1 Infertility and IVF Centre, Human Reproductive Medicine Unit, Alma Mater Studiorum, University of Bologna, Via Massarenti 13, 40138 Bologna and 2 Department of Internal Medicine and Gastroenterology, Alma Mater Studiorum, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy
3 To whom correspondence should be addressed. Email: ciottip{at}orsola-malpighi.med.unibo.it
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Abstract |
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Key words: embryo quality/first polar body/pregnancy rate/sperm injection
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Introduction |
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Materials and methods |
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Oocytes
Supernumerary oocytes are cryopreserved in our centre to avoid ethical, legal and religious problems related to embryo freezing. For this reason, a maximum of six oocytes per patient were inseminated while the rest were prepared for cryopreservation (Fabbri et al., 2001). All the oocytes were cultured in Universal IVF Medium (MediCult, Denmark) at 37°C in a 5% CO2 atmosphere. They were denuded by brief exposure to hyaluronidase at a final concentration of 40 IU/ml (MediCult) and mechanically cleaned from their surrounding cumulus cells by aspiration using a denuding pipette with a diameter of 170140 µl (Denuding Flexi-PetTM; Cook, Australia). Nuclear maturity was evaluated and metaphase II oocytes were randomly selected for insemination. The classification of 1st PB morphology was performed using an inverted microscope (Eclipse TE-300; Nikon, Japan) by the same operator as previously reported (Ebner et al., 1999
): round or ovoid with smooth surface=grade 1; round or ovoid with rough surface=grade 2; fragmented=grade 3; broken into two parts=grade 4; huge and extruded in a large perivitelline space=grade 5. In the analysis of the data reported, a simpler classification was used by grouping grades 1 and 2 as intact polar bodies and grades 3 and 4 as fragmented polar bodies (Figure 1) (Ebner et al., 2002
).
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Semen treatment
All semen samples were freshly ejaculated. The samples were collected in Flushing Medium (MediCult) supplemented by 0.5% human albumin (CAF-DCF, Belgium). After liquefaction and evaluation of the basal parameters, the samples were prepared by discontinuous density gradient centrifugation (Pure Sperm; Nidacon, Int. AB, Sweden). Concentration, motility and morphology were evaluated after treatment. The samples were maintained at 37°C until ICSI and utilized at a final concentration of 1 x 106/ml motile sperm.
Effect of the duration of in vitro culture on the 1st PB fragmentation
The 1st PB morphology was checked twice (1st observation at the moment of denudation and 2nd observation at the moment of injection) in a group of 180 randomly selected oocytes (49 cycles). For analysis, the time between the two observations was divided into three intervals: 1 h 20 min to 2 h 30 min; 2 h 31 min to 3 h 30 min; 3 h 31 min to 5 h 30 min. The percentage of intact 1st PB which showed fragmentation in the second observation was calculated (fragmentation rate).
ICSI and fertilization
ICSI was performed as previously reported (Van Steirteghem et al., 1993), utilizing an inverted microscope at x 20 magnification with a Hoffman modulation contrast (Eclipse TE-300 Nikon, Japan). 35° angled micropipettes were utilized (Cook, Australia). Oocytes were cultured individually after injection. ICSI was performed after a mean time of 6 h 02 min. The minimum time after retrieval was 2 h 40 min and the maximum time was 9 h 15 min. This time was divided into three intervals:
5, >5,
7, >7 h.
The fertilization assessment was performed 1618 h after ICSI and it was defined as the presence of two pronuclei and the 2nd PB.
Embryos
Zygotes and embryos were cultured individually in IVF Medium (MediCult, Denmark) in 4-well dishes (Nunclon; Nunc, Denmark). The embryo quality was evaluated prior to transfer (4042 h after ICSI) on day 2 (26 cells) and divided into five grades: 1=regular cells, no fragments; 2=regular or irregular cells, 010% fragments; 3=regular or irregular cells, 1130% fragments; 4=regular or irregular cells, 3145% fragments; 5=irregular cells, 4660% fragments. In the analysis of the reported data, embryos were grouped into three categories: A=grade 1 or 2; B=grade 3; C=grade 4 or 5.
