黃泓淵醫師,王馨世醫師,宋永魁醫師

Loss of embryo viability and developmental block in culture medium has restricted the length of time during which embryos may be cultured in vitro prior to transfer. Various types of coculture systems have been devised to overcome these problems with cellular monolayers being the most common. Uterine fibroblasts, human tubular cells, and Vero cells (an epithelial cell line derived from Green monkey kidney) have all been successfully used to enhance early in vitro human embryo development and implantation. Successful embryonic implantation requires an appropriately prepared uterus and a dialogue between the blastocyst and the maternal uterine surface, recent studies strongly suggested a critical role for paracrine cytokines. IL-1 is a family of polypeptides comprised of IL-1 agonist, IL-1 receptor antagonist (IL-1 ra) and two IL-1 receptors (IL-1 R) have been identified. Since Vero cell coculture has been shown to enhance embryonic development and the IL-1 system has been suggested to play a key role in fetal-maternal communication during implantation process, we examined cytokine mRNA expression in individual mouse embryos. In the present study, we used Vero cell coculture to enhance mouse embryonic development through blastocyst stage. Furthermore, we investigated the pattern of embryonic mRNA transcripts of b actin, IL-1 b, icIL-1 ra and IL-1 R type I in individu preimplantation embryos at multiple developmental stages retrieved from Vero cell coculture and control medium with a novel reverse transcription (RT), nested polymerase chain reaction (PCR) protocol.

Viable Vero cells (1x105/well) were plated into 24-well tissue culture plate 72 hours prior to the introduction of embryos. Two-cell embryos were obtained from stimulated female mice, then randomly transferred to Vero cell monolayer cultures or HTF medium only as a control group. The stages of embryo development were observed daily by inverted microscopy. Single embryos from different developmental stages were collected and examined for mRNA-levels of ß actin, IL-1 b, icIL-1 ra and IL-1R t I using RT followed by nested PCR with specific designed primers. 2% agarose gel electrophoresis was carried out for each PCR product (Figure 1).

The rate of both blastocyst formation and hatching were both significantly higher in Vero cell coculture compared with the embryos cultured in control medium by student t test (81.2 ± 2.6% vs. 42.2 ± 3.7%, p < 0.001; 75.6 ± 2.7% vs. 19.2 ± 6.2%, p< 0.001, respectively). A total of 292 embryos cocultured on Vero cell monolayers and 77 control embryos cultured with medium alone were examined by RT-PCR. The percentage of embryos cocultured in Vero cells positive for the expression of the IL-1 b and IL-1 ra mRNA increased progressively as development proceeded through the hatching blastocyst stage. The number of embryos expressing IL-1 R tI did not increase during in vitro development, remaining the same at 15 - 20%. Comparison of the number of embryo expressing IL-1 b mRNA at different developmental stages revealed a significant increase from compact morula to hatching blastocyst embryos (p<0.05). Twenty percent of embryos at compact morula stage expressed IL-1 ra mRNA expression with a significant increase in the number of IL-1 ra producing embryos at early expanded blastocyst stage and hatching blastocyst stage (p<0.001). 

The present study documents the developmental increase in cytokine expression and supports the hypothesis that IL-1 system provides important embryotrophic factors and molecular signals during implantation interactions between the embryo and maternal endometrium. It is interesting to hypothesize that the IL-1 system ”dialogue” during the embryonic-maternal communication may be relevant for embryo implantation. Our results may provide indirect evidence that IL-1 could be used as a marker to possibly investigate embryo in vivo viability. In summary, the purpose of this study was to document the expression of the IL-1 agonist, antagonist and receptor mRNA expression in individual mouse embryos during development from 8-cell stage through hatching blastocyst. These findings also support a putative autocrine-paracrine role for the IL-1 system in embryo-maternal communication during the implantation process.