Correlation of human in vitro fertilization with the hamster egg bioassay*

Vol. 40, No. I, July 1983 Printed in U.8A.

F'ERTIlJTY AND STERILITY Copyright ~ 1983 The American Fertility Society

Correlation of human in vitro fertilization with the hamster egg bioassay*

Don P. Wolf, Ph.D.t Joseph E. Sokoloski, B.A. Martin M. Quigley, M.D. Department of Obstetrics and Gynecology, The University of Texas Medical School at Houston, Houston, Texas

We compared fertility potential measurements by the zona-free hamster egg bioassay with the in vitro fertilization of human eggs. Sperm from 24 husbands participating in an in vitro fertilization-embryo transfer program were used in 27 inseminations of the wives' eggs and in simultaneous inseminations of hamster eggs. In seven cases, a positive fertility assessment was obtained in the absence of fertilization of the wives' eggs attributable to egg immaturity or in one case to equipment failure. In the remaining 20 cases, at least one egg was fertilized, for a level of 73%. In 90% of the husbands who fertilized their wives' eggs, a positive fertility q,ssessment was obtained. Two husbands gave false-negative assessments. To account for these, alternative culture conditions were examined; sperm exposure to longer preincubations or at higher concentrations usually improved assessments. These results indicate that fertility assessment with this bioassay is highly correlated with the fertilization of human eggs in vitro. Fertil Steril40:53; 1983

The evaluation of male infertility has previously centered on semen analysis describing sperm density, morphology, motility, and progression. l The results correlate poorly with fertility except in the extreme cases of azoospermia or asthenospermia. This poor correlation has forced attention to direct measurements of sperm function. Currently, several bioassays are available, including in vivo sperm penetration of cervical mu-

Received January 10, 1983; revised and accepted March 21, 1983. *Supported in part by a University of Texas Biomedical Research Support Grant. tReprint requests: Don P. Wolf, Ph.D., Department of Obstetrics and Gynecology, The University of Texas Medical School at Houston, 6431 Fannin, Suite 3270, Houston, Texas 77030. Vol. 40, No.1, July 1983

cus2 (the postcoital or Sims-Huhner test), the in vitro capillary tube penetration test3 , 4 (with bovine or midcycle human cervical mucus), and sperm-egg interaction systems. At least two in vitro sperm-egg interaction assays are available in addition to the fertilization of human eggs. In one, sperm are incubated with nonliving human oocytes, 5 and the ability of sperm to penetrate the zona pellucida is measured. The other assay utilizes the living zona-free hamster egg6 to measure sperm-egg fusion. The zona-free hamster egg bioassay has potential significance in the evaluation of human sperm function. The test has been adapted to sperm karyotyping 7 and to investigations of immunologic infertility8 and epididymal spermatozoan maturation. 9 Results from several laboratories indicate that the bioassay has application in the management of the infertile couple. lO - l3 Wolf et al. Hamster egg bioassay

53

Table 1. Semen Parameters and Fertility Status of Husbands Participating in a Human in Vitro Fertilization-Embryo Transfer Program Subject No.

1 2 3a 3b 4 5a 5b 6 7 8 9a 9b 10 11

12 13 14 15 16 17 18 19 20 21 22 23 24 Mean ± standard error of the mean Range

Semen analysisa Progression

Fertility index

Volume

Concentration

Motility

ml

x Uhml

%

4.5 5.5 2.5 3.5 4.2 4.5 4.3 2.7 5.3 2.8 2.0 3.2 5.5 2.5 2.6 1.6 2.8 4.8 3.3 4.0 5.6 2.6 3.5 5.2 3.6 2.3 3.3

59 56 75 129 34 57 12 94 99 106 30 24.5 17 67.5 84 63.5 163 21 133 102 114.5 307 136 125 28.5 66 114

53 57 61 60 59 61 63 48 55 55 64 60 55 49 56 67 56 58 62 62 57 51 49 60 59 58 61

3-3+ 3+ 3+ 3-3+ 3+ 3+ 3+ 3-3+ 3-3+ 2+-3 2+-3 3 3-3+ 3 3+-4 3-3+ 3-3+ 3+-4 3+-4 3+ 3+-4 3+-4 3-3+ 3+-4 3+ 1-2 3+-4

14.5 12.5 19.5 17.5 14.5 16.5 15.5 15.5 13.5 15 16 16 11.5 15 16 17.5 15.5 15 18 17.5 14 16 15.5 16 14.5 14 18

3.6 ± 0.2

86 ± 12

57.6 ± 0.9

3.5

15.6 ± 0.3

1.6-5.6

12-307

48-67

2-4

11.5-19.5

Prior fertility

Yes No No No No No No No No No No Yes No No Yes Yes No No Yes Yes Yes Yes Yes Yes

aMorphologic characteristics were normal in all subjects (~ 60% normal forms); progression is based on a subjective score with a 0-4 range; the fertility index is based on weighted averages for each semen parameter with a perfect reference of 20.

