Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm

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1 FERTLTY AND STERLTY Copyright <> 1992 The American Fertility Society Vol. 57, No. 1, January 1992 Printed on acid free paper in U.S.A. Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm Ariff Bongso, Ph.D.* Chui Yee Fong, B.Sc. Soon-Chye Ng, M.D. Shan Ratnam, M.D. Department of Obstetrics and Gynaecology National University of Singapore, Singapore Objective: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. Design: Prospective. Setting: Assisted reproductive technology (ART) program. Patients: Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. Results: Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1 %; P < 0.01). Zona-free oocytes had higher fertilization rates than zonaintact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). Conclusions: One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients. Fertil Steril1992;57: The ability to consistently achieve optimal fertilization rates is an essential prerequisite to the success of any assisted reproduction program. Fertilization rates in most successful in vitro fertilization (VF) programs range from 60% to 90%. However, it has been observed that 12.5% to 30% of oocytes will not fertilize after the first insemination, and in some patients there could be total fertilization failure. This could be because of male factors or suboptimal oocyte quality. 1 2 The male factors may include an inadequate sperm acrosome reaction and/ or poor curvilinear velocity, whereas the poor oocyte quality may be related to maturation and other unknown intrinsic problems. Total fertilization failure Received March 20, 1991; revised and accepted August 20, * Reprint requests: Ariff Bongso, Ph.D., Department of Obstetrics and Gynaecology, National University Hospital, Lower Kent Ridge Road, Singapore Vol. 57, No. 1, January hours after insemination poses a big problem for the embryologist and clinician to investigate and plan out the management of the next cycle for the childless couple. Reinsemination of the patient's oocytes in that particular cycle with her husband's fresh sperm has given varied results in terms of pregnancy rates n some patients, total fertilization failure occurs even after reinsemination. t is therefore necessary to investigate whether there is an intrinsic oocyte problem or the husband has an unidentified sperm problem, even when sperm count, motility, and morphology were normal. The hamster penetration assay and hemizona-binding assay are the currently available tests to predict the fertilizability of human sperm. 6 7 Both these tests, although useful, are indirect measures of assessment, require time and facilities for preparation, and are not always predictable. The direct fertilization of human oocytes with human sperm will be an ideal assay to not only evaluate sperm fertilizability but Bongso et al. Cytogenetics of reinseminated oocytes 129

2 also intrinsic problems in the oocyte. The use of fresh donor oocytes for such an assay would be unethical. A study was therefore undertaken to explore the possibility of using leftover 48-hour unfertilized human oocytes from an VF program, as an oocyte penetration assay to check sperm or oocyte fertilizability. The objective of the study was to find out if 48-hour unfertilized oocytes (from cohorts in which other oocytes had fertilization) could be fertilized by fertile donor sperm. The resulting reinseminated cleaved embryos were karyotyped to investigate the relationship between chromosome anomalies and oocyte aging in vitro. MATERALS AND METHODS Human Oocytes With Failed Fertilization A total of 497 metaphase oocytes that failed to fertilize 48 hours after insemination in 97 patients enrolled in an assisted reproduction program were used in this study. The oocytes comprised four groups: A, B, C, and D. Unfertilized oocytes of groups A and B were those from cohorts in which the remaining oocytes in the patient's cycle had fertilization. Group A oocytes were maintained zona-intact, whereas group B oocytes had their zonae removed for donor insemination. Group C and D oocytes were those from patients with total fertilization failure. Group C oocytes were maintained zona-intact, whereas group D were zona-free for donor insemination. Patients in all three groups were those admitted for gamete intrafallopian transfer, tubal embryo transfer at two-pronuclear stage, or VF, with histories of blocked tubes or idiopathic subfertility. The patients were on one of two stimulation regimens: (1) follicule-stimulating