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

Human Reproduction vol.13 no.4 pp.1014–1019, 1998 Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic ...
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Human Reproduction vol.13 no.4 pp.1014–1019, 1998

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

L.Rienzi1, F.Ubaldi, R.Anniballo, G.Cerulo and E.Greco Reproductive Medicine, European Hospital, Rome, Italy 1To

whom correspondence should be addressed

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 cumulus–corona– 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, ø3 h (18 cycles); group II, .3–ø6 h (52 cycles); group III, .6–ø9 h (14 cycles); and group IV, .9– ø12 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 χ2 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 (ø20% 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

Introduction 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 1014

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). © European Society for Human Reproduction and Embryology

Preincubation of oocytes may improve fertilization by ICSI

Trounson et al. (1982) postulated that preincubation of cumulus–corona–oocyte 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 Patients 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. Ovarian stimulation In all cycles ovarian stimulation was performed using a gonadotrophinreleasing hormone analogue (buserelin; Suprefact; Hoechst, Marion Roussel, Milan, Italy), menotrophins (Metrodin) and human chorionic gonadotrophin (HCG). Administration of the agonist was started on day 21 of the menstrual cycle, with a dose of 100 mg administered intranasally six times daily. When serum oestradiol concentrations were ø40 pg/ml and ovarian follicular structures on ultrasound were ,8 mm in diameter, ovarian stimulation was usually started with 2 ampoules/day (150 IU) follicle stimulating hormone (FSH) for 4 days. Thereafter, the dose of FSH was adapted individually according to the serum oestradiol increment and ultrasound measurements of follicular diameter (Smitz et al., 1992). From day 5 of the stimulated cycle onwards, blood samples were taken and assayed for oestradiol and progesterone. Ovulation was induced with 10 000 IU HCG when serum oestradiol concentrations exceeded 1000 pg/ml and when at least three follicles ù18 mm in diameter were recorded by ultrasound. Oocyte retrieval was carried out 36 h after HCG administration by ultrasound-guided transvaginal aspiration. Semen evaluation and preparation Semen samples were collected at the time of oocyte retrieval by masturbation after 3–7 days of sexual abstinence and were allowed to liquefy for at least 20 min at 37°C. Sperm concentration and motility were assessed by microscopy using a Makler counting chamber (Sefi-Medical Instruments Ltd, Haifa, Israel) according to World Health Organization (1992) criteria. Semen morphology was then assessed according to Kruger’s strict criteria (Kruger et al., 1986). First, sperm samples were washed by centrifugation at 1200 g for 10 min in HEPES-buffered medium supplemented with human serum albumin (Gamete 100; Scandinavian IVF Science AB, Gothenburg, Sweden). Then the resuspended pellet was layered onto a discontinu-

