Department of IVF, Obstetrics and Gynaecology, Reinier de Graaf Groep locatie Diaconessenhuis, PO Box 998, 2270 AZ Voorburg, The Netherlands
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Abstract |
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Key words: culture volume/group culture/human blastocyst formation/ICSI/IVF
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Introduction |
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Previously published data have shown that micro-cultures (Lane and Gardner, 1992; Gardner et al., 1997
), as well as the communal growth of embryos (Wiley et al., 1986
; Moessner and Dodson, 1995
; Almagor et al., 1996
), may lead not only to improved embryo development but ultimately to higher pregnancy rates. Autocrine or paracrine growth or survival factors released by the embryo might support itself and other embryos in their development (Paria and Dey, 1990
; O' Neill, 1998
). A smaller incubation volume could prevent a dilution of these specific embryo-derived factors. These studies, however, involve either mouse morula or blastocysts or human embryos until at most 48 h post-insemination.
The present study was designed to assess whether single or group culture, in a small or large incubation volume, influences the development of human day 3 embryos to blastocyst stage. In addition, though this was not the primary endpoint, pregnancy and implantation rates were evaluated.
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Materials and methods |
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Culture conditions
Throughout the entire culture period (i.e. from day 0 to day 5) only one type of culture medium was used: a mixture of Earle's and Ham's F10, without hypoxanthine and thymidine, as described previously (Rijnders and Jansen, 1998) with a pasteurized plasma solution (8.7%) as protein source. Oocytes, spermatozoa and embryos were incubated under strict controlled conditions (37.0°C; 5% CO2 in air; 90% humidity).
After the assessment of fertilization, all zygotes were cultured under the same conditions, i.e. in groups (24 embryos per droplet, 160200 µl) until day 3. On this day the embryo morphology was assessed as described previously (Rijnders and Jansen, 1998), based on which patients entered the study.
Randomization
On day 3 patients were prospectively randomized into four groups by blindly drawing lots on day 3 after insemination. The groups involved group or single embryo culture, and small or large culture volume. Group 1: group culture, volume adjusted to 5 µl per embryo; group 2: single culture, each embryo in 5 µl; group 3: single culture, each embryo in 160 µl; group 4: group culture, total culture volume of 160 µl. In the case of group culture in a small volume (group 1), the volume was adapted to the number of embryos cultured allowing all embryos to be cultured in one single culture drop (812 embryos), preventing a selection bias. On day 3 the medium was refreshed. Patients gave their informed consent regarding the transfer of blastocysts.
Blastocyst formation
On day 5 the morphology of all embryos was re-evaluated. Embryos were classified as blastocysts [blastocyst (B) and expanded blastocyst (EB)], morula/compaction stage (M/C), cleavage arrest (AR) or degenerated embryos (Deg.). The percentage of blastocyst formation in each group was determined. Preferably blastocysts were transferred. If these were not available, non-cavitating embryos were used. Two or three embryos were transferred, depending on the woman's age, previous infertility history and embryo quality.
Statistical analysis
Data are expressed as mean ± SD. Logistic regression analysis was done where the odds of having a blastocyst were modelled using the following factors: number of embryos on day 3, volume, culture and interaction between volume and culture. Categorical modelling was performed to relate the outcome in terms of blastocyst formation to embryo morphology (Collette, 1991; Agresti, 1996
).
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Results |
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Blastocyst formation
In total, 324 day 3 embryos were used for extended culture to day 5. The quality of the embryos on day 3 was determined by the number of blastomeres and percentage of fragmentation (Table I). A total of 75% of the embryos had 020% fragmentation (class 1 and 2 embryos), whereas 25% had more than 20% fragmentation (class 3 and 4 embryos). The percentage of class 1 and 2 embryos together was comparable between the four groups (77, 80, 71 and 73% respectively).
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Between group culture (groups 1 + 4) and single culture (groups 2 + 3), there was no significant difference in blastocyst formation: 35 versus 40%. The difference between small (groups 1 + 2) and large (groups 3 + 4) incubation volume also was not significant: 40% versus 36%.
Within the groups of single culture, there was no difference whether embryos were cultured in a small (group 2) or large (group 3) incubation volume: 45 and 36% respectively. In case of group culture, similar rates of blastocyst formation were observed when embryos were cultured in a small (group 1) or large (group 4) incubation volume: 35 versus 36% (Table II). Using a small incubation volume, there was no difference in blastocyst formation whether embryos were cultured in a group (group 1) or individually (group 2): 35 and 45% respectively.
