Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, Gothenburg, Sweden
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Abstract |
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Key words: early cleavage/embryo quality/good quality embryos/IVF/pregnancy
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Introduction |
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Embryo scoring is performed in different ways, with each centre having its own scoring system. However, since all current systems are based on morphological evaluation, most parameters evaluated are the same between centres, albeit with different emphases being placed on each parameter.
The current practice in most IVF laboratories is to score cleavage-stage embryos in connection with embryo transfer, i.e. on day 2 or 3 after oocyte retrieval, evaluating the grade of fragmentation, cytoplasmic appearance and number of blastomeres per embryo. These variables can be collectively counted as a cumulative embryo score (CES = grade x number of blastomeres) (Steer et al., 1992; Visser and Fourie, 1993
). In addition, variation in the zona pellucida thickness, presence of multinucleated blastomeres, location of fragments and size of blastomeres in relation to each other may be analysed (Cohen et al., 1989
; Giorgetti et al., 1995
; Pickering et al., 1995
; Ziebe et al., 1997
; Palmstierna et al., 1998
; Pelinck et al., 1998
; Alikani et al., 1999
; Hardarson et al., 2001
). Embryo quality has also been reported to correlate with oocyte and zygote morphology, e.g. appearance of the cytoplasm, pronuclei and polar bodies (Sadowy et al., 1998
; Scott and Smith, 1998
; Manor et al., 1999
; Tesarik and Greco, 1999
; Ebner et al., 2000
).
Another indicator of embryo quality that can easily be determined is the embryo cleavage rate. Until recently, the cleavage rate has mainly been documented at day 2 or 3, i.e. the number of cells at that time has been correlated with pregnancy and implantation rates. It has been reported (Shoukir et al., 1997; Sakkas et al., 1998a
) that early cleavage, i.e. the time of the first mitotic division, could also be correlated with embryo quality and pregnancy rates. However, both these reports were based on a small number of transfers and had a biased embryo selection at transfer, since early-cleaving embryos were chosen whenever possible. In addition, the transfers were sometimes mixed, with both early- and late-cleaving embryos being transferred together. This makes it difficult to draw any conclusions, since it is impossible to ascertain which of the embryos actually implanted.
The aim of the present study was therefore to examine, in a large sample size, whether the time of first cell cleavage influenced embryo quality, pregnancy, implantation and/or birth rates.
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Materials and methods |
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Only cycles that led to embryo transfer with embryos of identical cleavage score at early evaluation (i.e. one or two cells at 2527 h post insemination/microinjection) were included in the pregnancy, implantation and birth rate analysis. Each patient was only included once (their first cycle during this time period) and no mixed (early- and late-cleaving embryos together) transfers were included.
Ovarian stimulation was carried out by a desensitizing protocol using a gonadotrophin-releasing hormone agonist preparation in a long protocol in combination with recombinant FSH (Gonal-F; Serono, Geneva, Switzerland or Puregon; Organon, Oss, The Netherlands). Monitoring was carried out by measuring serum oestradiol concentrations and performing transvaginal ultrasonography. When an adequate stimulation was achieved (three or more follicles 18 mm diameter), human chorionic gonadotrophin (HCG) (Profasi; Serono) was administered (10 000 IU, s.c.). Oocytes were retrieved 3638 h following HCG using transvaginal ultrasound-guided puncture.
Conventional IVF or intracytoplasmic sperm injection (ICSI) was performed 35 h after oocyte aspiration. Only metaphase II oocytes were used for ICSI. The oocytes were cultured separately in media droplets under oil (IVF-20 and Ovoil; Scandinavian IVF Science, Gothenburg, Sweden).
Fertilization was determined by confirmation of two pronuclei (2PN) 1719 h after insemination/microinjection. The zygotes were checked again in the afternoon on day 1 (2527 h after insemination/microinjection), and the number of cells (one or two) was documented.
Embryos were transferred routinely on day 2. However, day 3 transfers were occasionally performed, mainly due to slowly cleaving (fewer than four cells on day 2) or fragmented embryos. It should be noted that embryos were not selected for transfer on the basis of early or late cleavage, but on morphology alone.
Pregnancy was defined as a positive HCG test in urine on day 19 post transfer. Implantation rate was defined as number of fetal sacs at ultrasound in gestational week 7, per number of transferred embryos.
