The predictive value of zona-free hamster egg sperm penetration assay for failure of human in vitro fertilization and subsequent successful zona drilling*

Vol. 53, No.6, June 1990

FERTILITY AND STERILITY

Printed on acid-free paper in U.S.A.

Copyright " 1990 The American Fertility Society

The predictive value of zona-free hamster egg sperm penetration assay for failure of human in vitro fertilization and subsequent successful zona drilling*

Monica Vazquez-Levin, Ph.D.t Paul Kaplan, Ph.D. Benjamin Sandler, M.D.

G. John Garrisi, Ph.D. Jon Gordon, M.D., Ph.D. Daniel Navot, M.D.

Department of Obstetrics, Gynecology, and Reproductive Science, Mount Sinai Medical Center, New York, New York

The value of various sperm parameters and the zona-free hamster egg sperm penetration assay (SPA) in predicting human in vitro fertilization (IVF) failure and subsequent successful fertilization with zona drilling was assessed. In 19 couples, throughout 31 IVF cycles, a total of 153 oocytes failed to be fertilized. In subsequent 12 cycles with zona drilling, 33 of 131 (25%) were fertilized. The incidence ofteratospermia and asthenospermia was significantly higher in the study group than in the control, 74% versus 32% and 42% versus 5%, respectively. Although the mean values for the performance of sperm in SPA and fertilization of human eggs after zona drilling were remarkably similar (28 ± 6 versus 28 ± 4), there was no correlation between individual parameters (r = 0.15). Thus, whereas male factor infertility is more likely to be associated with teratoasthenospermia, neither the SPA nor other sperm parameters have any predictive value for failure in IVF. In addition, no criterion of sperm function has yet been identified that would eliminate oligoteratoasthenozoospermic males from consideration of IVF with zona drilling. Fertil Steril53:1055, 1990

Human in vitro fertilization (IVF) and embryo transfer (ET) was originally developed for couples in whom sterility was due to absent or irreparably damaged fallopian tubes. 1 As IVF-ET evolved and became more commonly available, its indications widened. Currently, IVF is performed to treat unexplained and immunological infertility, 2 male factor infertility,3 and endometriosis. 4 Promising preliminary results have prompted many IVF centers to treat male infertility as one of the major indications for either IVF, gamete intrafallopian transfer, or zigote intrafallopian transfer procedures. 5-7 Received September 25, 1989; revised and accepted February 2,1990. * Presented at the 45th Annual Meeting of The American Fertility Society, San Francisco, California, November 13 to 16, 1989. t Reprint requests: Monica Vazquez-Levin, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Science, Mount Sinai Medical Center, New York, New York 10029. Vol. 53, No.6, June 1990

Although IVF may considerably enhance the fertilizing ability of subfertile sperm, the overall results with male factor infertility in IVF are disappointing. At present, screening tests available for miile infertility patients before IVF include conventional seminal fluid analysis, 8 automated computer-assisted semen analysis, 9 and several bioassays. The assay most widely used is the zona-free hamster egg sperm penetration assay (SPA). 10 There is controversy regarding the relative value of various parameters in predicting subsequent IVF success. Although several groups have found correlation between IVF and sperm motility, sperm morphology, and SPA, 11- 13 no test has proven to be predictive of success in IVF .14•15 Sperm from a subgroup of severe male infertility patients totally fail to penetrate human oocytes. For these individuals, gamete micromanipulation has been recently implemented; zona pellucida drilling, 16•17 subzonal insertion of sperm/8 and microinjection of sperm

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into oocytes 19 are the most promising procedures. Because these procedures are unlikely to be a universal treatment for all forms of male infertility, it is important to identify factors that predict a successful outcome. We have retrospectively analyzed a subgroup of infertile patients who exhibited complete failure of oocyte penetration in one or more IVF cycles. The predictive value of routine seminal analysis, computerized analysis, and SPA was evaluated in this population with an exceptionally poor IVF prognosis. The value of the parameters mentioned above was specifically assessed in relation to sperm fertilizing capacity of human oocytes subjected to zona drilling. MATERIALS AND METHODS Patient Selection

