It should be emphasized, nevertheless, that the precise lower limit of SPA penetration rate to distinguish fertile epididymal sperm from 1004
Rojas et al.
SPA using human epididymal sperm
sub fertile epididymal sperm remains to be defined. Studies using ejaculated sperm indicate that the conditions selected for sperm capacitation have a significant impact on the definition of normal ranges in SPA (17). Thus for example, use of TEST-yolk medium and prolonged incubation at low temperature have been shown to enhance the capacity of human sperm to penetrate hamster eggs, allowing the selection of a cutoff value as high as 20% (14, 19). It seems, therefore, that the definition of normal ranges for SPA using epididymal sperm awaits characterization of the optimal conditions for sperm preparation and capacitation. Selection of a proper cutoff point is expected to improve SPA scores concerning IVF performance. Also, a large number of cases need to be analyzed before definitive conclusions can be drawn on the value of SPA to predict the chances of achieving pregnancy by IVF using sperm aspirated from the epididymis. This is important because SPA yields no predictive information on the ability of the sperm to penetrate the ZP. In the meantime, our data provide new insights on the application of SPA for both clinical and basic research. Thus, the good overall association between SPA and human IVF strongly supports the use of SPA as an excellent bioassay to test laboratory experimental conditions for improving fertilizing potential of sperm aspirated from the human epididymis. In this view, SPA may be particularly important in evaluating different conditions for sperm preparation and capacitation of human epididymal sperm for clinical decision-making in IVF. Furthermore, the observation that zona-free hamster eggs can be penetrated by epididymal sperm provides the potential to use SPA as a powerful research tool to elucidate the mechanisms and factors controlling sperm maturation in the human. Ackrwwledgments. We thank Ines Moretti, Ph.D., Hitachi Chemical Research Center, Irvine, California, for critical reading of the manuscript and Ms. Elisa Hendrickson and Mr. Jeffrey Deutsch for manuscript preparation.
REFERENCES 1. Robaire B, Hermo L. Efferent ducts, epididymis, and vas
deferens: structure, functions, and their regulation. In: Knobil E, Neill, JD. The physiology of reproduction. New York: Raven Press, 1988:999-1080. 2. Temple-Smith PD, Southwick GJ, Yates CA, Trounson AO, De Kretser DM. Human pregnancy by in vitro fertilization (IVF) using sperm aspirated from the epididymis. J In Vitro Fert Embryo Transf 1985;2:119-22. 3. Silber SJ, BalmacedaJ, Borrero C, Ord T, Asch R. Pregnancy with sperm aspiration from the proximal head of the epididy-
Fertility and Sterility
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mis: a new treatment for congenital absence of the vas deferens. Fertil Steril 1988;50:525-8. Olar TT, La Nasa J, Dickey RP, Taylor SN, Curole DN. Fertilization of human oocytes by microinjection of human sperm aspirated from the caput epididymis of an individual with obstructive azoospermia. J In Vitro Fert Embryo Transf 1990;7:160-4. Jequier AM, Cummins JM, Gearon C, Apted SL, Yovich JM, Yovich JL. A pregnancy achieved using sperm from the epididymal caput in idiopathic obstructive azoospermia. Fertil SteriI1990;53:1104-5. Silber SJ, Ord T, Balmaceda J, Patrizio P, Asch RH. Congenital absence of the vas deferens. The fertilizing capacity of human epididymal speno. N Engl J Med 1990;323:1788--92. 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 1986;45:665-70. Coetzee K, Kruger TF, Menkveld R, Swanson RJ, Lombard CJ, Acosta AA. Usefulness of sperm penetration assay in fertility predictions. Arch AndroI1989;23:207-12. Kremer J, Jager S. The significance of the zona-free hamster oocyte test for the evaluation of male infertility. Fertil Steril 1990;54:509-12. Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence ofpentoxifylline in severe male factor infertility. Fertil Steril 1990;53:715-22. Ord T, Patrizio P, Marello E, Balmaceda JP, Asch RH. Minipercoll. A new method of semen preparation for
