Co-culture Of Embryos Research Articles

Partial purification of embryotrophic factors from human oviductal cells.

Embryotrophic factors from human oviductal cells were partially purified by liquid chromatographic methods. The conditioned medium from human oviductal cell culture was fractionated successively by concanavalin A (Con-A) affinity chromatography, ion-exchange chromatography and gel filtration. The presence of the embryotrophic activity in the eluates was determined by the stimulatory effects on the development of mouse embryos in vitro. The fraction that did not bind to the lectin Con-A possessed no embryotrophic activity. Ion-exchange chromatography separated the glycoproteins that bound to Con-A into five fractions. Three of them significantly enhanced blastulation as well as conceptus formation. Gel filtration further separated these embryotrophic fractions into five fractions. Three of them with molecular weights of 154 +/- 1, 164 +/- 0.2 and 207 +/- 0.3 kDa significantly stimulated blastulation of mouse embryos. The results of this study demonstrated that several embryotrophic factors with different biochemical properties contributed to the embryotrophic effect of the human oviductal cell/mouse embryo co-culture system.

Expression of insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA in embryos and endometrial stromal cells.

The objective of the present study was to investigate the effects of insulin-like growth factor-binding protein-1 (IGFBP-1) on the interaction between mouse embryos and endometrial stromal cells in co-culture systems. To explore quantitatively the changes in expression of IGFBP-1 mRNA from endometrial stromal cells cultured alone or co-cultured with embryos, a combination of reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting followed by a densitometric analysis was used. In co-culture systems, development of embryos was significantly improved and the production of IGFBP-1 from endometrial stromal cells was stimulated by the embryos. Quantitative analysis showed that expression of IGFBP-1 mRNA in endometrial stromal cells co-cultured with embryos was higher on days 3,5 and 8 (1.6-, 4.5- and 2.3-fold respectively) than in endometrial stromal cells cultured alone. In addition, intensity of PCR products for IGFBP-1 mRNA in endometrial stromal cells co-cultured with embryos was higher on day 5 than on days 3 and 8. However, the expression of IGFBP-1 mRNA in embryos cultured alone was very low. In conclusion, co-culture of embryos with endometrial stromal cells improved the development of embryos and may be associated with the production of IGFBP-1 by the co-cultured endometrial stromal cells. A combination of RT-PCR and Southern blotting followed by a densitometric analysis appeared to be a sufficiently quantitative method to determine changes in IGFBP-1 mRNA values.

Co-Culture of Rat Ova with Oviductal Cells in m199 FCS.

This experiment was designed to evaluate the ability of 4 different types of somatic cells to promote development of early cleavage in rat embryos. Embryos were collected from Wistar-Imamichi rats. Four and 8-cell rat embryos were co-cultured with rat granulosa, oviductal, uterine and kidney monolayer cells. The culture was performed in TCM 199 supplemented with 10% fetal calf serum, sodium lactate and pyruvate alone (m199FCS). Among the co-culture systems, morula and blastocyst development was better with granulosa, oviductal and uterine cells (98.6, 95.6 and 94.1%, respectively) than with m199FCS alone and kidney cells (77.6 and 73.7%, respectively). Similarly, 4-cell rat embryo development also followed the same trend and the development percentages with granulosa, oviductal, uterine, kidney cells and m199FCS alone were 32.0, 30.7, 29.3, 0 and 2.7%, respectively. Nevertheless, 8-cell embryos were developed to hatched blastocysts in co-culture with oviductal, uterine cells and in the conditioned medium of oviductal cells, 21.0, 19.0 and 2.1%, respectively. But co-culture with granulosa, oviductal and uterine cells represented the best physiologic approach and showed superiority to the other cell types. Forty-four of 184 1-cell embryos co-cultured with oviduct cells developed to the morula/blastocyst stage. The co-culture of early rat embryos in a medium with oviductal explants can support further development.