Nineteen patients who completely failed IVF in a total of 31 IVF cycles comprised the study population. An equal number of tubal infertility patients treated immediately after the study group served as a control group. The two groups of patients had similar stimulation regimens, and identical methodology was utilized for sperm, egg, and embryo handling. 20 In 12 cases, eggs were subjected to zona pellucida drilling after a procedure described previously .16 Semen Analysis

In each sample, volume (mL), concentration (number of sperm/mL), total count, and percent motility (number of sperm with progressive motility/total number of sperm) were determined. Sperm morphology was assessed by staining of sperm smears according to the method of Papanicolaou.21 Cervical mucus penetration was assessed by the bovine cervical mucus capillary test. 22 In addition, automated computer-assisted semen analysis was performed. Mean values of velocity, linearity, amplitude of lateral head displacement, as well as amplitude of lateral head displacement maximum, the ratio of sperm beat to cross frequency, and frequency distribution for velocity (Vc) and linearity (L) were determined by the CellSoft program9 {Cryo Resources Ltd., New York, NY). Hamster Penetration Assay

Hamster sperm penetration assay, using zonafree hamster eggs, was carried out as described previously.10 Sexually-mature Golden hamsters were 1056

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superovulated with pregnant mare's serum and human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO). Subsequent to recovery, oocytes were treated with hyaluronidase (Sigma) and trypsin (Sigma) to remove the cumulus cells and zona pellucida, respectively. Approximately 20 zonafree eggs were cultured in Biggers, Whitten, and Whittingham medium containing human serumalbumin (3.5% Fraction V; Sigma) under oil. The sperm were preincubated in Ham's FlO medium (Flow Laboratories, McLean, VA) containing serum albumin for 3 and 18 hours before the eggs were inseminated with 2.5 to 20 X 106 motile sperm/mL. Three hours later the eggs were removed, washed free of loosely adherent sperm, mounted on slides, and fixed. Sperm penetration was assessed on acetolacmoid-stained whole mounts at 400X with phase-contrast optics. The SPA rate is calculated as number of hamster oocytes penetrated/number of hamster oocytes inseminated. A penetration rate of ~20% was considered positive. Data Analysis

All data are presented as means ± SE. Student's t-test and Fisher exact test were used for statistical analysis as appropriate. Correlation between SPA and fertilization rate in human oocytes postzona drilling was assessed by Spearman rank-order correlation coefficient. Analysis of Vc and L between both groups was carried out using the Kolmogorov-Smirnov test for nonparametric data as described before. 23 RESULTS Seminal Fluid Analysis and Cervical Mucus Penetration

Conventional seminal fluid characteristics revealed 2 study patients with normal parameters, whereas 10 had single sperm abnormalities: 2 with asthenozoospermia and 8 with teratozoospermia. The rest of the patients (n = 7) had at least two abnormal parameters (oligoteratoasthenozoospermia). Details of conventional seminal fluid parameters and cervical mucus penetration assay are shown in Table 1. No statistical difference was observed between the seminal fluid volume in the study population and control group (Table 1). Although the study population as a whole had sperm concentration significantly lower than the control group (53 ± 8 X 106 versus 120 ± 19 X 106; P < 0.005); in only 3 cases (16%) was sperm con-

Hamster SPA predicting zona drilling

Fertility and Sterility

Table 1

Conventional Seminal Fluid Analysis and Cervical Mucus Penetration in the Study Population Compared With Control

Study population •

Parameter Semen volume (mL) Sperm concentration (no. sperm/mL) Sperm motility(%) Normal sperm morphology(%) Cervical mucus penetration (mm)

Percentage of study patients with abnormalityb

Control"

Percentage of control patients with abnormalityb

3.0 ± 0.4 (1.3 to 6.5)

11 (2/19)

2.8 ± 0.2 (0.4 to 4.1)

53 ± 8 X 106 c (0.4 to 120 X 106 ) 37 ± 5 (15 to 74)

16 (3/19) 42 (8/19)d

120 ± 19 X 106 c (15 to 358 X 106 ) 63 ± 4 (25 to 94)

32 ± 3 (14 to 54)

74 (14/19)•

45 ± 4 (20 to 72)

32 (6/19)•

36 ± 4 (4 to 61)

37 (7/19)'

50± 2 (31 to 65)

or

11 (2/19) 5 (1/19) 5 (1/19)d

• Values are means ± SE; ranges in parentheses. b Semen volume < 1.5 mL; sperm concentration < 20 X 106 sperm/mL; sperm motility< 40%; normal sperm morphology < 40%; cervical mucus penetration< 30 mm. c Sperm concentration is significantly lower in the study population with respect to control (P < 0.005). d Percentage of patients with abnormal sperm motility is significantly higher in the study population with respect to control (P<0.005).

