Centre for Reproductive Medicine and Gleneagles IVF Centre, Gleneagles Hospital, Singapore 258500
1 To whom correspondence should be addressed. e-mail: suresh.cccrm@pacific.net.sg
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Abstract |
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Key words: fertilization failure/oocytes/polar body/pronuclei/rescue ICSI
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Introduction |
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Oocytes have been observed to be fertilized 24 h after exposure to spermatozoa, and the second polar body is released in 90% of fertilized oocytes by 6 h (Chen and Sathanathan, 1986
; Plachot et al., 1986
; Nagy et al., 1994
; Payne et al., 1997
). We therefore proceeded to look for the release of the second polar body 6 h after initial insemination and subjected to ICSI those oocytes in which a second polar body was not evident. The outcome of this rescue attempt was compared with retrospective results of rescue ICSI performed 22 h after failed fertilization in a completely different patient group.
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Materials and methods |
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Ovarian stimulation
All patients used Suprefact (Buserelin 0.5 mg/day s.c.; Hoechst, Germany) for down-regulation of the pituitary commencing from cycle day 21 and continuing up to the day of HCG administration. When pituitary down-regulation was achieved [estradiol (E2) <25 pg/ml and ultrasound demonstrated ovarian follicles <0.5 cm], Metrodin HP 300 IU for 5 days (Serono, Switzerland) followed by Puregon 150 IU daily (Organon, The Netherlands) was administered until the day before HCG injection. Follicular development was monitored by ultrasound scanning and laboratory E2 and progesterone values. Patients received 10 000 IU of hCG when three or more follicles measured >1820 mm in diameter. Oocytes were then obtained by using transvaginal ultrasound under sedation 38 h after hCG injection.
Sperm preparation
Semen samples were collected in sterile containers by masturbation. After liquefaction, samples were analysed for sperm concentration, motility and morphology. Subsamples of 1 ml were placed in 6 ml tubes, overlaid with 1 ml of Medicult IVF medium (Medicult, Denmark), and incubated at 37°C under 5% CO2. After 3060 min, the supernatant was pelleted and washed twice at 600 g for 5 min. The sperm pellet was suspended in 0.51.0 ml of culture medium and then used for oocyte insemination.
Insemination
Oocytes were washed in culture medium and placed in groups of four in 0.5 ml of freshly equilibrated medicult IVF medium in four-well Nunc dishes. Oocytes of the 6 h group were exposed to 2030 x 103 spermatozoa 40 h post-hCG for 6 h only (until 46 h post-hCG), after which time they were checked for the presence of the second polar body. Those in which a second polar body was not evident were subjected to ICSI and checked for fertilization the next day.
The oocytes in the 22 h group were inseminated with 2030 x 103 motile spermatozoa 40 h post-hCG, incubated in culture medium and then checked for evidence of fertilization after 1820 h. Any corona cells that still remained attached to the oocytes were removed using finely drawn Pasteur pipettes to allow assessment of fertilization. ICSI was then performed on those oocytes that failed to fertilize i.e. those that did not show a second polar body or two pronuclei (2PN)
ICSI
For the procedure of ICSI, both holding and injection pipettes were obtained commercially (Humagen Fertility Diagnostics, USA), and the ICSI procedure was performed using Narashige micromanipulators (Narashige, Japan) under Hoffman modulation optics.
Just before the ICSI procedure the sperm suspension was placed in a 10 µl droplet of 10% polyvinyl-pyrrolidone (Medicult) at the 3 oclock position. Injection of the oocyte was performed in microdroplets of Medicult IVF medium under mineral oil (International Medical, The Netherlands).
A single motile morphologically normal spermatozoon that had migrated to the 9 oclock position was selected, immobilized by touching its tail with the injection micropipette, and then aspirated tail first into the pipette. The oocyte to be injected was secured with the holding pipette (9 o clock position) adjacent to the polar body (6 oclock position). The micropipette containing the sperm was then inserted through the zona pellucida and the oolemma into the ooplasm at the 3 oclock position of the oocyte. Penetration of the oolemma was confirmed by aspiration of some cytoplasm into the micropipette and the spermatozoon was then slowly injected. The pipette was withdrawn gently and the oocyte released from the holding pipette.
Assessment of fertilization and cleavage
Oocytes in the 6 h group were examined for fertilization 1618 h after ICSI, which was 24 h after oocyte retrieval. Cleavage of the oocytes was assessed on day 2 (48 h) and day 3 (72 h) before transfer into the uterus.