hormone + human menopausal gonadotropin (hmg) or (2) gonadotropin-releasing hormone agonist+ hmg. The protocol for follicular measurement and monitoring of cycles, oocyte aspiration, oocyte maturation, and insemination were as described by Ng et al. 8 After denuding of cumulus at 15 to 20 hours, each oocyte was examined for the presence of two or more pronuclei by phase-contrast inverted optics and then monitored for cleavage until 48 hours. Only oocytes with no observable pronuclei and absence of cleavage at 48 hours were used in this study. Preparation of Zona-Free Oocytes A total of 224 unfertilized oocytes (120 oocytes in group Band 104 in group D) were treated with 0.5% pronase (Sigma Chemical Co., St Louis, MO) in phosphate-buffered saline (GBCO, Grand sland, NY) for 5 minutes at 37 C in 5% C0 2 in air to remove the zona. Zona removal was monitored under phase-contrast inverted optics, and as soon as the zona was completing dissolution, the oocytes were transferred to culture medium supplemented with serum. Remnants of the zona were removed by gentle pipetting. Sperm Samples Semen samples were from fertile donors with normal sperm parameters (concentration, motility, and morphology) according to World Health Organization recommendations (WHO, 1987). 9 Preparation of sperm for donor insemination was by the swim-up method similar to that used for VF 10 and sperm motility in all recovered samples was rapid and linear (~grade 2, WHO). nsemination With Donor Sperm All zona-intact and zona-free unfertilized oocytes were inseminated with 100,000 motile sperm/oocyte per 1 ml of culture medium, freshly prepared by the swim-up method from known fertile donors. All oocytes were examined 15 to 20 hours after insemination for the presence of pronuclei and 48 hours later for cleavage. The quality of embryos (grade 3: regular blastomeres with slight to no fragments; grade 2: regular blastomeres with moderate fragments; grade 1: unequal blastomeres with many fragments) were recorded, and all cleaved embryos were subjected to chromosome analysis. Chromosome Analysis Forty-eight-hour zona-intact embryos at the two to five-cell stage were transferred to 1 ~tg/ml colcemid solution (GBCO) and incubated for 18 to 20 hours at 37 C in 5% C0 2 in air before fixation for chromosome analysis according to modifications of the methods of Tarkowski 11 and Bongso et at.l 2 Briefly, the embryos were placed in 1% chilled sodium citrate in watch glasses and kept at room temperature for 10 minutes. Each embryo was then transferred to a precleaned grease-free glass slide, excess citrate removed, and one drop of freshly prepared fixative (3:1, methanol:glacial acetic acid) in a 1-mL tuberculin syringe fitted with a 25-G needle was placed directly over the embryo to allow it to flatten. Excessive fixation was avoided to prevent artifactual scattering and loss of chromosomes. The slides were air dried, stained with Giemsa, and examined under bright field optics. Zona-free embryos 130 Bongso et al. Cytogenetics of reinseminated oocytes Fertility and Sterility

3 were not karyotyped. Approval for this study was obtained from the hospital ethical committee. Statistical Analysis The fertilization rates between groups A and C (zona-intact) and groups B and D (zona-free) were analyzed by the Student's t-test. Analysis of data involved comparing the effect of treatment for each patient. RESULTS The results of fertilization (all4 groups), cleavage, and chromosome analysis (groups A and C) for zonaintact and zona-free oocytes are shown in Table 1. Fertilization rates in oocytes of patients in which there was fertilization in the same cohort with their husbands' sperm (groups A and B) were significantly higher than those of patients with total fertilization failure (groups C and D) (33.2% to 60.9% versus 20.0% to 48.1 %; P < 0.01). Zona-free oocytes in both groups B and D had higher fertilization rates than zona-intact oocytes (groups A and C) (48.1% to 60.9% versus 20.0% to 33.2%). The incidence of multiple pronuclei (>2) was highest in zona-free oocytes (33.1% to 41.3%). From two to six pronuclei were observed per zona-free oocyte. All zona-intact oocytes showing evidence of two or more pronuclei cleaved to form two to five-cell embryos at 48 hours after insemination. Twenty-eight percent to 32% of good-quality embryos (grades 2 and 3) were observed in groups A and C. Cleavage occurred in all zonafree embryos containing two or more pronuclei, but the resulting blastomeres took irregular shapes and accumulated vacuoles in the absence of a zona. t was therefore not possible