Preimplantation genetic diagnosis
Encyclopedia
In medicine
and (clinical) genetics
pre-implantation genetic diagnosis (PGD or PIGD) (also known as embryo screening) refers to procedures that are performed on embryo
s prior to implantation, sometimes even on oocyte
s prior to fertilization
. PGD is considered another way to prenatal diagnosis
. When used to screen for a specific genetic disease, its main advantage is that it avoids selective pregnancy termination
as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology
, and requires in vitro fertilization
(IVF) to obtain oocytes or embryos for evaluation.
The term pre-implantation genetic screening (PGS) is used to denote procedures that do not look for a specific disease but use PGD techniques to identify embryos at risk. PGD is a poorly chosen phrase because, in medicine, to "diagnose" means to identify an illness or determine its cause. An oocyte or early-stage embryo has no symptoms of disease. They are not ill. Rather, they may have a genetic condition that could lead to disease. To "screen" means to test for anatomical, physiological, or genetic conditions in the absence of symptoms of disease. So both PGD and PGS should be referred to as types of embryo screening.
The procedures may also be called preimplantation genetic profiling to adapt to the fact that they are sometimes used on oocytes or embryos prior to implantation for other reasons than diagnosis or screening.
Procedures performed on sex cells before fertilization may instead be referred to as methods of oocyte selection
or sperm selection, although the methods and aims partly overlap with PGD.
(PCR) technology. Handyside and collaborators' first successful attempts at testing were in October 1989 with the first births in 1990 though the preliminary experiments had been published some years earlier. In these first cases, PCR was used for sex determination for patients carrying X-linked diseases.
implications. For example, in Germany the use of PGD had been prohibited by the Embryo Protection Act of 1990 http://www.bmj.bund.de/files/-/1147/ESchG%20englisch.pdf, though this prohibition was relaxed somewhat in July of 2011 http://www.dw-world.de/dw/article/0,,15217136,00.html.
In other countries PGD is permitted in law but its operation is controlled by the state. In the UK, the use of PGD is controlled by the HFEA
(http://www.hfea.gov.uk) - the UK regulator for fertility treatment and embryo research. The HFEA only permits the use of PGD where the clinic concerned has a licence from the HFEA and sets out the rules for this licensing in its Code of Practice (http://cop.hfea.gov.uk/cop). Each clinic, and each medical condition, requires a separate application where the HFEA check the suitability of the genetic test proposed and the staff skills and facilities of the clinic. Only then can PGD be used for a patient.
.
). PGD helps these couples identify embryos carrying a genetic disease or a chromosome abnormality, thus avoiding diseased offspring. The most frequently diagnosed autosomal recessive disorders are cystic fibrosis
, Beta-thalassemia
, sickle cell disease and spinal muscular atrophy
type 1. The most common dominant diseases are myotonic dystrophy
, Huntington's disease
and Charcot-Marie-Tooth disease
; and in the case of the X-linked diseases, most of the cycles are performed for fragile X syndrome
, haemophilia A
and Duchenne muscular dystrophy
. Though it is quite infrequent, some centers report PGD for mitochondrial disorders or two indications simultaneously.
PGD is also now being performed in a disease called Hereditary multiple exostoses
(MHE / MO / HME)
In addition, there are infertile couples who carry an inherited condition and who opt for PGD as it can be easily combined with their IVF treatment.
to perform embryo selection of an embryo that appears to have the greatest chances for successful pregnancy. However, as the results of PGP rely on the assessment of a single cell, PGP has inherent limitations as the tested cell may not be representative of the embryo because of mosaicism
.
A systematic review and meta-analysis of existing randomized controlled trial
s came to the result that there is no evidence of a beneficial effect of PGP as measured by live birth rate. On the contrary, for women of advanced maternal age, PGP significantly lowers the live birth rate. Technical drawbacks, such as the invasiveness of the biopsy, and chromosomal mosaicism are the major underlying factors for inefficacy of PGP.
When used for women of advanced maternal age and for patients with repetitive IVF failure, PGP is mainly carried out as a screening for detection of chromosomal abnormalities such as aneuploidy
, reciprocal and Robertsonian translocations, and few cases for other abnormalities such as chromosomal inversion
s or deletions. The principle behind it is that, since it is known that numerical chromosomal abnormalities explain most of the cases of pregnancy loss, and a large proportion of the human embryos are aneuploid, the selective replacement of euploid embryos should increase the chances of a successful IVF treatment. Comprehensive chromosome analysis methods include array-comparative genomic hybridization (aCGH), quantitative PCR and SNP microarrays. Combined with single blastomere biopsy on day-3 embryos, aCGH is very robust with 2.9% of tested embryos with no results, and associated with low error rates (1.9%).
