Journal of Postgraduate Medicine
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Year : 2010  |  Volume : 56  |  Issue : 4  |  Page : 317-320  

Preimplantation diagnosis of genetic diseases

SK Adiga1, G Kalthur1, P Kumar1, KM Girisha2,  
1 Division of Reproductive Medicine, Kasturba Medical College, Manipal University, Manipal, India
2 Genetics Clinic, Kasturba Medical College, Manipal University, Manipal, India

Correspondence Address:
K M Girisha
Genetics Clinic, Kasturba Medical College, Manipal University, Manipal


One of the landmarks in clinical genetics is prenatal diagnosis of genetic disorders. The recent advances in the field have made it possible to diagnose the genetic conditions in the embryos before implantation in a setting of in vitro fertilization. Polymerase chain reaction and fluorescence in situ hybridization are the two common techniques employed on a single or two cells obtained via embryo biopsy. The couple who seek in vitro fertilization may screen their embryos for aneuploidy and the couple at risk for a monogenic disorder but averse to abortion of the affected fetuses after prenatal diagnosis, are likely to be the best candidates to undergo this procedure. This article reviews the technique, indications, benefits, and limitations of pre-implantation genetic testing in clinical practice.

How to cite this article:
Adiga S K, Kalthur G, Kumar P, Girisha K M. Preimplantation diagnosis of genetic diseases.J Postgrad Med 2010;56:317-320

How to cite this URL:
Adiga S K, Kalthur G, Kumar P, Girisha K M. Preimplantation diagnosis of genetic diseases. J Postgrad Med [serial online] 2010 [cited 2023 Mar 27 ];56:317-320
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Diagnosis of genetic disorders early in pregnancy refers to prenatal diagnosis. A step further, preimplantation genetic testing is an early form of prenatal diagnosis, where genetic defects in embryos created in vitro are analyzed before implantation in the uterus. [1] This offers couples at risk of a genetic disease the chance to have an unaffected child, without facing termination of pregnancy. Two decades have elapsed since the first description of pregnancy, after application of this technique in sex selection of human embryos at risk of an X-linked disease. [2] To date, many studies have addressed the impact of preimplantation genetic screening in different groups of patients, however, its effectiveness has not been consistently proven. [3] Despite this, according to the recent European Society of Human Reproduction and Embryology (ESHRE) preimplantation genetic diagnosis (PGD) consortium data collection, the number of PGD cycles performed for aneuploidy screening, pregnancies, and babies reported annually have increased considerably. [4] Here we aim to briefly outline the scope of PGD and its applications in a clinical setting.

 Who Benefits from Preimplantation Testing?

Indications for preimplantation testing can be viewed from two perspectives. One, the couple is already known to be at risk of a genetic disease, usually a monogenic disease or chromosomal abnormality in the offspring, due to a balanced chromosomal rearrangement in one of the partners. Here the couple is averse to termination of the affected conceptions after an invasive prenatal diagnostic test. Although most of them can conceive naturally, they undergo in vitro fertilization (IVF) so that the embryos are selected for implantation only if they are found to be free of the genetic defect. Second, the couples undergo in vitro fertilization in view of infertility to achieve conception. This provides the couples a window of opportunity to test the embryos for common chromosomal aneuploidies. This group may also be at a higher risk of aneuploidy (especially trisomy 21) in view of advanced maternal age (> 35 years at the expected time of confinement). Often the term 'preimplantation genetic diagnosis (PGD)' is used for the first group and 'preimplantation genetic screening (PGS)' for the latter.

 Mendelian Disorders (Monogenic Disorders)

Single gene disorders, also known as Mendelian disorders are diagnosed by PCR-based techniques. If one of the couple carries a mutation for an autosomal dominant disease, they carry a 50% risk of transmitting the disease to the offspring. The couple is at 25% risk for an autosomal recessive disease in the child if both of them are carriers of the disease. In an X-linked recessive condition, with the female partner being carrier of the mutation, the risk to a male offspring is 50%. It may be noted that the occurrence and severity of the disease in the child are also determined by the penetrance and expressivity of the condition. Preimplantation genetic testing is being reported for many more single gene disorders. Expectedly, for all common diseases like beta thalassemia, cystic fibrosis, spinal muscular atrophy, myotonic dystrophy, Huntington disease, Marfan syndrome, and so on, preimplantation genetic diagnosis has been successfully done. Technically many of the monogenic diseases must be amenable to PGD. An option for the X-linked recessive disease is selection of only female embryos that are unlikely to be affected by the disease. Sexing of the embryo can be done by fluorescence in situ hybridization (FISH) or even polymerase chain reaction (PCR). This is a particularly useful alternative for specific diagnosis in X-linked mental retardation and X-linked retinitis pigmentosa, where a specific molecular defect is not identified or almost impossible to identify. Linkage analysis is an alternative to identify the unaffected males in some specific situations.