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IVF in severe male factor infertility. Hum Reprod 1990;5: 987-9. Ord T, Marello E, Patrizio P, Balmaceda JP, Silber SJ, Asch RH. The role of the laboratory in the handling of epididymal sperm for assisted reproductive technologies. Fertil Steril 1992;57:1103-6. Yanagimachi R, Yanagimashi H, Rogers BJ. The use of zonafree animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Bioi Reprod 1976;15:471-6. YangY-S, Rojas FJ, Stone SC. Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test. Fertil Steril 1988;50:954-9. Yanagimachi R. Zona-free hamster eggs: their use in assessing fertilizing capacity and examining chromosomes of human spermatozoa. Gamete Res 1984;10:178-232. Asch RH, Silber SJ. Microsurgical epididymal sperm aspiration and assisted reproductive techniques. Ann NY Acad Sci 1991;626:101-10. Bronson RA, Rogers BJ. Pitfalls of the zona-free hamster egg penetration test: protein source as a major variable. Fertil Steril1988;50:851-4. Polansky FF, Lamb EJ. Analysis of three laboratory tests used in the evaluation of male infertility: Bayes' rule applied to the postcoital test, the in vitro mucus migration test, and the zona-free hamster egg test. Fertil Steril 1989;51:215-28. Falk RM, Silverberg KM, Fetterolf PM, Kirchner FK, Rogers BJ. Establishment of TEST-yolk buffer enhanced sperm penetration assay limits for fertile males. Fertil Steril1990;54: 121-6.
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SPA using human epididymal sperm
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Vol. 58, No.5, November 1992
Copyright v 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization*
Francisco J. Rojas, Ph.D. t:j: Anh-Tuan La, B.S.t Terri Ord, B.S. t Pasquale Patrizio, M.D. t
Jose P. Balmaceda, M.D.t Sherman J. Silber, M.D.§ Richardo H. Asch, M.D. t
University of California, Irvine Medical Center, Orange, California, and St. Luke's Hospital, St. Louis, Missouri
Objectives: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). Design: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. Patients, Participants: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. Results: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71 %. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. Conclusions: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm. Fertil Steril 1992;58:1000-5 Key Words: Human epididymal sperm, zona-free hamster oocytes, sperm penetration assay, human in vitro fertilization, sperm maturation, sperm fertilizing capacity, human epididymis
Numerous reports in several mammalian species indicate that transport of sperm through most of the epididymis is a mandatory condition for the
Received May 7, 1992; revised and accepted July 27, 1992. * Presented at the 47th Annual Meeting of The American Fer-
tility Society, Orlando, Florida, October 21 to 24, 1991. t Division of Reproductive Endocrinology and Gynecology, Department of Obstetrics and Gynecology, University of California, Irvine Medical Center. :j: Reprint requests: Francisco J. Rojas, Ph.D., Department of Obstetrics and Gynecology, University of California Irvine Medical Center, 101 The City Drive, B-41, Orange, California 92668. § Department of Urology, St. Luke's Hospital.
1000
Rojas et al.