Oocytes in the 22 h group were examined for fertilization 1820 h after ICSI, i.e. on day 2 after oocyte retrieval (48 h). Cleavage of the fertilized oocytes was assessed on day 3 (72 h) just before their transfer into the uterus. The embryos were graded on a scale of 1 to 4 (Cummins et al., 1986). Grade 1 embryos were the best embryos, containing even-sized, symmetrical blastomeres with no obvious fragmentation; grade 2 had blastomeres of uneven size or the total cytoplasmic mass contained <10% fragmentation; grade 3 embryos had 1050% of their cytoplasm fragmented; and grade 4 showed >50% cytoplasmic fragmentation.
Patient -hCG was measured for diagnosis of pregnancy 910 days after embryo transfer and then measured serially to monitor the rise in its titre. Implantation was noted later by the appearance of the gestational sac in the uterus using transvaginal ultrasonography.
All data are expressed as mean ± SEM. Fertilization, implantation and pregnancy rates were compared between the 6 h group and the 22 h group, and tested for significant difference by the 2 test. A P value of <0.05 was considered statistically significant.
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Results |
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The 6 h group consisted of 245 oocytes, of which 226 (92.2%) were in metaphase II. Following rescue ICSI, evidently normal fertilization was observed in 159 oocytes (70.3%). This was significantly higher (P < 0.0001) than the fertilization rate obtained in the 22 h group, in which 167 (91.7%) of 182 oocytes were in metaphase II and 81 (48.5%) showed evidence of normal fertilization. The 1PN and 3PN rates in the 6 h group and the 22 h group were not significantly different (Table I). In the 6 h group 145 (91.1%) of the 159 embryos cleaved, while in the 24 h group 73 (90.1%) of the 81 embryos cleaved. There were more grade 1 embryos from the 6 h group (53.1%) than from the 22 h group (20.5%) of oocytes (P < 0.0008).
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All the patients in both groups had at least three embryos to transfer, except one patient in the 6 h group and two in the 22 h group, who had only two embryos to transfer. In total, 74 embryos were transferred in the 6 h group and 58 embryos were transferred in the 22 h group.
In the 6 h group there were 12 (48%) clinical pregnancies, whereas in the 22 h group there was only one (5%) singleton pregnancy (P < 0.0003), which resulted in a healthy baby. The 12 pregnancies in the 6 h group resulted in three sets of twins, eight singletons and one abortion. In this group the implantation rate was 20.2%, compared with 1.72% (P < 0.02) in the 22 h group (Table I). The results of IVF in the non-failed fertilization cohort of the 6 h and 22 h groups are given in Table II.
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Discussion |
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Even though oocytes can be rescued after 22 h, owing to the long fertilization window the percentage of grade 1 embryos (20.5%) is comparatively much less (P < 0.0008) than normally resulted from oocytes that were fertilized at the optimum time in the remaining cohort of the same group of patients (Table II). This shows clearly that there is a direct relationship between the time interval from oocyte retrieval to fertilization and the development of grade 1 embryos, with obvious effects on pregnancy rates. This may be due to the ageing of oocytes in culture, which results in increased cytogenetic abnormalities (Edwards and Broody, 1995), fewer normal viable embryos and low pregnancy rates. Another factor contributing to low pregnancy rates following rescue ICSI 22 h after initial insemination may be asynchrony between developmental stage of the embryo and the state of the endometrium. Pronuclei appeared on the following day (16 h post-ICSI) when ICSI rescue was performed after 6 h, thus coinciding with the occurrence of PN formation among normal ICSI cycles. In contrast, when rescue ICSI was performed 22 h after initial insemination, PN appeared 32 h after oocyte retrieval. Consequently embryos from the 22 h group were at the 24-cell stage when transferred on day 3, whereas the embryos from the 6 h group had 5 or more cells.
In the present study, rescue ICSI was performed 6 h after insemination in those oocytes, which failed to show the release of a second polar body. This resulted in 70% fertilization rate, which is comparable to the fertilization rate of male factor cases (De Croo et al., 2000) and significantly higher than we obtained previously when rescue ICSI was performed 22 h after initial insemination (48.5%) (P < 0.0001). Even though oocytes can be rescued after 22 h their ability to undergo fertilization decreases with their ageing.