 Aneuploidies and Structural Chromosomal Abnormalities

It is well known that chromosomal aneuploidies are an important cause of abnormal embryos and reproductive failures. Using probes for five chromosomes (X, Y, 13, 18, and 21), it is estimated that 70% of the embryos derived from IVF were abnormal for at least one of the chromosomal numbers. [5] Hence, it can be expected that incorporation of aneuploidy screening in embryos can improve the success of IVF. Additionally, as female partners of most of the couples seeking IVF have advanced age, they are at an increased risk of aneuploidy, specifically trisomy 21. Aneuploidy screening can also be an investigation to look into the causes of poor implantations and recurrent abortions of unidentified cause. Aneuploidy screening is by far the most common indication for preimplantation genetic testing. Second, the couple where one is a carrier of a balanced structural chromosomal abnormality may face the risk of abortion, stillbirth, or birth of a child with malformations and / or mental retardation. Usually these abnormalities are balanced translocations or inversions and rarely other rearrangements. FISH probes for the specific region of interest need to be deployed in these settings and may not be available at all the centers. If the female is a carrier, the polar bodies may be analyzed for chromosomal abnormality.

 Obtaining Cells from Early Embryo

Three methods have been developed to carry out PGD. The most widely used approach is testing the individual embryonic cells (blastomeres) obtained on the third day after IVF. One or two blastomeres are removed through a hole created in the zona pellucida, and the cells are analyzed. [6] Another approach is to use the non-embryo forming cells (trophoectoderm cells) of the embryos on day 5 of development. In both the cases, healthy embryos are subsequently transferred to the uterus on days 4, 5 or 6, after IVF. An alternative method for carrying out PGD examines the genetic material within the first and second polar bodies, the by-products of meiosis I and meiosis II, respectively. This method can be used in case of maternally derived dominant mutations, translocations, and aneuploidy. It cannot be used when paternally derived genetic information is critical for the diagnosis, such as, paternally derived dominant mutations, translocations, and aneuploidy.

 Blastomere Biopsy

Embryo biopsy is the removal of one or two blastomeres from the cleavage stage embryo. It is the most widely used biopsy procedure for PGD at present. Ideally a six-to-eight cell staged embryo is preferred for biopsy. By this time the embryo completes at least three mitotic divisions. Biopsy of one or two cells is performed on day 3 and the embryos are transferred on day 4 or 5. This allows enough time for the genetic investigations to be performed and for the transfer of genetically normal embryos, which have progressed till the blastocyst stage.

Embryos are generally incubated for a brief period in a medium deficient in divalent cation-like calcium, to disrupt the tight junctions and cell-to-cell interaction between the blastomeres. An opening in the zona (~20 mm) is created using the mechanical, enzymatic or laser drilling method. After creating the opening, the blastomeres are removed by aspiration, which is the most widely used method. An aspiration pipette with an inner diameter of 35 - 40 μm is introduced into the perivitelline space through the hole in the zona and the cells are completely aspirated or partially aspirated and pulled out from other blastomeres.

 Blastocyst (Trophoectoderm) Biopsy

The use of trophoectoderm (TE) cells as a source of biopsy material was first suggested two decades ago by Dokras et al. [7] The main advantage of this method is that a large number of cells are available for genetic analysis unlike in the blastomere biopsy, where only one or two cells are available.

