Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome

Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome

Vol. 51, No.4, April1989 FERTILITY AND STERILITY Printed in U.S.A. Copyright 1989 The American Fertility Society Hemizona assay: assessment of spe...

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Vol. 51, No.4, April1989

FERTILITY AND STERILITY

Printed in U.S.A.

Copyright<> 1989 The American Fertility Society

Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome* Sergio Oehninger, M.D.t Charles C. Coddington, M.D.t Richard Scott, M.D. Daniel A. Franken, Ph.D.§

LaniJ. Burkman, Ph.D. Anibal A. Acosta, M.D. Gary D. Hodgen, Ph.D.

The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical Sc1wo~ Medical College of Hampton Roads, Norfolk, Virginia

The hemizona assay (HZA) was used in a prospective, blinded study to assess the relationship between tight sperm binding in the HZA and sperm fertilizing ability in in vitro fertilization (IVF). In each controlled assay, the authors compared sperm binding of proven fertile men with that of patients undergoing IVF. Human oocytes stored in a salt solution were used in the study, and binding results were correlated with the fertilization rate of preovulatory oocytes during IVF. Patients with poor fertilization rates in IVF had significantly lower binding than those cases with successful fertilization (7 .3 ± 1.4 versus 62.1 ± 10.9, respectively; mean ± standard error, P < 0.02). Based on current standards, the HZA was able to predict fertilization accurately in 26 of 28 cases (sensitivity of 83%, specificity of 95%, positive predictive value of 83% ). The authors conclude that the HZA is a valuable tool for evaluating dysfunctional sperm-zona pellucida binding, with good predictive value for fertilization in vitro. Fertil Steril51:665, 1989

The hemizona assay (HZA) has been introduced as a new diagnostic test for the binding of human spermatozoa to human zonae pellucidae to predict fertilization potential. 1 In the HZA, the two matched zona hemispheres created by microbisection provide several advantages: (1) the two halves (hemizonae) are functionally (qualitatively) equal zona surfaces, allowing a controlled comparison of binding; (2) the limited number of available human oocytes is amplified, since an internally controlled Received August 12, 1988; revised and accepted December 19, 1988. • The opinions expressed herein are those of the authors and do not represent those of the United States Department of the Navy or United States Department of Defense. t Reprint requests: Sergio Oehninger, M.D., Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 825 Fairfax Avenue, Norfolk, Virginia 23507. :j: Present address: Portsmouth Naval Hospital, Portsmouth, Virginia. § Present address: Department of Obstetrics and Gynecology, University ofthe Orange Free State, Bloemfontein, South Africa. Vol. 51, No.4, April1989

test can be performed in a single oocyte; (3) ethical objections to possible inadvertent fertilization of a viable oocyte are eliminated by using nonliving human oocytes cut into halves. 1 Previous studies examined the feasibility of sperm binding to the hemizonae as well as the kinetics of tight sperm binding, showing maximal binding after 4 to 5 hours of coincubation. 1•2 Normal spermatozoa show equivalent binding to dimethylsulphoxide (DMSO; 2.0 M solution) frozenstored and salt (magnesium chloride, MgCl2 ; 1.5 M solution)-stored human hemizonae. Yanagimachi et al. 3 demonstrated earlier that the zona pellucida retains sperm binding potential following salt storage. Salt storage offers a simple and inexpensive means of accumulating and transporting human zonae pellucidae while maintaining functional properties for estimating sperm binding potential in theHZA. 2 Franken et al., 4 using DMSO-treated hemizonae, reported higher binding for patients achieving fertilization during in vitro fertilization (IVF) than Oehninger et al. Hemizona assay, in vitro fertilization

665

for those patients with failure of fertilization. Tight zona binding was significantly and positively correlated with the percentage of motile sperm, the percentage of normal morphology, and seminal sperm concentration. In this previous report, although patients used as controls in the HZA had sperm counts of ~20 X 106 /ml with motility of ~30%, the majority were infertile, and some had a slight degree of teratozoospermia. Here, a prospective, blinded study was designed for two purposes: (1) to further assess the relationship between tight binding in the HZA and IVF outcome, and (2) to determine the sensitivity, specificity, and predictive value of the HZA. For optimal results, two strict criteria were established: (1) only proven fertile donors with normal semen parameters were used as internal controls in each test and (2) only IVF patients with at least two mature preovulatory oocytes at retrieval were included in the study. This study further differed from previous work in that only one source of oocytes and one storage condition was used (human oocytes recovered from surgically removed ovaries and stored in salt solution) and that evaluation of male factor patients included a thorough workup, as described below.

