Influence of group culture and culture volume on the formation of human blastocysts: a prospective randomized study

P.M. Rijnders and C.A.M. Jansen

Department of IVF, Obstetrics and Gynaecology, Reinier de Graaf Groep locatie Diaconessenhuis, PO Box 998, 2270 AZ Voorburg, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The optimal culture conditions for embryos to reach the blastocyst stage are under investigation. One factor is the putative influence of autocrine or paracrine factors produced by the embryo itself. Studies in mice blastocysts showed a beneficial influence of micro-cultures and communal growth on pregnancy and implantation rates, attributed to these growth or survival factors. In humans, studies have only involved embryos up to day 2 or 3 without consistent results. Therefore a prospective, randomized study was designed to assess whether group culture or incubation volume would influence day 3 human embryos to develop into blastocysts. Embryos were initially cultured in groups until day 3, after which patients were randomly allocated to four groups. Group 1: group culture in a small volume; group 2: single culture in a small volume; group 3: single culture in a large volume; group 4: group culture in a large volume. No significant differences in blastocyst formation could be found between the four groups (35, 45, 36 and 36% respectively). Therefore any of the four modes can be used, whichever is most convenient. The only factor which made a statistically significant contribution was the number of embryos on day 3. An increase in the number of embryos by one embryo decreased the odds of obtaining a blastocyst by 6%. The overall pregnancy rate per transfer was 40%, and the implantation rate was 28%. Single culture in a small volume allows a direct and individual assessment of embryo morphology, and as such it seems preferable.

Key words: culture volume/group culture/human blastocyst formation/ICSI/IVF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Culturing embryos to the blastocyst stage is a promising new technique from which patients may benefit because of the higher implantation rates of these embryos. These higher rates could allow the transfer of fewer embryos, thus avoiding the risk of multiple gestations. Early reports (Bolton et al., 1991Go) showed limited success, but with the introduction of co-culture the percentage blastocyst formation in humans increased to 40–60%. Subsequently the pregnancy per embryo transfer rate and implantation rate (per number of embryos transferred) also increased to 34–44 and 18–20% respectively (Ménézo et al., 1992Go; Olivennes et al., 1994Go). Scholtes and Zeilmaker (1996) also reported high corresponding pregnancy and implantation rates (49 and 35% respectively) when only expanding blastocysts were transferred, all without the use of co-culture. Much work still needs to be done regarding the ideal composition of the culture medium and culture conditions necessary to obtain higher rates of blastocyst formation. Recently, a blastocyst formation rate of more than 60% has been reported (Gardner et al., 1998aGo) with the use of two sequential media that was presumed to meet the needs of the changes in embryo physiology and metabolism (carbohydrate levels and amino acids) during the embryo development in vivo.

Previously published data have shown that micro-cultures (Lane and Gardner, 1992Go; Gardner et al., 1997Go), as well as the communal growth of embryos (Wiley et al., 1986Go; Moessner and Dodson, 1995Go; Almagor et al., 1996Go), 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, 1990Go; O' Neill, 1998Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and treatment
A prospective, randomized study was carried out between October 1997 and January 1998. Embryo number and morphology on day 3 were used as inclusion criteria: only couples with at least six embryos containing <20% fragmentation, or 10 embryos of mixed quality, on day 3 after insemination, were included in this study. Ovarian stimulation consisted of human menopausal gonadotrophin (HMG) or follicle-stimulating hormone (FSH), with or without gonadotrophin-releasing hormone (GnRH) agonists, using either short or long protocol, as described previously (Rijnders and Jansen, 1998Go). Conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was performed, followed by an embryo transfer on day 5, preferably using blastocysts. As we had not seen a significant difference in the rate of blastocyst formation between IVF and ICSI in the period preceding the study, we did not distinguish between these modalities.

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, 1998Go) 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 (2–4 embryos per droplet, 160–200 µl) until day 3. On this day the embryo morphology was assessed as described previously (Rijnders and Jansen, 1998Go), 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 (8–12 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, 1991Go; Agresti, 1996Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient characteristics
A total of 25 patients was included in this study; seven patients in group 1 and six patients in each of the other groups. The mean age of the patients in the study groups 1–4 were 36.0 ± 2.8, 34.1 ± 2.5, 31.3 ± 3.9 and 35.8 ± 2.7 years respectively. All treatments were followed by a transfer on day 5. All but one patient had at least one blastocyst transferred.

