Protein-free chemically defined medium for human embryo handling and culture in assisted reproduction

Embryos have been cultured in basal salt solution containing proteins ever since embryo culture procedures were initiated decades earlier. The sources of protein were either of human (serum proteins, such as maternal serum or cord serum or pooled sera) or animal ( bovine serum albumin, fetal calf serum or calf serum, etc.) origin. Media employed in embryo culture and handling in human assisted reproduction procedure usually contain human serum albumin (HSA) obtained from commercial sources. In some laboratories bovine serum albumin (BSA) is also used as a source of protein in human embryo culture.

The use in culture medium of protein obtained from donors (human or animal origin) has the potential to transmit diseases to patients undergoing assisted conception treatment. In the past an epidemic of hepatitis B occurred in about 200 IVF patients that received embryos cultured in medium containing pooled sera contaminated with hepatitis B virus (van Os et al.),The influence of contamination of culture medium with Hepatitis B virus on the outcome of in vitro fertilisation pregnancies.source:American Journal Obst/Gynec. vol.165 pg152-159, 1991).

Recently the scientific community was confronted with the dilemma of having to inform their patients that a commercial preparation of a culture medium used for embryo culture and handling may be contaminated with albumin donated by a person who later died of CJD (Kemmann, Creutzfeldt-Jakob disease (CJD) and Assisted Reproduction Technology (ART). source: Human Reproduction.vol.13.pg1777, 1998). In spite of stringent purification and sterilization measures, one cannot eradicate with absolute certainty the possibility of transmission of unknown/known pathogens bound to serum proteins (Truyen et al., There is nothing permanent except change.The emergence of new viral diseases.source:Veterinary Microbiology. (vol 43, pgs 103-122. 1995).

With the advent of deadly diseases such as AIDS and CJD it became obvious that embryo culture systems must be made safe for IVF patients especially since deadly pathogens and prions could be transmitted through donor proteins used in embryo culture and handling media. Media products devoid of added donor proteins would prevent transmission of diseases to IVF patients. The availability of a chemically defined protein free medium for the generation of viable early cleavage stage human embryos will ensure the safety of patients undergoing assisted reproduction treatment as the need to use potentially hazardous donor protein can be eliminated. It is useful for studies into the metabolism and nutritional requirement of embryos.

Table 1:Quality of day 2 human sibling embryos when cultured in ultra micro-droplets of ART-7b protein-free medium and control medium (commercial) containing serum proteins

(Embryo grade: 4=excellent; 3=good;2=fair;1=poor)

* 5 arrested at zygote stage

** 17 arrested at zygote stage

There was no difference in the fertilization rate between the protein-free ART-7b medium and the control medium (obtained from commercial sources) containing protein. There was a statistically significantly (p=0.0142) higher developmental arrest at the 1-cell or zygote stage in embryos generated in medium containing proteins. The average blastomere score was significantly (p=0.0401) higher in embryos generated in the protein-free medium while the average embryo grade was statistically similar in both groups. The higher average blastomere score of embryos generated in the protein-free medium suggest that the development or growth of embryos in this medium were faster than those generated in the control medium containing protein. The faster development or growth of embryos in the protein-free medium is indicative of its better quality compared to the control medium. All of the above findings collectively suggest the protein-free medium to be not only comparable but significantly better than that of the medium containing protein (Table 1).

Summary of Clinical Trial

Table 2: Implantation and Pregnancy Rates for Day 2 Human Embryos Generated in Media Completely Devoid of Added Protein With and Without Co-Culture

It was later shown that reasonable pregnancy rates could be obtained when the protein-free medium, ART-7 was used to generate embryos with the use of “helper” cells such as the patient’s own cumulus cells. Cumulus cells are found attached to the egg when retrieved from mature follicles. Although the clinical pregnancy rate of 28.6% obtained in the cumulus co-culture system was above the world average of 22% (for 1995). Further research was directed at eliminating the dependancy for cumulus cells and also to further improve the quality of the protein-free medium. Co-culture is very tedious and demanding on staff time. Furthermore the chances of contamination with microorganism is also high in co-culture. This research effort resulted in the formulation of the protein-free medium, ART-7b. The implantation and clinical pregnancy rates obtained with embryos generated in the ART-7b medium were excellent at 37.8% and 61.5% respectively which is somewhat similar to blastocyst transfers.

The above pregnancies were obtained from oocytes fertilized by the intracytoplasmic sperm injection (ICSI) technique. In a separate study instead of ICSI, oocytes were inseminated in protein-free medium with spermatozoa prepared in the same medium. This technique is known as the conventional IVF technique. Fertilization rate was excellent (about 80%) and was statistically similar to that of sibling oocytes inseminated in medium containing protein by spermatozoa prepared in medium containing protein. The embryos generated after conventional IVF was identical in both the ART-7b (protein-free) and control (protein containing) media. About 5 patients received embryos generated by conventional IVF in the ART-7b protein-free medium. Of these five patients, 4 are confirmed clinically pregnant. The remaining patient did not become pregnant. This is a preliminary finding since the research on conventional IVF in protein free medium is in its early stages.

This page shall be updated when this study is completed.

In conclusion it clear that viable human embryos can be generated in culture media devoid of added protein. Transmission of protein-bound pathogens to IVF patients can now be prevented by the use of protein-free culture and handling media. This is probably the first report of human pregnancies from embryos generated in completely chemically defined conditions during oocyte retrieval, spermatozoa preparation, insemination and embryo culture.

Dr J. Ali (jaffarali@hmc.org.qa or jaffarali@excite.com) of the IVF laboratory of the Assisted Conception Unit has been actively engaged in research to formulate protein-free media for use in human assisted reproduction for the past few years. His work led to the formulation of protein-free embryo culture and handling media. These media have been successfully tested in animal and human studies with excellent results and human clinical pregnancy rates (Ali, 1997, Proceedings of the Annual Scientific Meeting of the Fertility Society of Australia, Adelaide, Australia; Ali et al. Formulation of a protein-free medium for human assisted reproduction. Human Reproduction. January 2000, Vol. 15, pages 145-156).

posted on March 29, 2000