PENETRATION OF HUMAN SPERMATOZOA INTO THE HUMAN ZONA PELLUCIDA AND THE ZONA-FREE HAMSTER EGG: A STUDY OF FERTILE DONORS AND INFERTILE PATIENTS*

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1 FERTILITY AND STERILiTY Copyright" 1980 The American Fertility Society ""I. 33, No.5, May 1980 Prinred in U.SA. PENETRATION OF HUMAN SPERMATOOA INTO THE HUMAN ONA PELLUCIDA AND THE ONA-FREE HAMSTER EGG: A STUDY OF FERTILE DONORS AND INFERTILE PATIENTS* JAMES W. OVERSTREET, M.D., PH.D.t* RYUO YANAGIMACHI, PH.D. DAVID F. KAT, PH.D.* KEIKO HAYASHI, M.D. FREDERICK W. HANSON, M.D.* Departments of Human Anatomy and Obstetrics and Gynecology. University of California School of Medicine, Davis, California 95616, and Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii An in vitro assay of human sperm function was devised to evaluate two initial stages of sperm-ovum interaction: penetration through the zona pellucida and sperm entry into the ooplasm. The former stage was assessed with immature human oocytes and the latter with mature zona-free hamster eggs. The two gamete populations were incubated together in the same suspension of capacitated spermatozoa. Spermatozoa from five of six fertile donors were able to penetrate through the human zona pellucida into the perivitelline space, and to enter the zona-free hamster egg. Spermatozoa from one fertile donor consistently penetrated the zona pellucida, but fertilized only 1 of 44 hamster eggs. Several types of sperm dysfunction during gamete interaction were observed in a group of 21 patients with suspected infertility: (1) to bind to the zona pellucida, (2) zona binding with to penetrate, (3) incomplete zona penetration, and (4) zona penetration with poor sperm entry into the ooplasm. Eleven of the twelve cases of apparent fertilization dysfunction would have been detected by the human oocyte assay alone, and eight of the twelve would have been diagnosed by incubation with only the zona-free hamster eggs. The success of gamete interaction in vitro could not be correlated with any of the classic parameters of semen quality. Some men with "normal" semen and unexplained infertility had apparent dysfunction of gamete interaction, while others gave positive results. These experiments indicate that, at the present time, in vitro tests of gamete interaction should be reserved for selected cases of unexplained infertility. In the future they may prove useful in identifying couples with male infertility as candidates for in vitro fertilization and embryo transfer. Fertil Steril33:534, 1980 Received November ; revised January 2, 1980; accepted January 8, *Supported in part by a grant from the International Planned Parenthood Federation (to R. Y.). tdepartment of Human Anatomy, University of California School of Medicine. *Department of Obstetrics and Gynecology, University of California School of Medicine. Recipient of National Institutes of Health Research Career Development Award,HD To whom reprint requests should be addressed. Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine. 534 At the present time, our most detailed knowledge of sperm physiology relates to the events of fertilization. It is not surprising, therefore, that many initial attempts to apply advances in basic research to the diagnosis and treatment of human male infertility have been focused in this area. 1-5 Direct application of mammalian in vitro fertilization systems to human fertility diagnosis will require attention to many unresolved ethical questions. At the present time two types of in vitro assay are being used as alternative systems. In one

2 Vol. 33, No.5 SPERM PENETRATION OF HUMAN ONA PELLUClDA AND ONA-FREE HAMSTER EGG 535 type of system, spermatozoa are incubated with nonliving human oocytes to assay the sperm's ability to penetrate the zona pellucida. 1 The application of this assay can be simplified by storing the oocytes in hypertonic medium 6 or at low temperature. A second, more widely applied, system uses mature, living, zona-free hamster ova to study sperm incorporation into the ooplasm. 2 Both systems probably measure the same physiologic processes involving sperm capacitation in vitro and the acrosome reaction, and functional abnormalities in the spermatozoa which interfere with these processes should be detected by both systems. However, each also measures additional specific sperm functions, i.e., zona penetration and sperm entry into the ooplasm. The latter is an initial event of fertilization, the physiology of which is assumed to be similar in hamster and human eggs. Since infertility could result from specific abnormalities of zona penetration or fertilization, some types of male infertility may be demonstrated by one test and not the other. The present study was undertaken to investigate these questions by comparing the results of the two assays when applied simultaneously to a