MATERIALS AND METHODS

Controls Four proven fertile donors, who had fathered a child in the last 2 years, provided semen used as an internal control in each test. All specimens used had a sperm concentration of ~80 X 106 , motility of ~50%, and normal morphology of ~14%. 5 ·6 Semen Analysis Semen evaluation was performed using an automated computerized system (Cell Soft Semen Analyzer, Labsoft Division of Cryo Resources, Ltd., New York, NY), as previously described. 4 Briefly, sperm images were analyzed at a rate of 15 frames/ second. Lower limits included (1) sperm velocity ~ 10 #Lm/second, (2) ~1 frame of data for determining cell motility, and (3) a sperm head size of 4 to 25 pixels. The pixel size was set at 0.688 microns. For specimens with very low sperm counts or decreased motility, the sperm concentration was calculated using a hemacytometer; percentage of motility was assessed by scoring 100 sperm/microscopic slide. After sperm liquefaction was coip.pleted, slides were carefully prepared for morphologic assessment. The slides were stained with Diff Quick (American Scientific Products, McGraw, IL), and morphology was evaluated according to the strict criteria presently used in the Norfolk program.5 •6 •10 IVF/Embryo Transfer Procedures

Patients A total of 28 men participating in the Norfolk IVF program (series 30 and 31, February through June 1988) were allocated to the study group. All presented after 2 or more years of infertility and had had at least three previous semen analyses. Seventeen patients had sperm concentrations of ~20 X 106/ml, motility of ~50%, and normal morphology.5.6 There were 11 male factor patients: 2 had oligozoospermia ( <20 million/ml), 2 had asthenozoospermia (<50% motility), 4 had teratozoospermia (:::::;14% normal forms), and 3 had asthenoteratozoospermia. All had tested negative for serum and semen antisperm antibodies and bacteriologic studies. 7 Five patients had been further evaluated by a hamster oocyte/human sperm penetration assay (SPA), according to published protocols using Biggers, Whitten, and Whittingham as the incubation medium and 3% bovine serum albumin as the protein source in a short incubation (6 hours) protocol.8•9 666

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Ovarian stimulation, oocyte retrieval, classification of oocytes, insemination, and embryo transfer were performed according to previously published protocols. 11- 13 All women had at least two preovulatory oocytes (metaphase I and II) at retrieval. The inseminating concentration varied from 50,000 sperm/ml/oocyte (non-male-factor patients) to 500,000 sperm/ml/oocyte (male factors).14 Oocyte Handling and Cutting Oocytes were obtained from surgically recovered ovarian tissue, following protocols already described.1·2·15 Specimens were obtained from women aged 25 to 45 years who were undergoing pelvic surgery for benign and malignant gynecologic conditions. Zona-intact, immature oocytes (prophase I) were denuded of granulosa cells and placed in small plastic vials containing 0.5 ml of 1.5 M MgC1 2 (Mallinkrodt Chemical Works, St. Louis, MO), 0.1% polyvinylpyrrolidone, and 40 mM of Hepes Fertility and Sterility

Buffer, and stored at room temperature (23.C) for 6to 30 days. Before the assay, the desired number of oocytes was removed from salt storage and rinsed for 15 minutes in Ham's F-10 culture medium (Gibco, New York, NY) containing 7.5% heat-inactivated human fetal cord serum. Narishige micromanipulators (Narishige, Tokyo, Japan) were mounted on a phase-contrast inverted microscope (Nikon Diaphot, Garden City, NY) and used for cutting the oocytes. A detailed description of the cutting procedure has been published. 1•2•4 Sperm Handling for HZA

An aliquot of semen (0.5 ml) was mixed with 1 ml of Ham's F-10 culture medium, supplemented with 7.5% human fetal cord serum, then centrifuged for 5 minutes at 400 X g. After a second wash, the final sperm pellet was overlaid with 0.5 ml of Ham's F-10, then incubated at 37•c in 5% C02 in air to achieve a swim-up separation of vigorously motile spermatozoa. After 1 hour, the supernatant was removed and the sperm were used for the HZA. Experimental Design