SPA using human epididymal sperm
sperm to mature and acquire fertilizing capacity (see review by Robaire and Hermo [1]). In contrast to animal studies, information on the fertilizing capacity of sperm from the human testes and epididymis is very limited. Recently, men with azoospermia because of congenital absence of the vas deferens have been treated for infertility by direct microsurgical aspiration of sperm from the epididymis, combined with in vitro fertilization (IVF) and embryo transfer (2, 3). These studies indicate that epididymal sperm can fertilize the human oocyte in vitro and result in pregnancies with livebirths. The reported fertility of these patients suggests that epFertility and Sterility
ididymal sperm, at least in the human, may not be totally infertile. Thus, the role of the human epididymis in sperm maturation and fertilization remains to be defined. It becomes, however, clear that microsurgical aspiration of sperm directly from the testes combined with assisted reproductive techniques may be applied successfully to the treatment of azoospermia caused by congenital absence of the vas deferens or by idiopathic epididymal obstruction, and in men with failed reversals of vasectomy or other nonoperable inflammatory obstructive azoospermia (2-6). The use of the zona-free hamster egg sperm penetration assay (SPA) is a method that has gained increasing acceptance as an assay of functional activity of human spermatozoa (7-9). The assay measures some crucial steps in human spermoocyte interaction and fertilization, including sperm capacitation, acrosome reaction, and fusion of sperm with the plasma membrane of the oocyte. The advent of microsurgical techniques to obtain epididymal sperm provides the unique opportunity to use the SP A to directly evaluate the penetration potential of these sperm cells. The purpose of this study was to assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes as determined by SPA. Also, we used single sperm samples to directly compare the performance of epididymal sperm in SPA with that in zona-intact human oocytes as determined by IVF. MATERIALS AND METHODS Epididymal Sperm
Twenty-nine couples in whom the male partner had congenital absence of the vas deferens underwent direct microsurgical aspiration of epididymal spermatozoa for attempted IVF of their partner's oocytes. Two couples underwent the procedures two times. Results of both attempts were included. Aspirations were performed in the most proximal regions of the epididymis where usually sperm with progressive motility are found. The details of the surgical sperm retrieval technique have been described elsewhere (6). Epididymal aspirates were incubated with 3 mM pentoxifylline (Sigma, Holmdel, NJ) for 30 minutes at room temperature and then washed with Hepes-buffered human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, CA), supplemented with 0.5% human serum albumin (fraction V; Sigma, Holmdel, NJ). Pretreatment of sperm Vol. 58, No.5, November 1992
with pentoxifylline was included in an attempt to improve fertilization rates (10). Sperm pellets were suspended in 0.3 mL of Hepes-buffered HTF medium and prepared for IVF using the mini-Percoll method (11), consisting of a discontinuous gradient formed by three layers of 0.3 mL each of 95%,70%, and 50% of isotonic Percoll (Pharmacia, Vppsala, Sweden). The gradient was centrifuged at 300 X g for 45 minutes. After centrifugation, the 95% Percoll layer was removed, and the recovered sperm were incubated with 3 mM 2' -deoxyadenosine (Sigma) for 30 minutes at 37°C (12). Sperm were then washed two times and suspended in 1 mL HTF medium plus 10% fetal cord serum and used for insemination in vitro. Controlled Ovarian Hyperstimulation (COH) and IVF
All the women were between 24 and 42 years of age. They had regular menstrual cycles and normal tubal patency on hysterosalpingography, laparoscopy, or both. Synchronization of the onset of their menses and COH were carried out as described in details elsewhere (6). The retrieval of oocytes was performed transvaginally with ultrasound guidance, 36 hours after the injection of human chorionic gonadotropin (hCG) (6). Oocytes were added to culture tubes containing sperm (0.4 to 3.3 X 106 sperm/mL) and incubated for 13 to 15 hours at 37°C in a humidified atmosphere of 5% CO 2 and air. Higher concentrations of sperm were added for some samples with poor motility in an attempt to achieve fertilization. After incubation, the oocytes were examined for signs of fertilization, indicated by the presence of two pronuclei, and were transferred to fresh growth medium for another 30 to 36 hours. Fertilization was considered confirmed only if cleavage occurred. Sperm Penetration Assay
Eggs were obtained from hamsters by inducing superovulation using the method described by Yanagimachi et al. (13). Briefly, adult female Golden Syrian Hamsters, 7 to 11 weeks old, were superovulated by intraperitoneal injection of 30 IV of pregnant mare serum gonadotropin (Sigma) on the postestrous morning during which a vaginal discharge was present. Fifty-five to 57 hours later, 30 IV of hCG (Sigma) was given to trigger ovulation. The animals were killed 15 to 17 hours after the hCG injection, and the oviducts were removed and placed in Biggers, Whitten, and Whittingham meRojas et al.