selected group offertile donors and male patients with suspected or proven infertility. MATERIALS AND METHODS Patient Population. Semen samples were obtained from 6 fertile donors and from 21 patients with suspected infertility. All of the donors had fathered a child within the past 2 years (Table 11. The patients were selected from a group with infertile marriages of at least 2 years' duration. Eight of these men had normal semen quality as assessed by sperm concentration (>20 x 106/mll, sperm motility (>40% motile), and sperm morphology (>50% oval heads) (Table 2). Of the 13 men with abnormal semen, 4 had abnormalities in all 3 parameters, 3 had abnormalities in 2, and 6 had a single abnormal semen parameter (Table 3). There was no evidence of reproductive dysfunction in the wives of 17 of the 21 patients (Tables 2 and 3). Four of these women had become pregnant within the previous year as a result of artificial insemination with donor semen. Ofthe remaining 13, 9 had received extensive infertility evaluations, 6 of which included laparoscopy. Six of the thirteen had had a previous pregnancy. Two of the wives had oligo-ovulation which responded to treatment with clomiphene citrate, one had pelvic endometriosis, and one had unilateral occlusion of a fallopian tube. Preparation of the Sperm Suspension. A single semen specimen was used from each of the 21 patients and from 4 ofthe fertile donors. Semen from two of the donors was tested on two occasions. All semen samples were used within 1 hour of collection. Following an initial assessment of sperm concentration and motility, the semen (liquefied) was divided into 0.5-ml aliquots, each of which was transferred to the bottom of a test tube (15 x 50 mm) containing 2 ml of Biggers, Whitten, and Whittingham medium 10 <BWW) supplemented with 0.3% bovine serum albumin. The constitution of this medium and the manufacturers of its constituent chemicals have been\ published previously.6 The tubes, sealed with Parafilm, were inclined at a 30 angle and incubated in room air at 37 C for 1 hour, allowing time for spermatozoa to swim upward out of the seminal plasma and into the BWW medium. The BWW layers from several tubes were combined and transferred to a centrifuge tube at 22 C, and then diluted to 10 ml with fresh BWW medium. This suspension was centrifuged for 5 minutes at 500 x g. The pellet was resuspended in 2 ml of a modified BWW medium. The centrifugation was then repeated and the pellet was resuspended in a small volume of modified BWW medium. The sperm concentration and percentage motility in this suspension were then determined. The composition of the modified BWW medium was the same as that of regular BWW medium except that the NaCI concentration was reduced from mm to mm, NaHC0 3 concentration was increased from mm to mm, and 0.3% bovine serum albumin was replaced with 3.5% human serum albumin (Fraction V, Sigma Chemical Co., St. Louis, Mo.). Semen samples from the fertile donors were used to determine the efficiency of gamete interaction in this system over a range of sperm concentrations. Each suspension of donor sperm was diluted serially to obtain three or four aliquots of decreasing sperm concentration, the lowest of Donor TABLE 1. Semen Quality of the Fertile Semen Donors in the Study Semen quality Most recent paternity Sperm concen- % Sperm q- Normal sperm tration/ml motility morphology A 1 yr 181 X B 6mo 282 X C 2 yr 136 X D 6mo 143 X E 1 yr 27 X F 1V2 yr 40 X

3 01 0') TABLE 2. Clinical Data on the Infertile Couples in Which the Male Had Normal Sperm Concentration, Sperm Motility, and Sperm Morphology Male Femalea Case no. Patient no. Duration of infertiity Sperm. concen- 'k Sperm 'k Normal trationlml motility sperm mor- Remarks Ovulation Cervical Tubal Remarks phology mucus patency X History and physical BBT, SP PCT, Lap noncontributory x History and physical BBT Pregnancy in prenoncontributory vious marriage x Brother is childless in 2 marriages BBT, SP PCT, Lap 6 Cycles of AIR x History and physical BBT, SP PCTnoncontributory CMCT, Lap Pregnant in 1 cycle of AID CMPT x History and physical BBT PCTnoncontributory CMCT CMPT x History and physical BBT, SP PCT, Lap 2 Pregnancies in noncontributory previous marriage x History and physical BBT, SP PCT, Lap 6 Cycles of AIR noncontributory x History and physical BBT, SP PCT- Pregnant in l! cynoncontributory CMCT cle of AID CMPT 0 < I:'J 6l ;3 I:'J I:'J..,.., I:'J > r abbt, biphasic basal body temperature; PCT, postcoital test (,normal, -, abnormal) 7 ;, normal hysterosalpingogram; SP, ovulatory levels of midluteal serum progesterone; Lap, no pathology at laparoscopy; CMCT, shaking sperm motility on cervical mucus contact tests; CMPT, normal mucus and abnormal semen at semen-mucus cross-penetration test 9 ; AIH, artificial insemination with husband's semen; AID, artificial insemination with donor semen....

