Co-culture of the early human embryo: Factors affecting human blastocyst formation in vitro: CO-CULTURE OF THE EARLY HUMAN EMBRYO

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Abstract

Co-culture systems have been designed to overcome the embryonic developmental arrest observed in vitro in conventional culture media. Oviduct and uterine epithelial cells can sustain embryonic development, as can trophoblastic tissue and transport epithelia of non-genital origin. Its benefits involve neither hormone dependency nor histo-specificity. Fibroblasts do not overcome the developmental arrest in most mammalian species, but whether they do in humans is still unsure. In all systems, the quality of the feeder cells and the co-culture medium are very important. Using the Vero cell line, 60% of human IVF embryos reach the blastocyst stage. The quality of the sperm seems to affect results. We have observed: For 10% of the patients with unexplained fertility, blastocyst stage is not attained; this probably involves a maternal (ovarian) problem. When at least one blastocyst is transferred, the pregnancy rate per transfer is 31%. The implantation rate in pregnant women is higher than after transfer at day 2. After repeated failures of transfer at early stages (2-6 cells), transfer at the blastocyst stage gives high pregnancy rates (40%). This indicates an in vitro selection. There is a strong paternal effect on blastocyst formation: poor quality sperm give lower rates of blastocyst. Co-culture helps to understand treatment failures related to male factors. Around 60% of the patients having spare embryos have had blastocysts frozen. Transfers of frozen-thawed blastocysts give a 20% pregnancy rate and an implantation rate per embryo of 11%. Co-culture is a new tool which has to be carefully evaluated in human IVF programs. It does not impair "a minima" embryo viability and it allows in vitro selection.(ABSTRACT TRUNCATED AT 250 WORDS)

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Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

W H Eyestone, N L First (1989)

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

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Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells.

F Gandolfi, R Moor (1987)

To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)

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Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose.

S A Schini, Barry D Bavister (1988)

The failure of hamster 2-cell embryos to develop in vitro (2-cell block) was examined with experiments in which concentrations of glucose and phosphate in the culture medium were varied. Embryos were cultured in a protein-free modified Tyrode's solution that normally contains 5.0 mM glucose and 0.35 mM sodium dihydrogen phosphate. In the presence of 0.35 mM phosphate but without glucose, 23% of 2-cell embryos reached the 4-cell stage or further after culture for 1 day and 27% after 2 days. Glucose inhibited embryo development even at 0.1 mM (4% development to greater than or equal to 4-cells after culture for 2 days); there was no dose-related inhibition above this glucose concentration. In a second experiment, phosphate levels were varied in the absence of glucose. Phosphate was highly inhibitory to development, with 97% of 2-cell embryos reaching the 4-cell stage or further after culture for 1 day in the absence of phosphate compared to 9-21% in the presence of 0.1-1.05 mM phosphate. After culture for 2 days, 26% of embryos reached the 8-cell stage or further when phosphate was absent compared to 0% development to 8-cells with 0.1 mM phosphate or higher. In a factorial experiment, phosphate blocked development when glucose was present or absent, whereas glucose did not block embryo development in the absence of phosphate. However, 2-deoxyglucose (a non-metabolizable analogue of glucose) inhibited embryo development in the absence of phosphate. These data show that the in vitro block to development of hamster 2-cell embryos is caused at least in part by glucose and/or phosphate. Deletion of these compounds from the culture medium eliminates the 2-cell block to development in virtually all embryos, and approximately 25-75% of embryos develop to the 8-cell or morula stages in vitro. The observations provide a possible explanation for the 2-cell and 4-cell blocks that occur in conventional culture media: stimulation of glycolysis by glucose and/or phosphate may result in inefficient adenosine triphosphate (ATP) production. The data indicate marked dissimilarities in the regulation of in vitro development of early cleavage stage hamster embryos compared with embryos of inbred mice, since the latter have an inactive glycolytic pathway prior to the 8-cell stage of development and will grow from 1-cell to blastocyst with both phosphate and glucose in the culture medium.