ous gradient (PureSperm; Scandinavian IVF Science AB) on two layers (45 and 90%) and centrifuged at 300 g for 30 min. The entire 90% layer was washed twice by adding 2 ml Gamete 100 and centrifuging at 1600 g for 5 min to remove silica gel particles. In the event of a very low sperm count, the semen was prepared by washing twice and centrifugation. The concentration of the prepared sperm suspension was, when possible, adjusted to 13106/ml. Samples were stored at room temperature until ready for ICSI. The incubation period of the prepared spermatozoa was approximately the same as that of the oocytes. Oocyte preparation During oocyte retrieval, the aspirated follicular fluid was passed to the adjoining laboratory. CCOC were identified in sterile plastic dishes (cat. no. 1029, Falcon; Becton-Dickinson Labware, Franklin Lakes, NJ, USA), rinsed, transferred in IVF-50 medium (Scandinavian IVF Science AB) and incubated at 37°C in an atmosphere of 5% CO2 in air until the ICSI procedure. This preincubation period lasted from 2 to 12 h. The 95 ICSI cycles performed were divided into four groups depending on the time lapse between oocyte retrieval and ICSI: group I, ø3 h (18 cycles); group II, .3–ø6 h (52 cycles); group III, .6–ø9 h (14 cycles); and group IV, .9–ø12 h (11 cycles). Immediately before the ICSI procedure the cumulus and corona cells were removed by a brief exposure to Gamete 100 (HEPESbuffered medium) containing 80 IU/ml hyaluronidase Fraction VIII (Hyase-10X; Scandinavian IVF Science AB). To enhance enzymatic removal of the cumulus and corona cells, the oocytes were aspirated in and out of a hand-drawn Pasteur pipette with an approximate inner diameter of 130 µm (Laboratory Pipette art. no. 11130; Sweemed Lab International AB, Va¨sta Fro¨lunda, Sweden). Denudation was performed in a four-well culture dish (cat. no. 45-176740; Nunc Bround Products, Kamstrup, Denmark). Denuded oocytes were washed twice and incubated in IVF-50 medium until ICSI was performed. Oocytes were then examined under an inverted microscope at 3200 magnification to assess integrity and the stage of maturation. Only morphologically normal-appearing mature oocytes with a visible first polar body were microinjected. Equipment for micromanipulation ICSI was performed using a Nikon (Nikon Ltd, Tokyo, Japan) Diaphot TMD microscope equipped with Hoffman Modulation contrast, a 37°C heating stage (cod. MS100; Linkam Scientific Instruments Ltd, Surrey, UK), two Narishige MM188 electric coarse movement controls, two MO188 3D oil hydraulic micromanipulators and two Narishige IM6 injectors for holding the oocyte and the injector pipette (Narishige Ltd, Tokyo, Japan). Microtools for ICSI The holding pipette used to hold the oocyte in position during ICSI had an outer diameter of 130 µm, an inner diameter of 15 µm and a distal tip angle of 35° (cat. no. K-HPIP-1035; Cook, Queensland, Australia). The microinjection needle pipette had an outer diameter of 7 µm, an inner diameter of 5 µm, a heat-formed spike and a distal tip angle of 35° (cat. no. K-MPIP-20135; Cook). ICSI procedure ICSI was performed in microdroplets under oil (Ovoil-150; Scandinavian IVF Science AB) using plastic culture dishes (cat. no. 1006, Falcon; Becton-Dickinson Labware) under a microscope at 3400 magnification. Immediately before microinjection, a 5 µl droplet of 10% polyvinylpyrrolidone (PVP) solution in IVF-50 medium (ICSI-100; Scandinavian IVF Science AB) was placed in the middle