Statistical analysis
Distribution of the variables are given as means, SD and ranges. For comparison between groups, the MannWhitney U-test was used for ordered and continuous variables, 2 test for dichotomous variables. In order to select independent predictors of pregnancy and birth/ongoing pregnancy, all variables in Table III
were entered into stepwise logistic regression analyses. All significance tests were two-tailed and conducted at the 5% significance level. In addition to the overall analysis including both ICSI and IVF transfers, two separate stepwise logistic regressions were also performed, for ICSI alone and for conventional IVF alone. The reason for this was that ICSI and conventional IVF may be considered as two separate populations in regard to the time of fertilization.
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Results |
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An analysis of all cycles leading to embryo transfer on day 2 and 3 is shown in Table II. The transfer of early-cleaving embryos resulted in significantly higher overall pregnancy (40.5 versus 31.3%, P = 0.0049) and implantation rates (28.0 versus 19.5%, P < 0.0001) than the transfer of late-cleaving embryos (Table II
). The twin pregnancy rate (confirmed by ultrasound in gestational week 7) was 33.3% in the early-cleaving group and 22.3% in the late-cleaving group (P = 0.032). The spontaneous abortion rate was significantly lower in the early-cleaving group (12.1 versus 20.2%, P = 0.036), and the birth rate significantly higher (34.3 versus 24.0%, P = 0.0009) compared with the late-cleaving group (Table II
). For those pregnancies that went to term, the rate of born males versus females was calculated. No difference was found in the sex ratio between born children from early-cleaving embryos compared with late-cleaving embryos (50/50 and 53/47% respectively).
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The results from the overall stepwise logistic regression analyses of all transfers (Table IV) showed that total number of good quality embryos and female age were independent predictors of both pregnancy and birth, while early/late cleavage was not. However, when stepwise analyses were performed separately for ICSI and IVF, it was found that for ICSI the variable early/late cleavage was an independent predictor of birth (Table IV
).
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Discussion |
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When comparing the development of all early- versus late-cleaving embryos, it was found that significantly higher numbers of early-cleaving embryos became good quality embryos (Table I), as well as giving significantly higher pregnancy, implantation and birth rates (Table II
). These results imply that early cleavage is indeed a good biological indicator of embryo potential. This was also confirmed in a univariate analysis where early cleavage, female age, day of transfer and total number of good quality embryos were shown to be positively correlated with pregnancy and birth (Table III
).
When performing a stepwise multivariate analysis including the variables of number of aspirated oocytes, female age, mean number of blastomeres per transferred embryo, total number of good quality embryos, early/late cleavage, day of transfer and fertilization method, early cleavage was found not to be an independent predictor of either pregnancy or birth/ongoing pregnancy (Table IV). However, when performing a similar stepwise logistic regression analysis with only the ICSI transfers included, early cleavage was found to be an independent predictor of birth, but not of pregnancy.
In contrast to the results of previous studies which stated that the time of first cleavage could be used for selection of embryos at transfer (Shoukir et al., 1997; Sakkas et al., 1998a
), no selection for the early-cleaving embryos was performed at embryo transfer in the current study. Instead, the embryos judged to be morphologically best at the time of transfer were chosen, irrespective of cleavage rate. Also of importance in the current study is that only results from those transfers where all embryos in each transfer showed the same cleavage rate, i.e. either early or late, were included.
Embryos derived after ICSI had a higher rate of early cleavage than those derived after IVF. This might be expected since the injected spermatozoa bypass many of the fertilization steps such as the acrosome reaction and binding to the zona pellucida, resulting in a shorter fertilization time (van Wissen et al., 1995; Nagy et al., 1998
). However, the percentage of good quality embryos derived from IVF were similar to those derived from ICSI. The question therefore arises; if IVF embryos had been checked for early cleavage at a slightly later time, would a similar rate of early cleavage as for ICSI embryos have been found? The reason why the separate analysis of ICSI/IVF transfers showed that early cleavage was an independent predictor of birth for ICSI transfers but not for IVF transfers is not known, but it may possibly be associated with the higher rate of early cleaved ICSI embryos. Hence, it may be better to screen IVF embryos later and/or to inseminate them earlier in relation to ICSI embryos, in order to make use of the early cleavage rate screening for IVF embryos. However, the possibility that there is an actual developmental difference in embryos that stem from different fertilization methods cannot be excluded.