In the 22 h group there is the possibility that some oocytes that were not fertilized after initial insemination were in the germinal vesicle or metaphase I stage when inseminated. Culture may have resulted in maturation to metaphase II, enabling fertilization by ICSI and subsequent clinical pregnancy.
Thus, rescue ICSI 6 h after insemination has advantages over rescue ICSI after 22 h. First, rescue ICSI after 6 h results in higher fertilization rates (P < 0.0001) and more grade 1 embryos compared with rescue ICSI after 22 h (P < 0.0008). Secondly, the time-course events of fertilization among early rescued oocytes (6 h) followed a pattern similar to those of oocytes that underwent ICSI at the normal time of fertilization in that PN formation was complete by 1618 h after ICSI, resulting in embryos that are at a developmental stage in synchrony with endometrial development. Thirdly, higher pregnancy and live birth rates resulted among patients in the 6 h group compared with the 22 h group (P < 0.003).
Although rescue ICSI after 22 h resulted in a higher percentage of 1PN and 3PN zygotes compared with rescue ICSI after 6 h, the difference was not statistically significant (Table I). Of the 15 3PN zygotes in the 6 h group, five had polar bodies fragmented, thus making it difficult to distinguish between first and the second polar body and whether the extra PN was contributed by a second spermatozoon. The remaining 3PN zygotes had only one polar body, thus confirming the retention of the second polar body. Usually with ICSI, 1PN and 3PN embryo formation are due to inadequate oocyte stimulation during injection of the spermatozoa or to cytoskeletal damage (Edwards and Broody, 1995). This results in disorganization of the spindle and cytoplasm and retention of the second polar body with resultant chromosomal anomalies (Edwards and Broody, 1995
). Interestingly, some oocytes in the 22 h group offered very little resistance to the ICSI needle compared with oocytes from the 6 h group, indicating that oocytes probably had already undergone cytoskeletal damage and disorganization. Nevertheless, there are some concerns that need to be addressed when considering early rescue ICSI.
The earliest indication of fertilization of oocytes is the extrusion of the second polar body. It has been shown that following ICSI, 22% of oocytes release their second polar body by 24 h and 66% at 4 h. Pronuclei appear by 6 h and 80% have two pronuclei by 8 h (Nagy et al., 1994
). Theoretically, by 8 h all oocytes would have released their second polar body if they have been fertilized. One of the concerns when performing rescue ICSI 6 h after insemination is the possible delay in the release of second polar body that occurs in
10% of the oocytes. Although this is a small number, the risk will be greatest if it occurs in all the oocytes in a given patient. The risk may be greatly reduced by performing rescue ICSI 8 h after initial insemination when all of the fertilized normal oocytes would be expected to have released the second polar body.
Performing rescue ICSI on oocytes that are already in the process of fertilization could lead to the formation of 3PN zygotes. During normal insemination, 3PN zygotes result from two spermatozoa participating in fertilization, probably after entering the oocytes simultaneously. It is not known whether a second sperm entering at a later time causes any abnormalities other than 3PN formation. In the event of 3PN zygote formation after rescue ICSI, microsurgical removal of the second male PN may be attempted, although it is not a routine practice. We have achieved a live birth from such microsurgical procedure after oocytes were fertilized with three PN following normal insemination (Kattera and Chen, 2003).
The second concern is when the first polar body in some oocytes becomes fragmented, making it difficult to distinguish from the second polar body when rescue ICSI is attempted after 6 h. Rescue ICSI on these oocytes may lead to the formation of 3PN zygotes. In such situations, it is advisable to look for the appearance of PN as early as possible, so that 3PN zygotes may be identified.
Finally, it is difficult to predict fertilization failure in IVF due to several contributing factors (Benadiva et al., 1999). Hence it is important to look for earliest indications of fertilization. We carry out our oocyte retrievals 38 h post-hCG, inseminate at 40 h (10 a.m.) post-hCG, check for the release of second polar body 46 h (4 p.m.) post-hCG and microinject those oocytes that do not show the second polar body.
In conclusion, the present study demonstrates that oocytes that failed to fertilize in IVF can be rescued by ICSI 6 h after insemination after checking for the extrusion of the second polar body. However, caution must be exercised when considering such rescue attempts bearing in mind the potential for 3PN zygote formation.
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References |
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Submitted on February 11, 2003; accepted on April 30, 2003.