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of a Petri dish and surrounded by eight droplets of 10 µl Gamete 100 medium. The droplets were then covered by pre-equilibrated mineral oil to avoid evaporation. Finally, 1–2 µl sperm suspension were added to the middle of the drop containing PVP and an oocyte was placed in each surrounding droplet. When the sperm suspension was added to the drop containing PVP, motile spermatozoa migrated into the viscous medium, which decelerated the spermatozoa allowing for careful observation and facilitation of aspiration. A single spermatozoon of apparently normal morphology was selected, immobilized by touching the tail and aspirated tail first in the injection pipette. The pipette containing the sperm cell was then moved from the PVP droplet into one of the peripheral droplets containing an oocyte. The oocyte was rotated to locate the first polar body at the 6 o’clock position, held by gentle suction on the holding pipette, and the equatorial plane located in focus. The spermatozoon was then ejected slowly, close to the tip and the edge of the pipette, and pushed gently horizontally from the 3 o’clock position deep into the ooplasm. To ensure that the spermatozoon was always deposited intracytoplasmically, gentle suction was carefully applied to break the oolemma membrane. The cytoplasmic organelles and the spermatozoon were ejected back into the cytoplasm slowly with the smallest amount of medium possible. Thereafter the injection pipette was gently withdrawn and the oocyte released from the holding pipette. This procedure was repeated for each oocyte. The injected oocytes were then rinsed in IVF-50 medium and placed one by one in an equilibrated culture dish (cat. no. 3802, Falcon; Becton-Dickinson Labware) containing eight droplets of 30 µl IVF-50 medium. Assessment of oocyte survival, fertilization and further development Oocytes were observed 12–18 h after ICSI to assess the presence of pronuclei (PN) and polar bodies. Fertilization was considered normal only when two distinct pronuclei containing nucleoli were present. Embryo cleavage of the two-pronuclear oocytes was evaluated after 24 h of in-vitro culture. For each embryo, the number and size of the blastomeres were recorded, as well as the percentage of anucleate fragments. Cleaved embryos with ,20% anucleated fragments and with equal-sized blastomeres were considered type A. If the percentage of anucleate fragments was between 20 and 50% the embryos were considered type B. If .50% anucleated fragments were present then the embryos were considered type C. Only type A and B embryos were eligible for transfer. The selected embryos were placed in 2 µl IVF-50 medium and transferred to a Frydman catheter (Frydman 5,5; Laboratoire CCD, Paris, France). They were then transferred to the uterine cavity. The number of embryos transferred depended on the age of the patient and embryo quality. Luteal phase assessment and establishment of pregnancy The luteal phase was supported using natural progesterone in oil, 50 mg/day i.m. (Gestone 100; Amsa, Barberino del Mungello, Italy), starting on the day after the HCG injection. Pregnancy was confirmed by a serial rise in serum HCG concentration on two consecutive occasions at least 12 days after embryo replacement. A clinical pregnancy was confirmed by detecting (using ultrasound) fetal cardiac activity at 7 weeks. Statistical evaluation All statistical tests were performed using the Statview 4.0 statistical package for the Macintosh (Abacus Concepts Inc., Berkeley, CA, USA). Tests were carried out (two-tailed) at the 5% level of significance. For comparison of the mean of the variables, the unpaired Student’s t-test was used. For comparisons of fertilization, cleavage,

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implantation and pregnancy rates between the groups, a χ2 analysis was used.

Results No statistically significant differences were observed between the four groups with regard to the mean age of the patients (33.1, 32.4, 30.8 and 33.6 years respectively). A total of 943 CCOC were retrieved. The mean numbers of CCOC retrieved were comparable between the four groups. After removing the cumulus and corona cells, 75 CCOC contained a germinal vesicle-stage oocyte (7.9%), 53 contained metaphase I (MI) oocytes (5.6%) and 753 contained MII oocytes (79.8%). In all, 62 CCOC (6.6%) contained no oocytes or were damaged following manipulation errors. Table I reports the maturational stages of the oocytes retrieved for each group. A total of 723 oocytes that had extruded a polar body were injected with a single spermatozoon from the patient’s partner. The proportions of oocytes remaining intact after ICSI were similar between the four groups (92.9, 96.6, 93.6 and 94.5% respectively; Table II). There was a significant difference (P , 0.05 using the χ2 test) in the fertilization rate according to the time lapse between oocyte retrieval and ICSI. The lowest fertilization rate of 52.3% was observed when the preincubation period of the oocytes was ,3 h (group I), while the fertilization rates were 66.8, 65.1 and 69.2% for groups II, III and IV respectively (Table II). There was no significant difference observed in the proportion of parthenogenetically activated oocytes. The mean percentage of unipronuclear oocytes was 1.7%. The percentages of oocytes displaying three or more pronuclei were similar for all groups (3.9, 2.9, 2.4 and 1.0% respectively). The mean percentage of good quality embryos (1–20% fragmentation, type A) was 37.0%. There was a significant difference in the percentage of good quality embryos (P , 0.05) between group I and groups II and IV. The lowest percentage of type A embryos was found in group I (24.2%), while in groups II, III and IV the percentages were 39.8, 36.5 and 39.6 respectively. There was also a significant difference (P , 0.05) in the percentage of fair quality embryos (embryos with 20–50% fragmentation, type B) in the four groups. The highest percentage of type B embryos was found in group I (43.9%). No significant differences were observed in the percentage of poor quality embryos (embryos with .50% fragmentation, type C) in the four groups. Therefore the percentages of transferable embryos (types A and B) were similar in all four groups (68.1, 69.7, 79.2 and 79.3% respectively; Table III). A total of 317 embryos were transferred (3.3 per patient, range 1–5): 51 in group I, 174 in group II, 48 in group III and 44 in group IV (Table IV). Rising serum HCG concentrations at least 12 days after embryo transfer were recorded in 33 cycles (34.7%); a clinical pregnancy was confirmed by ultrasound in week 7 in 27 cycles (28.4%). The frequency of sac formation per embryo transferred was 13.6%. 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), pregnancy rates per embryo transfer (22.2, 36.5,