• Percentage of patients with abnormal sperm morphology is significantly higher in the study population with respect to control (P < 0.01). f Percentage of patients with abnormal cervical mucus penetration is significantly higher in the study population with respect to control (P < 0.01).

centration lower than normal values (i.e., 20 X 106 sperm/mL). When the proportion of patients with abnormalities was compared in the study population and control groups, deficits in progressive motility (P < 0.005), percentage of normal morphological features (P < 0.01), and cervical mucus penetration (P < 0.01) were significantly more frequent in the study population (Table 1). None of the parameters derived from the computerized sperm motion analysis differed between groups. The average values found for maximum amplitude of lateral head displacement were 1. 78 ± 0.5 and 1.94 ± 0.3 in the study population and control, respectively. The average beat/cross frequency ratio was 14.1 ± 2.0 in the study group and 13.6 ± 1.6 for the control. Finally, the cumulative distributions for Vc and L, analyzed as described previously,23 were not significantly different (data not shown).

In nine patients, data was available on subsequent fertilization with oocytes subjected to zona drilling. Over 12 cycles a total of 131 oocytes were drilled, of which 33 (25%) were fertilized. Ifthe fertilization rate for each patient is considered as an equal contributor to calculation of an overall mean penetration rate, that rate is 28% ± 4% compared with 0% without zona drilling. If a similar overall mean penetration is calculated for the SPA the figure is remarkably similar: 28% ± 6% (Table 3). However, when the SPA rate is compared with the human oocyte penetration rate after zona drilling in each corresponding patient, no correlation can be found (r = 0.15).

Sperm Penetration Assay and Standard IVF

In Table 2, IVF performance and SPA results are compared between the study population and control groups. In the study population, 153 oocytes were retrieved in a total of 31 IVF cycles; a total failure of fertilization was observed. The control population exhibited normal fertilization rates, ranging between 50% and 100% (80 ± 4). Nevertheless, the SPA results were similar between the two populations in both mean penetration rate and range of penetration (Table 2). In the study population, one patient had complete lack of hamster egg sperm penetration, whereas three additional cases scored in the infertile range (<20%). In the control group, four patients scored below 20%. Vol. 53, No.6, June 1990

DISCUSSION

A variety of tests have been previously invoked in an effort to predict the fertilizing capacity of sperm in IVF. The impetus for this effort is based on the invasive nature of the IVF procedure, and Table 2 Comparison of IVF and Zona-Free Hamster Egg SPA in the Study Population Versus the Control Group Human oocytes fertilized in vitro/ No. of oocytes cycles inseminated

SPA" %

Study population (n = 19) Control group (n = 19)

31 19

0/153 (O)

31 ± 4 (O to 70)

87/111 (50 to 100) 38 ± 5 (3 to 100)

• Values are means ± SE; ranges in parentheses.

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Table 3 IVF Performance in Nine Patients From the Study Population Whose Oocytes Were Subjected to Zona Drilling Throughout 12 Cycles; Interrelation Between Zona Drilling and Zona-Free Hamster SPA

Patient no.

No. of oocytes subjected to zona drilling

No. of oocytes fertilized

h-IVF rate postzona drilling

ZD1 ZD2 ZD3 ZD4 ZD5 ZD6 ZD7 ZD8 ZD9

10 12 32 5 13 29 13 15 2

1 2 6 1 3 8 5 6 1

10 17 19 20 23 28 39 40 50

28 64 31 0 23 33 8 36 25

9

131

33

28± 4"

28± 6"

SPA rate

%

a

Values are means ± SE.