There are two approaches for collecting the TE cells from the blastocyst. In the first approach, the zona drilling is done in the blastocyst, exactly opposite the inner cell mass (ICM). After four to ten hours of culture, few TE cells herniate through the zona on which the biopsy can be performed. In the second approach, the hole is made in early embryos on the third (6 - 8 cell stage embryos) or fourth day (morula stage embryo) of development. The rationale of this approach is to drill a hole in the zona with minimum damage to the adjacent cells, as at the blastocyst stage the perivitelline space is almost non-existent. In this approach the zona drilling is done on all the available embryos and they are cultured till they reach the balstocyst stage. The TE cells, which are closer to the hole on the zona pellucida approach, can be used for biopsy. In both cases, the biopsies are obtained by securing the blastocyst with a holding pipette, with the herniated cells located in the 3 o'clock position. Usually two to nine TE cells are gently aspirated with a biopsy pipette (internal diameter 30 μm) and three to five laser pulses are then applied in order to detach them from the blastocyst. The cells obtained are finally ejected from the biopsy pipette and are ready to be processed. The major limitation of this approach is the requirement of sufficient number of embryos reaching up to the blastocyst stage, the very short time available for genetic assessment, and that the TE cells may not reflect the true genetic feature of cells of the ICM.

 Polar Body Biopsy

The polar bodies are considered as waste nuclear material after the meiotic division and do not have any physiological role to play in the development of embryos. By establishing whether there is an abnormal gene or chromosome arrangement in the polar bodies, it is possible to infer the maternal genetic contribution to the embryo. Therefore, they are considered excellent material for genetic analysis of the oocyte. The polar bodies are located in the perivitelline space enclosed by the zona pellucida, and to gain access to these materials, an opening in the zona has to be created either by chemical or laser drilling.

The polar body biopsy technique is advantageous because it maintains the embryo's integrity, as only meiotic by-products and not blastomeres are used to assess the genetic integrity of the developing embryo. Hence, any possible effect of the procedure on the developmental potential of the embryo and its polarity is thought to be the least. Nevertheless, it is hampered by the impossibility of diagnosing paternally-derived defects, and those originating after fertilization or first cleavage events. In recent times, the accuracy and reliability of the results obtained from the genetic analysis of the polar body is enhanced by investigating both the polar bodies. The use of this strategy permits chromosomal abnormalities associated with the second meiotic division to be identified, and in case of single-gene mutations, the occurrence of crossing-over between homologous chromosomes can be verified.

 Genetic Testing of Biopsied Cells

In addition to accuracy, the important attributes of the techniques for preimplantation genetic testing include robustness and rapidity. Usually, preimplantation genetic testing uses either a PCR-based technique or FISH for the detection of genetic defects.

 Polymerase Chain Reaction

The polymerase chain reaction amplifies the region of interest in the DNA of a biopsied cell. It may be noted that a polar body contains a single copy of the DNA (haploid) and the cell biopsied at the cleavage stage has double copies of the DNA (diploid). This is a very demanding PCR, as only a single cell is the source of DNA. The cell is lysed and the region of interest in the DNA strand is amplified using specific primers. The amplified fragment may be analyzed subsequently for the size, digestibility by a restriction enzyme or sequencing, to infer the presence of the defect (mutation). This technique demands that the defect has already been identified in the couple or their previous child, and is normally applied for the diagnosis of single gene disorders. Multiplex PCR, real time PCR, and quantitative fluorescent PCR have been added to the technical armamentarium recently.

 Fluorescence In situ Hybridization

Fluorescence in situ hybridization involves the use of fluorescent labeled probes that bind to the target DNA in the biopsied cell. Both probe and target DNA are denatured and complementary sequences are allowed to hybridize with each other under optimal chemical and temperature conditions. When an excess probe is washed, the number of fluorescent signals corresponds to the presence and number of target sequences. In other words, if the probe used is specific for a unique region on chromosome 21, two signals indicate the presence of two normal copies of chromosome 21 and three signals imply trisomy 21 in the biopsied embryo. The common probes used are: X, Y, 13, 18, and 21. The number of chromosomes tested in a single FISH is limited by the number of fluorochromes available. However, mixing of colors, several rounds of FISH on the same cell, and comparative genomic hybridization (CGH), enhance the possibility of detecting the abnormalities in several chromosomes / regions. Obviously, FISH is the technique employed for detection of aneuploidies of autosomes, and sex chromosomes and chromosomal rearrangements.

