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Human Reproduction Vol.16, No.12 pp. 2652–2657, 2001 Early embryo cleavage is a strong indicator of embryo quality in human IVF K.Lundin1, C.Bergh and T.Hardarson Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, Gothenburg, Sweden 1To whom correspondence should be addressed at: IVF-lab, Reproductive Medicine, SU/Sahlgrenska, SE-413 45 Göteborg, Sweden. E-mail: [email protected] BACKGROUND: In order to decrease multiple birth rates without decreasing birth rates overall, it is important to increase the capability of selecting the most optimal embryos for transfer. It has been shown that human embryos which cleave early, i.e. complete the first mitotic division within 25–27 h post insemination, provide higher pregnancy and implantation rates. METHODS AND RESULTS: In this prospective study, an evaluation of 10 798 scored embryos showed that early cleavage resulted in a significantly higher proportion of good quality embryos compared with late cleavage (62.5 versus 33.4%, P < 0.0001). When examining both day 2 and day 3 transfers together, earlycleaving embryos (306 transfers) gave rise to significantly higher rates of pregnancy/transfer (40.5 versus 31.3%, P ⍧ 0.0049), implantation (28.0 versus 19.5%, P ⍧ 0.0001) and birth/ongoing pregnancy (34.3 versus 24.0%, P ⍧ 0.0009) than did late-cleaving embryos (521 transfers). A stepwise logistic regression of all data showed that the total number of good quality embryos and female age were independent predictors of both pregnancies and birth. For intracytoplasmic sperm injection (ICSI) embryos, early cleavage was found to be an independent predictor of birth. CONCLUSIONS: Early embryo cleavage is a strong biological indicator of embryo potential, and may be used as an additional embryo selection factor for ICSI embryos. Key words: early cleavage/embryo quality/good quality embryos/IVF/pregnancy Introduction One of the greatest problems in assisted reproduction today is the high multiple pregnancy rate, associated mainly with the number of embryos transferred and embryo quality (Puissant et al., 1987; Steer et al., 1992; Shulman et al., 1993; Hu et al., 1998; Strandell et al., 2000). In order to be able to transfer two, or even one, embryo(s) without markedly lowering the pregnancy rate, it is important to increase our knowledge of how to select the optimal cleavage-stage embryos or blastocysts. 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 ⫻ 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 2652 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 latecleaving 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 © European Society of Human Reproduction and Embryology Early cleavage and embryo quality influenced embryo quality, pregnancy, implantation and/or birth rates. Table I. Analysis of all zygotes/embryos screened at 25–27 h post insemination/microinjection (n ⫽ 10 798) Early cleavage Late cleavage 3046 (28.2) 1903 (62.5)a 1909 (34.7)c 1137 (21.5)d 7752 2593 3596 4156 Materials and methods This prospective study was initiated in January 1997 at the IVF unit, Sahlgrenska University Hospital, Göteborg, Sweden. Data from this time until June 2000 were analysed. All zygotes that were normally fertilized (two pronuclei) and checked for early embryo cleavage within 25–27 h post insemination/microinjection were included in an evaluation of cleavage rate and embryo quality. Embryos either transferred or frozen on day 2 or 3 (grade 1 or 2, with 4–6 blastomeres on day two, or 6–8 blastomeres on day 3) were considered to be good quality embryos. Embryos of a lower quality were very seldom transferred. Only cycles that led to embryo transfer with embryos of identical cleavage score at early evaluation (i.e. one or two cells at 25–27 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 (earlyand 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 艌18 mm diameter), human chorionic gonadotrophin (HCG) (Profasi; Serono) was administered (10 000 IU, s.c.). Oocytes were retrieved 36–38 h following HCG using transvaginal ultrasound-guided puncture. Conventional IVF or intracytoplasmic sperm injection (ICSI) was performed 3–5 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) 17–19 h after insemination/microinjection. The zygotes were checked again in the afternoon on day 1 (25–27 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 Mann–Whitney U-test was used for ordered and continuous variables, χ2 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 twotailed 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. Cleaved embryos Good quality embryos Cleaved embryos from ICSI treatment Cleaved embryos from IVF treatment (71.8) (33.4)b (65.3) (78.5) Values in parentheses are percentages. aversusb, P ⬍ 0.0001. cversusd, P ⬍ 0.0001. Results During the study period, a total of 18 120 oocytes were normally fertilized (2PN). Due to laboratory schedules and availability of personnel, only 10 798 of these zygotes were checked for early cleavage at 25–27 h post insemination/ microinjection and included in the evaluation of cleaving rate and embryo quality (Table I). Of the early cleaving embryos, 1125 were transferred and 778 were frozen, i.e. a total of 62.5% good quality embryos were obtained from this group. From the late-cleaving embryo group, 1613 were transferred while 980 were frozen, giving a total of 33.4% good quality embryos in this group (P ⬍ 0.0001). ICSI embryos had a higher rate of early cleavage than IVF embryos (Table I), although the percentage of good quality embryos derived with either technique did not differ (39.7% for IVF versus 42.8% for ICSI). 