Premature progesterone elevation spares blastulation but not pregnancy rates in in vitro fertilization with coculture

Objective: To clarify whether embryo development to the blastocyst stage may be affected by premature P elevation during controlled ovarian hyperstimulation (COH) for IVF-ET with embryo coculture. Design: Retrospective study. Setting: Tertiary care infertility center. Patient(s): One hundred thirty-one women undergoing 153 IVF-ET cycles with embryo coculture. Intervention(s): Patients underwent COH with GnRH agonist and hMG. Embryos were cocultured up to the blastocyst stage. According to plasma P levels on the day of hCG, two groups were defined: low P (P ≤ 0.9 ng/mL; conversion factor to SI unit, 3.180) and high P (P > 0.9 ng/mL). Main Outcome Measure(s): Blastulation (number of blastocysts/number of noncavitating embryos × 100) and pregnancy rates (PRs). Result(s): Blastulation rates were similar in the low and high P groups (51% and 48%, respectively). Moreover, patients included in the high P groups achieved significantly lower clinical and ongoing PRs (12% versus 29% and 7% versus 25%, respectively). Conclusion(s): The lack of difference in blastulation rates between the groups further supports the hypothesis that premature P elevation does not alter oocyte and embryo quality. Hence, the observed decrease in PRs is likely to reflect impaired endometrial receptivity in the high P group.

Coculture of human embryos with buffalo rat liver cells for women with decreased prognosis in in vitro fertilization

OBJECTIVE: The coculture of human embryos with epithelial cells may improve both embryo quality and pregnancy rates. In this current study we tested the efficacy of coculture with the buffalo rat liver cell line on pregnancy rates in women with a potentially poor prognosis for success with in vitro fertilization (previous in vitro fertilization failure, advanced maternal age, increased early follicular follicle-stimulating hormone levels, and anovulation). STUDY DESIGN: This prospective controlled study evaluated a total of 203 women (135 coculture, 68 controls) undergoing in vitro fertilization. Implantation rates per embryo, clinical pregnancy rates, and continuing/delivered pregnancy rates were analyzed. RESULTS: Buffalo rat liver cells, which are commercially available, are stable in coculture. Implantation rates (number of sacs with fetal heart motion per embryos transferred) were similar for coculture (19%) and control (18%) embryos. No difference in the rate of continuing/delivered pregnancies per retrieval was noted (17% coculture vs 14% control) in the group with advanced maternal age, but coculture caused a trend toward improved pregnancy rates in the group with ovulatory dysfunction (43% coculture vs 14% control) and the group with previous in vitro fertilization failure (34% coculture vs 28% control). CONCLUSION: This is the first published controlled study to our knowledge that reports the use of the buffalo rat liver cell coculture for human in vitro fertilization in a large number of patients. Our data support consideration of buffalo rat liver coculture for in vitro fertilization for women with previous in vitro fertilization failure and possibly for patients with oocyte or ovulatory dysfunction. (Am J Obstet Gynecol 1997;177:358-63.)

Evaluation of mitomycin-treated vero cells as a co-culture system for IVM/IVF-derived bovine embryos

Support of the in vitro development of IVM/IVF-derived bovine embryos by Vero cells was evaluated by comparing the following treatment groups: 1) proliferating (Unt-Vero) vs nonproliferating (Mit-Vero) cells; 2) supplementation of medium with estrous cow serum (ECS) vs bovine serum albumin (BSA); 3) Mit-Vero cells vs bovine oviduct epithelial cells (BOECs); and 4) addition of leukemia inhibitory factor (LIF) to Mit-Vero cell co-cultures at Day 1 vs Day 4. Mit-Vero cells stimulated higher rates of blastocysts (Day 7, 40 vs 27%) and hatched blastocyst (Day 10, 38 vs 12%) formation than Unt-Vero cells. These rates were comparable to those obtained with BOECs; blastocyst hatching was slightly higher following co-culture with Mit-Vero cells (36%) than BOECs (29%). Blastocyst formation was similar in ECS- vs BSA-supplemented medium; however, hatching was greatest (37%) during co-culture in medium +10% ECS. While the addition of LIF throughout the co-culture period was ineffective, addition of the cytokine beginning at Day 4 slightly increased blastocyst formation rates. Evaluation of LIF secretion using ELISA revealed detectable levels of the cytokine in Mit-Vero-conditioned medium (50 pg/10 5 cells); this may explain the minimal influence of exogenous LIF during embryo co-culture. Mit-Vero cells provided comparable support of bovine embryo development when used even up to 2 wk after establishment as monolayers. In conclusion, Mit-Vero cells provide a readily-available, safe and easy-to-use co-culture method which is at least as supporting of bovine embryo development as BOECs. One contribution of these cells may be secretion of the cytokine LIF.

