Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 5, 1987

Effect of Growth Factors in Culture Medium on the Rate of Mouse Embryo Development and Viability in Vitro CATRIONA M. CARO, 1,2 ALAN TROUNSON, 2 and CAROL KIRBY 2

Submitted: January 15, 1987 Accepted: June I1, 1987 (Central Editorial Office)

There is a need to develop culture media which enable embryos to achieve their full developmental potential at rates expected in vivo if the success rates of in vitro fertilization (IVF) are to be maximized. Mouse embryos will develop from the two-cell stage to the blastocyst stage in media with or without protein or amino acid supplementation (5,6) but their rate of cleavage and development to blastocysts is retarded compared to those in vivo. In the present study, we have examined the rate of development of two-cell mouse embryos to blastocysts in vitro and their viability when transferred to recipient mothers. The routine culture medium (Whittingham's T6 medium) was supplemented with the growth factor insulin or the cocktail of growth factors, hormones, and other substances in Nu-Serum, which has been used as a serum replacement in media for the growth and maintenance of a variety of cell types in vitro.

The development of mouse embryos from two cells to blastocysts in Whittingham's T6 medium containing the growth factor insulin or the serum replacement NuSerum was studied and compared with development in vivo. The rate o f embryo development to blastocysts was increased by the addition of Nu-Serum to T6 and there was a significant increase in the implantation rate of embryos grown in T6 + Nu-Serum compared with T6 alone or with insulin. However, the rate o f embryo development in vitro was retarded compared with that in vivo and the number of normal fetuses following transfer to recipients o f blastocysts grown in T6 + Nu-Serum was not different from the number for those grown in T6 alone or with insulin. The addition of neither insulin nor NuSerum to T6 optimizes embryo development or viability. KEY WORDS: embryo culture; embryo viability; growth factors; serum replacement.

INTRODUCTION The d e v e l o p m e n t of h u m a n e m b r y o s may be achieved in a wide range of culture media despite very different chemical compositions (I-3). There appears to be no agreement on the most appropriate medium for human embryo culture and it is apparent that human serum which is normally added to basic media can be successfully replaced by human serum albumin (2). Even the complete omission of all protein and amino acid has no significant effect on embryo development or viability (4).

MATERIALS AND METHODS Six-hundred two-cell mouse embryos were collected from superovulated F~ hybrid (CBA x C57) females, 40 hr after injection with human chorionic gonadotropin (hCG) as previously described (6). Embryos were flushed from the oviducts in Dulbecco's phosphate-buffered saline, and any abnormal or degenerate embryos discarded. Whittingham's T6 (T6) medium was prepared as described before (4) and used without any protein supplementation was used as the primary control medium in the present study. Experimental culture media consisted of T6 with 10 ~g/ml bovine insulin (Cat. No. 40205, Collaborative Research, Lexington, MA) and 4 mg/ml bovine serum albumin

t To whom correspondence should be addressed at the Infertility Medical Centre, Epworth Medical Centre, Erin Street, Richmond, Victoria, Australia, 3121. 2 Centre for Early Human Development, Monash University, Queen Victoria Medical Centre, Melbourne, Australia, 3000. 265

0740-7769/87/1000-0265505.00/0 9 1987 Plenum Publishing Corporation

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CARO, TROUNSON, AND KIRBY

(BSA V, Sigma Pharmaceuticals) or T6 supplemented with 10% Nu-Serum (Collaborative Research, Lexington, MA). The BSA was a necessary additive to prevent loss of the insulin activity because of binding to the culture vessels. Previous e x p e r i m e n t s (6) have shown that BSA-supplemented medium does not significantly improve the rate of embryo development of two-cells to blastocysts compared with protein-free medium. NuSerum is a consistent formulation of epidermal growth factor, endothelial cell growth supplement, insulin, transferrin, triidothyronine, progesterone, estradiol-1713, testosterone, hydrocortisone, selenium, O - p h o s p h o r y l e t h a n o l a m i n e , glucose, amino acids, vitamins, bovine serum, and cell nutrients as determined by the manufacturer. Normal two-cell mouse embryos were washed in the medium in which they were to be cultured and placed in 0.5-ml droplets of either T6, T6 + BSA, T6 + BSA + Insulin, or T6 + Nu-Serum in plastic tissue culture dishes (Kayline, Adelaide), and the droplets covered with light-weight paraffin oil (BDH) which had been equilibrated with T6 at 37~ for at least 48 hr. Embryos were incubated in a humidified atmosphere under 5% CO2 in air at 37~ and the stages of development of all embryos recorded at 24, 48, and 72 hr after the commencement of culture. The rate of development in vivo relative to that in vitro in the three different media was determined by flushing embryos from superovulated, mated F 1 hybrids at equivalent times post-hCG injection. In the second part of the study, a further 600 two-cells were cultured in the three different media, and at 72 hr after the initiation of culture, blastocysts which had developed were transferred to pseudopregnant recipients (6) to assess their viability. A maximum of six blastocysts was transferred to each uterine horn of pseudopregnant recipient females 3 days after mating with vasectom i z e d males. The r e p r o d u c t i v e tracts o f all recipient mice were examined 12 days after embryo transfer, and the numbers of normal, abnormal, and resorbing fetuses recorded. Data were analyzed by chi-square tests to determine the level of significance.