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 IGo). A total of 75% of the embryos had 0–20% 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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Table I. Embryo morphology on day 3 and 5 post-fertilization
 
On day 5, the embryos in the four study groups were evaluated for blastocyst formation in relation to their culture volume and their culture conditions (group or single). In group 1 (group culture in small volume) 28 out of 81 embryos (35%) developed into blastocysts, whilst 47 arrested cleavage or degenerated (58%). Group 2 (single culture in small volume) had 45% blastocyst formation and 53% cleavage arrest or degeneration. Blastocyst formation as well as the percentage of cleavage arrest/degeneration was similar for group 3 (single culture in large volume) and group 4 (group culture in large volume), i.e. 36% blastocyst formation and ~60% cleavage arrest/degeneration (Table IGo).

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 IIGo). 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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Table II. Blastocyst formation in relation to group or single culture and culture volume
 
The results of the logistic regression analysis are summarized in Table IIIGo. Neither culture (P = 0.124) nor volume (P = 0.809) had a significant impact on the odds of obtaining a blastocyst. There was no interaction either (P = 0.531). However as the number of embryos on day 3 had a negative impact, this variable was included in the regression analysis. Controlling for the number of embryos on day 3 revealed no significant differences either. The only factor that made a statistically significant contribution to predicting the outcome of a blastocyst was the number of embryos on day 3 (P = 0.0001, OR 0.97, 95% CI: 0.95—0.98). The odds of obtaining a blastocyst on day 5 decreased by 6% with each extra embryo on day 3. Categorical modelling using embryo morphology on day 3 revealed that the only variable which just met the criterion was class 4 embryos. However here again, as previously published, morphology appeared to be of limited predictive value (Rijnders and Jansen, 1998Go).


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Table III. Blastocyst formation in relation to communal culture and culture volume: summary of the results from the logistic regression analysis
 
Pregnancy and implantation rate
This study was designed to assess whether group culture and the incubation volume could influence the blastocyst formation; however, it could not discriminate statistically concerning pregnancy and implantation rates. Table IVGo shows the number of embryos transferred and their developmental stage, the pregnancies in each group and pregnancy rate per embryo transfer, as well as the implantation rate per number of embryos transferred.


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Table IV. Outcome in relation to group or single culture and culture volume
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been reported that communal growth, as well as minimizing the incubation volume may facilitate blastocyst formation in murine embryos (Wiley et al., 1986Go; Lane and Gardner, 1992Go; Gardner et al., 1997Go) as well as in sheep (Gardner et al., 1994Go). Communal growth of embryos up to day 2 after insemination in human may also influence cleavage and/or pregnancy rates (Moessner and Dodson, 1995Go; Almagor et al., 1996Go). These effects were attributed to a putative embryo-derived growth or survival factor. It is especially noteworthy that Paria and Dey (1990) suggested that the primary effect was operational between the 8-cell/morula and blastocyst stage (Paria and Dey, 1990Go). Since no such studies have been performed up to the blastocyst stage in humans, this prompted us to perform a prospective, randomized study on these variables in the human embryo for culture between day 3 and day 5.

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, 1998Go). 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., 1995Go); 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., 1997Go). 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., 1998bGo). In the day 5 group, embryos were cultured in groups (3–4) 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, 1996Go). It has recently been reported that the biological age rather than the chronological age influences blastocyst formation (Scholtes and Zeilmaker, 1998Go). 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, 1994Go; Jones et al., 1998bGo) as well as the technique that has been used, IVF or ICSI (Shoukir et al., 1998Go). 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, 1998Go). 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, 1995Go; Almagor et al., 1996Go), 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., 1998aGo). 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., 1998bGo). 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., 1998aGo) also reported a similar rate of blastocyst formation (30–52%) as our own group (Rijnders and Jansen, 1998Go), 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.


    Acknowledgments
 
The authors thank Nedjad Losic PhD, for his help in performing the statistics.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Agresti, A. (1996) An Introduction to Categorical Data Analysis. John Wiley, Bristol

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Submitted on August 12, 1998; accepted on June 16, 1999.