Twenty-eight HZA tests were performed. In 17 assays, infertile men with normal semen parameters were evaluated, while in 11 assays male factor patients were studied. In each of the 28 experiments, one salt-stored oocyte cut into halves provided one hemizona that was coincubated with the patient's semen; the matching hemizona was coincubated with the sperm of the fertile control. All patients were asked to provide a second semen sample for the HZA at 48 hours after insemination of the oocytes for IVF. Personnel carrying out the HZA had no prior knowledge of the patients' clinical history or IVF outcome. During the IVF attempt, successful fertilization occurred in 22 cases, while poor or failed fertilization was observed in 6 cases (all male factor patients). A threshold value of 65% fertilization rate of preovulatory oocytes in IVF was used to distinguish group 1 (successful fertilization) from group 2 (poor fertilization). This cut-off value represents the 95% confidence interval of 112 couples undergoing IVF simultaneously, with a diagnosis of tubal factor and normal semen parameters, and fertilization of at least two preovulatory oocytes (mean fertilization rate, 90.1%). All assays were done with 100 JLl of the processed semen with a motile concentration of 500,000 Vol. 51, No.4, April1989

sperm/mi. All sperm drops were held under oil in 35 mm X 10 mm plastic dishes (Falcon Plastics, Oxnard, NJ). The hemizonae were removed after 4 hours of incubation, and each was rinsed by quickly pipetting five times to dislodge loosely associated spermatozoa. The number of tightly bound spermatozoa on the outer surface then was counted with a phase-contrast microscope (200X). 1•2•4 Statistical Analysis

All results are expressed as mean ± standard error of the mean. The two-tailed Student's t-test was used to compare the mean number of spermatozoa bound to the hemizonae, the hemizona index1 (number of spermatozoa bound for patients/controls X 100), and semen parameters for patients with successful versus poor fertilization. As discussed above, a threshold value of 65% fertilization rate of preovulatory oocytes in IVF was used to distinguish these two groups. Group 1 had successful fertilization of ~65% of preovtilatory oocytes. Group 2 had poor fertilization of <65% of preovulatory oocytes. In the Norfolk program, patients with tubal infertility, 16 endometriosis, 17 diethylstilbestrol exposure, 18 and unexplained infertility19 have a fertilization rate of >65% of preovulatory oocytes. Within each of the two groups, comparison of the mean number of spermatozoa bound to the hemizonae from patients versus each internal control (proven fertile donors) was made by a paired t-test. RESULTS

Table 1 presents sperm parameters (concentration, percentages of motility, and morphology) and HZA results (number of tightly bound sperm and HZA index) in patients and controls according to IVF outcome (group 1: ~65% fertilization rate; group 2: <65% fertilization rate). In the group with poor fertilization in vitro (n = 6), four patients had failure of fertilization (0% fertilization rate). In two cases, 50% of the preovulatory oocytes were fertilized. As we have noted in our laboratory, in these cases of failed fertilization, low numbers of spermatozoa were observed at the level of the zona pellucida, with no sperm cells in the perivitelline space. Of the classic sperm parameters, groups 1 and 2 differed significantly only in the percentage of motility (P < 0.01) (Table 1, Part A). The mean number of tightly bound sperm in the HZA and the HZA index were significantly lower in the patients with poor fertilization rates in IVF (P < 0.02). Part Oehninger et al. Hemizona assay, in vitro fertilization

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Table 1

Sperm Parameters and HZA Results According to IVF Outcome Group2 (Fertilization rate of preovulatory oocytes < 65%) (n=6)

Group 1 (Fertilization rate of preovulatory oocytes :2:: 65%) (n = 22)

p

116.0 ± 16.6 71.3 ± 3.3 9.7 ± 1.3

0.1 >P> 0.05 P<0.01 0.1 >P> 0.05

49.0 ± 14.5 31.5 ± 6.3 4.3 ± 2.1

93.3 ± 54.2 ± 62.1 ± 120.4 ±

0.2>P>0.1 P<0.02 P<0.02 P<0.02

51.0 ± 33.1 ± 7.3 ± 29.5 ±

Part A Patients' IVF samples Concentration Percent motility Percent normal morphology Patients' HZA samples Concentration Percent motility HZAbinding HZAindex PartB Controls' HZA samples Concentration Percent motility HZAbinding

13.9 3.4 10.9 18.3

150.5 ± 13.3 64.6 ± 1.7 59.5 ± 11.2

P>0.5 P>0.5 P>0.5

12.3 1.4 1.4 7.5

142.8 ± 16.6 66.8± 3.5 40.6 ± 11.9°

• P < 0.02 (Controls' versus patients' HZA binding within group 2, paired t-test).