Preincubation of oocytes may improve fertilization by ICSI

Table I. Maturity stage of the cumulus–corona–oocyte complexes (CCOC) retrieved in relation to the preincubation period between oocyte retrieval and intracytoplasmic sperm injection Parameter

No. No. No. No. No. No.

of of of of of of

Total

cycles CCOC retrieved metaphase II oocytes (%) metaphase I oocytes (%) germinal vesicle oocytes (%) empty zona (%)

95 943 753 53 75 62

Preincubation period (h)

(79.8) (5.6) (7.9) (6.6)

ø3

.3–ø6

.6–ø9

.9–ø12

18 153 126 5 10 12

52 499 391(78.3) 37 (7.4) 36 (7.2) 35 (7.0)

14 171 142 6 19 4

11 120 94 (78.3) 5 (4.2) 10 (8.3) 11 (9.2)

(82.3) (3.3) (6.5) (7.8)

(83.0) (3.5) (11.1) (2.3)

Table II. Survival and fertilization rates after intracytoplasmic sperm injection in relation to the preincubation period Parameter

Total

No. of injected oocytes No. of intact oocytes Percentage of intact oocytes Pronuclear status of injected oocytes No. of 2PN oocytes (%) No. of 1PN oocytes (%) No. of 3PN oocytes (%)

Preincubation period (h) ø3

.3–ø6

.6–ø9

.9–ø12

723 688 95.2

126 117 92.9

380 367 96.6

126 118 93.6

91 86 94.5

465 (64.3) 12 (1.7) 20 (2.8)

66 (52.3)a – 5 (3.9)

254 (66.8)a 6 (1.5) 11 (2.9)

82 (65.1)a 4 (3.2) 3 (2.4)

63 (69.2)a 2 (2.2) 1 (1.0)

PN 5 pronuclear. aP , 0.05 using the χ2 test.

Table III. Embryo development rates after intracytoplasmic sperm injection in relation to the preincubation period Parameter

Total

No. of 2PN oocytes Embryo quality of cleaved 2PN oocytes Type A (%) Type B (%) Type C (%)

Preincubation period (h) ø3

.3–ø6

.6–ø9

.9–ø12

465

66

254

82

63

172 (37.0) 165 (35.5) 93 (20.0)

16 (24.2)a 29 (43.9)a 13 (19.7)

101 (39.8)a 30 (36.5) 76 (29.9)a 35 (42.7) 57 (22.4) 14 (17.1)

25 (39.6)a 25 (39.6)a 9 (14.3)

PN 5 pronuclear. aP , 0.05 using the χ2 test.

Table IV. Implantation and pregnancy rates after intracytoplasmic sperm injection in relation to the preincubation period Parameter

No. of cycles No. of embryos transferred Mean no. of embryos per transfer No. of pregnancies (HCG1) Pregnancy rate per cycle (%) No. of clinical pregnancies Clinical pregnancy rate (%) No. of gestational sacs Implantation rate (%) No. of ongoing pregnancies Ongoing pregnancy rate per cycle (%)

Total

95 317 3.3 33 33/95 (34.7) 27 27/95 (28.4) 43 43/317 (13.6) 22 22/95 (23.2)

Preincubation period (h) ø3

.3–ø6

.6–ø9

.9–ø12

18 51 2.8 4 4/18 3 3/18 6 6/51 3 3/18

52 174 3.3 19 19/52 (36.5) 15 15/52 (28.8) 23 23/174 (13.2) 11 11/52 (21.1)

14 48 3.4 5 5/14 4 4/14 5 5/48 3 3/14

11 44 4 5 5/11 (45.4) 5 5/11 (45.4) 9 9/44 (20.4) 5 5/11 (45.4)

(22.2) (16.6) (11.7) (16.6)

(35.7) (28.5) (10.4) (21.4)

HCG 5 human chorionic gonadotrophin.