In addition to screening for specific abnormalities, techniques are in development that can avail for up to full genome sequencing
, from which genetic profiling can score the DNA patterns by comparing with ones that have previously been found among embryos in successful or unsuccessful pregnancies.
(HLA) typing of embryos, so that the child's HLA matches a sick sibling, availing for cord-blood stem cell donation. The child is in this sense a "savior sibling
" for the recipient child. HLA typing has meanwhile become an important PGD indication in those countries where the law permits it. The HLA matching can be combined with the diagnosis for monogenic diseases such as Fanconi anaemia or b-thalassemia in those cases where the ailing sibling is affected with this disease, or it may be exceptionally performed on its own for cases such as children with leukaemia. The main ethical argument against is the possible exploitation of the child, although some authors maintain that the Kantian imperative is not breached since the future donor child will not only be a donor but also a loved individual within the family.
even before implantation, and may therefore be termed preimplantation sex discernment. Potential applications of preimplantation sex discernment include:
, although the use of natural cycles and in vivo fertilization followed by uterine lavage was attempted in the past and is now largely abandoned. In order to obtain a large group of oocytes, the patients undergo controlled ovarian stimulation (COH). COH is carried out either in an agonist protocol, using gonadotrophin-releasing hormone (GnRH) analogues for pituitary desensitisation, combined with human menopausal gonadotrophins (hMG) or recombinant follicle stimulating hormone (FSH), or an antagonist protocol using recombinant FSH combined with a GnRH antagonist according to clinical assessment of the patient’s profile (age, body mass index (BMI), endocrine parameters). hCG is administered when at least three follicles of more than 17 mm mean diameter are seen at transvaginal ultrasound scan. Transvaginal ultrasound-guided oocyte retrieval is scheduled 36 hours after hCG administration. Luteal phase supplementation consists of daily intravaginal administration of 600 µg of natural micronized progesterone.
Oocytes are carefully denudated from the cumulus cells, as these cells can be a source of contamination during the PGD if PCR-based technology is used. In the majority of the reported cycles, intracytoplasmic sperm injection
(ICSI) is used instead of IVF. The main reasons are to prevent contamination with residual sperm adhered to the zona pellucida and to avoid unexpected fertilization failure. The ICSI procedure is carried out on mature metaphase-II oocytes and fertilization is assessed 16–18 hours after. The embryo development is further evaluated every day prior to biopsy and until transfer to the woman’s uterus. During the cleavage stage, embryo evaluation is performed daily on the basis of the number, size, cell-shape and fragmentation rate of the blastomeres. On day 4, embryos were scored in function of their degree of compaction and blastocysts were evaluated according to the quality of the throphectoderm and inner cell mass, and their degree of expansion.
The biopsy procedure always involves two steps: the opening of the zona pellucida
and the removal of the cell(s). There are different approaches to both steps, including mechanical, chemical (Tyrode’s acidic solution) and laser technology for the breaching of the zona pellucida, extrusion or aspiration for the removal of PBs and blastomeres, and herniation of the trophectoderm cells.
of the oocyte are extruded at the time of the conclusion of the meiotic division
, normally the first polar body is noted after ovulation
, and the second polar body after fertilization
. PB biopsy is used mainly by two PGD groups in the USA and by groups in countries where cleavage-stage embryo selection is banned. They have been used for diagnosing translocations and monogenic disorders of maternal origin, as well as for PGS.
The first PB is removed from the unfertilised oocyte, and the second PB from the zygote, shortly after fertilization. The main advantage of the use of PBs in PGD is that they are not necessary for successful fertilisation or normal embryonic development, thus ensuring no deleterious effect for the embryo. One of the disadvantages of PB biopsy is that it only provides information about the maternal contribution to the embryo, which is why cases of autosomal dominant and X-linked disorders that are maternally transmitted can be diagnosed, and autosomal recessive disorders can only partially be diagnosed. Another drawback is the increased risk of diagnostic error, for instance due to the degradation of the genetic material or events of recombination that lead to heterozygous first PBs. It is generally agreed that it is best to analyse both PBs in order to minimize the risk of misdiagnosis. This can be achieved by sequential biopsy, necessary if monogenic diseases are diagnosed, to be able to differentiate the first from the second PB, or simultaneous biopsy if FISH is to be performed.
In Germany, where the legislation bans the selection of preimplantation embryos, PB analysis is the only possible method to perform PGD. The biopsy and analysis of the first and second PBs can be completed before syngamy, which is the moment from which the zygote is considered an embryo and becomes protected by the law.
The main advantage of cleavage-stage biopsy over PB analysis is that the genetic input of both parents can be studied. On the other hand, cleavage-stage embryos are found to have a high rate of chromosomal mosaicism, putting into question whether the results obtained on one or two blastomeres will be representative for the rest of the embryo. It is for this reason that some programs utilize a combination of PB biopsy and blastomere biopsy. Furthermore, cleavage-stage biopsy, as in the case of PB biopsy, yields a very limited amount of tissue for diagnosis, necessitating the development of single-cell PCR and FISH
techniques.