However, the diagnostic value of the zona-free hamster egg bioassay is not fully accepted, because the specificity and sensitivity of the assay require further definition and substantiation. The availability of a human in vitro fertilization-embryo transfer program gave us a unique opportunity to further assess the accuracy of this bioassay by comparing the results directly with observations of human sperm-human egg interaction in a controlled setting. Herein we present evidence that male fertility assessment with a hamster egg bioassay correlates highly with the fertilization of human eggs in vitro. MATERIALS AND METHODS FERTILIZATION OF HUMAN EGGS

All women in the study had absent or irreparable fallopian tubes with no other major infertility factors. The patients ovulated predictably, as evi54

Wolf et al. Hamster egg bioassay

denced by regular menstrual periods and luteal phase serum progesterone levels (:;;=: 12 ng/ml) in spontaneous cycles. In a treatment cycle, patients received clomiphene citrate (50 or 150 mg/day, days 5 to 9) and underwent daily ultrasonographic assessment of follicular growth beginning on cycle day 10 or 11. When the mean diameter of the largest follicle reached at least 20 mm, the patient received 2500 to 5000 IU of human chorionic gonadotropin to induce egg maturation; and follicular aspiration was performed 36 hours later, provided peripheral levels of luteinizing hormone were basal. 14 The culture medium used was a modified Ham's F-I0 (GIBCO, Grand Island, NY) supplemented with heat-inactivated maternal serum. 15 Eggs, recovered from follicular aspirates, were rinsed in culture medium and preincubated for 5 hours in organ culture dishes (#3037, Falcon Plastics, Oxnard, CA) in a humidified atmosphere of 5% CO 2 , 5% O2 , 90% N 2 . Fertility and Sterility

1B). All of the eggs fertilized in this study were transferred to the donor's uterus. FERTILIZATION OF HAMSTER EGGS AND FERTILITY ASSESSMENT

Figure 1 Human pronuclear egg and cleaving embryo. A, Pronuclei can be observed readily 16 to 20 hours postinsemination after mechanical dispersal of the corona radiata. B, Approximately 24 hours later the embryo is usually at the 4-cell stage of development. Phase contrast (original magnification, x 100).

Semen was collected by masturbation after 48 hours of abstinence, and a routine semen analysis was performed after 30 minutes of liquefaction. These results are summarized in Table 1. All the husbands were considered as potentially fertile; those with proven fertility are so indicated in Table 1. After liquefaction, aliquots of semen (usually 1.0 ml) were diluted with three volumes of Ham's F-10 containing 7.5% maternal serum and centrifuged for 10 minutes at 500 x g; the supernatants were removed by aspiration, and the pellets were washed again with three volumes of Ham's F-10. The final sperm pellet from 1 ml of semen was suspended in 1 ml of culture medium and pre incubated for 5 to 6 hours before the wife's eggs were inseminated. Evidence for fertilization included both the presence of two pronuclei at 16 hours postinsemination (Fig. lA) and cleavage to the 2-, 4-, or 8-cell stage by 40 hours postinsemination (Fig. Vol. 40, No.1, July 1983

Sexually mature Golden hamsters were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin and the eggs were processed as described previously.16 Zona-free eggs (15 to 20) were cultured in 200-1...1 drops of Biggers, Whitten and WhittinghamI7 medium containing 3.5% human serum albumin (Fraction V, Sigma Chemical Co., St. Louis, MO) under silicone oil. The husbands' sperm were preincubated in Ham's F-10 medium containing 7.5% maternal serum for 3 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 scored on acetolacmoid-stained whole mounts at 400 x with phase-contrast optics (Fig. 2). Sperm progression and motility was monitored before and after the insemination by microscopic analysis. Fertility assessment scores were compared by Student's t-test. I8 A positive score was defined as 10% penetration. To relate fertility assessments obtained with the zona-free hamster egg test to the fertilization of human eggs, aliquots of the same sperm sample used for insemination of the wives' eggs were used for inseminations of hamster eggs (Table 2).

