Pre-implantation genetic testing.

 

We continue to receive several inquiries from the interested public concerning genetic diagnosis. For this reason and more, I am fit to give a simplified detail of the processes involved in Preimplantation genetic testing (PGT). 

This presentation should create awareness of the use of PGT for helping couples with the sickle cell trait. We employ the technique of trophectoderm biopsy, which is an improvement over our earlier publication that biopsied embryos on day 3 with one single blastomere taken out of the eight blastomere cells for analysis. In comparison, trophectoderm biopsy is performed on a Day-5 embryo called a “blastocyst.” We removed 4 to 5 cells from the embryo’s external layer and analyzed the extracted cells for abnormalities. This new process has a higher degree of specificity. And a significant percentage increase in pregnancy success, making it more acceptable and accessible. It is, therefore, noteworthy to bring into the public domain for those who may require such therapy.

The technique of PGT is in conjunction with Assisted Reproductive Technology (ART). ART refers to conception requiring the complex handling or manipulation of male and female gametes in vitro to facilitate pregnancy. 
WHAT IS IVF?
IVF means Fertilization achieved outside of the body. It involves ovulation induction, oocyte retrieval, sperm preparation, oocyte stripping, insemination and Fertilization in a culture dish, and embryo transfer. Oocyte retrieval and embryo transfer processes are under ultrasound guidance. It is also essential to know that gamete handling is under strict temperature control. Without the development of IVF, preimplantation genetic testing (PGT) would not be possible.

WHAT IS PGT?

Preimplantation genetic testing, as the name implies, involves testing for specific genetic defects in the DNA code before embryo implantation—targeted screening of a known genetic abnormality in the couple. PGT was first performed in the early 1990s as a way for couples to prevent the pregnancy of a child with a congenital disease. 

At Medical Art Center, we offer PGT for genetic conditions, including sickle cell anemia and chromosome aneuploidies. We collaborate with the Reproductive Genetic Institute of Chicago, a world-renowned genetics institute and pioneers of PGT for inherited genetic abnormalities.

DOES PGT REPLACE PRENATAL TESTING?
No, PGT does not. PGT is a research-based test allowing for a similar diagnosis to those available by prenatal testing. However, prenatal testing, such as chorionic villus sampling (CVS) or amniocentesis, is recommended. These widely used and well-accepted tests remain the gold standard of modern obstetrics and are necessary for this setting should a pregnancy ensue.
HISTORY OF PGT
In humans, PGT was developed in the United Kingdom in the mid-1980s as an alternative to current prenatal diagnoses. In 1989, the first unaffected child born after PGT performed for an X-linked disease was reported in London. PGT became increasingly popular during the 1990s. When used to determine a handful of severe genetic disorders, such as sickle-cell anemia. Others include Tay-Sachs disease, Duchenne’s muscular dystrophy, and Beta-thalassemia.  In 2010, Oladapo Ashiru et al. used PGT with FISH (Fluorescent in situ Hybridization) for chromosomal abnormalities and family balancing. And in 2013, published their first success with using PGT to deliver a sickle cell-free baby boy. (Nigerian Medical Journal 2014). As of 2006, more than 15,000 PGT cycles have been reported. PGT is currently available for the most known genetic mutations. Although the indications for PGT are well established, it is a relatively new and evolving technique in Africa.

INDICATIONS FOR PGT IN NIGERIA

Autosomal dominant disorders: This is the situation where one of the partners is a carrier of the genetic defect (e.g., Dwarfism)

Autosomal recessive disorders: This is a situation whereby couples are carriers of a genetic defect (e.g., Sickle Cell Disease)

X-linked disorder( Disorder associated with the X chromosome of XX or XY): This is a situation whereby one partner is a carrier of an X-linked genetic defect (e.g., Hemophilia)

Structural chromosomal abnormality: One partner is a carrier of a chromosome abnormality (translocation, inversion, deletion, insertion)

Human leukocyte antigen (HLA) matching: Allogeneic hematopoietic stem cell transplantation (HSCT) treats some acquired and congenital diseases. An essential factor in the procedure’s outcome is the degree of human leukocyte antigen (HLA) compatibility between patient and donor. The more compatible the donor is with the recipient, the higher the chances of success. HLA identical siblings, therefore, provide the best opportunity for the recipient. PGT can be used to select HLA-compatible embryos if there is no sibling. It is for bone marrow transplants in sickle-cell-affected children.