O-241. Co-culture of embryos on homologous human endometrial epithelial cells in patients with repeated implantation failures

O-241. Co-culture of embryos on homologous human endometrial epithelial cells in patients with repeated implantation failures

Interest of co-cultures for embryos obtained by in-vitro fertilization: a French collaborative study.

Co-cultures of human embryos, particularly with Vero cells, are used by several French groups, mainly in cases of successive failures of implantation. In most cases co-culture is continued until the blastocyst stage, expanded if possible. A total of 1603 co-cultures have been performed by 11 groups over a 2-year period. Of these, 41.8% of cleaved eggs (day 2) reached the blastocyst stage at day 5 or day 6. The mean pregnancy rate and implantation rate per transfer were 32.9 and 24.8% respectively, which represented a significant improvement compared to the transfer of 2 day old embryos. The rate of multiple pregnancies remained high (29.1%), which implies that there should be transfer of not more than two blastocysts. The rate of anomalies perceived at birth or in utero was not different from the rate observed in the general population, taking account of the maternal age.

Preparation of animals with a high degree of chimerism by one-step coculture of embryonic stem cells and preimplantation embryos.

A single-step coculture procedure has been developed that can generate chimeric mice with high efficiency and reproducibility. The procedure involves culture of embryonic stem (ES) cells with 8- to 16-cell embryos in microwells to provide conditions for effective cell-cell-embryo contact. A suspension of ES cells is layered over the microwells, followed by transfer of an embryo without zona pellucida into each microwell. Following overnight culture, the blastocysts are transferred into pseudopregnant recipients. The method has several advantages due to its simplicity and reproducibility: (i) Over 90% of ES-cell contribution in new-borns can be obtained frequently and most of the chimeras display germ-line transmission. (ii) The procedure does not require specialized skills or expensive instruments. (iii) All the steps of embryo manipulation can be completed in a relatively short period of time; therefore, a large number of embryos can be manipulated simultaneously. The method was tested with three independent ES cell lines and two different strains of mice with similar results. The technique may be an alternative to microinjection of DNA into zygotes to prepare transgenic lines of small and large mammalian species.

Modifications made to culture medium by bovine oviduct epithelial cells: changes to carbohydrates stimulate bovine embryo development.

Co-culture remains a common method to support the development of bovine embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo remains undetermined. This study therefore analysed the changes made to the culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined the effects of specific changes in medium composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, amino acids, NH4+, H+ and the gas tensions of O2 and CO2 were measured in TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and directly following 48 h incubation periods with each cell type. All three somatic cell types modified the carbohydrate composition of the media in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and pyruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine the relevance of such changes in carbohydrate concentrations on bovine embryo development, modifications were made to carbohydrate levels in synthetic oviduct fluid (SOF) medium and their effect on blastocyst development in vitro assessed. In SOF medium supplemented with amino acids and BSA (SOFaa), significantly more zygotes developed to the blastocyst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 (11%). Interestingly, when the levels of carbohydrates in SOFaa mimicked those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was ascribed to the reduction in glucose and increases in D/L-lactate and pyruvate concentrations in the culture system. Results from this study demonstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are present in equimolar amounts. As such, culture media in which D/L-lactate syrup is used actually contain only 50% biologically active lactate meaning that all D/L-lactate concentrations are reported at twice the effective concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24-36%) until the total D/L-lactate was present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained low (60.1 +/- 6.9 - 78.5 +/- 6.6) until a total D/L-lactate concentration of 3.3 mM. This data reinforces that embryo morphological appearance is not sensitive enough to be used as the sole criterion for assessing embryo development.

Modifications made to culture medium by bovine oviduct epithelial cells: Changes to carbohydrates stimulate bovine embryo development

