Methods: The early embryo following in vitro fertilization is tested for

preimplantation genetic diagnosis. At the 6-10 cell stage (day 3 of development),

one or two cells (blastomeres) are removed from the preimplantation embryo
during biopsy, allowing intact embryos to be implanted into the uterus.
ICSI (intracytoplasmic sperm injection), rather than standard IVF, is used. Sperm
that remain attached to the egg membrane during standard IVF can cause
problems. The following are the steps to perform PGD on the embryos before
transferring them to the uterus:
Egg fertilization: Instead of using typical IVF, ICSI (intracytoplasmic sperm
injection) is used to fertilize the eggs. During traditional IVF, this reduces the
danger of sperm remaining attached to the egg membrane. The research center
followed recognized methods for ovarian stimulation, oocyte extraction, and the
ICSI process. Human chorionic gonadotropin (hCG) was given once the follicles
had matured (follicle size 15–20 mm in diameter, as determined by
ultrasonography). After 36 hours, the eggs were extracted via aspiration.
Embryo culture: On day 3, the embryos are cultured until they have 6–8 cells, at
which time they are removed. It's also feasible to continue the culture until the
embryo reaches the blastocyst stage, which is made up of two cell groups: the
inner cell mass (gives rise to the embryo) and the trophectoderm (protects the
embryo; form the placenta). The rates of fertilization, oocyte cleavage, and
embryo cleavage, as well as the morphology, were measured after oocyte
insemination. The counts of two pronuclear oosperm as well as the embryo
development stage were observed and recorded on days 2, 3, 5, and 6 following
ICSI. On day 4 or 5, following ICSI, the Gardner blastocyst grading system and the
Puissant embryo grading system were transplanted.
Embryo biopsy: Using a laser or chemical components, a small hole is created in
the zona pellucida, and one or two cells (embryos on day 3) or a small collection
of trophectoderm cells are recovered through it (blastocyst on day 5).It is
important to see that the tested cells have a single nucleus (where the DNA is
stored) to avoid altering the results. A double needle holder was used to secure
the fixation, biopsy, and cutting needles on the micromanipulator, and the biopsy
embryo was placed in a G-Mops microdroplet containing 5% human serum
albumin (HSA). A biopsy needle was utilized to aspirate out the blastomere
containing the nucleus after cutting the zona pellucida in a favorable direction
with a mechanical cutting method. Each embryo was fixed using a single
nucleated blastomere.
Tubing: consists of depositing, with special delicacy, the cells obtained from the
embryo in a tube specialized for this process.
Genetic analysis: Molecular biology tools such as PCR, FISH, CGH array, and NGS
are used to examine the cells in the tube. Hypotonic therapy and blastomere
fixation are required prior to FISH. Following that, a fluorescence probe was
chosen based on the karyotype of the patient. The slide containing the fixed
sample was then dehydrated, denatured, hybridized, eluted, and negative stained
according to the kit's instructions. The slices can be studied right away or
preserved for a long time at - 20 C in the dark.