WHAT IS SICKLE CELL ANAEMIA?
Sickle cell anemia is a hereditary genetic condition in which a mutated form of hemoglobin distorts the red blood cells from a healthy disc shape into a crescent body. The disease is associated with acute and chronic health problems, such as severe infections, attacks of excruciating pain (“sickle-cell crisis”), and an increased risk of death. Seventy percent of the world’s children with sickle cell disease are born in sub-Saharan Africa.
According to the World Health Organization (WHO), in Africa, the highest prevalence of sickle-cell trait occurs between latitudes 15° north and 20° south, ranging between 10% and 40% of the population in some areas. In countries such as Cameroon, the Republic of Congo, Gabon, Ghana, and Nigeria, the prevalence is between 20% and 30%, while in some parts of Uganda, it is as high as 45%. In countries where the trait prevalence is above 20%, the disease affects about 2% of the population. The geographic distribution of the sickle-cell trait is very similar to that of malaria.

PGT FOR SICKLE CELL:

STEPS INVOLVED 
Preimplantation genetic testing can distinguish between genetically normal or affected embryos. Currently, this is the only way to determine the genetic condition of an embryo before pregnancy.
Preliminary work before PGT: Probe preparation
To go through PGT for sickle cell anemia, DNA samples from family members must be obtained to build a probe. This probe, which contains specific genetic information from the collected DNA, will be used to test the cells biopsied from the embryos. DNA samples from only direct family members (siblings or the parents of the two partners) are required, so there is no need to involve extended relatives. PGT probe preparation takes 8 to 10 weeks.
Oocyte Retrieval
The female partner gets medications to create multiple follicular development and ovulation. Once these follicles are well developed, they are retrieved from the patient under conscious sedation using a unique ultrasound-guided technique. The oocytes are then isolated from the follicles and rest in the incubator for 3-4 hours before insemination. We prepare the sperm for IVF at the interval. 
ICSI or Fertilization by “brute force.”
For PGT, insemination is by ICSI. Gianpiero Palermo developed the technique at the Vrije Universiteit Brussel in the Center for Reproductive Medicine, headed by Paul Devroey and Andre Van Steirteghem in 1992. Jacques Cohen also developed the method in 1992. Oladapo Ashiru also used this technique at the University of Illinois, Chicago, in 1995. 

Under a very specialized microscope, one sperm cell is aspirated from the very few ones and is injected directly into the egg cytoplasm. It ensures Fertilization in significantly high numbers. The inseminated eggs are kept in the incubator in the IVF laboratory for about three days in earlier cases but currently for five or 6days. The fertilized embryo will rapidly divide into the 2-cell, 4-cell, 8-cell, and blastocyst stages, at which point embryo biopsy can occur.

HOW IS PGT DONE?

We remove Cell (s) out of the embryo for this diagnosis. The process of taking out these cells is called biopsy.

Embryo Biopsy.

For cleavage biopsy, we evaluate embryos on the morning of day 3, and a biopsy of 1-2 blastomeres is done on the embryos showing 6 – 8 cells, respectively. We perform trophectoderm biopsy by removing the cells at the blastocyst stage. They are embryos on day 5 or 6 after Fertilization. Trophectoderm biopsy involves the removal of the trophectoderm component (cells that form the placenta) of the embryo. It is much less traumatic, and the resilient blastocyst cells recover quickly. Since the embryo has many more cells at the blastocyst stage (about 100 cells) than at the cleavage stage (6-10), we can remove about 4-5 cells (trophectoderm cells) from a blastocyst with little or no impact on its developmental potential.