 Limitations of Laboratory Techniques and Challenges

The major challenges of PGD are relatively short interval of time available for analysis and few numbers of cells available for analysis, compared to the hundreds of cells obtained via amniocentesis or chorionic villi sampling. Technical difficulties that may arise during PCR amplification of target DNA sequences include failure of amplification, contamination with external DNA, and more specifically the 'allele drop-out'. This refers to the situation where one of the alleles in a diploid cell (either normal or abnormal) fails to amplify and the results are interpreted on the basis of the other allele that is amplified. This can result in both false positive and false negative results. It is estimated that the risk of transferring an affected embryo mistakenly identified as normal by PGD is approximately 2% for recessive disorders and 11% for dominant disorders. [8] Follow-up testing by prenatal or postnatal sampling can confirm the exact incidence of such errors.

When FISH is the technique of screening for aneuploidy in the embryo, failure to hybridize, poor signal intensity, split or fused signals may give rise to false results. As only a single cell is analyzed, mosaicism is not accurately picked up by this technique. This is especially important, as there may be a trisomy rescue and embryos that are diagnosed to have a trisomy may eventually develop into a fetus with normal chromosomal content. It is estimated that misdiagnosis in aneuploidy screening after biopsy of one blastomere is 7%, with 6% due to mosaicism. [9]

 Counseling for Preimplantation Genetic Testing

The couples have to undergo an informed counseling addressing the risk of a genetic disease, IVF, issues related to the technique and the cost of IVF and PGD. They have to understand the risk of a genetic disease in their offspring (may be a monogenic disorder or a chromosomal abnormality), the natural history of the condition, and the options available to treat or prevent the condition. They are are informed about the advantages and disadvantages of prenatal diagnostic options as compared to preimplantation testing. The options must include choosing not to proceed with IVF and PGD and sometimes accepting donor ovum / sperm. Accuracy and error rate of the techniques also need to be discussed before the couples opt for prevention of the genetic disease by selecting the method that suits them best.

 Pregnancy Outcome

Although there are no published data providing the number of centers performing PGD, in Europe, according to the European Society of Human Reproduction and Embryology (ESHRE) PGD consortium data collection for the year 2004, where 45 centers participated, 3358 egg collections have been performed: 1192 in cycles with PGD, 2087 with PGS, and 79 cycles with social sexing. The indications were chromosomal abnormalities for 559 cycles, X-linked disorders for 113 cycles, and monogenic diseases for 520. In cycles with PGD for chromosomal abnormality indications, 76% embryos were biopsied; among them, 93% provided a diagnosis, of which 25% were transferable. In cycles with PGD for monogenic diseases, 71% embryos were biopsied; 88% provided a diagnosis, of which 52% were transferable. In total, 69.6% of the cycles led to embryo transfer. [10] In France, 70% of the biopsied embryos provided a diagnosis, of which 60% were transferable. It has been shown that the PGD implantation rate is 17%, which is less than the implantation rate observed in non-PGD ART cycles. [10] The clinical pregnancy rate is reported as 18% per oocyte retrieval and 25% per embryo transferred, leading to 679 pregnancies and 528 babies born. The pediatric follow-up of PGD babies has shown a similar mental and psychomotor developmental outcome at age two, when compared with children conceived after IVF-ICSI. [11] Embryo biopsy does not seem to affect the course of pregnancy, the baby's characteristics at birth (birth weight, length, gestational age at delivery), or the rate of malformations at birth. [12],[13],[14]

 Ethical Issues

Many a time parents may opt to select the sex of the fetus by opting for preimplantation genetic testing, for social reasons. As per the Preconception and Prenatal Diagnosis Act of 1994, this is illegal in India and many other countries. Couples may select a human leukocyte antigen (HLA)-matched embryo for a previous child with a genetic disease treatable by bone marrow transplantation. In case of adult onset diseases like Huntington disease and cancer predisposition syndromes, the possibility of a new therapy becoming available and the burden of preventive measures need to be taken into account, when preimplantation genetic testing is considered. Ethical issues in preimplantation testing are not completely the same as in prenatal diagnosis, as the former concerns the selection of unaffected embryos and the latter, the termination of affected pregnancies.


Preimplantation genetic diagnosis is only offered at a few select centers worldwide compared to prenatal diagnosis, but the number of centers is growing steadily along with the number of diseases that can be diagnosed. PGD needs a close collaboration between obstetricians, fertility specialists, IVF laboratories, and human geneticists. It has gained a place among the choices offered to couples at risk of serious disease transmission. PGD is an important alternative to standard prenatal diagnosis, for genetic disorders. Low pregnancy and birth rates and the high cost of the procedure, however, are important considerations.


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