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35.7 and 45.4% respectively), clinical pregnancy rates per embryo transfer (16.6, 28.8, 28.5 and 45.4% respectively) and miscarriage rates per pregnancy (25.0, 31.6, 40.0 and 0.0%) (Table IV).

Discussion The aim of this study was to analyse the possible effects of preincubation of CCOC before ICSI on fertilization and embryo development rates. Because fertilization failure after ICSI can be caused by cytoplasmic immaturity of the treated oocytes, a specific programme of in-vitro maturation, such as a longer preincubation period before microinsemination (as suggested previously by Calafell et al., 1991) could help to obtain a higher percentage of oocytes with mature cytoplasm, thus resulting in a higher activation rate after ICSI. All the oocytes were collected 36 h after HCG administration. At the time of recovery it was not possible to ascertain whether the oocytes were completely mature. The timing of the HCG injection provides some information about the length of time that the follicles are exposed to the stimulus for resumption of meiosis. However, because the capacity of the follicles to bind and thus respond to HCG can be variable, and because human oocytes are collected from follicles of different size, some oocytes may be cytoplasmically immature despite having reached MII. In this study, the time of preincubation of the CCOC between oocyte retrieval and ICSI was random and did not depend on the size of the follicles or the aspect of the cumulus and corona cells. We analysed and compared the fertilization potential and embryo development of the oocytes microinjected with a single spermatozoon after four different periods of preincubation: ø3, .3–ø6, .6–ø9 and .9– ø12 h. The oocytes were incubated with the cumulus–corona cells that were removed immediately before the injection. At the time of enzymatic and mechanical removal of the cumulus and corona cells, no differences were observed between the four groups concerning the percentage of oocytes at MII or the percentage of immature oocytes (MI and germinal vesicle stages). Nagy et al. (1996) postulated that after the removal of cumulus–corona cells, in-vitro maturation of 50–70% of the oocytes at the germinal vesicle stage normally occurs 30– 32 h after oocyte retrieval, while only 30–50% of MI oocytes complete maturation 1–5 h after oocyte retrieval, depending on the culture conditions. Immature oocytes were not used for the patients in our study, but a future option may be to inject them after maturation following in-vitro culture (Van Steirteghem et al., 1996) or even at an immature stage (Hamberger, 1996). The fact that we did not find a decrease in the percentage of MI oocytes even after a long preincubation period (9–12 h) may indicate that the percentage of MI oocytes that mature in vitro if incubated with cumulus and corona cells could be lower. It is not clear what the explanation for this observation can be. One possibility is that oocytes, which are enclosed in cumulus and corona cells, do not have the capability to undergo meiotic maturation, perhaps as a result of the blocking effect of the neighbouring cells. In this respect it would be interesting to test whether meiotic maturation would 1018

