Human Reproduction April 1993 [antikvár]

OPINION Are failed-fertilized human oocytes useful? N.J.Winston, P.R.Braude^ and M.H.Johnson' Embryo and Gamete Research Group, Department of Anatomy, University of Cambridge, Downing Sn-eet, Cambridge CB2 3DY and ^Assisted Conception Research Unit, United Medical Dental Schools of Guy's St Thomas's, Department of Obstetrics and Gynaecology, St Thomas's Hospital, London SEl 7EH, UK 'To whom correspondence should be addressed Key words: aged human oocyte/reinsemination/research/thera-peutie value Introduction Guidelines from the Human Fertilization and Embryology Authority (HFEA) require that patients undergoing treatment by in-vitro fertilization (TVF) give consent for use of their oocytes in research. Since these guidelines include aged failed-fertilized oocytes, there is an implication that they may still have some potential use in the creation of embryos in vitro or in a therapeutic context. Indeed, since there is a relatively poor pregnancy outcome following IVF treatment, and a large physical, psychological and often financial investment made by the patient, clinics wish to maximize the chances of successfully establishing a pregnancy after each treatment cycle. Reinseminating failed-fertilized oocytes seems to be a simple approach to achieve this objective. However, the value of oocyte reinsemination in human rVF therapy is not clear. In this report we review the data reporting reinsemination of failed-fertilized oocytes, present the results of a UK nationwide survey into the practice of reinsemination in current IVF therapy, and examine whether failed-fertilized human oocytes might be a usefiji source of material for research purposes. Can failed-fertilized oocytes produce pregnancies? Any assessment of the value of failed-fertilized oocytes must address whether all such oocytes were maWre at insemination. If they were not, their fertilization at reinsemination might simply reflect the completion of maturation in vitro. There is clear evidence that a proportion of cumulus-oophorus complexes recovered are inmiature, although their state of matarity, as judged by cumulus expansion, need not reflect that of the oocytes within them (Diedrich et al. ,1983; Laufer et al., 1984; Plachot et al, 1985). Cultare of immature cumulus complexes in vitro can lead to the maturation of their oocytes (Testart et al., 1983b; Boldt etal., 1987). The proportion of immature oocytes recovered is affected by the method of ovarian stimulation, being © Oxford University Press higher with use of clomiphene citrate and/or human menopausal gonadoO-ophin (HMG) because of the more asynchronous follicular growth than is observed with use of gonadotrophin releasing hormone (GnRH) superactive analogues combined with HMG (Cabau and Bessis, 1981; Testart et al., 1983a). The number of immature oocytes can also be reduced if administtation of human chorionic gonadotrophin (HCG) is delayed until more large follicles (>18 mm) are present. As a result the incidence of fertilization at first insemination rises, leaving fewer oocytes to be reinseminated and a lower overall contribution by them to the fertilized pool of oocytes (Trounson et al., 1982; Ben-Rafael et al., 1986; Boldt et al., 1987; Conaghan et al., 1989; Dimitry etal, 1991;FahmyetaZ., 1991; Pampiglione ef a/., 1990). For example, in an unpublished study (K.Dawson, N.Winston, R.Margara and R.M.L.Winston) the HCG injection was withheld until three follicles >17 mm were seen by ultrasound scan and the oocytes were inseminated 5-6 h after their collection. Reinsemination of 323 oocytes which had shown no sign of fertilization at 18 h post-insemination (hpi), and where no apparent male problem was present, resulted in fertilization of only 11 of them, an increase in the overall fertilization rate of 3%. These 11 fertilized oocytes were transferred to seven patients. A slight rise in /3HCG over background was observed in one patient, but no clinical pregnancies occurred. Taken together, these results suggest that reinsemination may be simply a 'rescuing' procedure for poor superovulation protocols and immattirity of oocytes at collection. If oocytes are allowed to marnre fully in vivo, the initial rate of fertilization is high and reinsemination is both unsuccessful and unnecessary. The presence of less mature oocytes may also complicate interpretation of reinsemination data in another way. If some immature oocytes complete maturation during the first few hours after exposure to spermatozoa, they might show delayed fertilization. If they are then examined for signs of fertilization at the same time as other more mature oocytes, usually at 18 to 24 h post-insemination, pronulei may not be evident and fertilization could be missed. Trounson and Webb (1984) showed that when oocytes with no sign of fertilization at 12 to 14 hpi were retained in culture without reinsemination, a significant proportion had formed pronuclei by 24-28 hpi. Therefore, if reinsemination is to be attempted as a therapeutic procedure, perhaps oocytes should be examined routinely at 18 hpi and any that are unfertilized should be inspected again at 24 hpi before reinsemination is attempted. If oocytes were genuinely mature at or shortly after recovery, they will have been cultured in vitro for upwards of 20 h by the time of their reinsemination. Is their potential for fertilization affected? The reports cited above, of poor fertilization of 503