RESULTS IN VITRO FERTILIZATION OF HUMAN EGGS

Sperm from 24 husbands were used in a total of 27 inseminations of their wives' eggs. In seven cases, no eggs were fertilized (5a, 8,11,13,20,21, and 23; Table 2). Fertilization failure was attributable to egg immaturity in six cases, where eggs were recovered from small follicles (15 to 20 mm in mean diameter)I4 and were abnormal by gross morphologic assessment. Equipment failure was responsible for fertilization failure in case 11. In the remaining 20 inseminations, at least one egg was fertilized, for a fertilization rate of 73% (33 of 45). Two husbands with low sperm densities (5b and 10 in Tables 1 and 2) also fertilized their wives' eggs but only at relatively high sperm conWolf et al. Hamster egg bioassay

55

The observed range in sperm penetration varied from 0% to 100%, with a mean ± standard error of the mean of 50.4 ± 6.5%. Since the zonafree hamster egg does not possess polyspermy prevention mechanisms, the total number of spermegg fusions or the degree of polyspermy is also relevant to fertility assessment. The husbands, as a group, yielded a polyspermy level of 1.0 ± 0.2 sperm/egg. When more than one sperm concentration was tested, the highest value was employed in this calculation. During the course of the hamster egg inseminations, we also used controls for changes in sperm motility and progression because these parameters have an impact on fertility assessment; motility and progression scores for washed sperm were comparable for all husbands, and they did not decline appreciably over the 6-hour culture period employed here (3-hour preincubation plus 3-hour insemination). These results indicate that fertility assessment with the zona-free hamster egg test is highly correlated with the fertilization of human eggs in vitro. EFFECT OF CULTURE CONDITIONS ON FERTILITY ASSESSMENT SCORES

Figure 2 Human male pronuclei in zona-free hamster eggs. A, Phase contrast x 400. Note the presence of the penetrating sperm tail and the intact sperm on the egg surface. B, Nomarski interference contrast x 200. This egg contains three pronuclei.

centrations; zero of two eggs fertilized at 1 x 105 motile sperm/ml, while three of four eggs fertilized at a concentration of 5 x 105/ml. FERTILITY ASSESSMENT

In 89% (16 of 18) of the husbands whose sperm fertilized their wives' eggs, a positive fertility assessment was obtained (~ 10% penetration) utilizing the zona-free hamster egg test. A false-negative assessment was obtained fo~ two husbands (4 and 9 in Tables 1 and 2). A repeat assessment on patient 9 approximately 3 months later was also negative_ However, limited penetration of hamster eggs (7.7%) was observed when sperm from this patient were pre incubated for 24 hours prior to insemination (Table 2). 56

Wolf et al. Hamster egg bioassay

Since the culture conditions for fertility assessment have been defined empirically, we were interested in evaluating whether or not altered conditions could improve the response of previously studied false-negative cases (proven fertile men who scored poorly). The sperm concentration dependency in penetration for a "typical" fertile donor pre incubated for 3 hours is depicted in Figure 3; much longer pre incubations are usually associated with a decline in penetration scores. 19 In contrast, a false-negative donor, comparable to husbands 4 and 9, failed the fertility assessment when his sperm were preincubated for 3 hours and used to inseminate hamster eggs over the motile sperm concentration range of 1 x 106 to 1 X 10 7 . A positive test result was obtained only at a very high sperm concentration of 3.5 x 10 7 (P < 0.05). When sperm from this donor were subjected to longer preincubation times, the concentration dependency was now comparable with the 3-hour preincubated "typical" donor. Therefore, higher sperm concentrations and longer preincubation times can occasionally improve fertility assessment scores. We conclude from these observations that the incidence of false-negative assessments in proven fertile men can be reduced or Fertility and Sterility

Table 2. Correlation Between Fertilization of Human Eggs and Penetration of Zona-Free Hamster Eggs in Vitro Fertility assessment Human in vitro fertilization Subject no_

Eggs inseminated

Eggs fertilized

Inseminating motile sperm concentration

Penetration Percentage

Sperm/egg

82 95 15 100 6 71 13 100 40 48 69 0 0 7.7 33 38 88 40 100 100 25 67 100 47 19 21 77 31 12 13 5 22 14 60