Although theoretically PB biopsy and blastocyst biopsy are less harmful than cleavage-stage biopsy, this is still the prevalent method. It is used in approximately 94% of the PGD cycles reported to the ESHRE PGD Consortium. The main reasons are that it allows for a safer and more complete diagnosis than PB biopsy and still leaves enough time to finish the diagnosis before the embryos must be replaced in the patient’s uterus, unlike blastocyst biopsy.
Of all cleavage-stages, it is generally agreed that the optimal moment for biopsy is at the eight-cell stage. It is diagnostically safer than the PB biopsy and, unlike blastocyst biopsy, it allows for the diagnosis of the embryos before day 5. In this stage, the cells are still totipotent and the embryos are not yet compacting. Although it has been shown that up to a quarter of a human embryo can be removed without disrupting its development, it still remains to be studied whether the biopsy of one or two cells correlates with the ability of the embryo to further develop, implant and grow into a full term pregnancy.
TE biopsy has been shown to be successful in animal models such as rabbits, mice and primates. These studies show that the removal of some TE cells is not detrimental to the further in vivo development of the embryo.
Human blastocyst-stage biopsy for PGD is performed by making a hole in the ZP on day three of in vitro culture. This allows the developing TE to protrude after blastulation, facilitating the biopsy. On day five post-fertilization, approximately five cells are excised from the TE using a glass needle or laser energy, leaving the embryo largely intact and without loss of inner cell mass. After diagnosis, the embryos can be replaced during the same cycle, or cryopreserved and transferred in a subsequent cycle.
There are two drawbacks to this approach, due to the stage at which it is performed. First, only approximately half of the preimplantation embryos reach the blastocyst stage. This can restrict the number of blastocysts available for biopsy, limiting in some cases the success of the PGD. Mc Arthur and coworkers report that 21% of the started PGD cycles had no embryo suitable for TE biopsy. This figure is approximately four times higher than the average presented by the ESHRE PGD consortium data, where PB and cleavage-stage biopsy are the predominant reported methods. On the other hand, delaying the biopsy to this late stage of development limits the time to perform the genetic diagnosis, making it difficult to redo a second round of PCR or to rehybridize FISH probes before the embryos should be transferred back to the patient.
, embryo development and pregnancy outcomes.
(FISH) and Polymerase chain reaction
(PCR) are the two commonly used, first-generation technologies in PGD. Other approaches have been proposed or are currently in development (such as whole genome amplification and comparative genomic hybridization
) . PCR is generally used to diagnose monogenic disorders and FISH is used for the detection of chromosomal abnormalities (for instance, aneuploidy
screening or chromosomal translocations). Over the past few years, various advancements in PGD testing have allowed for an improvement in the comprehensiveness and accuracy of results available depending on the technology used. Recently a method was developed allowing to fix metaphase plates from single blastomeres. This technique in conjunction with FISH, m-FISH can produce more reliable results, since analysis is done on whole metaphase plates
The type and number of probes that are used on a sample depends on the indication. For sex determination (used for instance when a PCR protocol for a given X-linked disorder is not available), probes for the X and Y chromosomes are applied along with probes for one or more of the autosomes as an internal FISH control. More probes can be added to check for aneuploidies, particularly those that could give raise to a viable pregnancy (such as a trisomy 21). The use of probes for chromosomes X, Y, 13, 14, 15, 16, 18, 21 and 22 has the potential of detecting 70% of the aneuploidies found in spontaneous abortions.
In order to be able to analyse more chromosomes on the same sample, up to three consecutive rounds of FISH can be carried out. In the case of chromosome rearrangements, specific combinations of probes have to be chosen that flank the region of interest. The FISH technique is considered to have an error rate between 5 and 10%.
The main problem of the use of FISH to study the chromosomal constitution of embryos is the elevated mosaicism rate observed at the human preimplantation stage. A meta-analysis of more than 800 embryos came to the result that approximately 75% of preimplantation embryos are mosaic, of which approximately 60% are diploid–aneuploid mosaic and approximately 15% aneuploid mosaic. Li and co-workers found that 40% of the embryos diagnosed as aneuploid on day 3 turned out to have a euploid inner cell mass at day 6. Staessen and collaborators found that 17.5% of the embryos diagnosed as abnormal during PGS, and subjected to post-PGD reanalysis, were found to also contain normal cells, and 8.4% were found grossly normal. As a consequence, it has been questioned whether the one or two cells studied from an embryo are actually representative of the complete embryo, and whether viable embryos are not being discarded due to the limitations of the technique.
conceived PCR
in 1985 as an in vitro simplified reproduction of the in vivo process of DNA replication
. Taking advantage of the chemical properties of DNA and the availability of thermostable DNA polymerase
s, PCR allows for the enrichment of a DNA sample for a certain sequence. PCR provides the possibility to obtain a large quantity of copies of a particular stretch of the genome, making further analysis possible. It is a highly sensitive and specific technology, which makes it suitable for all kinds of genetic diagnosis, including PGD. Currently, many different variations exist on the PCR itself, as well as on the different methods for the posterior analysis of the PCR products.
