1. Faculdade de Medicina da Universidade de Coimbra 2009/2010 Cellular Molecular Biology II Work done by: -Iolanda Coutinho; -Inês Cunha; -Joana Abrantes; -Joana Couto; -João Barros.

3. What is PGD? It is a technique used to identify genetic defects in embryos created through in vitro fertilization before pregnancy. This technique refers specifically to when one or both genetic parents has a known genetic abnormality and testing is performed on an embryo to see if it also carries a genetic abnormality It is a technique used to identify genetic defects in embryos created through in vitro fertilization before pregnancy. This technique refers specifically to when one or both genetic parents has a known genetic abnormality and testing is performed on an embryo to see if it also carries a genetic abnormality

4. PGD is used to search the presence of genetic sequences linked to a variety of conditions and characteristics. A cell is extracted from an embryo at its eight-cell stage and analyzed. However, PGD is increasingly being used for other reasons. These include social sex selection and the creation of “savior siblings”

5. Treatment Cycle 1. Stimulation of the ovaries with hormones. 2. She is monitored during this time with blood tests and ultrasounds 3. The eggs are removed in a process known as an egg-retrieval

6. 4. The injected eggs are inspected under the microscope to determine wich have fertilized 5. Each of these eggs will then have a single sperm injected into them: ICSI – Intracytoplasmic sperm injection 6. Removal of two cells from each embryo (biopsy) for testing.

7. 7. Embryo testing and selection of unaffected embryos. 8. Transfer of the unaffected embryos into the woman’s uterus. 9. Pregnancy test 12 days later.

8. PGD Scheme

9. One or two cells are removed from the embryo at 6-8 cell stage (after3 days), from the blastocist. It’s important that embryos have reached the 8 cell stage - Blastomeres. Each one contains identical chromosomal information Extracted cells are analysed for the specific genetic condition in question.

10.  The developing embryo is placed back into the culture media and returned to the incubator where it can resume its normal growth and development.

11. PGD should be done in two groups of patiens: First group couples with a high risk of transmitting an inherited condition Second group couples with poor success in FIV

12. MONOGENETIC DISORDER X-linked disorders Autossomal Dominant Reciprocal & Robertsonian translocations CHROMOSSOMAL STRUCTURAL ABERRATION Autossomal recessive

13. Embryos are screened for chromosome aneuploidies

19. Technique that allows the synthesis of multiple copies from a chromosomal DNA fragment, with or without altering that original sequence; In PCR, the original DNA is copied by an enzyme called DNA polymerase, that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. Using primers, PCR can copy only a small part of interest from the DNA strand: target DNA.

20. Primers are short strands of nucleic acid that serve as a starting point for DNA replication, by binding to the beginning of the target DNA sequence.

21. The technique is based on thermal cycling, which consists of repeated cycles of heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. This process consists of several cycles, which contemplate the following steps:

22. In this step the DNA chain is separated into two single strands by DNA polymerase Primers bind to DNA and simple DNA polymerase binds to primers that are already paired The DNA polymerase creates a complementary DNA strand

23. Used only with clinical information that is important to know what type of probes we should use, for which chromosome and region. Biopsy; Cells are fixed on a microscope; Utilization of fluorescent probes will hybridize with a chromosome that have complementarity with it; With a fluorescent microscopy we can identify the number of copies of chromosomes.  Use of probes for chromosomes: X/Y/13/14/15/16/18/21/22 (detecting 70% of the aneuploidies).

24.  Each chromosome that we want to study can be labelled with different colors of flourocromes.  Each chromosome will have some unique areas of DNA that no other chromosome has.  The application of this technique has also identified that chromosomal mosaicism is common at the cleavage stage of development. A discovery that has very important implications on the diagnosis of dominant single gene disorders, monosomies and trisomies.

25. Some limitations of FISH expensive; Elevated mosaicism rate observed at the human pre-implantation stage Cannot detect maternal contamination Very specific testing. Analysis is often limited to only 5 chromosomes due to a limited number of flourocromes.

27. This is a molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA of tumor cells or a subject’s DNA. This method implies a pre-amplified DNA from a single test cell labelled with one fluorochrome, red—and then mixed with pre-amplified DNA from a control sample labelled with a different fluorochrome, green—with which it is compared, being applied to a normal metaphase spread, and the colour ratio measured.

28. On array, with the use of epifluorescence microscopy and quantitave image analysis, regional differences in the fluorescence ratio of gains/losses vs. reference DNA can be detected and used for identifying abnormal regions in the genome: Red areas: indicates that the test sample contained more of this genetic material; Green areas: indicates that the test sample contained lesser quantities of this genetic material.

29. Advantages: the whole chromosome complement is analyzed, though polyploidy and balanced translocations cannot be detected; Disadvantages: the whole procedure takes about 72 h, which is why the method is either applied to polar bodies, leaving 5 days for analysis, or to cells obtained at the cleavage stage of development, after which embryos are cryopreserved and transferred in a later cycle if identified as healthy.

34. Misdiagnosis are rare and variable : –1 in a series of 57 pregnancies -trisomy 21 (Munné et al., 1999) –1 in a series of 18 pregnancies -spinal muscular atrophy (Pickering et al., 2003) – 8 in a series of 451 pregnancies (ESHRE PGD, 2001): Duchenne muscular dystrophy Retinitis pigmentosa ß thalassaemia Cystic fibrosis Myotonic dystrophy Translocation [11;22] Trisomy 21

35. PGD is a safe method to avoid the birth of children with genetic defects (Strometal., 2000) Outcome of pregnancy is comparable to IVF (ESHRE, 2001) Clinical pregnancies resulted in: – 62 of 368 (17%) after testing for structural chromosomal abnormalities, including translocations – 41 of 254 (16%) after sexing – 119 of 575 (21%) after testing for monogenic diseases – 199 of 799 (25%) after PGD – AS (ESHRE, 2001) – 700 of 3000 (24%) PGD – 5% of babies had some kind of abnormality (International WGPG, 2001)

37. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1B-4CCNBK5- X&_user=2459680&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000057391&_version =1&_urlVersion=0&_us0&md5=cf0e16feb9512afa9bc285999821f1ea Chakravarti A, et al. Preimplantation Genetic Diagnosis. Genetics and Public Policy Center Advisory Board, 2004. Cabelo G. Diagnóstico de doenças Genéticas: métodos de rastreamento [on-line]. Laboratório de Genética Humana -Departamento de Genética. [retrieved17-04-09] fromhttp://www.ghente.org/ciencia/genetica/index.htm Wikipédia. Diagnóstico pré-implantatório[on-line]. Janeiro 2009 [retrieved17-04-09] fromhttp://pt.wikipedia.org/wiki/Diagn%C3%B3stico_gen%C3%A9tico_pr%C3%A9- implanta%C3%A7%C3%A3o OgilvieCM, BraudePR, ScrivenPN. Preimplantationgeneticdiagnosis–anoverview. J HistochemCytochem2005;53: 255 –260.