Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection.

Human Reproduction Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection L.Rienzi 0 F.Ubaldi 0 R.Anniballo 0 G.Cerulo 0 E.Greco 0 0 Reproductive Medicine, European Hospital , Rome , Italy 1To whom correspondence should be addressed European Society for Human Reproduction and Embryology - The aim of this study was to examine the relationship between different preincubation periods of oocytes and the outcome of intracytoplasmic sperm injection (ICSI). We analysed retrospectively 95 ICSI treatment cycles performed to alleviate severe male-factor infertility. Oocyte collection was performed ~36 h after human chorionic gonadotrophin administration. The cumuluscorona oocyte complexes obtained were incubated until the moment of ICSI. Fertilization, embryo development and implantation rates were analysed in four groups, which were divided according to the time lapse between oocyte retrieval and ICSI: group I, 3 h (18 cycles); group II, .36 h (52 cycles); group III, .69 h (14 cycles); and group IV, .9 12 h (11 cycles). Immediately before ICSI the cumulus and corona cells were removed from the oocytes. A total of 723 metaphase II oocytes were injected: 126 from group I, 380 from group II, 126 from group III and 91 from group IV. The fertilization rates obtained were 52.3, 66.8, 65.1 and 69.2% respectively [P , 0.05 (using the c2 test) between group I and groups II, III and IV]. Cleavage rates were similar in all groups (68.1, 69.7, 79.2 and 79.3% respectively), but the proportion of good quality embryos (20% fragmentation) was significantly lower (P , 0.05) in group I (24.2%) compared with groups II (39.8%) and IV (39.6%). However, no statistically significant differences were observed between the four groups with regard to implantation rates (11.7, 13.2, 10.4 and 20.4% respectively). The results suggest that a preincubation period between oocyte retrieval and ICSI can improve the fertilization rate and embryo quality. This period might be necessary for some oocytes to reach full cytoplasmic maturity, leading to a higher activation rate upon microinjection. Key words: assisted reproduction/fertilization/ICSI/microinjection/oocyte maturity Intracytoplasmic sperm injection (ICSI) is the most widely applied type of assisted fertilization treatment for infertility involving mainly severe male-factor cases (Palermo et al., 1992; Van Steirteghem et al., 1993a,b). It has been established that the success rate of ICSI is unrelated to sperm concentration, motility and morphology (Nagy et al., 1995b). It has also been suggested that this technique can be successful in the treatment of globozoospermia (Lundin et al., 1994) and complete retrograde ejaculation (Gerris et al., 1994). Furthermore, it has been shown that high fertilization and pregnancy rates can be obtained using epididymal (Silber et al., 1994; Tournaye et al., 1994) or testicular spermatozoa (Schoysman et al., 1993; Nagy et al., 1995a; Silber et al., 1995). In addition, with the advent of ICSI, infertility caused by complete fertilization failure after conventional in-vitro fertilization (IVF) as a result of gamete interaction problems (Lanzendorf et al., 1988a,b) can be treated successfully. However, there are several aspects of ICSI that require clarification. One is the cellular mechanism that leads the injected metaphase II (MII) oocytes to activation, fertilization and syngamy. Meiotic maturation of the oocyte and subsequent activation of the egg by a spermatozoon are two separate events which are absolute prerequisites for normal fertilization. The oocytes are considered to be meiotically mature after extrusion of the first polar body, which is a characteristic of MII. However, nuclear and cytoplasmic maturation are acquired independently during oocyte maturation (Eppig et al., 1994). It has been observed that MII mouse oocytes gradually develop the capacity for activation after they have reached MII (Kubiak, 1989). After intracytoplasmic injection, the fertilizing spermatozoon makes two important contributions to the oocyte: (i) it contributes paternal DNA; and (ii) it is the trigger that activates the oocyte to complete the second meiotic division. It has been demonstrated recently that the spermatozoon releases the factor responsible for starting oocyte activation. This factor seems to be heat-sensitive, intracellularly active and not a species-specific substance, with an activity that is not identifiable in dead sperm cells (Dozortsev et al., 1995). However, Flaherty et al. (1995) found that many unfertilized oocytes after ICSI had a correctly injected spermatozoon that had undergone partial or complete nuclear decondensation. Fertilization failure could be caused by either the unsuccessful release of the activation signal by the spermatozoon or the lack of a response of the oocyte to the activation signal. Some authors (Tesarik and Sousa, 1995) have reported that the major cause of fertilization failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability of the oocyte to transduce the signal delivered by the injected spermatozoon could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterized by release from MII arrest and extrusion of the second polar body, followed by pronuclear formation. Both oocyte maturation and egg activation are apparently regulated by levels of intracellular free Ca21 (Homa et al., 1993). Trounson et al. (1982) postulated that preincubation of cumuluscoronaoocyte complexes (CCOC) before conventional insemination improves the outcome of IVF. They proposed that a period of culture in vitro is beneficial for the completion of oocyte maturation and to allow for high rates of fertilization and embryo development in vitro. No information is as yet available about the possible effect of preincubation of oocytes before performing ICSI. To evaluate whether the timing of ICSI has an effect on outcome, we analysed and compared ICSI cycles performed with different oocyte preincubation periods. In particular we were interested whether MII oocytes needed a preincubation period to reach cytoplasmic maturity and also the length of this period before fertilization and embryo development rates were affected. Materials and methods The outcomes of 95 consecutive ICSI cycles in which ejaculated semen was used for microinjection were analysed retrospectively. Couples were selected for ICSI treatment according to the following criteria: (i) total absence or ,20% normal fertilization after standard IVF; and (ii) ,500 000 progressively motile spermatozoa in the ejaculate. Before starting ICSI treatment the patients signed a consent form which included giving permission for a prenatal diagnosis by amniocentesis. In all cycles ovarian stimulation was performed using a gonadotrophinreleasing ho (...truncated)