Preimplantation Genetic Screening (PGS) is a laboratory technique in which the pre-implantation embryo is tested for chromosome abnormalities prior to being transferred into the uterus. The aim is to increase the likelihood that a healthy embryo will implant and reduce the possibility of early miscarriage. PGS would also be expected to reduce the incidence of fetal abnormality and birth defects related to chromosomal disorders.
This page will provide background information on
- Chromosome abnormalities in embryos
- How the testing is performed
- Practical points relating to benefits and risks of the procedure
Chromosome Abnormalities in Embryos
Chromosomes are string-like structures found in the centre of the cell, the nucleus. Chromosomes contain genes that are made of DNA. Therefore, the genetic information we inherit from our parents is housed on the chromosomes. Normal human cells (including those in the embryo) contain 46 chromosomes arranged in 23 pairs. We receive 23 chromosomes from each parent. The first 22 pairs of chromosomes are the same for men and women and are numbered in order of largest to smallest as chromosomes 1 to 22. The 23rd pair determines our sex. A female has 2 “X” chromosomes whereas a male has an “X” and a “Y” chromosome. As such, the woman can only pass an X in her egg to her child. The man passes either an X or a Y in his sperm therefore determining the sex of the child.
If an error occurs leading to the egg or sperm having an extra or missing chromosome, the embryo created from that egg or sperm would have an extra or missing chromosome. This situation of an abnormal number of chromosomes is called aneuploidy. Having one extra chromosome is known as trisomy (tri = three of the chromosome) and having a chromosome missing is known as monosomy (mono = one of the chromosome). If the aneuploidy involves the larger chromosomes, the embryo may not attach to the wall of the uterus or may stop developing soon after attaching and miscarry. However, if the aneuploidy involves other chromosomes such as the 13, 18, 21, X or Y, the pregnancy may still carry on until birth, even though the pregnancy has a chromosomal disorder. The most common of these is an extra number 21, known as Down’s syndrome or trisomy 21 (three 21 chromosomes). Other common aneuploidies are Kleinfelter’s syndrome (XXY), trisomy 13 and trisomy 18. The features of the chromosome condition will depend upon which chromosome is extra or missing, but can include physical differences and mental retardation.
Risk of Aneuploidy and Maternal Age
As a woman advances in age, the chance of aneuploidy in her pregnancies increases. This association occurs because a woman’s eggs are as old as she is. Females are born with all the eggs they will have in their lifetime. So a 40 year old woman has 40 year old eggs. In men, sperm are made every 3 months therefore the sperm are always “young”. Therefore, the theory is that as women get older the chromosomes in the egg are less likely to divide properly leading to the egg having an extra or missing chromosome. The risk of aneuploidy increases with maternal age. The chances to deliver an affected child are 1/385 at 30 years, 1/179 at 35 years, 1/63 at 40 years and 1/19 at the age of 45. However, the occurrence of aneuploidy in embryos is much higher than what would be expected looking only at affected live born children. In fact almost two thirds of embryos from women in the age range from 35 to 39 have chromosome aneuploidies and over three quarters of embryos from women 40 or older are affected. This difference in percentages of abnormalities in embryos versus live born is due to the fact that a pregnancy with aneuploidy is less likely to attach to the uterus or go to term. Most will be miscarried. As such, the percentage of affected pregnancies is reduced over the course of the pregnancy due to the affected pregnancies that are lost. Any embryo with a missing chromosome (monosomy) will usually cease to grow before implantation (except monosomy X and 21), and only a few of those carrying an extra chromosome (trisomy) will go to term. The lack of implantation and loss rate of aneuploid embryos are believed to be the main reasons why the pregnancy rate in women over 40 is so low. It also explains why the miscarriage rate is much higher in women of older reproductive age. The purpose of preimplantation genetic screening for aneuploidy therefore is to select for transfer only chromosomally normal embryos so as to achieve more pregnancies, reduce the number of pregnancy losses, and reduce the number of affected children.
How PGS Is Performed?
We would recommend patients undergoing a PGS cycle to fertilise their eggs using ICSI rather than standard IVF. Embryos that have been created using IVF often have excess sperm bound to their shell, known as the zona pellucida. By performing ICSI where a single sperm is injected into the egg this will eliminate the risk of contamination to the biopsied cell from excess sperm.
Biopsy can be performed on either day 3 of embryo development or on day 5 at the blastocyst stage.
Biopsy of embryos on Day 3
Embryo biopsy is performed on the third day of embryo development when the embryo has between 6 and 10 cells.