1.2 3.8 0.2 3.5 0.1 1.1 0.1 2.4 0.4 0.5 0.9 0 0 0.1 0.3 0.4 2.1 0.4 3.7 5.8 0.3 0.7 1.3 0.5 0.2 0.2 0.9 0.3 0.1 0.1 0.1 0.2 0.1 0.6

x I0 6/ml

1 2 3a 3b 4 5a 5b 6 7

1 2 3 3 3 1 4 2 2

1 1 1 2 2 0 2 1 2

8 9a 9b

1 3 3

0 3 2

10 11 12 13 14

2 1 4 1 2

1 0 4 0 2

15 16 17 18

2 1 1 2

2 1 1 1

19 20

2 2

2 0

21

1

0

22 23 24

1 2 1

1 0 1

10 3.9 10.4 19.8 4.8 8.4 3 10.4 8.5 6.5 4.8 13.2a 6.3 7.9a 6.8 14 22 11.7 23.8 11.9 9 17.3 19.2 15.8 7.9 3.9 . 2.5 7.3 3.6 9.5 4.7 3.6 13.3 23.3

"Twenty-four-hour preincubation.

eliminated by further optimizing the conditions and/or time employed for sperm capacitation.

DISCUSSION

The bioassays available for the functional evaluation of human sperm are limited. The in vitro fertilization of human eggs is, of course, impractical for numerous reasons. The ability of sperm to bind to and penetrate zonae pellucidae has been measured in nonliving ov.arian oocytes5 obtained during autopsy or following surgical removal of the ovary for medical reasons; and although long-term storage of human zonae in concentrated salt solutions20 is feasible, this approach is cumbersome. The most promising and practical of available bioassays is the zona-free hamster egg test. Eggs are available in virtually Vol. 40, No.1, July 1983

unlimited supply and can be stored frozen. 21 The test measures the ability of sperm to undergo capacitation, an acrosome reaction, and fusion with the egg. 6 , 22 Hence, it should be capable of detecting fertilization-related abnormalities. Previous reports have correlated hamster egg penetration with the ability of sperm to penetrate the human zona. 5 , 11 We have extended these studies to correlate hamster egg penetration with the ability of sperm to penetrate and fertilize a human egg in vitro. Our criterion for fertilization of human eggs was pronuclear formation and cleavage within defined time intervals. While none of the embryos transferred during the course of this study resulted in a pregnancy, clinical pregnancies were established in 13% of these couples during subsequent treatment cycles. The latter involved the use of a lower dose of clomiphene citrate. 23 Wolf et al. Hamster egg bioassay

57

•

10

•

c

=50 ......

o

CD C CD

a..

014-----------------~----------

6

7 Log Motile Sperm Concentration

Figure 3 Motile sperm concentration dependency in human sperm penetration of zona-free hamster eggs. Open circles, "normal" donor sperm pre incubated for 3 hours; closed circles, 3-hour pre incubated sperm from a proven fertile man who scores poorly under standard conditions; quadrangles, same donor after 24-hour preincubation.

Despite the fact that the hamster egg test does not monitor postfertilization events, a correlation, albeit indirect, also exists between test scores and conception. Thus, scores for clinically infertile and proven fertile populations are distinctly different. Means for the respective populations are 5% and 60%, with ranges that approach each other around 15%.10-13 Parenthetically, we do not support the notion of others 10. 12 that there exists a cut-off value that always distinguishes between these populations. The test has been validated for inter- and intra-sample variability, and recent efforts have been devoted to standardizing assay conditions whereby results from different laboratories can be compared. 19 . 24, 25 The most significant parameter in this regard is sperm capacitation. The kinetics of capacitation vary for proven fertile donors l9 ,26 and are dependent upon the concentration and type of albumin used as the capacitating agent. As indicated here, fertility assessments are conducted with 3-hour capacitated sperm, although occasional men score higher after longer preincubation, suggesting that they may be slow capacitators. The appropriateness of conducting assessments at two different capacitation times is therefore clear. As exemplified by the false-negative scores observed with two husbands in the present studies 58

