PGS-Trophoectodermal biopsy : Zona free embryo transfer

 Q.1: What is the role of Zona Pellucida—“ the guard wall of a sea full of jewels” ?? Ans: The role of zona includes A) initial binding of sperm, B) induction of acrosome reaction, C) and prevention of multiple sperm penetration and D) of blastomere scatter with E) avoidance of direct contact with toxic by-products. But , after all these process are completed that the zona is no longer essential for continuing of normal development in vitro, once compaction has been achieved. It has been theorized that protease-like enzyme is released in vivo either by the blastocyst itself or by the endometrium which aids hatching and subsequent implantation.

In view of the above-mentioned theory, assisted hatching was practiced by drilling small holes in the zona pellucide with acid Tyrode mechanically and more recently with laser (laser-assisted hatching [LAH]). In current times with advent of preimplantation genetic screening (PGS), which requires laser drilling, it was thought to improve IVF results more folds, but somehow PGS and LAH have yet not proven drastically to improve IVF outcomes.

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. What is assisted hatching?? Assisted zona hatching has been used in “in vitro fertilization (IVF)”- programs for several years. Successful hatching of the embryo from the zona pellucida is a prerequisite for implantation in the uterus. Assisted zona hatching came into limelight in IVF cycles to breach the zona pellucida and promote the natural process of hatching. IN the initial yrs of ART , embryo transfer(ET) programs started off with day 2 transfer; success rates were then usually much lower (10%—20%). IVF had a major breakthrough when its successful blastocyst culture dramatically increased the success rates to around 40%. Blastocyst transfer was not only more physiological but also the strongest of embryos which bypass the 4—8 cell block and survived blastocyst and hatching stages were transferred. Although human blastocyst expands readily in vitro, its blastocyst rate very heavily depends on laboratory environment, culture media, and techniques.

Q. 3: How exactly PGS is done??  Ans: Blastocyst embryos is placed in 10 pL of Quinn’s advantage medium with HF.PKS (Ca/Mg free) (sage, CT, USA) under light mineral oil and subjected to biopsy. A hole  is  made in the zona pellucida exactly opposite to the inner cell mass of the each blastocyst using the LYKOS, (Hamilton Thorne, Beverly, MA, USA) applying gentle suction with the biopsy pipette (Origio, Denmark).

Trophectoderm cells were encouraged to herniate from the zona pellucida and 5-7 trophectoderm cells are  dissected from each of the blastocyst. The biopsied cells were placed in DNase-RNase-free PCR tubes containing 2.5 pL phosphate-buffered saline. All biopsied embryos are then placed into CSC medium and incubated overnight in HERA cell incubator. PGS is done using next genome sequencing method. In the next day, PGS report will exhibit normal and abnormal embryo. The normal blastocyst will completely hatched out; and then loaded in embryo transfer catheter (Cook Medical, USA) and transferred .