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Discussion |
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In our study, however, we found no significant influences of communal growth or incubation volume on embryo development from day 3 embryos to blastocyst stage. Therefore, culturing embryos to blastocysts can be performed using any of the four modes; whichever is most convenient. In this study, neither the incubation volume nor group culture influenced blastocyst formation from day 3 embryos, although the single culture in a small volume yielded apparently the highest, though not significant, rate of blastocyst formation. Culturing embryos individually in a small volume has a practical advantage: one can perform a direct and individual assessment of embryo morphology. This, therefore, is the method we prefer. From our study, we cannot rule out influences of `putative embryo-derived growth factors'. However, if present, these influences may be small. Some explanations as to why we did not find any difference include: firstly, the possibility that even 5 µl droplets are not small enough to let these factors influence the growth; secondly, it has recently been suggested that autocrine survival factors in mice only are required on or before the 2-cell stage (O'Neill, 1998). If extrapolation to humans is warranted it may be that from day 2 onwards these factors have ceased to influence the development of the embryos. This is not in agreement with the findings of Paria and Dey (1990), who detected epidermal growth factor (EGF) receptors on the embryonic cell surface first at 8-cell/morula and blastocyst in mice, suggesting the beneficial effects of group culture on embryo development to occur at or after the 8-cell stage. In humans, the presence of transcripts for EGF has been detected in embryos between 8-cell and blastocyst stages (Chia et al., 1995
); therefore one might expect an influence of group culture after the 8-cell stage.
Lane and Gardner (1992) did not find a significant difference in blastocyst formation in mice in relation to the incubation volume when embryos were cultured individually. Culturing embryos in groups, they found a significant increase in blastocyst formation in the 5 µl and 320 µl group (but not 20 µl) with increasing embryo concentration. Later Gardner et al. (1997) noticed a higher rate of blastocyst formation in mice when reducing the embryo:incubation volume ratio (Gardner et al., 1997). However, as in the group with the smallest incubation volume (2 µl), 10 embryos were placed in a 20 µl droplet, one cannot make the distinction between group culture and small incubation volume. Recently, Gardner et al. (1998b) published results of a randomized study comparing day 3 with day 5 transfer in patients where at least 10 oocytes were to be expected (Gardner et al., 1998b
). In the day 5 group, embryos were cultured in groups (34) in a large volume (1 ml) and the overall blastocyst formation rate was 46.5%.
It has been suggested that maternal age is the most important factor concerning blastocyst development and embryo viability (Janny and Ménézo, 1996). It has recently been reported that the biological age rather than the chronological age influences blastocyst formation (Scholtes and Zeilmaker, 1998
). Group 3 in our study consisted of relatively young patients (mean 31.3 years; not significant) but this did not influence the results.
The rate of blastocyst formation might be dependent on paternal factors (Janny and Ménézo, 1994; Jones et al., 1998b
) as well as the technique that has been used, IVF or ICSI (Shoukir et al., 1998
). No significant difference was noted in blastocyst formation between IVF and ICSI in the period preceding this study. However, taking into account that this rate could have been lower in ICSI, we reanalysed our data. If the ICSI patients were left out, the blastocyst formation rate in the four study groups became more nearly equal. Therefore, whether or not ICSI was performed did not affect the conclusion of this study. It should be noted that our study group consisted of a selected population in terms of number of embryos at the start, therefore the study was not designed to draw conclusions on maternal and paternal effects.
It is noteworthy that the odds of blastocyst formation per embryo diminished in relation to the number of embryos: per extra embryo the chance decreased by 6%. However, it should be remembered that in a natural cycle just one oocyte is selected for ovulation.
Not all patients may benefit from prolonging embryo culture to the blastocyst stage. Since it is not known what risks exist to the embryo between days 3 and 5, patients with a limited number of good quality embryos may end up without blastocysts. It is conceivable, however, that the suboptimal culture conditions created using artificial media during this period may impose additional hazards. Only 50% of the good quality embryos and 20% of the lesser quality embryos, based on day 3 morphology, reached blastocyst stage (Rijnders and Jansen, 1998). For this reason the patients' entry into the study could only be confirmed on day 3 when embryo morphology was assessed; until this moment all embryos were cultured under the same conditions (in groups). This is in contrast to previously published work (Moessner and Dodson, 1995
; Almagor et al., 1996
), where the study started at the zygote stage and ended at the 4-cell stage.
The introduction of a new culture medium introduces a second variable in addition to prolonging the culture duration. In our study the type of culture medium was not changed during the culture period, except for one refreshment on day 3. Gardner et al. (1998a) reported a high rate of blastocyst development (more than 60%) when using two sequential media (G1 and G2) (Gardner et al., 1998a). This study included only eight patients in total in the day 5 group (101 embryos divided over two groups, each with a different culture medium between day 1 and 3), and the patients were not randomized. As there may have been a selection bias for patients that were allowed to proceed to day 5 in comparison with the ones that had an embryo transfer on day 3, the resulting pregnancy and implantation rates may have been skewed. In a subsequent prospective, randomized study, significant increased implantation rates were shown after the transfer of blastocysts (Gardner et al., 1998b
). The pregnancy rates, however, were the same after transfer on day 3 and day 5 as fewer embryos were transferred on day 5. In this study, the blastocyst formation rate was 46%, which is comparable to the blastocyst formation rate in an equivalent set of patients in our study. Jones et al. using another type of sequential medium (mHTF/EG1 and EG2) (Jones et al., 1998a
) also reported a similar rate of blastocyst formation (3052%) as our own group (Rijnders and Jansen, 1998
), where a single medium system was used. It is clear that much further work needs to be performed regarding the composition of the ideal culture medium, if such a medium exists, to improve blastocyst development and viability.
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Acknowledgments |
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Notes |
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References |
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Submitted on August 12, 1998; accepted on June 16, 1999.