to grade such embryos. The overall incidence of chromosome anomalies for zona-intact embryos (groups A and C) was 48% to 54% (Table 1). The common chromosome anomalies observed were mosaicism, aneuploidy, and chromosome fragmentation (pulverization). DSCUSSON t is clear from the results of this study that the 48-hour unfertilized zona-intact and zona-free human oocyte is still fertilizable. This was true for patients whose remaining oocytes fertilized with their husband's sperm as well as for those with total fertilization failure. The low fertilization rate of 20% to 33.2% in 48-hour zona-intact oocytes in the present study (compared with a mean fertilization rate of 70% observed in our VF program) is suggestive of a zona-hardening process in vitro that limits fertilization. The fact that fertilization rates were lower in oocytes of patients with total fertilization failure (20.0% to 48.1 %) when compared with the oocytes of patients who had fertilization with their husband's sperm (33.2% to 60.9%) suggests that a major cause of total fertilization failure may be because of intrinsic oocyte problems. This is also confirmed by the fact that many patients who had total fertilization failure to their husband's sperm also had total fertilization failure to donor insemination with fertile sperm. Additionally, even though zona-free oocytes (groups B and D) had higher fertilization rates than zona-intact oocytes (groups A and C), zonafree oocytes in patients with total fertilization failure showed significantly lower fertilization rates than those in group B. These intrinsic oocyte problems therefore appear to be confined to both the zona and oolemma in those patients with total fertilization failure. The findings in the present study demonstrate that both zona-intact and zona-free unfertilized oo- Table 1 Fertilization, Cleavage, and Chromosome Anomalies of Resulting Embryos in Donor-nseminated 48-Hour Unfertilized Oocytes Fertilization Cleavage No. of No. of Chromosome Group patients oocytes 2 pronuclei >2 pronuclei Total Grade 3" Grade 2" Grade 1" anomalies A (zona-intact) B (zona-free) C (zona-intact)d D (zona-free)d Grade 3, regular blastomeres, no or few fragments; grade 2, regular blastomeres, moderate fragments; grade 1, irregular blastomeres, many fragments. b Group A significantly different from group C (P < 0.01, t-test). % 33.2b < 20.0b ' Group B significantly different from group D (P < 0.01, t-test). d Total fertilization failure with husband's sperm. Vol. 57, No.1, January 1992 Bongso et al. Cytogenetics of reinseminated oocytes 131

4 cytes may be of some value in investigating sperm fertilizability or intrinsic oocyte problems in patients with total fertilization failure after VF. There is usually a fair proportion of unfertilized oocytes generated from busy VF programs that are usually disposed of. The results also confirm earlier suggestions that insemination of unfertilized human oocytes with donor sperm may serve as a useful diagnostic test in cases of suspected male infertility or repeated failure of VF Other methods of assessing the fertilizability of sperm include the hamster penetration assay, hemizona, and complete zona binding assay These three methods are indirect and are not as reliable as assessment via direct fertilization of human oocytes with human sperm. Moreover, if zona-free human oocytes are used, low concentrations of sperm from oligozoospermic males can be tested. Although the hamster penetration assay reflects mainly the ability of sperm to undergo the acrosome reaction, 16 it can be still useful as a complementary test to the fertilization of 48-hour unfertilized human zona-intact or zona-free oocytes. Despite the long in vitro aging period (48 hours) of the unfertilized human oocyte, the ooplasm has been shown to be well preserved with no apparent alterations of cell organellesp t also appears that although there may be a certain degree of zona hardening with in vitro aging, the 48-hour zona is not resistant to the penetration of spermatozoa. However, the in vitro aging process reduces the penetration of zona-intact oocytes with more than one sperm because the incidence of polyspermy rates in such oocytes was much lower than that usually observed with fresh oocytes (1.0% to 2.1% versus 5%) when the same sperm concentrations of 100,000 sperm/oocyte per 1 ml of medium are used. Conversely, the removal of the zona allows penetration by multiple sperm even in the 48-hour oocyte. This was observed in the present study in which up to six pronuclei were seen in single 48-hour zona-free oocytes. This was also reported by Tesarik 17 where a mean of 7.3 ± 2.4 pronuclei per zona-free oocyte were observed when donor sperm was used for insemination. A detailed analysis of sperm penetration of human zona-free oocytes related to time of oocyte aging has been documented. 