the consequent importance of identifying those patients most likely to be successfully treated. In several reports, reduced sperm motility was found to have the highest correlation with fertilization failureY·14 Other studies have suggested that abnormal sperm morphology is the most reliable index of sperm fertilizing ability. 12 The advent of zona drilling and related micromanipulation methodologies adds a new perspective to this problem, since fertilization and pregnancies can be obtained from otherwise inadequate samples. 16•17 It is especially important to identify the best candidates for this still empirical and inherently less efficient form of treatment. Moreover, exposure of sperm directly to the oocyte after zona pellucida manipulation approximates more closely the SPA assay, in which zona-free hamster eggs are exposed to sperm. This analogy makes the SPA a particularly attractive procedure for possible prediction of IVF failures after zona drilling. These considerations prompted the present retrospective analysis, directed toward identification of factors that would predict IVF performance after zona drilling. Because of the similarity between zona-free hamster penetration and fertilization of drilled human oocytes, special attention was paid to the SPA as a possible predictive test. However, no sperm parameter could be identified that was indicative of subsequent performance in IVF augmented by zona drilling. With regard to semen analysis, it was not surprising to find that mean values for sperm concentration, percent motility, percentage of abnormal forms, and performance in the cervical mucus penetration test were all significantly reduced relative to controls. However, perhaps the most significant 1058

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of these figures is sperm morphology. Not only was the overall sperm quality reduced, but the percentage of patients with abnormal morphology was high (14/19, 73%; Table 1). These findings are in agreement with studies showing that morphology, followed by motility, were the most important indicators of infertilityY· 14·24 Moreover, whereas cervical mucus penetration was reduced in the infertile group, the value obtained was still within the normal range. However, none of these parameters studied was predictive of success after zona drilling, in which all patients exhibited similar increase in the oocyte penetration rate (Table 3). When the infertile males were compared with controls with respect to the SPA assay, again no differences were found, despite a total failure of the study group's sperm to penetrate zona-intact human oocytes in standard cycles of IVF (Table 2). These results are in agreement with those reported by Margalioth et al. 15 who found no correlation between the SPA and human IVF outcome in cases of oligoasthenozoospermia. However, the SPA measures the ability to penetrate the oolemma, whereas IVF measures the ability of sperm to bind to the zona pellucida, undergo the acrosome reaction, penetrate the zona most likely using acrosin, and fuse with the egg. For this reason, it was hoped that the SPA would correlate better with success after zona drilling, a procedure in which sperm bypass the zona and interact directly with the egg surface.25 The data show that the SPA is not predictive of IVF success, even after zona drilling. This point is underscored by cases 5 and 9. In these instances the SPA was negative, but oocytes were penetrated after zona drilling (Table 3). It is of interest to consider possible explanations for the inability of the SPA to predict IVF performance with or without zona drilling. The results indicate that the zona-free hamster egg is not the same as the zona-free human oocyte. Our own view is that penetration of hamster ova by human sperm, while resembling in some respects true fertilization, may actually involve mechanisms distinct from intraspecific sperm:egg interactions. Perhaps sperm are occasionally incorporated into hamster eggs by mechanisms other than normal sperm: egg fusion, but once incorporated, decondense in a manner consistent with interpretation as a positive penetration. Such mechanisms may not necessarily involve specific molecular interactions which probably characterize true fertilization. In the circumstance where this less specific mechanism fails, a negative SPA could be obtained, but might not predict failure of true fertilization