24 hr was significantly (P < 0.01) higher in T6 + Nu-Serum than in any other medium tested or than in vivo. Development to compaction in T6 + BSA + Insulin was similar to that in T6 + BSA, and both were significantly better than that in T6 alone. The proportion of embryos developing from compacted morulae at 24 hr to the early blastocyst stage at 48 hr was significantly higher (P < 0.05) in T6 + Nu-Serum, T6 + BSA, and T6 + BSA + Insulin than in the T6 alone. There was no significant difference between these three media with additives in the proportion of embryos developing from compacted morulae to the early blastocyst stage. There was a significantly higher (P < 0.001) proportion of embryos developed in vivo to the blastocyst stage at this time than in any of the in vitro culture groups. The only significant difference (P < 0.05) in the proportion of early blastocysts at 48 hr developing to expanded and hatching blastocysts at 72 hr in the test media was between T6 + Nu-Serum and T6 alone. The proportion of in vivo controls at the hatched blastocyst stage after 72 hr was only just significantly higher (P < 0.05) than that in T6 + Nu,Serum. The small number of expanded and hatching blastocysts recovered in vivo indicates that a large proportion of embryos had already begun to implant. These results show that T6 + Nu-Serum may afford a slightly faster rate of development in vitro than the other media tested but does not equate with the capacity of embryo development in vivo. The viability of embryos grown from two-cells to blastocysts is shown in Table II. There was no significant difference in the proportion of blastocysts that developed in T6 alone, T6 + BSA + Insulin, or T6 + Nu-Serum, which developed to normal fetuses. However, there were more abnormal and resorbing fetuses in recipient mice transferred with embryos grown in T6 + Nu-Serum, resulting in a significantly (P < 0.05) higher implantation rate of transferred embryos. This result is concerning because the improvement in the rate of embryo development of two-cells to blastocysts in medium supplemented with Nu-Serum is not accompanied by an increase in embryo viability.

RESULTS

DISCUSSION

The rate of develoment of embryos in culture and in vivo (controls) from the two-cell stage is shown in Table I. The proportion of compacted morulae at

It has been reported that the presence of insulin in culture medium stimulates methylglucose transport in mouse embryos and also increases their rate

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 5, 1987

GROWTH FACTORS IN CULTURE AND MOUSE EMBRYO DEVELOPMENT

267

Table I. The Rate of Development of Mouse Embryos in T6 and Supplemented Media in Vitro Culture medium Factor Total number of embryos cultured Number of compacted morulae at 24 hr (%) Number of embryos with a blastocoel at 48 hr (%) Number of expanded and hatching blastocysts at 72 hr (%)

T6

T6 + BSA

T6 + BSA + insulin

T6 + Nu-Serum

187

82

206

182

8 (4)

14 (17)

37 (18)

55 (30)

0/284 (0%)*

39 (21)

16 (20)

62 (31)

73 (40)

322/374 (86%)

141 (75)

60 (73)

168 (82)

167 (92)

In vivo controls

52/52 (100%)

" Ninety-six percent uncompacted eight-cell embryos.

of development in vitro (7). The results of the present study support the observation of an increased rate of embryo development during culture in medium containing insulin. A further increase in the rate of embryo development was observed in medium supplemented with Nu-Serum, which contains growth factors and hormones in addition to insuliri. There was, however, no increase in viability with embryos cultured in medium containing insulin. An increase was observed in the rate of implantation, but not in the proportion of normal fetuses when embryos were cultured in medium supplemented with Nu-Serum. These results demonstrate the inadequacy of depending on development to blastocysts for assessing embryo viability. There is a possibility that even though preimplantation development is stimulated, the components of NuSerum may be detrimental for complete embryo viability. It is possible to eliminate serum from medium used to culture many cell lines in vitro by substituting a combination of hormones and other speTable II. Viability of Mouse Embryos Cultured in T6 and Supplemented Media in Vitro Culture medium T6 Total number of embryos transferred 177 Number of embryos transferred to pregnant recipients 155 Number of normal fetuses (%) 63 (41) Total number of normal, abnormal, and resorbing fetuses (%) 92 (59) * P < 0.05.