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dium (BWW; Irvine Scientific) containing 0.3% bovine serum albumin (BSA). The cumulus masses were then retrieved and treated with 0.1% hyaluronidase (Sigma) in BWW medium to free the eggs. The eggs were collected, washed in BWW medium twice, and then transferred to a 0.1 % trypsin (Sigma) in BWW medium to remove the zona pellucida (ZP). Once the zona was dissolved, the eggs were washed three times in fresh BWW medium (14). In an effort to minimize differences between SPA and IVF in both sperm preparation and conditions for sperm capacitation, SPA was performed with the same sperm sample used for IVF. This included pretreatment with pentoxifylline/deoxyadenosine, mini-Percoll gradient, and incubation for 13 to 15 hours at 37°C in a humidified atmosphere of 5% CO 2 and air. Also, SPA was performed with a sperm concentration (0.5 to 1.0 X 106 sperm/mL) that was within the range of that used in IVF. Sperm were transferred in 100-IlL drops to the center of a 35 X lO-mm Petri dish and covered with warmed (37°C) paraffin oil (Fisher Scientific Company, Santa Clara, CA). Twenty to 30 zona-free hamster oocytes were added to each drop of sperm and incubated at 37°C for 3 hours. Then all eggs were collected and washed in BWW medium containing 0.3% BSA to remove the unattached sperm and were mounted under a coverslip supported by two solid lines of a 10:1 vaseline/paraffin mixture. The eggs were examined under a phase-contrast microscope at X400 for the presence of decondensing sperm heads and their visible tails in the cytoplasm, recording the percentage of penetrated eggs. Ejaculated sperm (fresh samples) from two fertile donors was prepared as described above and included for every run as an internal control. These internal controls were used at a sperm concentration equivalent to that used for patient samples. RESULTS Ability of Human Epididymal Sperm to Penetrate Zona-Free Hamster Oocytes
Penetration capacity of human epididymal sperm as determined by SPA is shown in Table 1. A wide range of penetration values was observed in the 31 cases analyzed. Penetration of at least one oocyte occurred in 22 of them. This represented 71 % of cases. However, penetration values were in general low and did not exceed 30% (Table 1). In contrast to epididymal sperm, internal controls using ejaculated, mature sperm from fertile donors 1002
Rojas et al.
SPA using human epididymal sperm
Table 1 Penetration of Zona-Free Hamster Oocytes by Sperm Aspirated From the Human Epididymis Penetration rate*
No. of cases
%
o 1 to 6 to 11 to 16 to 21 to
5 10 15 20 30
9 (29) t 2 (6)
5 (16) 6 (19) 7 (23) 2 (6)
* Percentage of ova penetrated; results are divided according to categories of penetration rates. t Values in parentheses are percentages of the total number of 31 cases.
showed penetration rates in SPA that ranged from 30% to 71 %, with a mean (±SD) value of 58% ± 12% for 15 determinations (data not shown). These data indicate that ejaculated sperm from fertile men penetrated zona-free hamster eggs to a greater degree compared with epididymal sperm. Penetration of Zona-Free Hamster Oocytes Using Human Epididymal Sperm Compared With HumanIVF
Table 2 shows the rate of fertilization as determined by IVF and the penetration scores as determined by SPA for each of the cases using epididymal sperm. Cases in which no fertilization in IVF was observed are presented in first place. In general, no fertilization was associated with either no penetration or low penetration values in SPA. As fertilization rate increased, penetration values also increased. A good association was more evident at higher fertilization rates. Thus, the highest penetration values were observed in cases showing the highest fertilization values (Table 2). Penetration rates in SPA expressed as percentage of ova penetrated ranged from 0% to 30%. On the other hand, the range of fertilization rates in IVF was wider, with the lowest value being 0% and the highest 94% (Table 2). The association between percentage of oocytes penetrated and the percentage of oocytes cleaved in IVF is illustrated in Figure 1. Linear regression analysis indicated a positive correlation that was highly significative (P < 0.001). The equation line intercepted the y-axis (0% fertilization rate) at 6% penetration rate in SPA (Fig. 1). Assuming that ~10% penetration rate in SPA represents potential fertility, a cutoff value used by other investigators (8, 15), a positive SPA was associated with a positive Fertility and Sterility
Table 2 Penetration of Zona-Free Hamster Oocytes Using Human Epididymal Sperm Compared With the Results of Human IVF
Case no.