4 TABLE 3. Clinical Data on Infertile Couples in Which the Male Had Abnormal Semen Quality Male Case no. Patient no. Duration of infertility Sperm con- 'k Sperm 'k Normal centrationlml motility sperm mor- Remarks Owlation mucus phology X yr abuse of mepro- BBT PCT bamate; 46% tapering morphology X % tapering mor- BBT PCT phology x Left varicocele BBT x Left varicocelectomy BBT PCT x History and physical BBT, SP PCTnoncontributory x History and physical BBT, SP PCTnoncontributory x Left varicocelectomy BBT PCT X Left varicocelectomy BBT, SP PCT x yr industrial expo- BBT PCT sure to trichloroethylene, sodium cyanide x % tapering mor- PCT phology x Vasovasostomy 2 yr ago; BBT 60% of sperm have coiled tails x History and physical PCTnoncontributory x % tapering morphology; 9 yr of infertility in previous marriage afor meaning of abbreviations see footnote to Table 2., BBT, SP Cervical Female" Tubal patency, Lap, Lap, Lap Remarks Pregnancy in this marriage 8 yr ago Endometriosis treated with danazol Pregnant in 1 cycle of AID 6 Cycles of AIH Pregnant in 1 cycle of AID Left tube occluded by fibroid; 4 cycles of AIR 2 Spontaneous abortions in this marriage 3 yr ago Oligoovulation, treated with Clomid Normal history and physical examination Oligoovulation, treated with Clomid 7 cycles of AIH; pregnant in this marriage 4 yr ago Pregnant in previous marriage =:c is:: til ti!i 0-,3 0 0 "'l a > til C (j 8 > IS 0 "'l ti!i ::z:: > is:: ti!i =:c ti!i 8 tl1 -.J

5 538 OVERSTREET,ET AL. May 1980 which ranged from 0.4 to 1.4 x 10 6 motile sperm/ ml. Suspensions of washed spermatozoa obtained from the patients' semen were used in most cases without further dilution. Collection and Preparation of the ona-free Hamster Eggs and Immature Human Oocytes. Mature, unfertilized eggs were collected from the oviducts of superovulated golden hamstersll within 5 hours of ovulation. The ova were freed from the surrounding cumulus cells by treating them for 15 minutes at 22 C with 0.1% bovine testicular hyaluronidase (300 USP units/mg, ICN Pharmaceuticals, Inc., Cleveland, Ohio) in BWW medium. The zona pellucida was removed by treating the eggs for 2 to 3 minutes at 22 C with 0.1 % bovine pancreatic trypsin (2 x crystallized, 10 4 BAEE units/ml, Sigma Chemical Co.) after which the zona-free eggs were thoroughly rinsed in BWW medium. Ovarian tissue was obtained after surgical removal for gynecologic indications and was stored at 0 C to 4 C until processed. Immature oocytes were recovered in phosphate-buffered saline (PBS, ph 7.2) within 24 hours of surgery as described previously.! All oocytes with an intact zona pellucida were retained, and there was no selection of oocytes on the basis ofthe condition of their granulosa cell investment or vitellus. Washed oocytes were diluted four times at 10-minute intervals with PBS containing dimethyl sulfoxide (DMSO, Sigma Chemical Co.) at 0 C such that the final concentration of DMSO was 2 M. Pairs of oocytes were loaded into 2- to 3-cm glass capillary tubes (cut from a 100-I.Ll pipette; Drummond Scientific Co., Broomall, Pa.) in 30 to 40 ILl of2 M DMSO in PBS. Both ends of the tube were sealed with Critoseal (Sherwood Medical Industries, Inc., St. Louis, Mo.) and placed directly in a freezer at - 80 C for storage. The oocytes were recovered from storage (period of storage ranged from 1 week to 6 months) by warming the sealed capillary tubes in three stages ( - 80 C to - 15 C, - 15 C to 10 C, Donor no. TABLE 4. Penetration by Spermatozoa from Fertile Donors into the ona Pellucida of Human Oocytes and Ooplasm of ona-free Hamster Eggs Gamete interaction Spenn concentration: motile spermlml Sperm bindin to zona pelluci a Sperm entry into zona pellucida Sperm penetration through zona pellucida Fertilization of zona free hamster eggs A 9.1 X 10 6a /25 (40%) 5.6 X / /8 (88%) 0.4 X /2 2/ /10 (20%) B 4.5 X /1 1/ /12 (75%) 2.9 X /5 (60%) 1.0 X /11 C b 7.4 X /3 3.2 X /3 2.6 X / X /2 2/2 2/2 0/7 0.8 X / X (11%) D 7.7 X 10 6a /26 (69%) 5.0 X /16 (94%) 2.9 X 10 6a /24 (71%) 0.5 X /25 (20%) Eb 18.0 X /10 (100%) 16.1 X /12 (100%) 8.8 X 106 2/2 2/2 2/2 14/15 (93%) 5.4 X /24 (100%) 1.4 X 106 2/2 2/ /15 (80%) 0.7 X 10 6a 2/2 2/2 2/2 37/39 (95%) 0.5 X /4 3/4 3/4 12/14 (86%) F 8.4 X /17 (88%) 2.2 X /21 (67%) 1.3 X / /18 (22%) 0.5 X /27 (33%) apooled results from two dishes prepared from the same sperm suspension. btwo sets of experiments were performed on different days.