The reason why early cleavage should yield better quality embryos is not known. It can be speculated that early-cleaving embryos stem from oocytes in which cytoplasmic and nuclear maturation are better synchronized and/or have a higher metabolic fitness, i.e. the availability and competence of, for example, ATP, mRNA, mitochondria, etc. Animal studies have shown (Grisart et al., 1994) that bovine embryos have a lag-phase (i.e. a lengthened cell cycle) either early (4- to 7-cell stage) or late (8-cell stage). It was found that embryos with a late lag-phase had a higher blastocyst rate than those with an early lag-phase. These authors speculated that the lag-phase is related to the amount and/or quality of RNA or proteins stored in the oocyte and to the transcriptional activity of the embryo. A premature transcription resumption, indicated by a pause in early growth, might thus harm further development.
Another variable that has to be considered is the contribution of the spermatozoa, i.e. the addition of paternal factors. In humans, the centrioles that control the first mitotic divisions of the oocyte are introduced by the spermatozoa (Sathananthan et al., 1991; Palermo et al., 1994
; Sathananthan, 1998
), and the quality of the spermatozoa could thus be one additional factor influencing the early cleavage. In addition, the DNA status of the spermatozoa will influence that of the embryo (Sakkas et al., 1998b
; Obasaju et al., 1999
; Larson et al., 2000
).
It has been shown that cells having an aneuploid chromosomal status cleave more slowly in general (Paton et al., 1974; Barrenäs et al., 2000
). This was also found in our IVF programme when investigating 3PN zygotes, which had a rate of early cleavage of 12.0% (own unpublished observations), as compared with the overall 26.9% of 2PN zygotes shown in Table I
. The rate of early cleavage might therefore be one indicator of the chromosomal status of the embryo. This is supported by the observation that unevenly cleaved embryos (those with a large difference in blastomere size) have been shown to have a higher rate of aneuploidy than embryos with blastomeres of equal size, as well as a significantly lower rate of early cleavage and lower pregnancy and implantation rates (Hardarson et al., 2001
).
Although the overall results showed significant differences between early- and late-cleaving embryos in pregnancy, implantation and birth rates, it is important to note that there was a significant difference in blastomere number per embryo between the groups; many of the later-cleaving embryos were still slow on the day of transfer (Table II). This indicates that embryos with a slow start have a lower rate of development in general, and that this influences the success rate. With regard to the rate at which cells cleave, a number of reports have been published showing that embryos having fewer than four cells at day 2 or fewer than six cells at day 3 have a significantly lower pregnancy and implantation rate than faster-cleaving embryos (Lewin et al., 1994
; Söderlund et al., 1998
; Van Royen et al., 1999
). This may correlate with the increased chromosomal abnormality rate for slow/fragmented embryos (Munne et al., 1995
).
It has been found in several studies that mammalian male embryos may cleave faster than female embryos (Tsunoda et al., 1985; Xu et al., 1992
; Pergament et al., 1994
; Ng et al., 1995
; Peippo and Bredbacka 1995
; Tarin et al., 1995
). These data originate from studies of in-vitro-cultured embryos analysed from the 4-cell to the blastocyst stage. In the present study, no difference was found when analysing the sex ratio of delivered babies originating from either early- or late-cleaving embryos (Table I
). This implies that the possible difference in cleaving rate between male/female human embryos is something that does not occur until later, possibly coinciding with the onset of genomic transcription (Braude et al., 1988
).
It can be concluded from the results of the current study that early cleavage is a highly significant biological indicator of embryo potential, resulting in overall significantly improved embryo quality as well as pregnancy, implantation and birth/ongoing pregnancy rates. Since a separate logistic regression analysis of ICSI transfers showed that early cleavage was an independent predictor of birth, it is suggested that early cleavage may be an additional factor for selecting embryos with a higher potential for resulting in birth. Further studies are needed to investigate whether early cleavage might also be used as a screening variable for embryos derived from conventional IVF if the time interval from insemination to screening is increased.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 20, 2001; accepted on August 21, 2001.