Hamster SPA predicting zona drilling

Fertility and Sterility

once sperm are exposed to the human oocyte. Similarly, a positive SPA resulting from such a nonspecific process might falsely suggest that fertilization will occur in IVF. With the advent of increasingly aggressive efforts to treat male infertility by IVF, it is of great importance to develop or identify assays that give an indication of subsequent IVF performance, especially when IVF failure indicates oocyte micromanipulation. Unfortunately, the currently available methods for evaluating semen leave very much to be desired. The SPA, despite its attractiveness as a functional assay and its analogy to zona -drilled human oocytes, could not predict either IVF failure or successful fertilization subsequent to zona drilling. Based on these findings, we suggest that while patients should not be unduly optimistic after a positive SPA, they should also not be eliminated from consideration for IVF with zona drilling on the basis of negative SPA results. Acknowledgment. The authors thank Svetlana Goldberg, M.S., for her technical expertise in the zona-free hamster egg sperm penetration assay. REFERENCES 1. DeCherney AH: Tubal disease: surgery and in vitro fertilization. In Principles and Practice of Clinical Gynecology, Edited by NG Kase, AB Weingold. New York, John Wiley and Sons, Inc., 1983, p 461 2. Navot D, Schenker JG: The role of in vitro fertilization in unexplained and immunological infertility. In Immunology and Immunopathology of Reproduction, Edited by V Toder, AE Beer, Basel, Karger, 1985, p 160 3. Cohen J, Edwards R, Fehilly C, Fishel S, Hewitt J, Purdy J, Rowland G, Steptoe P, Webster J: In vitro fertilization: a treatment for male infertility. Fertil Steril43:422, 1985 4. Matson PL, Y ovich JL: The treatment of infertility associated with endometriosis by in vitro fertilization. Fertil Steril46:432, 1986 5. Cohen J, Fehilly CB, Fishel SB, Edwards RG, Hewitt J, Rowland GF, Steptoe PC, Webster J: Male infertility successfully treated by in vitro fertilization. Lancet 1:1239, 1984 6. Matson PL, Yovich JM, Bootsma BD, Spittle JW, Yovich JL: The in vitro fertilization of supernumerary oocytes in a gamete intrafallopian transfer program. Fertil Steril 47: 802, 1987 7. Devroey P, Staessen C, Camus M, Wisanto A, Bollen N, De Grauwe E, Van Steirteghem AC: Results of zygote intrafallopian transfer. (Abstr.) Presented at VI World Congress in In Vitro Fertilization and Alternate Assisted Reproduction, Jerusalem, Israel, April2 to 7, 1989, p 155

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8. Mahadevan MM, Baker HWG: Assessment and preparation of semen. In Clinical In Vitro Fertilization, Edited by C Wood, AO Trounson. Berlin, Springer-Verlag, 1984, p 83 9. Mack SO, Wolf DP, Tash JS: Quantitation of specific parameters of motility in large numbers of human sperm by digital image processing. Biol Reprod 38:270, 1988 10. 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. Biol Reprod 15:105, 1979 11. Talbert LM, Hammond MG, Halme J, O'Rand M, Fryer JG, Ekstrom RD: Semen parameters and fertilization of human oocytes in vitro: a multivariable analysis. Fertil Steril48:270, 1987 12. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S: Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril49:112, 1988 13. Ausmanas M, Tureck RW, Blasco L, Kopf GS, Ribas J, Mastroinanni 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 14. Mahadevan MM, Trounson AO: The influence of seminal characteristics on the success rate of human in vitro fertilization. Fertil Steril 42:400, 1984 15. 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 Steril45:665, 1986 16. Gordon JW, Grunfeld L, Garrisi GJ, Talansky BE, Richards C, Laufer N: Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil Steril 50:68, 1988 17. Malter HE, Cohen J: Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 51:139, 1989 18. Gordon JW: Use of micromanipulation for increasing the efficiency of mammalian fertilization in vitro. Ann NY Acad Sci 541:601, ~988 19. Oshumik K, Katagiri C, Yanagimachi R: Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs. J Exp Zool237:319, 1986 20. Laufer N, Tarlatzis BC, Naftolin F: In Vitro Fertilization: state of the art. Semin Reprod Endocrinol2:197, 1984 21. Papanicolaou GN: A new procedure for staining of vaginal smears. Science 5:483, 1942 · 22. Gaddum-Rosse P, Blandau RJ, Lee WI: Sperm penetration into cervical mucus in vitro. II. Human spermatozoa in bovine mucus. Fertil Steril 33:644, 1980 23. Vantman D, Banks SM, Koukoulis G, Dennison L, Sherins RJ: Assessment of sperm motion characteristics from fertile and infertile men using a fully automated computerassisted semen analyzer. Fertil Steril51:156, 1989 24. Comhaire FH, Vermeulen L, Hinting A, Schoonjans F: Accuracy of sperm characteristics in predicting the in vitro fertilizing capacity of semen. J In Vitro Fert Embryo Transfer 5:326, 1988 25. Conover JC, Gwatkin RBL: Fertilization of zona-drilled mouse oocytes treated with monoclonal antibody to the zona glycoprotein, ZP3. J Exp Zool247:113, 1988

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