IVF -oocytes cleaved
SPA-oocytes penetrated
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0/6 (O)* 0/22 {OJ 1/16 {OJ 0/27 {OJ 0/10 {OJ 0/11 {OJ 0/50 (OJ 0/16 (OJ 0/34 (OJ 0/9 (OJ 0/18 (OJ 1/27 (4) 1/26 (4) 1/20 (5) 2/21 (9) 2/18 (11) 4/38 (11) 2/13 (15) 4/27 (15) 6/13 (16) 3/16 (19) 5/21 (24) 3/12 (25) 8/20 (40) 3/7 (43) 17/36 (47) 7/12 (58) 29/38 (76) 10/11 (91) 10/11 (91) 15/16 (94)
0/22 {OJ 0/25 {OJ 0/22 {OJ 0/25 {OJ 0/25 {OJ 0/25 {OJ 1/23 (4) 1/20 (5) 2/24 (8) 2/23 (9) 4/25 (16) 0/24 (OJ 2/20 (10) 0/20 (OJ 0/21 (OJ 3/24 (13) 4/20 (20) 4/29 (14) 2/25 (8) 5/24 (21) 2/22 (9) 5/27 (19) 4/24 (17) 5/26 (19) 3/22 (14) 4/33 (12) 3/25 (12) 3/22 (14) 5/29 (17) 5/25 (20) 6/20 (30)
* Values in parentheses are percents.
IVF in 15 of 20 cases (75%). On the other hand, a negative «10% penetration rate) SPA was associated with a negative IVF in 10 of 11 cases (91%). Table 3 summarizes the SPA results using a cutoff point of 10% penetration rate, compared with the ability of human sperm aspirated from the epididymis to fertilize the human oocytes in vitro. DISCUSSION
In this study, we report the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes determined by SPA. Demonstration of penetration capacity of these cells is consistent with previous clinical studies that indicated the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies (2, 3). Also, we report that human epididymal sperm penetrate zona-free hamster oocytes with a wide range of penVol. 58, No.5, November 1992
l-30
•
R -0.87 P < 0.001 N -31
..--~~--~~--.--.--.--.~
o~
OW.
30
•
•
•
~
•
•
~
IVF - Oocytes cleaved (%) Figure 1 Correlation between the zona-free hamster egg SPA and human IVF using sperm aspirated from the epididymis. The line equation was y = 0.18x - 5.89.
etration scores. At least one oocyte was penetrated in most (71%) cases. However, penetration rate, as expressed as percentage of ova penetrated, was low compared with ejaculated sperm and failed to exceed 30%. Fertilization rates in IVF, on the other hand, showed values ranging from 0% to 94%. Incidence of high fertilization rates, nevertheless, was low; table 2 indicates that only 5 of the 31 cases had a fertilization rate in IVF higher than 50%. Similar data have been reported in previous studies (6). Thus, our observations using SPA agree with data on IVF showing that epididymal sperm present low fertilization rates per oocyte compared with rates observed with ejaculated sperm (16). Diminished penetration rate of zona-free hamster oocytes suggests that human epididymal sperm has reduced functional competence. For a sperm to pen-
Table 3 Correlation Between the Results of SPA and the Outcome of IVF Using Human Epididymal Sperm Results *
No. of cases
+SPA/+IVF +SPA/-IVF -SPAj+IVF -SPA/-IVF
15 (75)t 1 (9)t 5 (25)t 10 {9I)t
* A cutoff of 10% penetration rate in SPA was considered to represent potential fertility. t Percentage of positive IVF (20 cases). t Percentage of negative IVF (11 cases). Rojas et al.