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 earlycleaving 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). The results from the univariate correlation analysis are shown in Table III. Female age, day of transfer, total number of good quality embryos available and early cleavage were all correlated with both pregnancy and birth (Table III). 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). 2653 K.Lundin, C.Bergh and T.Hardarson Table II. Analysis of all cycles with early- or late-cleaving embryos that led to embryo transfer on days 2 or 3 No. of cycles Aspirated oocytesa Good quality embryosa Range Blastomeres per transferred embryo day 2 Blastomeres per transferred embryo day 3 Mean no. of embryos transferred Female age (years)a Range Pregnancies per transfer Implantation rate Spontaneous abortionsb Birth ratec Born male/female (%) Early cleavage Late cleavage P 306 13.1 ⫾ 6.31 3.8 ⫾ 2.55 0–18 4.1 (1.00) 7.8 (1.06) 1.92 32.2 ⫾ 4.0 21.9–40.8 124 (40.5) 165/589 (28.0) 15/124 (12.1) 105/306 (34.3) 50/50 521 11.4 ⫾ 5.55 2.3 ⫾ 1.84 0–12 4.0 (0.87) 6.0 (1.46) 1.97 33.2 ⫾ 3.9 22.2–41.4 163 (31.3) 200/1027 (19.5) 33/163 (20.2) 125/521 (24.0) 53/47 0.0004 ⬍ 0.0001 0.0010 ⬍ 0.0001 NS ⬍ 0.0001 0.0049 ⬍ 0.0001 0.036 0.0009 Values in parentheses are percentages. aMean ⫾ SD; b% per pregnancy; c% per transfer. NS ⫽ not significant. Table III. Factors analysed for univariate correlation with pregnancy and birth/ongoing pregnancy Variable Pregnancy Birth/ongoing pregnancy Yes (n ⫽ 292) No. of aspirated oocytesa Female age (years)a No. of blastomeresa No. of good quality embryosa 12.32 32.21 4.81 3.51 ⫾ ⫾ ⫾ ⫾ 5.74 3.78 1.48 2.32 No (n ⫽ 535) 11.86 ⫾ 5.94 33.16 ⫾ 4.02 4.82 ⫾ 1.44 2.57 ⫾ 2.13 Yes (n ⫽ 238) P NS 0.0014 NS ⬍ 0.0001 Day of embryo transfer Day 2 Day 3 223 (38) 69 (29) 363 (62) 172 (71) – Fertilization method ICSI IVF 160 (36) 132 (35) 290 (64) 245 (65) Cleavage Early Late 124 (41) 163 (31) 182 (59) 358 (69) 12.55 32.3 4.86 3.51 ⫾ ⫾ ⫾ ⫾ 5.97 3.88 1.43 2.29 No (n ⫽ 589) 11.80 ⫾ 5.83 33.1 ⫾ 4.11 4.80 ⫾ 1.50 2.65 ⫾ 2.18 P NS 0.0042 NS ⬍ 0.0001 178 (30) 60 (25) 408 (70) 181 (75) – – NS 133 (30) 105 (28) 317 (70) 272 (72) – NS – 105 (34) 125 (24) 201 (66) 396 (76) – 0.0006 0.0034 0.0050 0.0012 Values in parentheses are percentages. aMean ⫾ SD. NS ⫽ not significant. Discussion Although many factors influence the result of an IVF cycle (e.g. stimulation response, endometrial receptivity, oocyte maturity, culture conditions, paternal contribution), embryo morphology is regarded as one of the most important. Morphological embryo variables are easy to evaluate, and have thus been the determining factors when the selection for embryo transfer is made. Much effort has been made in trying to find an optimal scoring system. A number of reports have been published concerning the importance of different factors for embryo development and pregnancy rate, including the appearance of the cumulus cells, the position of the polar body(ies) in relation to the pronuclei, the morphology and size of the pronuclei, and the number and size of nuclei in the blastomeres (Edwards and Beard, 1997; Scott and Smith, 1998; Manor et al., 1999; Tesarik and Greco, 1999; Ebner et al., 2000). An ideal scoring system may start at the oocyte stage, continue 2654 through the zygote stage, and conclude with a morphologybased selection either at the cleavage or blastocyst stage. However, the advantage of a scoring system with many observations must be weighed against the impairment of the environment for the embryo. 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 Early cleavage and embryo quality Table IV. Variables independently predictive of pregnancy or birth, adjusted for all other variables Variable Pregnancy Birth Odds ratio (95% CI) Adjusted P-value Odds ratio (95% CI) Adjusted P-value All transfers Female age No. of good quality embryos 0.942 (0.91–0.98) 1.240 (1.15–1.33) 0.0023 ⬍ 0.0001 0.954 (0.92–0.99) 1.169 (1.10–1.25) 0.017 0.0001 Only ICSI transfers No. of good quality embryos Early cleavage 1.297 (1.16–1.45) – ⬍ 0.0001 – 1.243 (1.12–1.39) 1.744 (1.12–2.73) ⬍ 0.0001 0.015 Only IVF transfers Female age No. of good quality embryos 0.929 (0.87–0.99) 1.139 (1.04–1.25) 0.018 0.0045 – 1.106 (1.01–1.21) – 0.029 CI ⫽ confidence interval. 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 ‘lagphase’ (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 2655 K.Lundin, C.Bergh and T.Hardarson 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 fastercleaving 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 latecleaving 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. Acknowledgements The authors thank Nils-Gunnar Pehrsson for advice and statistical analyses and Graham Coull for valuable criticism of the manuscript. 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Received on February 20, 2001; accepted on August 21, 2001 2657