The embryo has to show a distinct inner cell mass (ICM) and trophectoderm layer. ICM forms the embryo correctly and must be avoided during the biopsy process. Assisted hatching is performed on day three or the morning of day five and uses laser beams under a microscope to create a small hole in the embryo’s outer shell. Blastocysts are considered ready for biopsy when three or more trophectoderm cells are seen hatching out. Using a suitable micropipette and more laser beams, herniating trophectoderm cells are detached and used for diagnosis. At the blastocyst stage, the embryo is ready to implant and cannot be left to continue growing in the incubator, hence the need for embryo freezing. All biopsied embryos are immediately frozen.

At Medical Art Center, we mainly perform trophectoderm biopsy as this provides enough cells for the analysis while being much less traumatic to the embryo.  

EMBRYO CRYOPRESERVATION (FREEZING)

Embryos that have been biopsied are preserved in cryo devices. They are frozen by a technique called vitrification, which is described as very rapid cooling at very low temperatures. They are appropriately labeled and stored in liquid nitrogen and can be thawed. The embryos have been safe in nitrogen tanks for years. Our first sickle cell-free delivery was a boy conceived from a frozen embryo transferred to his mother. 

PGT ANALYSIS

The first process involved is termed PCR (Polymerase Chain Reaction). Kary Mullis conceived PCR in 1985 as an in vitro simplified reproduction of the in vivo function of DNA replication. Taking advantage of the chemical properties of DNA and the availability of thermostable DNA polymerases, PCR allows for the enrichment of a DNA sample for a specific sequence. PCR allows for many copies of a particular stretch of the genome, making further analysis possible. It is a highly sensitive and specific technology, making it suitable for all genetic diagnoses, including PGT.

Once PCR has amplified the DNA in trophectoderm cells, they can now be analyzed by a technique known as comparative genomic hybridization. CGH looks at the entire set of chromosomes to ensure the correct number of chromosomes is present. CGH also ensures a proper balance of chromosome sets, providing the genetic integrity of embryos from patients.

With our collaborating center, we usually get the results of the PGD test within ten days. The results indicate which embryos are HbAA, HbAS, HbSS, or otherwise. A consult is had with the patient to discuss the outcome and to plan and prepare for embryo transfer.

EMBRYO TRANSFER.

It is a more straightforward process for the patient. The patients are given medications to build up the endometrium to receive the embryos.  Patients are evaluated to ensure we eliminate several implantation defects. We increase the uterus receptivity for the embryos and support the early phase of placental development. We use ultrasound guidance to transfer the embryos (usually 1 or 2) into the patient’s uterine mid-cavity. Once the endometrium develops to our accepted standard. Patients are now given implantation support and asked to return in 2 weeks for the pregnancy test.

CONCLUSION

In Nigeria and West Africa, sickle cell carrier couples can screen their embryos before conception, avoiding a sickle cell baby. Today, we have been able to help several couples who are carriers of the sickle-cell gene have babies that are free of the disease.

Even though relatively high, the cost of this process compensates for all the stress of dealing with sickle cell babies. It is hoped that various groups, including the government, can come to the aid of couples in need of this technique but cannot afford it. In the long run, it will save a lot of money on the health management of complications of this disease.                      

More research is required to improve the speed of diagnosis and make the process less expensive, especially with implantation. It is the only significant gap in knowledge for us to achieve close to 95% success in IVF. Many scientists have predicted that by 2025, IVF’s success rate should be 90% or more.

Finally, in our experience, we have preliminary evidence to suggest that environmental toxins and diet significantly reduce fertility and possible IVF success. Most of these stem from my earlier basic research in reproductive toxicology. The use of Mayr therapy to detoxify the body and get rid of toxins such as heavy metals like mercury, arsenic, antimony, and lead, as well as such toxins and allergies from lousy eating, has, in general, enhanced our success in IVF and PG. Elimination of these factors helps in increasing the pregnancy rate.