6 Vol_ 33, No.5 SPERM PENETRATION OF HUMAN ONA PELLUCIDA AND ONA-FREE HAMSTER EGG 539 and 10 C to 22 C), each of which required a minimum of 20 minutes. The thawed oocytes were then rinsed through five different aliquots of BWW medium over a 45-minute interval. Gamete Interaction in Vitro. A 0.3-ml aliquot of the sperm suspension was placed under mineral oil in a plastic Petri dish (10 x 30 mm), and the zona-free hamster eggs and immature human oocytes were introduced into this same suspension. The number of eggs and oocytes per dish varied depending on the number of tests being run and the number of gametes available. The range was 4 to 26 hamster eggs and 1 to 4 human oocytes per dish. A total of 711 hamster eggs and 73 human oocytes were used in the study. The preparations were incubated for 4 hours in the dark at 37 C in room air. At the end of in cubation, sperm motility in the medium was again assessed. The hamster eggs and human oocytes were then mounted on glass slides under cover glasses supported by four pillars of Vaselineparaffin. The hamster eggs were observed with phase-contrast microscopy and classified as "fertilized" when swollen sperm heads were present. When excess spermatozoa were present on the zona surface, the human oocytes were aspirated in and out of a small-bore pipette several times before mounting. Each human oocyte was repeatedly rolled between the slide and cover glass to assess sperm penetration into the thickness of the zona pellucida and/or into the perivitelline space. RESULTS Fertile Donors. Spermatozoa from all six of the fertile donors were able to penetrate into and through the zona pellucida of human oocytes in every experiment, i.e., at sperm concentrations ranging from 0.4 to 18.0 x 10 6 motile sperm/ml (Table 4). Sperm numbers in the zona pellucida ranged from 1 to more than 100, and the number of spermatozoa in the perivitelline space ranged from 1 to 50. There was a tendency for greater sperm numbers to be present in the zona pellucida and perivitelline space as the concentration of motile sperm in the medium increased (data not shown). In experiments with four of the six donors, at least 20% of the zona-free hamster eggs were fertilized at all sperm concentrations tested (Table 4). No fertilization could be detected when spermatozoa from donor B were tested at a concentration of 1 million motile sperm/ml. Spermatozoa from donor C consistently failed to fertilize the zona-free hamster eggs. For this donor, only 1 of 44 eggs was penetrated in 6 incubations carried out with 2 different ejaculates, in spite of the fact that the zonae pellucidae of human oocytes in the same dishes were consistently penetrated (Table 4). There was a clear trend toward higher fertilization rates as the concentration of motile spermatozoa in the medium increased (Table 4). Infertility Patients. Of the eight patients with "normal" semen quality, four gave results in the mixed gamete assay which were indistinguishable from those obtained with semen from fertile donors (Table 5). The spermatozoa from one patient (case 1) traversed none of the zonae pellucidae of three oocytes, although 56% of the hamster eggs in the same dish were penetrated. Spermato- Case no. TABLE 5. Characteristics of the Sperm Suspension and Egg/Oocyte Penetration o(patients with "Normal" Semen Quality Sperm suspension Gamete interaction Sperm Preincubation Postincubation Sperm bindinl Sperm entry Sperm penetration Fertilization of concentration % motility % motility to zona pelluci into zona pellucida through zona zona-free pellucida hamster eggs Penetration x /3 3/3 0/3 13/23 (56%) Fertilization x /2 2/2 2/ (5%) Penetration and fertilization x /2 2/2 0/2 4/26 (15%) x /3 0/3 0/3 0/8 Normal x (40%) x /2 2/2 2/2 18/20 (80%) x /2 2/ /16 (37%) x /6 (33%)