Wolf et al. Hamster egg bioassay

and by published results for proven fertile men,!l, 13 a limited population of men fail fertility assessments as currently conducted. Consequently, we consider a positive fertility assessment a specific indicator of fertility but do not interpret a low score as an absolute indicator of infertility. Follow-up studies in infertility patients support this restricted interpretation, because some patients who score poorly subsequently father offspring. 25 In addition, manipulation of sperm concentration, preincubation time, and culture medium composition may all affect fertility assessment scores. 27 The possibility of falsepositive results with the hamster egg bioassay was not evaluated in the present study because failure of human fertilization when it occurred was attributable to egg immaturity or technical problems. The treatment of male factors by in vitro fertilization-embryo transfer may provide the opportunity to evaluate this possibility. Fertility assessment with the hamster egg bioassay is important diagnostically in elucidating infertility of unknown cause. Screening husbands with this bioassay might allow identification of those with subnormal sperm function when conventional parameters of semen analysis are normal. Test results should assist in the management of these patients. A negative fertility assessment in repeat testing under varied conditions would be an indicator for donor artificial insemination. A positive assessment might identify candidates for an in vitro fertilization-embryo transfer program or an artificial insemination homologous protocol where the husband's sperm are capacitated in vitro prior to an intrauterine insemination. In this mode, we intend the bioassay to assist in the development of new treatment modalities for the subfertile man. The results of this series of experiments, utilizing human in vitro fertilization candidates, indicate a good correlation between the zona-free hamster egg bioassay and human sperm-egg interaction. These data provide additional evidence of the worth of this bioassay in the evaluation of the subfertile man. Acknowledgments. The authors express appreciation to Dr. Berel Held for his valuable editorial suggestions, and to Ms. Nancy Mann for her secretarial assistance. REFERENCES 1. Smith KD, Rodriguez-Rigau LJ, Steinberger E: Relation between indices of semen analysis and pregnancy rate in infertile couples. Fertil Steril 28:1314, 1977

Fertility and Sterility

2. Jaszezak S, Hafez ESE: Post-coital test. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St. Louis, C. V. Mosby, 1976, p 375 3. Ulstein M, Fjallbrant B: In vitro tests of sperm penetration in cervical mucus. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St. Louis, C. V. Mosby, 1976, p 383 4. 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 5. Overstreet JW, Hembree WC: Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro. Fertil Steril 27:815, 1976 6. 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 7. Rudak E, Jacobs PA, Yanagimachi R: Direct analysis of the chromosomal constitution of human spermatozoa. Nature 274:911, 1978 8. Haas GG, Sokoloski J, WolfDP: The interfering effect of human IgG antisperm antibodies on human sperm penetration of zona-free hamster eggs. Am J Reprod Immunol 1:40,1980 9. Hinrichsen MJ, Blaquier JA: Evidence supporting the existence of sperm maturation in the human epididymis. J Reprod Fertil 60:291, 1980 10. Rogers BJ, Van Campen H, Ueno M, Lambert H, Bronson R, Hale R: Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil Steril 32:664, 1979 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. Karp LE, Williamson RA, Moor DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: a useful test in the evaluation of male fertility. Obstet Gynecol 57:620, 1981 13. Zausner-Guelman B, Blasco L, Wolf DP: Zona-free hamster eggs and human sperm penetration capacity: a comparative study of proven fertile donors and infertility patients. Fertil Steril 36:771, 1981

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14. Quigley MM, Wolf DP, Maklad NF, Dandekar PV, Sokoloski JE: Follicular size and number in human in vitro fertilization. Fertil Steril 38:678, 1982 15. Lopata A, Johnston IWH, Hoult IJ, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33: 11 7, 1980 16. Binor Z, Sokoloski JE, Wolf DP: Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33:321, 1980 17. Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, Freeman, 1971, p 86 18. Snedecor GW, Cochran WG: Statistical Methods, Sixth edition. Ames, Iowa, Iowa State University Press, 1967, p 59 19. Wolf DP, Sokoloski JE: Characterization of the sperm penetration bioassay. J Androl 3:445, 1982 20. Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA, Nicolson GL: Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril 31:562, 1979 21. Fleming AD, Yanagimachi R, Yanagimachi H: Fertilizability of cryopreserved zona-free hamster ova. Gamete Res 2:357, 1979 22. Talbot P, Chacon RS: Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes. Fertil Steril 37:240, 1982 23. Quigley MM, Maklad NF, Wolf DP: Unpublished data 24. Tyler JPP, Pryor JP, Collins WP: Heterologous ovum penetration by human spermatozoa. J Reprod Fertil 63: 499, 1981 25. Cohen J, Weber RFA, van der Vijver JCM, Zeilmaker GH: In vitro fertilizing capacity of human spermatozoa with the use of zona-free hamster ova: interassay variation and prognostic value. Fertil Steril 37:565, 1982 26. Perreault SD, Rogers BJ: Capacitation pattern of human spermatozoa. Fertil Steril 38:258, 1982 27. WolfDP, Sokoloski JE: Unpublished data

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