Blastomere Biopsy for cleavage stage embryo
welcome dears, we are here to share some knowledge about blastomere biopsy in cleavage-stage D3 for genetic examination. Blastomere biopsy for PGT is an invasive procedure that involves disruption of cell adhesion and breaching of the zona pellucida followed by aspiration of one blastomere from cleavage-stage embryos. First and before doing anything we should check the laser system to be sure it is calibrated. blastomere biopsy in cleavage-stage preferred to be in early D3. the first step is how to select the embryos to perform blastomere biopsy, In D3, the embryo selected to blastomere biopsy should be embryo grade A or at least grade B Why? in general, because that embryos theoretically we highly expect to develop into good blastocysts embryo at day 5 and be available for transfer. what are the criteria of a selected embryo? it should be embryo at least have a 6 cells blastomere without fragmentation or as a maximum 5-10% fragmentation. Embryo with symmetrically arranged blastomeres and almost equal in size. and with a clear visible nucleus inside the blastomeres. and finally, the embryo has a regular shape now, the next step is to select the best cell that will be extracted? from this picture, we have a very nice embryo with 8 cells symmetrically arranged, and most of them equal in size each blastomere have a clear single nucleus. Also, we can find there is a little free space between blastomeres, that can help us when we perform a laser beam to make a hole and make the withdrawing the selected blastomere easier without any damage for another blastomere. now we are select the ideal blastomere, then we are ready to start with the embryo needle holder and blastomere biopsy needle, if necessary we can use Ca, Mn free medium to breakdown the conjugated links between blastomeres. we have placed the selected blastomere at 3 or 4 o'clock position and starting a laser beam carefully from the free space area toward the selected blastomere slowly and carefully to avoid any damage for blastomeres during making a hole process. as we see now the selected blastomere was placed at 4 o'clock position, The laser beam started at 3 o'clock at the empty area and come down toward the selected blastomere slowly and carefully to be closer. and then starting to remove all Z.P from this area to form a suitable hole by using medium laser beam force. a strong laser beam can damage more area and blastomere by excess heat without noting that. Also using a small weak laser beam the making hole process can take more time than usual maybe affect the development of embryo especially if we used media without buffer or Ca, Mn free media. what is a suitable hole size? a medium size hole, nither not small nor huge. it is like the size of the needle biopsy tip. if the small hole maybe affects the withdrawing of blastomere and may be ruptured also maybe not comfortable for the needle to withdraw the blastomere and need extra force and stress to enter inside the embryo if a huge hole in size, that means extra blastomere can come out during withdrawing or during embryo handling, washing and transfer to the final culture dish now as we see we just make the needle tip closer to the blastomere edge to check we will spend the suction right for the right cell without making negative pressure or stress inside the embryo, now we are starting gentle and smooth suction and withdrawing slowly in the same time, don't withdraw fully blastomere inside the needle it will be rupture and we will lose it. as we see slowly and gently no suction just minimal to keep needle catch the blastomere. now it is out safely. now let the embryo go free gently by decrease holding needle pressure slowly to avoid a negative suction pressure can withdraw the free blastomere inside the holding needle. again finally checks the final shape of the embryo, and the extracted blastomere to confirm the presence of a single clear visible nucleus before fixation or tubing process and sending it to the genetic lab. General recommendation doesn't use Ca, Mn free media if not necessary don't expose the embryo for Ca Mn free media more than 7 min for that I also highly recommend that don't treatment more than three embryo in the same dish handling stripper needle size is important, please use large size when you deal with embryos that underwent of blastomere extraction again the timing plays a critical role in this process as usual in all IVF lab steps. don't forget the right time and duration for each step. I hope this video is helpful and fruitful thanks for the attention and see you again in another videoes