To remove a cell from an embryo, an opening is made in the zona of the embryo. A single cell is removed via aspiration with a fine glass pipette. Only this removed cell will be analysed. The remaining embryo is then replaced into the incubator to continue to develop while the analysis is being performed. Meanwhile the single cell which has been removed is washed and placed into a small tube ready for analysis. Only a single cell is analysed and not the whole embryo. However, with a few exceptions (see below) the chromosomes of the single cell are the same as those of the whole embryo.
The image above shows a Single Cell Embryo Biopsy where a hole has been made in the outer coat of the embryo (zona pellucida) and a single cell is being aspirated into the biopsy pipette.
The removed cell is analysed in the genetics laboratory. The results are available within 24-36 hours enabling the normal embryos to be transferred into your uterus on day 4 or 5 of development.
Blastocyst biopsy is performed on day 5 of embryo development. Several cells are taken from the trophectoderm layer of the developing blastocyst. These are the cells that will form the placenta in pregnancy.
Only blastocysts that have fully expanded can be biopsied. Trophectoderm cells extrude through the opening made in the zona on day 3. Approximately 3 to 5 of these cells are aspirated into a fine glass pipette and removed using the precision laser. Studies show that the removal of some of the trophectoderm cells is not detrimental to the further development of the embryo.
Following biopsy the blastocyst is returned to the incubator and the biopsied cells are washed and placed in a small tube ready for analysis.
The image above shows a Trophectoderm biopsy where several cells are aspirated into the biopsy pipette.
One advantage of blastocyst biopsy is that several cells may be taken giving a higher accuracy in the genetic analysis. There is also evidence that biopsy at the later stage may be safer for the continued development as the removed cells have already differentiated into the trophectoderm layer (future placenta) rather than taking cells from the developing embryo itself.
However there are also drawbacks to this approach, due to the stage at which it is performed. Only approximately half of the preimplantation embryos reach the blastocyst stage. The percentage may be significantly less than this in women over the age of 39 years. This can restrict the number of blastocysts available for biopsy, limiting in some cases the success of the analysis. Delaying the biopsy to this late stage of development limits the time to perform the genetic diagnosis. The genetic analysis normally takes 24 hours. If the blastocysts are fully expanded first thing in the morning on day 5 then it is possible to obtain the results and perform the embryo transfer on day 6 of development. Otherwise it may be necessary to freeze the blastocysts after biopsy and await the results before performing a frozen embryo transfer of a screened normal blastocyst in a subsequent cycle.
Analysis for aneuploidy
The biopsied cells are analysed using a technique called array comparative genome hybridization (aCGH). The DNA from the removed cells within the tubes is amplified and then fixed onto the specialised microarray slide. This slide is coated with fluorescently labelled control DNA and the ratio between the test sample and control can be analysed to provide clear diagnosis of the number and amount of chromosome material in the biopsied cells.
Advantages of PGS
Increased chance of becoming pregnant
It is well known that the pregnancy rate after in-vitro fertilization decreases dramatically with maternal age. Aneuploid embryos (those with abnormal numbers of chromosomes) have much lower implantation rates than normal embryos, and half of them (the ones missing a chromosome) seldom implant. It appears likely that the decrease in pregnancy rates with maternal age is mostly caused by a corresponding increase in aneuploid embryos.
By performing PGS for aneuploidy and transferring only chromosomally normal embryos, we may be able to increase the pregnancy rates.
Reduction in miscarriages
In women 35 and older, approximately 35% of pregnancies are lost through miscarriage. Aneuploidy is the cause in 50% or more of these losses. By transferring only chromosomally normal embryos, the number of pregnancies going to term should increase.
Risks of PGS
Risk of the embryo and blastocyst biopsy
While PGS is a relatively new procedure in IVF, the micromanipulation or biopsy techniques required to perform the procedure have been in use for many years. The risk of accidental damage to an embryo or blastocyst during removal of the cell(s) is very low (approximately 0.6% that is just over 1 in 200). If an embryo or blastocyst was damaged by the technique it will stop growing and would not be suitable to be transferred into the uterus.
Effect of removal of a cell on the development of the embryo
When an embryo is 3 days old each of the cells has the ability to develop into a whole and perfectly formed baby. Removing a single cell at this stage merely delays continued cell division for a few hours, after which the embryo reaches the same number of cells as before and continues its normal development. A similar situation occurs in IVF programmes when frozen embryos are transferred. These embryos are transferred even if they have lost up to half of their cells during the freezing and thawing procedure and no adverse effect has ever been demonstrated in babies born over the past 20 years from frozen embryos. An unanswered question is whether biopsied embryos implant less than ones which have not been biopsied. Data regarding this is incomplete. Potentially, embryo biopsy may lower implantation rates slightly while selection of chromosomally normal embryos using PGS may increase it. The balance between potential biopsy damage and beneficial effects of PGS seems to be positive.
Limitations of PGS
Problems of Diagnosis
There are 2 areas in which problems of diagnosis may occur.