Statistical analysis
Mean values, SD and frequencies were used as descriptive statistics. The MannWhitney test, the KruskalWallis test, the MantelHaenszel linear by linear 2-test, the Yates corrected
2-test, and the Spearman rank correlation were applied. Statistical analyses were performed by running the SPSS/PC+ statistical package on a personal computer. Two-tailed P<0.05 was considered statistically significant.
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Results |
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A total of 449 zygotes (75.3% fertilization rate) were produced and 429 embryos developed (95.5% cleavage rate). Of these, 244 embryos were of quality A (56.9%), 116 of quality B (27.0%) and 69 of quality C (16.1%). The 1st PB morphology (P=0.301), the fertilization rate (P=0.465), the cleavage rate (P=0.497) and the embryo quality (P=0.525) were not significantly related to the age of the patients. Moreover, no significant relationship was found between the 1st PB morphology and the fertilization rate (P=0.703), the cleavage rate (P=0.055) and the embryo quality (P=0.673) (Table I).
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The results of pregnancy and implantation rates according to the 1st PB morphology were evaluated by dividing the 165 embryo transfers into three groups: embryos developed from oocytes with intact 1st PB, embryos developed from oocytes with fragmented 1st PB, and embryos developed from oocytes with mixed 1st PB morphology (Table III). No significant relationship was found between 1st PB morphology and either pregnancy or implantation rate (P=0.201, P=0.511 respectively).
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Aging effect of in vitro culture on 1st PB morphology
In the group of 180 oocytes which were checked twice, 20 (11.1%) and 41 (22.8%) 1st PB were fragmented at the first and second observations respectively. Therefore, the fragmentation rate in this time interval was 13.1% (21/160). Furthermore, a significantly positive relationship (P=0.006) was found between the frequency of the 1st PB fragmentation and the time elapsed between denudation and ICSI (Table IV); in particular, the fragmentation rate of the oocytes in which the time interval was >3 h 30 min was 26.7%. No significant relationship between the embryo quality and the 1st PB morphology (evaluated both at the 1st and the 2nd observation) was found in this subgroup of 180 oocytes (P=0.571 and P=0.896 respectively).
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Discussion |
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The prognostic value of 1st PB morphology for embryo quality obtained after ICSI was assessed alone or in combination with other criteria. In the literature, it is emphasized that synchrony between cytoplasmic and nuclear maturity is a fundamental aspect for the successful development and vitality of the embryos (Xia et al., 1997). Furthermore, it is reported that higher quality embryos are obtained from oocytes having the 1st PB intact and a normal perivitelline space as if to suggest that oocytes with these characteristics are those with the best synchrony between cytoplasmic and nuclear maturation (Xia et al., 1997
). Furthermore, the encouraging results of Ebner et al. (1999
, 2000)
suggest a significant prognostic role of the 1st PB morphology.
This evidence, together with the simplicity and feasibility of the evaluation of the 1st PB morphology, has stimulated us to evaluate this parameter with reference to ICSI outcome. Since only a few oocytes are inseminated at our centre and the supernumerary oocytes are stored, the finding of a simple oocyte feature predicting embryo quality would be of great relevance. Our centre has routinely used this strategy for >3 years, but recently a law in Italy on assisted fertilization, which allows for the insemination of a maximum of three oocytes per patients, has been approved. All centres will have to follow this procedure and, therefore the early identification of prognostic parameters to be able to identify the oocytes with the greatest probability of implantation will become an absolute necessity.
Our results show that the age of the patients is not related to the distribution of 1st PB morphology. The age of patients is neither related to the fertilization and cleavage rates nor to the embryo quality, pregnancy rate and implantation rate.
No relationship between 1st PB morphology, fertilization and cleavage rates and embryo quality was found.
The pregnancy and implantation rate was also not related to the 1st PB morphology when the embryo transfers were divided on the basis of the classification of the 1st PB morphology obtained at the 1st observation.