SPA using human epididymal sperm
1003
etrate the oocyte, it must complete capacitation, undergo an acrosome reaction, develop a fusogenic region in its plasma membrane, collide with and bind to the oocyte surface, complete membrane fusion, and begin decondensation of the nucleus (15). Low penetration rate, therefore, indicates limited capacity of epididymal sperm to undergo these reactions. Altogether, our data support the concept that the fertilizing capacity of human sperm retrieved from an obstructed epididymis is lower than that of ejaculated sperm. In addition, although a population of sperm retrieved directly from the epididymis has fertilizing potential and can produce successful pregnancy, our results demonstrating reduced penetration rate in SPA suggest that sperm must pass through a certain length of the human epididymis to become fully mature. We have also compared the performance of epididymal sperm in the SPA with the results of IVF. Because performance of SPA has been shown to significantly depend on the conditions selected for sperm preparations and capacitation (17), we carried out SPA on the same sperm sample used for the IVF procedure. This minimized variations in sperm preparations and conditions for sperm capacitation. We found a good association between penetration scores of zona-free hamster oocytes in SPA and fertilization of zona-intact human oocytes in IVF. Linear regression analysis indicated that at zero fertilization rate in IVF, the penetration rate in SPA was 6% (Fig. 1, y-intercept). Interestingly, assuming that ~10% penetration rate in SPA may represent potential fertility, we found that the overall sensitivity of SPA, i.e., its true-positive rate (18) was 75%, and the overall specificity of the assay, i.e., its true-negative rate (18) was 91 %. These observations indicate that a positive SPA (~1O% penetration) was a good indicator of successful fertilization by human epididymal sperm and that the false-positive rate was low. Similarly, a negative SPA correlated remarkably well with failure of human epididymal sperm to fertilize human oocytes in vitro. This would be compatible with studies in ejaculated sperm, indicating that SPA score is a good indicator of sperm function in IVF and of chances to achieve pregnancy (7-9). Accordingly, in one of largest clinical studies, a positive SPA has been reported to predict subsequent in vitro cleavage in 91 (85%) of 107 cases, whereas a negative SP A predicted an overall fertilization failure in 78% of the cases (7). It should be emphasized, nevertheless, that the precise lower limit of SPA penetration rate to distinguish fertile epididymal sperm from 1004
Rojas et al.
SPA using human epididymal sperm
sub fertile epididymal sperm remains to be defined. Studies using ejaculated sperm indicate that the conditions selected for sperm capacitation have a significant impact on the definition of normal ranges in SPA (17). Thus for example, use of TEST-yolk medium and prolonged incubation at low temperature have been shown to enhance the capacity of human sperm to penetrate hamster eggs, allowing the selection of a cutoff value as high as 20% (14, 19). It seems, therefore, that the definition of normal ranges for SPA using epididymal sperm awaits characterization of the optimal conditions for sperm preparation and capacitation. Selection of a proper cutoff point is expected to improve SPA scores concerning IVF performance. Also, a large number of cases need to be analyzed before definitive conclusions can be drawn on the value of SPA to predict the chances of achieving pregnancy by IVF using sperm aspirated from the epididymis. This is important because SPA yields no predictive information on the ability of the sperm to penetrate the ZP. In the meantime, our data provide new insights on the application of SPA for both clinical and basic research. Thus, the good overall association between SPA and human IVF strongly supports the use of SPA as an excellent bioassay to test laboratory experimental conditions for improving fertilizing potential of sperm aspirated from the human epididymis. In this view, SPA may be particularly important in evaluating different conditions for sperm preparation and capacitation of human epididymal sperm for clinical decision-making in IVF. Furthermore, the observation that zona-free hamster eggs can be penetrated by epididymal sperm provides the potential to use SPA as a powerful research tool to elucidate the mechanisms and factors controlling sperm maturation in the human. Ackrwwledgments. We thank Ines Moretti, Ph.D., Hitachi Chemical Research Center, Irvine, California, for critical reading of the manuscript and Ms. Elisa Hendrickson and Mr. Jeffrey Deutsch for manuscript preparation.
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deferens: structure, functions, and their regulation. In: Knobil E, Neill, JD. The physiology of reproduction. New York: Raven Press, 1988:999-1080. 2. Temple-Smith PD, Southwick GJ, Yates CA, Trounson AO, De Kretser DM. Human pregnancy by in vitro fertilization (IVF) using sperm aspirated from the epididymis. J In Vitro Fert Embryo Transf 1985;2:119-22. 3. Silber SJ, BalmacedaJ, Borrero C, Ord T, Asch R. Pregnancy with sperm aspiration from the proximal head of the epididy-
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