1 Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford and 2 Assisted Conception Unit, Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK
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Abstract |
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Key words: co-culture/embryo/IVF/morphology/pregnancy rate
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Introduction |
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Using mouse, sheep and cow embryos, a number of studies have demonstrated that both the number and the rate at which blastocysts form in vitro is increased by increasing the number of embryos cultured in a given volume of medium or by reducing the volume of medium in which a given number of embryos are cultured (Wiley et al., 1986; Paria and Dey, 1990
; Canseco et al., 1992
; Lane and Gardner 1992
; Gardner et al., 1994
; Kato and Tsunoda, 1994
; Keefer et al., 1994
). This suggests that such preimplantation embryos produce autocrine factors that promote development.
Two small studies have supported the notion of a beneficial effect of group culture in the human, in terms of increased cleavage rate and improved morphology (Moessner and Dodson, 1995), and in terms of the potential of embryos to implant and develop into viable pregnancies following embryo transfer (Almagor et al., 1996
). In contrast, human embryos generated by IVF and cultured with ageing unfertilized oocytes developed less frequently to the morula stage or beyond than embryos cultured alone and, moreover, those blastocysts which did develop contained fewer cells than controls (Salahuddin et al., 1995
). This suggests that factors released by degenerating, unfertilized oocytes have a detrimental effect on preimplantation embryogenesis.
The present prospective randomized study extends these earlier investigations by comparing embryo morphology and IVF outcome in 159 cycles of IVF and embryo transfer during which preimplantation human embryos were cultured either individually or in groups.
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Materials and methods |
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IVF and embryo culture
Ovarian stimulation and IVF were performed as described previously (Bolton et al., 1989; Waterstone and Parsons, 1992
), except that the culture medium [Earle's balanced salt solution (EBSS) containing 0.11 mg/ml pyruvate] was supplemented with 0.001% (v/v) synthetic serum replacement (SSR) and 1% (v/v) human serum albumin (HSA; Medicult Universal IVF Medium, Medicult, Copenhagen, Denmark). Oocytes were examined at 1820 h post insemination (hpi), and those which had developed two pronuclei (2PN) were transferred individually (IC) or in groups of 35 (GC) into pre-equilibrated 20-µl drops of medium overlaid with light paraffin oil and incubated at 37°C in an atmosphere of 5% CO2 in air. Those cycles in which fewer than five oocytes developed 2PN were excluded from the study (n = 151, of which 72 would have been in the IC, and 79 in the GC group).
Morphological scoring system
Embryos were examined at 4244 hpi and assigned a grade between 4 and 1 according to their morphology at the level of the light microscope, with grade 4 embryos having perfect, spherical blastomeres with no extracellular fragmentation, and grade 1 embryos having barely defined blastomeres and >50% fragmentation, as described previously (Bolton et al., 1989). Each embryo was then given a morphological `score', derived from the product of the number of blastomeres and the grade of the embryo (Steer et al., 1992
). Thus, the minimum score was `0', assigned to necrotic embryos, and the maximum score was 24, assigned to 6-cell-stage grade 4 embryos.
Cycle outcome
The two or three cleavage-stage embryos with the highest morphological score were selected for transfer, which was performed between 46 hpi and 49 hpi. A qualitative test for human chorionic gonadotrophin (HCG) in urine was performed 14 days following embryo transfer using a commercial kit (Clearview, Unipath, Bedford, UK), and clinical pregnancy was confirmed 3 weeks later by the identification of one or more beating fetal hearts using ultrasound.
Statistical analysis
Statistical analysis was performed to compare the two groups in terms of implantation rate using the 2 test, and to compare all other parameters using the non-parametric MannWhitney test. Significance was set at P < 0.05 for both tests.
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Results |
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Discussion |
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Although all the studies were randomized prospectively, both the earlier studies included a relatively small number of treatment cycles (55 and 91 respectively), compared with 159 in the present study. Moreover, while one study reported the effect of the two culture conditions on embryogenesis in vitro prior to embryo transfer, but not on pregnancy outcome following embryo transfer (Moessner and Dodson, 1995), the other reported primarily the outcome following embryo transfer and gave only vague descriptions of embryo morphology (Almagor et al., 1996
). Indeed, in the latter study, between three and five embryos were transferred, yet the authors do not report whether or not there were differences between the two groups in the mean or median number transferred, making interpretation of the results impossible.
It is surprising that the differences seen in animal experiments (Paria and Dey, 1990) are not found in the human in the present study. The earlier studies both used Ham's F10 medium supplemented with 15% heat-inactivated human serum for embryo culture, compared with EBSS supplemented with 0.001% SSR and 1% HSA in the present study, but it is unlikely that this would explain the different findings.
It could be argued that the use of blastocyst formation as the end-point of the animal studies may have led to artefactual results (Bavister, 1995). However, this argument is based on the fact that prolonged culture may allow any slow-cleaving embryos to `catch up' with more developmentally competent, faster-cleaving embryos, thus failing to identify differences between different experimental groups. Since the mouse studies of Paria and Dey (1990) did demonstrate differences in blastocyst formation rates between different experimental groups, this argument is not valid. In terms of prolonged culture, it is more likely that the benefits of group culture identified by the animal studies are only manifested by culture beyond the 2- and 4-cell stage, and that prolonged culture of human embryos, beyond the 24 h in the present study, would demonstrate similar advantages.
This explanation cannot be invoked, however, to explain the differences between the findings of the present and previous human studies. The most marked difference in experimental design of these studies is in the volume of culture medium used. Given the beneficial effect of using smaller volumes of culture media suggested by Paria and Dey (1990), we chose to use 20-µl drops of medium, in contrast to the 1000-µl and 700-µl drops used in the studies of Moessner and Dodson (1995) and Almagor et al. (1996) respectively. Thus, it is possible that any autocrine factor(s) secreted by human embryos are already sufficiently concentrated to have a growth-enhancing effect in a drop of 20 µl containing a single human embryo, and that higher levels of the same factor(s) in the presence of additional embryos will have no effect. Indeed, the mouse studies found that the growth-enhancing effect of exogenous epidermal growth factor (EGF) was maximal at a concentration of 10 ng/ml; at 200 ng/ml, EGF did not further improve embryo development (Paria and Dey, 1990). If, however, the factor(s) secreted by a single embryo cultured in a drop of 700 µl or 1000 µl are diluted to the point of having a reduced effect, it can be seen that the presence of additional embryos would increase the concentration of the postulated factor(s), thereby restoring their stimulatory effect. Although this seems a feasible explanation, the mouse experiments upon which this study was based used drops of 25 µl containing 1, 5 or 10 mouse embryos (Paria and Dey, 1990
), and it is difficult to explain why human and mouse embryos should differ so greatly in the levels of secretion of putative autocrine stimulatory factor(s).
In conclusion, the present study fails to support the suggestion from animal experiments, and from limited data from human studies, that the development of preimplantation human embryos in vitro is enhanced when embryos are cultured in groups rather than individually. Further studies using the same experimental design but with embryos cultured in drops of larger volumes will be undertaken.
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Notes |
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References |
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Submitted on May 6, 1998; accepted on October 5, 1998.