1. No diagnosis or partial diagnosis
In certain situations it might not be possible to obtain a diagnosis i.e. we cannot tell if the cell(s) has a normal or abnormal number of chromosomes. Reasons for this could include:
problems with preparing the cell(s) prior to analysis or that the removed cell actually lacks a nucleus (the structure in the cell containing the chromosomes). Trophectoderm
- biopsy in blastocysts takes several cells and so no diagnosis due to lack of nucleus is unlikely in this technique.
- the DNA contained within the biopsied cell(s) may degrade and the DNA amplification process may be unsuccessful leading to a diagnosis not being obtained.
In these cases we would still be able to transfer the embryo into your uterus but all the potential benefits of PGS will not apply for this particular embryo. We are unable to replace embryos diagnosed as normal with those with no diagnosis. We can only replace an embryo with no diagnosis if there were no normal embryos to replace.
We would normally expect up to 5% of embryos to have no diagnosis. However, it would be very rare for a patient not to have a diagnosis for all of their analysed embryos.
2. False positive and false negative results
A false positive result is one where the biopsy result indicates that there are an abnormal number of chromosomes but the embryo is actually normal. In practice there are not likely to be any adverse consequences of this as we would not transfer this embryo into your uterus.
A false negative result is one where the biopsy suggests that the embryo is normal but actually the embryo does have aneuploidy. This is more significant because we could well transfer this embryo into your uterus. This may occur due to mosaicism. Typically all the cells of an embryo have the same chromosome make-up because they all originate from the same egg. Some embryos are mosaics i.e. some of their cells have different numbers of chromosomes to other cells in the same embryo. If we analyse a cell that has a normal chromosome number but another cell within the embryo has an abnormal number, we may erroneously label the embryo as normal.
Due to the chance of misdiagnosis we recommend all patients who are pregnant after PGS should undergo prenatal testing in pregnancy. (See below)
A more accurate analysis is obtained with blastocyst biopsy as several cells are analysed and the chances of mosaicism are less than day 3 embryos.
The error rate of preimplantation genetic diagnosis using CGH is not currently known. Consequently, in the event of an ongoing pregnancy conventional prenatal cytogenetic analysis should be performed to establish the fetal karyotype. The intention of PGS using CGH is to detect aneuploidy involving loss and gain of whole chromosomes. Abnormalities involving smaller chromosomal pieces may not be detected. Additionally some instances of chromosomal mosaicism may go undetected by CGH. CGH does not detect abnormalities involving the loss or gain of an entire set of chromosomes although these types of abnormalities are not able to result in a viable pregnancy.
There may be circumstances in which no PGS embryos are available for transfer.
- Some couples may not produce embryos that develop to contain a minimum of 6 cells by the 3rd day of development. If this was to arise embryo biopsy could not be performed but the unscreened embryos could be transferred. The benefits of PGS would not apply in this case.
- Trophectoderm biopsy may only be performed on expanded, good quality blastocysts. If there are no suitable blastocysts to biopsy then unscreened early or poorer quality blastocysts may be transferred but again the advantages of PGS would not apply.
- It may be necessary to freeze all biopsied blastocysts rather than performing a fresh transfer to allow time for the screening to take place. Although freezing of blastocysts and survival after thawing is very successful (between 70-80%) with the technique of vitrification, we cannot guarantee that the blastocysts will survive the thawing procedure and be available for transfer.
- If all the embryos that undergo PGS are found to be aneuploid, embryo transfer would not occur.
Reduced number of embryos for freezing
It is usual that some of the embryos analysed will be shown to be aneuploid and they would not be suitable to be frozen. If you had not undergone PGS it is possible that we would have frozen these embryos for your future use. PGS screening therefore will reduce the number of embryos available for freezing although this is actually beneficial because these embryos could not have developed into normal offspring.
Effect of patients age on results of PGS
It must be realized that as women become older in reproductive terms the number of abnormal embryos increases significantly. For example for women of 40 years and above we expect that at least 50% are likely to be abnormal (occasionally 100% of embryos). However in women below 35 years of age there is accumulating evidence that PGS can significantly increase pregnancy rates following single embryo transfer of a chromosomally normal blastocyst.
Follow Up Of Pregnancies from PGS Programme
Because of the possibility of misdiagnosis we recommend that all patients who become pregnant following PGS undergo prenatal screening. This is something which you would need to discuss with your Obstetrician when you are pregnant. Prenatal screening is usually performed by the non invasive prenatal diagnostic test (Harmony Test). This involves taking a sample of maternal blood after 10 weeks of pregnancy. The fetal cells are isolated and chromosome analysis is performed on the fetal chromosomes. Test results are normally available within 2 weeks and are 99.9% accurate.