47. Modern diagnostic and scientific approaches

Question 1

Modern diagnostic and scientific approaches

Answer 1

1. Micromanipulation of cells (cell engineering): fusion, injection
   into cytoplasm, experimental chimerism.
2. Transfer of nuclei from somatic cells into oocytes
   (animal cloning).

Question 2

Micromanipulation of cells

Answer 2

Micromanipulation of Cells:
- Uses a micromanipulator to separate cells or early embryos under a high-power inverted microscope.
- Involves microscopic treatment of individual eggs, sperm, or embryos to improve fertilization and/or pregnancy rates (e.g., ICSI, IMSI, ROSI techniques).

Intracytoplasmic Sperm Injection (ICSI):
- Developed to treat severe male factor infertility.
- Achieves fertilization by injecting a single sperm into the cytoplasm of the egg.

Preimplantation Genetic Diagnosis (PGD) and Preimplantation Genetic Screening (PGS):
- Used during in vitro fertilization (IVF) to test embryos for genetic disorders before transfer to the uterus.

Question 3

Cell Fusion

Answer 3

Cell Fusion:
Definition: Process where two or more cells merge to form a single entity, retaining the genetic information and organelles of the constituent cells.

Syncytium Formation:
- Same Type Cells: Fusion yields a syncytium.
- Common Locations: Placenta, bone, cartilage, muscle, and multi-nucleated giant cells.

Heterokaryon Formation:
- Different Types Cells: Fusion yields multi-nucleated cells called heterokaryons.
- Observed Fusions: Bone marrow cells with hepatocytes, cardiomyocytes, and Purkinje neurons.

Electrical Cell Fusion Method:
- Cell Preparation: Harvest and equalize the number of cells to ensure equal charge. Mix the cells in a fusion chamber.
- Alignment: Align cells in opposite pairs using dielectrophoresis, bringing them together.
- Membrane Fusion: Apply an electrical pulse voltage to allow cell membrane fusion; the nuclei remain separated initially.
- Post-Alignment: The cells are further aligned until nuclei fuse.
- Culturing: Transfer the fused cells to a cell culture dish for further growth.

Question 4

Applications of cell fusion

Answer 4

PICSI (Physiological ICSI)
- uses behaviour and property of sperm in natural conditions to
attach to hyaluronic acid
- mature spermatozoa bind to hyluronic acid (-> major component of egg’s coat) and one of them succeds at fertilization
- sperm capable of binding to hyaluronic acid are less likely to carry chromosomal abnormalities and greater structural DNA integrity.

LAH - Laser-assisted Hatching
- used to help “hatch” from its
zona pellucida, and to encourage implantation in uterine wall after embryo transfer.
- opening is made in the area w microneedle, using Tyrode’s acid solution, or with a laser (best technique).
- assisted hatching is performed immediately before the transfer of the embryos into the womb.

3 parent IVF - maternal spindle transfer
- healthy nuclear DNA removed from patient’s egg cell, leaving behind faulty mitochondrial DNA
- patients nuclear DNA transplanted into donor egg w healthy mitochondrial DNA
- Reconstructed egg cell fertilised w sperm in lab and implanted into patient. Resulting embryo has 3 genetic parents

3 parent IVF - pronuclear transfer
- mother’s egg w faulty mitochondrial DNA fertilised w father’s sperm in lab
- parents’ genes transplanted into donor egg w healthy mitochondrial DNA
- reconstructed embryo then is implanted back into mother. resulting baby has 3 genetic parents