Preimplantation genetic diagnosis “in a nutshell”

by Jim Meriano , BSc., RT., MSc.,Embryology Laboratory Director

LifeQuest Centre for Reproductive Medicine

Spring 2008

 

Preimplantation genetic diagnosis (PGD) has been used in research, and clinically in humans since 1990. Dr. Robert Edwards first introduced PGD in 1960 after performing sex selection on rabbit embryos and PGD was introduced to human clinical use in 1990 (Handyside et al., Verlinsky et al).

 

PGD allows couples to have their embryos screened during their ART cycle for known familial genetic disorders and other abnormalities. Embryos, free of disorders are transferred to the uterus while affected embryos are not. Depending on the disorder, patient counseling, and informed consent, an affected embryo may sometimes be considered for transfer if no “normal embryos’” are identified.

 

Preimplantation genetic screening (PGS) is another form of PGD; it screens embryos for chromosomal number abnormalities which have been shown to be the cause of nearly 50% of unexplained miscarriages (Stephenson et al,2002, Jobanputra et al. 2002). Also, it has been demonstrated (Munne et al. 2005) that in a recurrent miscarriage situation, even though the embryos “look” normal to the embryologist, up to 70% of these embryos may have an abnormal chromosomal number.

 

PGD allows for testing of the embryo before implantation occurs therefore allowing the option of transferring only genetically normal embryos. When first introduced it was thought that PGD could be done instead of prenatal testing. However, the PGD patient also undergoes prenatal testing after PGD to rule out the rare chance of a misdiagnosis. There is only a minor chance (4.2% Munne et al 2005) of a misdiagnosis inherent in the testing. The “error” rate of the procedure is proportional to the experience of the genetics lab doing the testing as well as biological factors.

 

Some disadvantages of PGD are misdiagnosis, the limited chromosomal number analyzed and the added cost of the procedure. The ethics of the procedure has sometimes resulted in very colorful debates between scientists, ethicists and patients. However the choice for PGD remains the collaboration between patient, genetic counselor and their physician.

PGD practice guidelines (2004) recommend that patients be counselled by qualified genetic counselors as part of their pretreatment before their cycle. The patient is usually seeing a genetic counselor for a particular disorder when the counselor will refer them to an infertility clinic to discuss the need for PGD. Many issues are on the table when a couple opts for PGD as part of their infertility treatment, or PGD without needing infertility treatment. Some of the issues routinely discussed are the reproductive options and alternatives to PGD, the number of embryos which will be affected, the reliability of PGD, the risk of premature birth and importance of follow up for PGD children.. IVF treatment counseling and informed consent is also part of the process. Occasionally a patient with a genetic disorder will wish to become pregnant using the PGD technology when the infertility technology is not needed (meaning this patient is not infertile).

 

Depending on the genetic disorder, a pre-cycle workup may be necessary. Once the patient is informed of all pertinent information , the cycle preparations begin. Depending on the disorder, the genetics lab may need white blood cells from the couple to test for and detect the disorder. It is important to work with labs that have extensive experience. During this time, the IVF plan with the IVF team is being designed and put into motion, so that when it comes to retrieval date all is in place and organized for the IVF and the PGD.

 

PGD is done as part of an IVF cycle; embryos are derived from parental gametes. The couple will come through the IVF clinic for ICSI/IVF and have embryos produced from their cycle as usual. The embryos are biopsied on day 3 of the cycle and one cell from the embryo will be sent for genetic diagnosis. Intracytoplasmic sperm injection (ICSI) is recommended to avoid contamination and is the preferred method of insemination for PGD genetic disorder cycles (Best practice guidelines, PGD consortium, UK, 2004). In the situation of genetic testing the single blastomere is placed into a small special tube, and sent to the genetics lab. When testing for an abnormality (recurrent loss, translocation, and advanced maternal age) the cell is placed onto a microscope slide and sent to the genetics lab for testing. Generally PGD is done during a day 5 transfer protocol. Since the biopsy is done on day three and the testing is done in a day or two, day five blastocyst transfer is ideal for the procedure. The day before or the day of transfer, genetic results are received, and suitable embryos are selected; only normal embryos are selected for transfer.

This question has not been completely answered yet. We do know that most of the time it does not, since many normal and healthy pregnancies and births have been achieved around the world using this technology. The amount of cytoplasmic volume that the embryo loses does not seem to affect the embryo. Generally one cell is removed and a second, only if needed as long as overall cell number is greater than 6. (Vandervors et al 2000) One may look at it in the following way: when the embryologist freezes embryos at day 3, sometimes upon thaw one, two or more cells may be lost due to damage during freeze or thaw, however pregnancy rates with day 3 frozen embryos are generally still very respectable, notwithstanding the infertility diagnosis and the freezing process. This concern is generally overshadowed by the actual reason that the patient needs PGD. Utmost care and strict procedure is followed in the embryology lab during the biopsy so that the blastomere removed and tested, remains identified to that particular embryo. 

 

Two types of PGD exist; genetic disorder testing and PGS. Genetic testing is recommended where one or both partners carry a serious genetic disorder that they do not wish to pass on to their child. Testing is done on the single cell sent to the genetics lab from the IVF lab for that particular disorder. The genetics lab then reports to the IVF lab with the genetic results of embryos. The labs can test for up to 1000 known mutations and some unknown in certain circumstances. Some disorders tested are: cystic fibrosis, muscular dystrophy and hemophilia, just to name a few.