The interval of time during which a human oocyte is fertilizable in vivo is limited and, therefore, the time that it spends in culture before being fertilized in vitro is an important factor. Analysing the results of our study on the basis of this factor, we see that there was a wide span of time between retrieval and ICSI and the majority (60%) of ICSI were performed 13 h later than recommended (Ebner et al., 1999, 2000
). It has been reported in the literature that there are no differences in results when the ICSI is carried out rapidly (12 h after retrieval) or later (56 h after retrieval) (Van de Velde et al., 1998
). In a previous study in which the 1st PB morphology was not considered, we found no significant differences in the fertilization rate, cleavage rate, embryo quality, pregnancy rate and implantation rate when comparing two groups of patients in which ICSI was performed within or after 4 h after retrieval (Ciotti et al., 1997
). In another study, poorer results using oocytes having an immature polar body were reported (Xia et al., 1997
). Recently it was reported that the ability for complete activation and normal development was achieved by human oocytes during the MII arrest stage. MII oocytes require further time for complete ooplasm maturation in order to be readily activated by the sperm (Eichenlaub-Ritter et al., 2003
; Balakier et al., 2004
).
In the present study, no correlation was found between fertilization rate, cleavage rate and embryo quality according to the time elapsed from retrieval until ICSI, considering all the oocytes together or dividing them according to their 1st PB morphology. Instead, there was a significant difference in the percentage of pregnancies correlated with time passed between the retrieval and the ICSI, considering three time intervals (5, >57,
7 h). The best result was obtained in the middle group and the worst in the group having the longest time interval. From our data it can be deduced that it is beneficial to wait a few hours for the complete cytoplasmic in vitro maturation of the oocytes before ICSI, and an interval of optimal waiting can be identified. The lower pregnancy rate at early ICSI could be explained by studies which utilize the novel spindle imaging method (Oldenbourg, 1996
, 1999
). In fact, human oocytes with a polar body can show the absence of a birefringent spindle because they are still at telophase I or prometaphase I stage (Eichenlaub-Ritter et al., 2003
). By contrast, it does not seem to be advisable to wait too many hours from retrieval in order to prevent in vitro ageing and all the negative consequences which ensue. At times, for various reasons, it is difficult to ensure that the injection is done within a suitable time interval such as, for example, cases in which the male has difficulty in producing a seminal liquid. Serious delays in carrying out the ICSI might compromise the final outcome of the procedure. This is a very important element which must be taken into account when dealing with microinjection of thawed oocytes because the time spent in vitro before freezing has to be added to that spent after thawing. Certainly in the near future, the employment of the spindle imaging method will be of crucial value to define the correct injection time of each single fresh or thawed oocyte.
We also verified the ageing effect of in vitro culture on 1st PB morphology and we found a positive relationship with the time elapsed in culture. The morphology of the 1st PB changes after a few hours of in vitro culture, and thus it can vary according to the moment in which the observation is carried out. However, we did not find any relationship between either the first or the second observation and embryo quality. Therefore, we could not find any predictive value for the morphology of the 1st PB.
In conclusion, our data do not agree with previous results which report a better fertilization rate, embryo quality, pregnancy and implantation rate in relation to 1st PB morphology (Ebner et al., 1999, 2000
). Our results agree in part with a recent study (Ebner et al., 2002
) in which no significant difference was found between 1st PB morphology and the fertilization rate. The authors believed that this was due to the simpler classification utilized (intact and fragmented PB). In addition, it should be noted that our data were also analysed with a five-grade classification (Ebner et al., 1999
) but our results did not change (data not shown). Moreover our study is completely in agreement with a recent study (Verlinsky et al., 2003
) in which PB morphology is not correlated with the percentage of fertilization, embryo quality, blastocyst survival and outcome. Furthermore, PB morphology does not show a correlation with the genotype analysed for aneuploidy in patients who underwent preimplantation genetic diagnosis.
The analysis of the literature shows that this topic is controversial and the results reported are often conflicting probably due to the small number of samples in each study.
From our results, we cannot conclude that 1st PB morphology is a prognostic factor of embryo quality and subsequent pregnancy and implantation rate. Even though a larger study should be performed, we believe that 1st PB morphology is a fragile parameter because it is susceptible to ageing and, in our opinion, using 1st PB morphology as primary prognostic factor of oocyte quality is hazardous but, as suggested (Ebner et al., 2003), it may be good practice to include such an evaluation among the several morphological features that aid embryologists in the difficult work of embryo selection at the different stages of preimplantation development.
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Acknowledgements |
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References |
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Submitted on August 1, 2003; resubmitted on April 28, 2004; accepted on July 1, 2004.
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