Pre-implantation Genetic Diagnosis (PGD) Fertilization Technology Agriculture Biotechnology / stage four Alaa kamil Abdulla (2017-2018) .. Lec.9

B- Newly applied methods 1- Flow-cytometric sorting of Semen. 2- Pre-implantation Genetic Diagnosis (PGD)

1- Flow-cytometric sorting of Semen. It now is possible to sex semen of most mammalian species with greater than 90% accuracy with an instrument called a flow cytometry /cell sorter . Unfortunately, the process is relatively slow. The flow cytometry is another method used to sort sperm. However, because flow cytometry-based sperm sorting often uses fluorescent dyes that often stain DNA, the safety of this technique in human reproductive medicine is a matter of scientific discussion.

1- Flow-cytometric sorting of Semen. The flow cytometry is the only currently used technique able to determine the sex of future progeny by measuring DNA content of individual sperm cells. It evaluates if they contain the larger X chromosome (giving rise to a female offspring) or smaller Y chromosome (leading to male progeny). It then allows separation of X and Y sperm.

1- Flow-cytometric sorting of Semen. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the "female" (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than its male (Y-chromosome bearing) counterpart. As a consequence, when exposed to UV light during flow cytometry, X spermatozoa fluoresce brighter than Y spermatozoa.

1- Flow-cytometric sorting of Semen. As the spermatozoa pass through the flow cytometry in single file, each spermatozoon is encased by a single droplet of fluid and assigned an electric charge corresponding to its chromosome status (e.g. X to positive charge, Y to negative charge). The stream of X- and Y- droplets is then separated by means of electrostatic deflection and collected into separate collection tubes for subsequent processing.

1- Flow-cytometric sorting of Semen.

2- Pre-implantation Genetic Diagnosis (PGD) The genetic analysis of a single cell from an eight-cell embryo done in conjunction with in vitro fertilization (IVF) to improve the chances of a “normal” pregnancy.

The General Method of IVF Monitor egg maturation in the ovary Ultrasound Hormone levels Collect eggs (mother’s own or from donor) - Injection of human chorionic gonadotropin (hCG) and follicle stimulating hormone (FSH) to time egg ripening - Transvaginal aspiration using hollow needle

Egg retrieval

The General Method of IVF 3. Obtain sperm from father (or donor) and assess quality 4. Combine eggs and sperm in vitro or using intracytoplasmic sperm injection (ICSI) if sperm is low quality 5. Nurture embryo growth by incubating in medium containing various nutrients and hormones - If doing PGD, remove one cell after 2-3 days (6-8 cell stage) for testing 6- Transfer embryos (usually 3-6 d) to uterus, artifically removing zona pellucida if necessary (“hatching”)

An 8-cell embryo: 2-3 days zona pellucida

6d embryo at time of implantation (blastocyst)

Why consider PGD in addition to IVF? 1. recurrent abortion 2. one child already affected with a genetic disease 3. family history of inherited disease 4. maternal age older than 38 5. prior failure with IVF 6. family “balancing” for sex

The Methods of Preimplantation Genetic Diagnosis 1- Remove a single cell from the 6-8-cell embryo using a fine glass needle to puncture the zona pellucida and aspirate the cell In skilled hands, this generally does not harm the developing embryo. Each cell is called a blastomere. 2- Prepare a metaphase spread of chromosomes to assess karyotype (number and integrity of each chromosome)

Blastomere removal for PGD testing

The Methods of Preimplantation Genetic Diagnosis 3. Two types of assessment techniques are common: a. chromosome “painting” Fluorescence in situ hybridization ( FISH) using fluorescent probes specific for each chromosome. These allow number and size of each chromosome to be checked.

Fluorescence in situ hybridization (FISH)

b. Genetic testing for specific disease loci (PCR or gene chips) Polymerase chain reaction (PCR) amplification of DNA specific to a gene of interest (family history guides choice of genes)

Examples of genetic disorders detectable via PCR-based tests: - Tay Sachs (autosomal recessive; ~98% accuracy) - Cystic fibrosis (autosomal recessive; ~85% for common allele mutation) - Huntington’s disease (autosomal dominant) - Thalassemias (autosomal recessive blood disorder) - Duchenne muscular dystrophy (X-linked recessive) - Spinal muscular atrophy As more genetic tests are developed as diagnostic tools, more will be used for predictive purposes in PDG.

Risks to the child conceived via IVF/PGD: 1. Low birth weight 2. premature birth 3. Developmental delays 4. Urogenital problems 5. Cerebral palsy 6. Certain cancers which may be related to ICSI (Note: The vast majority of children born of IVF appear normal. Low birth weight, cognitive delays, and cerebral palsy are more common for any multiple-birth situation, longitudinal studies have compare IVF to natural.)

Biological status of the embryo A- at the stage of PGD, the embryo consists of 6-8 viable cells. Cells are the smallest units of life, as defined by biologists.It is generally believed that, at this stage, a single cell does not possess the potential to develop into a normal fetus.

B- At the stage of implantation, the embryo consists of roughly 200 cells arranged in a hollow ball. Inside is an inner cell mass, the cells used to generate embryonic stem cell lines, which are cells that have the potential to develop into any tissue but not into a complete organism. C- Excess embryos that are not implanted are generally frozen for a time and eventually may be discarded.