 

There have been several cases in which a couple has a “sick” child who has a genetic disease and needs a bone marrow transplant but no matches have been identified in the family. Some couples choose to have a another baby through IVF and PGD to select a positive tissue match for the sibling, so that the cord blood from the umbilical cord may be used to treat and cure the sibling of the disorder.

 

For patients who have suffered recurrent pregnancy loss, , testing is also available. The incidence of recurrent loss. is observed in 2-5% of couples and is defined as 3 or more consecutive losses. (Usually before 20 wks of gestation) (Stephenson 1996,Findikli et al 2006). Using PGD for recurrent fetal loss to remove the possibility of transferring chromosomally abnormal embryos has increased live birth while decreasing spontaneous abortions. (Simon et al 1998, Pellicer et al 1999). In a recent controlled trial it was shown that using the PGS technology for those patients that have had more than 3 consecutive spontaneous abortions the loss rate was reduced to 16.7%, significantly lower than the compared value of 37%. ( Munne et al 2005)

 

Advanced maternal age is another indication for testing the embryo which has met with some modest controversy.   It has become well known that the incidence of chromosomal aberrations is directly proportional to advanced maternal age, as are decreased ovarian reserve and reduced fertility potential (Findikli et al 2006). Few reports have shown that the use for PGS in the advanced maternal age patient has not helped the implantation rate when used is conjunction with blastocyst transfer. However many more recent publications have shown the contrary; the elimination of chromosomally abnormal embryos from the transfer cohort using PGS has significantly increased live birth rates. ( Kuliev et al 2003, Rubio et al 2005, Munne et al 2005 Verlinsky 2005. ) . Similarly it was shown that fetal chromosomal abnormalities in women aged 40 yrs and older were as high as 64% when compared to a similar group aged 30-40yrs (33%). (Carp et al 2001). Given a related study that demonstrated that patients aged more than 35yrs and with a history of miscarriage, benefited positively from increased implantation rates, the use of PGS for recurrent miscarriage, and advanced maternal age seemed logical for those patients affected by these situations.

 

PGD, like ICSI, is a tool “ designed” for a specific group of patients to maximize the results for these patients, given certain chromosomal challenges that may affect their actual fertility potential or the health of the offspring they wish to have. For those patients with genetic disorders in their family, PGD is a good and safe tool for their health care providers to use to help them achieve their goal of having a child free of the genetic disorder, if they so wish. As well, for those patients whom have had many miscarriages, PGS is also a tool we may use to help these patients finally have a live birth. These technologically advanced methods should be treated like all methods of similar risks and benefits . . . with respect. We should use them when needed and monitor their results strictly and responsibly. While using these methods we should ensure complete informed consent on the patient’s part, and also ensure that the patient can confidently approach us for help if they need it.

 

Albert Einstein once wrote in a letter to his son that “life is like riding a bicycle, to maintain balance you must keep moving.” This can be said of scientific advancement as well, since the forward movement should be not erratic but responsible, smooth and safe.

References:

Carp HJA., et al.,2001Karyotype of abortus in recurrent miscarriage.Fertil. Steril., 75 678-682.

 

Findikli N., Kahraman S., et al   2006 Embryo aneuploidy screening for repeated implantation failure and unexplained recurrent miscarriage. RBMonline Jul (vol 13 ) 1. 1-16

 

Handyside AH, Kontogianni EH et al , 1990 Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification Nature 344, 768-770.

 

Jobanputra V Sobrino A., et al. 2002 Multiplex interphase FISH as a screen for common aneuploidiesin sponteneas abortions. Human Reproduction, 17, 1166-1170.

 

Kuliev A., Verlinsky Y et al 2003, The role f preimplantation diagnosis in women of advanced maternal age. Currt opinion in OB and Gyn. 15, 233-238.

 

Munne et al. 2005 PGD reduces pregnancy loss in women aged 35 yrs and older with a history of recurrent misscariages. Fert ad Steril. 84, 331-335.

 

Pellicer et al., 1999 IVF plus PGD in patients with recurrent miscarriage: an analysis of chromosomal abnormalities. Fert. Steril., 71, 1033-1039.

 

Rubio C., et al 2005 FISH screening of aneuploidies in preimplantation embryos to improve outcomes. RBM Online 11, 497-506.

 

Simon C., et al 1998 Increased chromosomal abnormalities in preimplantation embryos after IVF in patients with recurrent miscarriage. Reproductive fertility and development.1, 87-92.,

Stephenson MD., et al., Frequency of factors associated with habitual abortion in 197 couples 1996, Fertil.Steril.66, 24-29.

 

Stephenson MD., et al., 1996 Frequency of factors associated with habitual abortionin 197 . Fertil.Steril., 66,24-29. Stephenson MD, Awartani KA, Robinson WP. 2002 Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod. Feb;17(2):446-51.

 

Thornhill A.R. et al ., ESHRE PGD Consortium ‘Best practice guidelines for clinical preimplantation genetic diagnsis (PGD) and PGS, 2004, Human Reproduction, 20, No.1 35-38.

 

Vandervors M., etal., 2000 The Brussels’ experience of more than 5 yrs of clinical PGD. Human Reprod. Update Jul-Aug; 6(4):364-73.

 

Verlinsky Y., et al 1990Analysis of the first polar body: preconception genetic diagnosis. Human Reproduciton 5, 826-829.

 

Verlinsky Y., et al., 2005, Preimplantation testing for chromosomal disorders improves outcomes of poor prognosis patients RBM Online, 11,219-225