B of Table 1 shows sperm parameters and HZA binding for the fertile controls used in the HZA for groups 1 and 2. Paired analysis showed significantly lower HZA binding in infertile patients than in controls within the group with poor IVF results (P< 0.02). Objective comparisons of the matching hemizonae data are possible by calculating the ratio of the HZA index of tightly bound sperm. The scattering of data provided a natural breakpoint at an HZA index value of 36, allowing us to evaluate the HZA as a predictor of IVF results. Table 2 depicts the sensitivity (83%), specificity (95%), and the positive and negative predictive values (83% and 95%, respectively) of the HZA based on the present results. The HZA results according to sperm quality are shown in Table 3. Sperm concentration, percentages ofmotility20 and morphology,5 •6 mean number of sperm bound in the HZA, and the HZA index differed significantly when infertile patients with normal semen values were compared with male factor patients. Six of the 11 male factor patients correspond to the group with poor IVF results ( <65% fertilization rate). Table 2

Ability of the HZA to Predict IVF Outcome

HZA index

Poor fertilization

Good fertilization

Total

:s;36 >36

5 1

1

21

6 22

6

22

28

Total

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Oehninger et al. Hemizona assay, in vitro fertilization

Table 4 compares HZA, SPA, and IVF results in five of the male factor patients and in one non-male-factor infertile patient. In this small sample, the HZA results correlated better than the SPA with IVF outcome. DISCUSSION

This prospective, blinded study provides further evidence of the ability of the HZA to predict IVF outcome. With a sensitivity of 83%, a specificity of 95%, and an incidence of one false positive and one false negative in 28 experiments, the HZA discriminated by identifying patients at risk for poor or failed fertilization. More experiments are needed to establish the threshold HZA index value and the intra-assay variation. The threshold HZA index Table 3 HZA Results According to Sperm Quality in Patients with Normal Semen and in Male Factor Patients

Concentration Percent motility Percent normal morphology HZAbinding HZAindex

Normal sperm parameters • (n = 17)

Malefactor patients (n= 11)

126.0 ± 75.5 ± 12.1 ± 72.2 ± 124.5 ±

64.2 ± 42.9 ± 3.1 ± 16.7 ± 59.2 ±

18.2 2.5 1.2 17.6 23.2

17.6b 6.5° 0.9° 5.0d 13.0b

• Sperm concentration> 20 million/ml, motility> 50%, 20 and :2::14% normal morphology. 5•6 b P<0.05. • P
Fertility and Sterility

Table 4

Comparison of the Mean Number of Tightly Bound Sperm in the HZA, HZA Index, SPA, and IVF Results

Patient

Diagnosis

HZAbinding

HZAindex

SPA patient versus control

Fertilizat~on rate of preovulatory oocytes in IVF

%

%

1

Asthenozoospermia Teratozoospermia Oligozoospermia Oligozoospermia Normal sperm

4 7 11 13 68

14 35 64 16 340

7versus54 23versus43 6versus 32 Oversus65 Oversus 76

0 0 0 50 80

2

3 4

5

value selected here (36) was chosen arbitrarily, considering the natural scattering of data found here. In addition, to maximize application of the HZA and its power of discrimination, it may be necessary to establish a minimal number of tightly bound sperm (probably 20 to 30, according to our current information) 21 for the fertile control specimen, thus assuring an acceptable oocyte binding capacity. Fertilization of human oocytes is a complex process. Binding of spermatozoa to the zona pellucida represents an early, critical stage requisite for oocyte fertilization. Our results show a positive correlation between HZA results and IVF outcome. However, it is also important to realize that other physiologic steps follow the binding of sperm to the zona and are necessary to achieve fertilization and development: sperm passage through the zona, binding to the oolema, penetration into the oocyte, sperm decondensation, and finally pronuclei formation and cleavage. Thus the observation of false positive results should be an expected finding. False negative results could be the consequence of inter-egg variation (variation in the binding ability of the oocytes) or from variation in interassay handling. Establishment of a minimal binding threshold for the control specimen may overcome its occurrence. The interaction of human sperm and zonae pellucidae, as evaluated by the HZA, thus can provide important information, together with the percentage of motility and normal forms of a semen sample, in anticipating IVF outcome. In a previous study, 4 we showed a positive correlation between the percentage of normal forms and the number of tightly bound sperm during the HZA. Ongoing experiments are underway to establish the minimal threshold for the total motile fraction of a semen sample required to perform and validate the HZA and to make the test results reliable in severely oligospermic patients. The sperm penetration assay (SPA) tests for Vol. 51, No.4, April1989