take place more readily if oocytes were incubated without cumulus and corona cells. Because semen was collected and processed at the same time as oocyte retrieval was performed, the preincubation time period for the spermatozoa corresponded approximately to the preincubation period of the cumulus–oocyte complexes. However, this varying incubation period of spermatozoa probably has no important influence on the outcome of ICSI because it is known that after the selection procedure spermatozoa retain their fertilizing capacity for a longer time. This has been observed by Palermo et al. (1993), who used spermatozoa after a 24 h incubation period for fresh oocyte injection, and by Nagy et al. (1993), who also used spermatozoa retrieved 24 h earlier to inject IVF failed-fertilized oocytes. There is, however, one situation when the longer incubation period of spermatozoa might be disadvantageous: when the motility of the spermatozoa is strongly impaired. In this case the motility of spermatozoa might be completely lost, which would strongly decrease the fertilization rate after ICSI. Only MII oocytes were injected. Even if no differences were observed in the percentage of meiotically mature oocytes, after the injection differences were observed in the normal 2pronuclear (2PN) fertilization rates for the four different preincubation periods. This finding could mean that a delay of insemination may allow the oocyte to complete cytoplasmic maturation, resulting in a higher activation rate after ICSI. It seems that oocytes injected soon after extrusion of the first polar body are unable to proceed through the second meiotic division. Similar observations have been made previously in mouse IVF experiments (Kubiak, 1989). These findings are also similar to those reported by Trounson et al. (1982), who showed that relatively short periods of in-vitro culture (5– 5.5 h) following the recovery of human oocytes, before performing conventional IVF, were beneficial for the completion of oocyte maturation, thus promoting higher rates of fertilization. Haploidy, resulting from failure in the fertilization process, was observed only after .3 h of in-vitro culture. In these cases the oocytes had activated after injection, ejected their second polar bodies and formed large pronuclei with nucleoli. It has been shown that ageing oocytes are much more sensitive to activation than fresh oocytes (Plachot and Crozet, 1992); therefore these 1PN oocytes might represent a population of overmature oocytes which activate very readily after ICSI but are unable to induce or support male pronucleus development (Flaherty et al., 1995). However, the absence of parthenogenetically activated oocytes in group I could also be a result of the small number of oocytes analysed. Some oocytes also developed three pronuclei after injection. It has been speculated by others (Palermo et al., 1993) that the extra chromosome set of digynic triploid oocytes is of maternal origin and may be derived from retention of the second polar body. This aetiology seems to be associated with poor gamete quality or damage to the oocyte’s cytoskeleton during injection. In this study the percentage of haploid and triploid oocytes appeared to be unrelated to the preincubation period of the oocytes. Even 9–12 h of in-vitro culture of the oocytes before ICSI did not seem to impair oocyte quality, resulting in an unchanged

Preincubation of oocytes may improve fertilization by ICSI

percentage of digynic oocytes after ICSI. However, Tesarik et al. (1994) postulated that a longer preincubation period could affect oocyte quality. The capacity of the cytoplasm of MII oocytes to decondense sperm DNA, resume meiosis and promote the evolution of a male pronucleus appears to decline progressively 24 h after oocyte retrieval (Nagy et al., 1993). Comparison of the percentage of good quality embryos (embryos with 0–20% fragmentation) with the different preincubation periods of the oocytes has shown that better quality embryos are more usually generated after a delay in insemination (of at least 3 h). These data suggest that the invitro incubation of oocytes could also have an influence on the developmental capacity of a microinjected oocyte. However, no statistically significant differences have been observed between the different groups regarding implantation and pregnancy rates. This might be due to the relatively small number of cases analysed. Our findings suggest that human oocytes gradually develop the ability to activate during their arrest at MII. Furthermore, a long period of in-vitro culture (9–12 h) before ICSI does not appear to impair oocyte quality or an oocyte’s capacity to form a viable embryo. Therefore the optimum time range for more successful ICSI (in terms of fertilization and embryo development rates) seems to be between 3 and 12 h after oocyte retrieval, when oocytes are likely to achieve full cytoplasmic maturation. References Calafell, J.M., Badenas, J., Egozcue, J. et al. (1991) Premature chromosome condensation as a sign of oocyte immaturity. Hum. Reprod., 7, 1017–1021. Dozortsev, D., Rybouchkin, A., De Sutter, P. et al. (1995) Human oocyte activation following intracytoplasmic sperm injection: the role of sperm cell. Hum. Reprod., 10, 403–407. Eppig, J.J., Schultz, R.M., O’Brien, M. et al. (1994) Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev. Biol., 164, 1–9. Flaherty, S.P., Payne, D., Swann, N.J. et al. (1995) Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum. Reprod., 10, 2623–2629. Gerris, J., Van Royen, E., Mangelschots, K. et al. (1994) Pregnancy after intracytoplasmic sperm injection of metaphase II oocytes in a man with complete retrograde ejaculation. Hum. Reprod., 9, 1293–1296. Hamberger, L. (1996) Intracytoplasmic sperm injection in Scandinavia. Hum. Reprod., 11 (Suppl. 1), 77–79. Homa, S.T., Carrol, J. and Swann, K. (1993) The role of calcium in mammalian oocyte maturation and egg activation. Hum. Reprod., 8, 1274–1281. Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, 1118–1123. Kubiak, J.Z. (1989) Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest. Dev. Biol., 136, 537–545 Lanzendorf, S.E., Maloney, M.K., Veek, L.L. et al. (1988a) A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil. Steril., 49, 835–842. Lanzendorf, S., Maloney, M., Ackerman, S. et al. (1988b) Fertilization potential of acrosome-defective sperm following microsurgical injection into eggs. Gamete Res., 19, 329–337. Lundin, K., Sjogren, A., Nilsson, L. et al. (1994) Fertilization and pregnancy after intracytoplasmic sperm microinjection of acrosomeless spermatozoa. Fertil. Steril., 62, 1266–1267. Nagy, Z.P., Jorris, H., Staessen, C. et al. (1993) Intracytoplasmic sperm injection of 1 day-old unfertilized human oocytes. Hum. Reprod., 8, 2180–2184. Nagy, Z.P., Liu, J., Janssenswillen, C. et al. (1995a) Using ejaculated, fresh, and frozen–thawed epididymal and testicular spermatozoa gives rise to