T6 + BSA + insulin T6 + Nu-Serum 180

193

141

153

47 (33)

62 (41)

77 (55)

108 (71)*

cific factors such as transferrin (8). The specific hormonal requirements vary with the individual cell type and it is likely that preimplantation mouse embryos may also require a specific combination of h o r m o n e s and g r o w t h f a c t o r s to e n a b l e the cleavage rate and development to match those occurring in vivo. The a p p a r e n t stimulation o f cleavage rate in vitro obtained in the present study suggests that it may be worthwhile to investigate a range of different combinations of hormones and growth factors in order to improve further the embryo cleavage rate. Even under conditions defined as optimum in vitro blastocysts cultured from the two-cell stage are 18 to 24 hr behind Day 4 blastocysts developing in vivo (9). Alteration of the ionic composition of culture medium to resemble more closely that of oviductal secretions increased the percentage of blastocysts but had little effect on the cleavage rate, taking 5 days for one-cell embryos to develop to blastocysts (3). The culture of two-cell mouse embryos in human amniotic fluid (10) increased the numbers of blastocysts hatching from zonae pellucidae after 72 hr culture in vitro but a comparison of cell numbers and viability of blastocysts grown in vitro and in vivo was not available in this study. Human amniotic fluid contains a large number of hormones, growth factors, and other substances not normally present in embryo culture media. F u r t h e r studies are required to assess whether this complex physiological fluid enables embryo development and viability to approach those obtained in vivo. The present study has attempted to improve further the simple balanced salt solutions used for mouse embryo culture while avoiding the addition of serum, which has been shown to have little benefit from embryo development or viability (4,6) and may at times even have detrimental effects (11).

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The addition of insulin or the cocktail of growth factors in Nu-Serum had only a minor effect on promoting embryo development when compared to the potential rate of development that occurs in vivo. Further studies are under way to determine the conditions in vitro which may enable this potential developmeiat to be achieved in vitro.

REFERENCES

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8.

1. Trounson A: In vitro fertilization and embryos preservation. In In Vitro Fertilization and Embryo Transfer, A Tmunson, C Wood (eds). Edinburgh, Churchill Livingstone, 1984, pp 111-130 2. Menezo Y, Testart J, Perrone D: Serum is not necessary for human in vitro fertilization, early embryo culture and transfer. Fertil Steril 1984;42:750-755 3. Quirm P, Kerin JF, Warnes GM: Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-498 4. Caro CM, "fi'ounson A: Successful fertilization, embryo development, and pregnancy in human in vitro fertilization

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(IN'F) using a chemically defined culture medium containing no protein. J Vitro Fert Embryo Transfer 1986;3:215-217 Cholewa JA, Whitten WK: Development of 2-cell mouse embryos in vitro. III. The effect of fixed nitrogen source. J Exp Zool 1965;158:69-78 Caro CM, Trounson A: The effect of protein on preimplantation mouse embryo development in vitro. J Vitro Fert Embryo Transfer 1984;1:183- 187 Gardner HG, Kaye PL: Effects of insulin on preimplantation mouse embryos. In Proceedings of the Australian Society of Reproductive Biology, 1984, Abstr 107 Hayashi I, Sato GH: Replacement of serum by hormones permits growth of ceils in a defined medium. Nature 1976;259:132-134 Harlow GM, Quinn P: Development of preimplantation mouse embryos in vivo and in vitro. Aust J Biol Sci 1982;35:187-193 Gianaroli L, Seracchioli R, Ferraretti AP, Trounson A, Flamigni C, Bovicelli L: The successful use of human amniotic fluid for mouse embryo culture and human in vitro fertilization, embryo culture and transfer. Fertil Steril 1986;46:907913 Shirley B, Wortham JWE, Witmyer J, Condon-Mahony M, Fort G: Effects of human serum and plasma on development of mouse embryos in culture medium. Fertil Steril 1985;43:129-134

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 5, 1987