7 540 OVERSTREET ET AL. May 1980 TABLE 6. Characteristics of the Sperm Suspension and Egg/Oocyte Penetration of Patients with Abnormal Semen Quality Case no. Sperm suspension Spenn Preincubation Postincubation concentration % motility % motility Penetration x x Fertilization x x Penetration and fertilization x x x x x Normal x x x x Gamete interaction Spenn bindin Sperm entry into Sperm penetration Fertilization of to zona pelluci a zona pellucida. through zona zona-free pellucida hamster eggs 2/2 2/2 0/2 5/8 (63%) 2/2 2/2 0/2 4/12 (33%) 2/2 2/ (8%) 2/2 2/ /9 (22%) 0/2 0/2 0/2 0/ / / /2 0/4 3/3 0/3 0/3 0/ /1 0/20 2/2 2/ /11 (55%) /10 (100%) 2/2 2/2 2/ (69%) 2/2 2/2 2/2 3/6 (50%) zoa from patient 2 penetrated both human oocytes but fertilized only 5% of the 26 hamster eggs present. Spermatozoa from patients 3 and 4 performed poorly in both hamster egg fertilization and human oocyte penetration. In the case of patient 4 the latter was associated with a complete of sperm attachment to the zona pellucida. This was also associated with a decrease in percentage motility during the incubation, but the two events did not appear to be related causally; that is, the number of motile sperm in the medium at the end of incubation (3 x 10 6 /ml) should have been adequate' for zona penetration. Four of the thirteen men with abnormal semen also gave positive results in the mixed gamete assay (Table 6). Spermatozoa from 2 of the 13 (cases 9 and 10) failed to traverse the zona pellucida but were able to fertilize the hamster eggs. In two cases (cases 11 and 12) zona penetration was observed but fertilization levels were low. The spermatozoa of five men in this group gave negative results in both assays. In all these cases there was a complete of fertilization. In one instance there was an associated of sperm to bind to the zona pellucida (case 13), and in another (case 16) sperm binding occurred but there was no penetration into the zona pellucida. DISCUSSION Having considered the results of these experi- ments, we conclude that in vitro tests of gamete interaction can be usefully applied for fertility diagnosis in only a few selected cases. The mixed gamete assay described in this paper permitted discrimination between several types of sperm dysfunction which could lead to fertilization. In fact, all of the hypothetical abnormalities we anticipated (zona binding, zona penetration, passage through the zona, and incorporation into the ooplasm) are represented in this series. It should be noted that 11 of the 12 cases of apparent fertilization dysfunction would have been detected by the human oocyte assay alone, and that 8 of the 12 would have been diagnosed by incubation with only the zona-free hamster eggs. However, in the majority of cases (cases 4 and 9 to 17), the tests of gamete interaction merely confirmed a previous diagnosis of male infertility and did not contribute to the choice of therapy. The tests were useful clinically in assessing the five cases of unexplained infertility included in this study. In three of these (cases 1 to 3) the results indicated a spermrelated fertilization dysfunction, and in the others (cases 6 and 7) no abnormalities were apparent. We consider of zona penetration and/or fertilization in the mixed gamete assay to be an indication for artificial insemination with donor semen as therapy for these couples. The physiologic functions of the sperm cell as related to its fertilizing capacity include those in-

8 Vol. 33, No.5 SPERM PENETRATION OF HUMAN ONA PELLUCIDA AND ONA-FREE HAMSTER EGG 541 Case no Cervical mucus penetration test 5 Cervical mucus penetration test Cervical mucus penetration test 9 Semen evaluation 10 Semen evaluation 11 Semen evaluation 12 Semen evaluation 13 Semen evaluation 14 Semen evaluation 15 Semen evaluation 16 Semen evaluation 17 Semen evaluation 18 Semen evaluation 19 Semen evaluation 20 Semen evaluation 21 Semen evaluation apregnancy by AID while paper was in press. bpregnancy by husband while paper was in press. TABLE 7. Probable Cause of Infertility in the Couples Studied Male infertility Probable cause of Diagnoaed by other Diagnosed by hamster Diagnoaed by human infertility tests eggs oocytes Fertilization dysfunction Fertilization dysfunction Fertilization dysfunction: genetic etiology?a Fertilization dysfunction: immunologic' Failure of sperm transport: immunologic Unexplained b Unexplained Failure of sperm transport: immunologic Fertilization dysfunction: drug toxicity, Fertilization dysfunction endometriosis in female Fertilization dysfunction: varicocele? Fertilization dysfunction?: varicocele Fertilization dysfunction Fertilization dysfunction a Fertilization dysfunction: varicocele Fertilization dysfunction: varicocele Fertilization dysfunction: environmental toxin Ovulation dysfunction in female male contribution? Sperm transport? female factor? Ovulation dysfunction in female male contribution a Sperm transport? postfertilization? volved with (1) its transport and survival in the female, (2) its penetration of the ovum vestments, and (3) its incorporation into the ooplasm at fertilization. Two of these functions were at least partially evaluated in this study. We know that infertility may be caused by many other sperm-related abnormalities involving "postfertilization" events such as activation of the ovum, pronucleus formation, and syngamy. Abnormal embryonic development with consequent postfertilizationreproductive may result from genetic abnormalities in the fertilizing spermatozoon. Obviously, none of these potential sources of male reproductive dysfunction can be evaluated by observing the initial events of gamete interaction in vitro. This uncertain interpretation of a positive result (falsepositive) is illustrated by three cases in this series (cases 5, 8, and 21) in which a male contribution to the couple's infertility seems very likely, but was not diagnosed by either test of gamete interaction. Another practical problem with these tests is the. possibility of frequent false-negative results, that is, of fertile sperm to give positive results under standard, well-controlled conditions. This is illustrated by results obtained with semen from donor C in the hamster egg system. It seems very unlikely that this is a case of secondary infertility in view of the positive results obtained with the semen in the other diagnostic tests. A more likely cause is variation among the donors in requirements for sperm capacitation in vitro, although this logic leads one to question the equivalence of "capacitation" as assayed by the human oocyte and hamster egg systems.

9 542 OVERSTREET ET AL. May 1980 In conclusion, these experiments indicate that the mixed gamete assay is more likely to detect fertilization dysfunction than either of the two component assays when used alone. It also has the advantage of more critically identifying specific abnormalities of sperm function. However, our experience suggests that, at the present time, in vitro tests of gamete interaction cannot be rationally applied for routine screening of male fertility potential but should be reserved for cases of un explained infertility in couples for whom artificial insemination with donor semen is an acceptable therapeutic approach. In the future, these tests may prove useful in identifying couples with male infertility who are candidates for in vitro fertilization and embryo transfer. REFERENCES 1. Overstreet JW, Hembree WC: Penetration ofthe zona pellucida of non-living human oocytes by human spermatozoa in vitro. Fertil Steril 27:815, 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, Barros C, Gonzalez J, Herrera E, Bustos-Obregon E: Fertilizing capacity of human spermatozoa evaluated by actual penetration offoreign eggs. Contraception 17:87, Barros C, Gonzalez J, Herrera E, Bustos-Obregon E: Human sperm penetration into zona-free hamster oocytes as a test to evaluate the sperm fertilizing ability. Andrologia 11:197, 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, Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA, Nicholson GL: Retention of biologic characteristics of zona pellucida in highly concentrated salt sol ution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril 31:562, Davajan V: The postcoital tests. J Reprod Med 18:132, Kremer J, Jager S: The cervical mucus contact test: a preliminary report. Fertil Steril 27:335, Katz DF, Overstreet JW, Hanson FW: A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 33:179, Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JD Daniel. San Francisco, Freeman, 1971, p Yanagimachi R: In vitro capacitation of hamster spermatozoa by follicular fluid. J Reprod Fertil 18:275, 1969