Co-culture remains a common method to support the development of bovine embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo remains undetermined. This study therefore analysed the changes made to the culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined the effects of specific changes in medium composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, amino acids, NH4+, H+ and the gas tensions of O2 and CO2 were measured in TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and directly following 48 h incubation periods with each cell type. All three somatic cell types modified the carbohydrate composition of the media in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and pyruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine the relevance of such changes in carbohydrate concentrations on bovine embryo development, modifications were made to carbohydrate levels in synthetic oviduct fluid (SOF) medium and their effect on blastocyst development in vitro assessed. In SOF medium supplemented with amino acids and BSA (SOFaa), significantly more zygotes developed to the blastocyst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 (11%). Interestingly, when the levels of carbohydrates in SOFaa mimicked those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was ascribed to the reduction in glucose and increases in D/L-lactate and pyruvate concentrations in the culture system. Results from this study demonstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are present in equimolar amounts. As such, culture media in which D/L-lactate syrup is used actually contain only 50% biologically active lactate meaning that all D/L-lactate concentrations are reported at twice the effective concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24–36%) until the total D/L-lactate was present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained low (60.1 ± 6.9 – 78.5 ± 6.6) until a total D/L-lactate concentration of 3.3 mM. This data reinforces that embryo morphological appearance is not sensitive enough to be used as the sole criterion for assessing embryo development. Mol Reprod Dev 46:146–154, 1997. © 1997 Wiley-Liss, Inc.

Effect of culture in rabbit oviduct and of duration of in vivo culture on development of prepubertal goat embryos

Culture of pig embryos obtained prior to the four-cell stage has been difficult to accomplish. The ‘in vitro developmental block’ at the four-cell stage can be overcome by a number of methods to allow complete development of pig embryos from the one-cell stage to the blastocyst stage in vitro. Mouse oviducts in organ culture have been shown to provide a suitable environment for pig embryo development. Co-culture of pig embryos with oviductal cells or supplementation of culture medium with pig oviductal fluid results in improved embryonic development in vitro. Modifications to simple culture media have demonstrated that glutamine can serve as the sole exogenous energy source for development from the zygote to the blastocyst stage in vitro. Glucose was not inhibitory to the development of pig embryos in vitro. Addition of taurine and hypotaurine to the medium further increased the degree of embryo development in vitro. A number of different media have been reported that support pig embryo development in vitro. Although the nature of the ‘in vitro developmental block’ is not known, a number of methods now exist to circumvent this problem in the pig.

Bovine oviductal epithelial cells: their cell culture and applications in studies for reproductive biology.

Epithelial cells of the mammalian oviduct play an important role in reproductive and developmental events that occur there. Oviductal epithelial cells from several mammalian species can be isolated and cultured in serum or serum-free medium in vitro and cell culture of bovine oviductal epithelial cells (BOEC) has been described by many investigators. Cultured BOEC show a wide variety of secretory activities and these secretory factors may influence early embryonic development or sperm function. Monolayer cultures of BOEC have been widely used for in vitro co-culture of bovine preimplantation embryos. The use of BOEC co-culture systems has improved embryonic development in nearly all the studies conducted. In addition, interaction of bovine spermatozoa with BOEC, in a similar manner to that observed for spermatozoa in vivo, induced specific changes in sperm capacitation and consequently improved the fertilizing capacity of bovine spermatozoa in vitro. Thus co-culture systems with BOEC may not only offer an excellent model for studying the mechanisms of capacitation and acrosome reaction of bovine spermatozoa but also provide a useful tool for the improvement of embryo development in vitro.

Culture with Matrigel inhibits development of mouse zygotes.

It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. Matrigel may be a deleterious addition to embryo culture or coculture systems.

In-vitro fertilization and embryo transfer in women aged 40 years and over.

The decline of fertility with age and its possible causes are discussed; in particular the effect of ageing of oocytes and the uterus, and the effect of the ageing processes on the results of in-vitro fertilization (IVF) and embryo transfer in women aged > or =40 years. The role of prestimulation testing in older women is considered together with the importance of screening and counselling these patients about the likelihood of achieving a live birth. The potential problems that they may face should they become pregnant are reviewed, together with the role of oocyte donation as an alternative treatment for patients with reduced ovarian reserve. Possible ways of improving the chances of achieving a live birth in older women using their own oocytes are reviewed, including the use of more effective stimulation protocols, assisted embryo hatching and co-culture and high order embryo transfer. The outcome of pregnancies in older women and some of the ethical problems relating to their treatment are also discussed.

Co-culture of embryos and feeder cells.

Co-culture of embryos and feeder cells.

Maternal age effect on early human embryonic development and blastocyst formation.

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([blastocysts/embryos on day 2]* 100) and the blastocyst expansion rate ([expanded blastocysts/blastocysts]* 100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (< 30 years, 82%; > or = 40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo.

Secretion of leukaemia inhibiting factor by human endometrium alone and in co-cultures with embryos.

