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

Transcription

1 F'ERTIlJTY AND STERILITY Copyright ~ 1983 The American Fertility Society Vol. 40, No. I, July 1983 Printed in U.8A. 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, *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 cus 2 (the postcoital or Sims-Huhner test), the in vitro capillary tube penetration test 3, 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 egg 6 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

2 Table 1. Semen Parameters and Fertility Status of Husbands Participating in a Human in Vitro Fertilization-Embryo Transfer Program Semen analysis a Subject No. Fertility index Prior fertility Volume Concentration Motility Progression ml x Uhml % Yes No 3a No 3b No 5a No 5b No No No 9a No 9b No No Yes No No Yes Yes No No Yes Yes Yes Yes Yes Yes Mean ± standard 3.6 ± ± ± ± 0.3 error of the mean Range 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 evi- 54 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% N2. Fertility and Sterility

3 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. 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 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

4 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 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

5 Table 2. Correlation Between Fertilization of Human Eggs and Penetration of Zona-Free Hamster Eggs in Vitro Fertility assessment Human in vitro fertilization Inseminating motile Penetration sperm concentration Subject no_ Eggs inseminated Eggs fertilized Percentage Sperm/egg a 3 1 3b a 1 0 5b a 3 3 9b "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 oocytes 5 obtained during autopsy or following surgical removal of the ovary for medical reasons; and although long-term storage of human zonae in concentrated salt solutions 20 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 x I06/ml a a 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

6 -c 10 = CD C CD a.. o ~ 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% Parenthetically, we do not support the notion of others 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 , 25 The most significant parameter in this regard is sperm capacitation. The kinetics of capacitation vary for proven fertile donorsl9,26 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 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, Wolf et al. Hamster egg bioassay Fertility and Sterility

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