comparable results after intracytoplasmic sperm injection. Fertil. Steril., 63, 808–815. Nagy, Z.P., Liu, J., Joris, H. et al. (1995b) The results of intracytoplasmic sperm injection are not related to any of the three basic sperm parameters. Hum. Reprod., 10, 1123–1129. Nagy, Z.P., Liu, J., Janssenswillen, C. et al. (1996) Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. Fertil. Steril., 65, 1047–1050. Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic sperm injection of a single spermatozoon into an oocyte. Lancet, 340, 17–18. Palermo, G., Joris, H., Derde, M.P. et al. (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil. Steril., 59, 826–835. Plachot, M. and Crozet, N. (1992) Fertilization abnormalities in human invitro fertilization. Hum. Reprod., 7 (Suppl. 1), 89–94. Schoysman, R., Van der Zwalmen, P., Nijs, M. et al. (1993) Pregnancy after fertilization with human testicular sperm. Lancet, 342, 1237. Silber, S., Nagy, Z.P., Liu, J. et al. (1994) Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum. Reprod., 9, 1705–1709. Silber, S., Van Steirteghem, A., Liu, J. et al. (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicular biopsy. Hum. Reprod., 10, 148. Smitz, J., Van Den Abbeel, E., Bollen, N. et al. (1992) The effect of gonadotrophin-releasing hormone (GnRH) agonist in the follicular phase on in-vitro fertilization outcome in normo-ovulatory women. Hum. Reprod., 7, 1098–1102. Tesarik, J. and Sousa, M. (1995) More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil. Steril., 63, 343–349. Tesarik, J., Sousa, M. and Testar, J. (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum. Reprod., 9, 978–980. Tournaye, H., Devroey, P., Liu, J. et al. (1994) Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil. Steril., 61, 1045–1051. Trounson, A.O., Mohr, M.R., Wood, C. et al. (1982) Effect of delayed insemination on in-vitro fertilization, culture and transfer of human embryos. J. Reprod. Fertil., 64, 285–294. Van Steirteghem, A.C., Liu, J., Joris, H. et al. (1993a) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum. Reprod., 8, 1055–1060. Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993b) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066. Van Steirteghem, A., Nagy, P., Joris, H. et al. (1996) The development of intracytoplasmic sperm injection. Hum. Reprod., 11 (Suppl. 1), 59–72. World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. 3rd edition, Cambridge University Press, Cambridge, UK. Received on October 13, 1997; accepted on January 27, 1998

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