During our research programme on implantation failures after in-vitro fertilization (IVF), we investigated the expression of leukaemia inhibitory factor (LIF) by endometrial cells in women who have experienced several implantation failures after embryo transfer. Their embryos appeared to be morphologically normal and were either fresh or cryopreserved. Our intention was to investigate the role of LIF in regulating human preimplantation embryo development. In animals, the addition of 1000 IU/ml of recombinant human LIF (rhLIF) to culture medium, in comparison with control medium, significantly decreased the degeneration of ovine embryos (9 versus 27%), enhanced embryo hatching (64 versus 16%), and improved rates of implantation (50 versus 16%) (Fry et at., 1992). In the same way, co-culture cells that express LIF (including Vero cells and human embryonic fibroblasts) seemed to be superior to cells that did not express LIF (including human placental villous core mesenchymal cells) for the support of the early preimplantation development of mouse embryos (83-86% blastocysts versus 71 %) (Kauma and Matt, 1995). In our study, we first assessed LIF expression in endometrial cells by taking three uterine biopsies in one natural cycle in a group of women. Biopsies were taken in the luteal phase, i.e. early (2-4 days after ovulation), mid (6-7 days) and late (10-l2 days). LIF expression was checked by immunocytochemistry using double labelling with both anti-cytokeratin antibodies (fluorescein) and anti-LIF antibodies (rhodamine). Three observations were made: (i) endometrial cells (epithelial and stromal) contained LIF immunoreactivity in the luteal phase (3-11 days after ovulation); (ii) labelling was more intense for epithelial than stromal cells, indicating that differences existed in their LIF secretion; (iii) labelling was stable during the luteal phase in epithelial cells whereas it increased in stromal cells during the luteal phase. In a second experiment, embryo co-culture was performed on endometrial cell monolayers. The endometrial cells were obtained from biopsies carried out 3 days after ovulation in the preceding menstrual cycle; 5 days after beginning co­ culture, blastocysts were transferred into the uterus of patients and the conditioned media from the embryo cultures were assayed for LIF secretion. The endometrial

The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro.

Co-culture of human embryos with cell layers has generally shown that blastocyst formation rates are improved compared to routine culture in medium alone. In order to assess this further, we have additionally classified resulting blastocysts according to their morphology and secretion of human chorionic gonadotrophin (HCG). A total of 70 supernumerary human embryos from 15 patients were divided equally and randomly between two culture conditions: (i) co-culture with Vero cells; and (ii) culture in our routine medium. Embryo development and morphology were recorded for up to 14 days in culture. The results showed that embryos on Vero cells had a significantly higher blastocyst formation rate (P < 0.02) by or on day 6 of development than those in routine culture medium alone (77 and 46% respectively). For HCG analysis, the culture medium was changed in both culture systems on days 5, 7, 9, 12 and 14 of embryo development and analysed. Most embryos began to produce HCG between days 7 and 9, with HCG secretion being significantly higher from embryos on Vero cells between days 9 and 12 than from embryos in routine culture (P < 0.03). The morphology of the blastocysts obtained was related to their ability to hatch and produce HCG but was not significantly better for one type of culture system than for the other.

Fertilization and early embryology: Effect of different co-culture systems in early human embryo development

The objective of this study was to examine the effects of different culture systems on the development of early human embryos in vitro. A total of 460 fertilized oocytes from 82 cycles of patients was transferred into one of four systems: (1) into droplets of Ham's F10 medium + 12% normal human serum (NHS); (2) co-cultured on a human granulosa monolayer; (3) co-cultured with bovine oviductal epithelial cells (BOEC); or (4) co-cultured with bovine uterine epithelial cells (BUEC). The percentage of cleavage and the morphological appearance of embryos were recorded daily for 72 h in each system using an inverted phase-contrast microscope. The results showed that the proportions of the fertilized oocytes which developed to the four-cell stage 48 h after retrieval were, by culture system: (1) 70% (84/120); (2) 74% (85/115); (3) 78% (91/117); and (4) 76% (82/108). At 72 h after retrieval, the proportions of the eight-cell stage were, by culture system: (1) 45% (38/84); (2) 62% (53/85); (3) 75% (68/91); and (4) 70% (57/82). We concluded that a higher proportion of fertilized oocytes developed to embryos at the eight-cell stage in systems 2, 3 and 4 than in system 1. This indicates the beneficial effect of co-culture of human embryos with granulosa cell, BOEC and BUEC monolayers, which may be due to various factors.