18 t thus appears that the receptor sites on the oolemma membrane are not affected by oocyte aging. The incidence of chromosome anomalies in embryos generated from oocytes reinseminated with donor sperm in this study (48% to 54%) was much higher than the percentage observed in embryos after first insemination with husband's sperm in VF programs (29% to 30%). 19 Mosaicism and aneuploidy were the most commonly observed anomalies in the embryos generated from aged oocytes in the present study. n vitro aging of oocytes has been shown to increase chiasma frequency, 20 and chromosome analysis of 48-hour unfertilized oocytes showed higher values of aneuploidy as compared with 24- hour unfertilized oocytes. 21 Further, in vitro aging was also shown to make the pronuclei in such oocytes lack the ability to achieve normally their deoxyribonucleic acid synthesis. 17 Maternal age was shown to significantly increase the rate of aneuploidy in fresh stimulated oocytes as well as those that failed to fertilize in VF programs Nondisjunction with anaphase lagging in vivo was postulated as the cause of such aneuploidy and perhaps the same mechanisms occur in vitro as the oocyte ages through 48 hours, resulting in mosaicism and aneuploidy. Chromosome pulverization or fragmentation is usually the result of degenerative changes in vitro. The low pregnancy rates after transfer of reinseminated embryos 3 and the increase in chromosome anomalies in such embryos because of oocyte aging do not warrant their replacement. The results of this study have helped us to some extent in the management of couples with total fertilization failure admitted to our VF program. Figure 1 outlines the steps undertaken in our program OOCYTE >Good nsemination in vitro (M) Good SPERM + Fails + HPA/HZPA/HZFPA/HZBA +ve -ve MST Evaluate AR (FPA) Fails Poor Oocyte donation nduce AR (Cal) 1/2 / M ' 1/2 MST "-Fails/ Spern1 donation Figure 1 Flow chart for management of patients with failed fertilization. AR, acrosome reaction; Cal, calcium ionophore; FP A, fluorescein peanut agglutinin; HP A, hamster penetration assay; HZBA, human zona binding assay; HZFPA, human zona-free penetration assay; HZPA, human zona penetration assay; M, macroinsemination; MST, microinsemination sperm transfer. 132 Bongso et al. Cytogenetics of reinseminated oocytes Fertility and Sterility

5 to diagnose and counsel patients with total fertilization failure. f there is total failure of fertilization after first insemination or reinsemination with the husband's sperm, an immediate investigation into whether there is a sperm or intrinsic oocyte problem is undertaken. The unfertilized oocytes of that particular cycle will be inseminated with sperm from a known fertile donor. f the oocytes fertilize, an intrinsic oocyte problem may be ruled out. The husband's fresh or frozen-thawed sperm will then be investigated by using it for insemination with zonaintact or zona-free unfertilized oocytes from another patient who was shown to have previous fertilization. f the patient's zona-intact oocytes did not fertilize, but spare zona-free unfertilized oocytes fertilized to proven donor sperm, then a zonal problem is suspected, and the patient will be admitted for microinjection of her husband's sperm into the perivitelline space of her oocyte by bypassing the zona. We have also attempted to induce the acrosome reaction with calcium ionophore in males who have a suspected sperm problem if they do not fertilize donor oocytes. t is only if all these avenues fail that we consider sperm or oocyte donation. The human zona-intact or zona-free assay may thus provide some information on fertilization for VF centers that do not have the facilities and staff for the hamster penetration assay. Acknowledgments. The authors thank Shivani Vasuthevan, B.Sc., and Mrs. Helen Mok, Department of Obstetrics and Gynaecology, National University of Singapore; Singapore, for their kind help and all clinicians involved in the department's assisted reproduction program for their assistance. Appreciation is extended to the National University of Singapore for facilities. The secretarial assistance of Miss Harjeet Kaur is gratefully acknowledged. REFERENCES 1. Lopata A: Concepts in human in vitro fertilization and embryo transfer. 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