sperm capacitation and acrosome action, as well as for penetrating ability and decondensation in a heterologous system. The HZA, using a homologous system, gives additional valuable information on spermatozoa-zonae pellucidae interaction. It is interesting that in a small sample of our population, the HZA could better predict IVF results than the SPA. This may suggest that dysfunctional sperm-zona binding may be the underlying cause in cases of failed fertilization, both in IVF and in natural reproduction. In IVF, sperm abnormalities are associated with a high number of cases of failed fertilization. 22 In the current context of modern techniques for assisted fertilization (microinjection of a single spermatozoon into an oocyte23 or under the zona pellucida24 and zona drilling2 5), the HZA may provide physiopathologic support for their use. When a dysfunctional sperm-zona interaction is diagnosed, these techniques-if their success is finally validated-may constitute a logical therapeuticalternative to be offered to patients. In conclusion, in the HZA, male factor patients and patients with poor IVF rates have lower binding ability than selected fertile controls. The HZA is a valuable tool for evaluating dysfunctional sperm-zona binding, with good predictive value for IVF results. Acknowledgments. We thank Deborah Johnson, M.A., for technical assistance with the HZA and Charlotte Schrader, Ph.D., for editorial assistance.

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14. Oehninger S, Acosta AA, Morshedi M, Veeck LL, Swanson RJ, Simmons K, Rosenwaks Z: Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe morphology abnormalities. Fertil Steril 50:283, 1988 15. 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 16. Acosta AA, van Uem J, Ackerman SB, Mayer JF, Stecker JF, Swanson RJ, Pleban P, Yuan J, Chillik C, Brugo S: Estimation of male fertility by examination and testing of spermatozoa. In In Vitro Fertilization-Norfolk, Edited by HW Jones, Jr, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p 126 17. Oehninger S, Acosta AA, Kreiner D, Muasher SJ, Jones HW Jr, Rosenwaks Z: In vitro fertilization and embryo transfer: an established and successful therapy for endometriosis. J In Vitro Fert Embryo Transfer 5:249, 1988 18. Muasher SJ, Garcia JE, Jones HW Jr: Experience with diethylstilbestrol-exposed infertile women in a program of in vitro fertilization. Fertil Steril 42:20, 1984 19. Navot D, Muasher SJ, Oehninger S, Liu H-C, Veeck LL, Kreiner D, Rosenwaks Z: The value of in vitro fertilization for the treatment of unexplained infertility. Fertil Steril49: 854,1988 20. World Health Organization: Laboratory Manual for the Examination of Human Semen and Semen -Cervical Mucus Interaction. Cambridge, University Press, 1987, p 27 21. Hodgen GD, Burkman LJ, Coddington CC, Franken DR, Oehninger SC, Kruger TF, Rosenwaks Z: Hemizona assay (HZA): finding sperm that have the "right stuff." J In Vitro Fert Embryo Transfer 5:311, 1988 22. Oehninger S, Acosta AA, Kruger TF, Veeck LL, Flood J, Jones HW Jr: Failure of fertilization in IVF: the "occult" male factor. J In Vitro Fert Embryo Transfer 5:181, 1988 23. Lanzendorf S, Maloney MK, Veeck LL, Slusser J, Hodgen GD, Rosenwaks·Z: A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil Steril 49:835, 1988 24. Laws-King A, Trounson A, Sathananthan H, Kola I: Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida. Fertil Steril 48:637, 1987 25. Gordon JW: Use of micromanipulation for increasing the efficiency of mammalian fertilization in vitro. Ann NY Acad Sci 541:601, 1988

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