When using PCR in PGD, one is faced with a problem that is inexistent in routine genetic analysis: the minute amounts of available genomic DNA. As PGD is performed on single cells, PCR has to be adapted and pushed to its physical limits, and use the minimum amount of template possible: one strand. This implies a long process of fine-tuning of the PCR conditions and a susceptibility to all the problems of conventional PCR, but several degrees intensified. The high number of needed PCR cycles and the limited amount of template makes single-cell PCR very sensitive to contamination. Another problem specific to single-cell PCR is the allele drop out (ADO) phenomenon. It consists of the random non-amplification of one of the alleles present in a heterozygous sample. ADO seriously compromises the reliability of PGD as a heterozygous embryo could be diagnosed as affected or unaffected depending on which allele would fail to amplify. This is particularly concerning in PGD for autosomal dominant disorders, where ADO of the affected allele could lead to the transfer of an affected embryo.
In the case of FISH, in some centres only embryos are replaced that are found to be chromosomally normal (that is, showing two signals for the gonosomes and the analysed autosomes) after the analysis of one or two blastomeres, and when two blastomeres are analysed, the results should be concordant. Other centres argue that embryos diagnosed as monosomic could be transferred, because the false monosomy (i.e. loss of one FISH signal in a normal dipoloid cell) is the most frequently occurring misdiagnosis. In these cases, there is no risk for an aneuploid pregnancy, and normal diploid embryos are not lost for transfer because of a FISH error. Moreover, it has been shown that embryos diagnosed as monosomic on day 3 (except for chromosomes X and 21), never develop to blastocyst, which correlates with the fact that these monosomies are never observed in ongoing pregnancies.
Diagnosis and misdiagnosis in PGD using PCR have been mathematically modelled in the work of Navidi and Arnheim and of Lewis and collaborators. The most important conclusion of these publications is that for the efficient and accurate diagnosis of an embryo, two genotypes are required. This can be based on a linked marker and disease genotypes from a single cell or on marker/disease genotypes of two cells. An interesting aspect explored in these papers is the detailed study of all possible combinations of alleles that may appear in the PCR results for a particular embryo. The authors indicate that some of the genotypes that can be obtained during diagnosis may not be concordant with the expected pattern of linked marker genotypes, but are still providing sufficient confidence about the unaffected genotype of the embryo. Although these models are reassuring, they are based on a theoretical model, and generally the diagnosis is established on a more conservative basis, aiming to avoid the possibility of misdiagnosis. When unexpected alleles appear during the analysis of a cell, depending on the genotype observed, it is considered that either an abnormal cell has been analysed or that contamination has occurred, and that no diagnosis can be established. A case in which the abnormality of the analysed cell can be clearly identified is when, using a multiplex PCR for linked markers, only the alleles of one of the parents are found in the sample. In this case, the cell can be considered as carrying a monosomy for the chromosome on which the markers are located, or, possibly, as haploid. The appearance of a single allele that indicates an affected genotype is considered sufficient to diagnose the embryo as affected, and embryos that have been diagnosed with a complete unaffected genotype are preferred for replacement. Although this policy may lead to a lower number of unaffected embryos suitable for transfer, it is considered preferable to the possibility of a misdiagnosis.
(PGH) is a new clinical method of Preimplantation genetic diagnosis (PGD). PGH was first developed in 2006 at London's Guy's Hospital and greatly advances PGD by using DNA fingerprinting rather than identifying the actual genetic signature (such as point mutations).
is usually performed on day three or day five post-fertilization, the timing depending on the techniques used for PGD and the standard procedures of the IVF
centre where it is performed.
With the introduction in Europe of the single-embryo transfer policy, which aims at the reduction of the incidence of multiple pregnancies after ART, usually one embryo or early blastocyst is replaced in the uterus. Serum hCG is determined at day 12. If a pregnancy is established, an ultrasound examination at 7 weeks is performed to confirm the presence of a fetal heartbeat. Couples are generally advised to undergo PND
because of the, albeit low, risk of misdiagnosis.
It is not unusual that after the PGD, there are more embryos suitable for transferring back to the woman than necessary. For the couples undergoing PGD, those embryos are very valuable, as the couple's current cycle may not lead to an ongoing pregnancy. Embryo cryopreservation
and later thawing and replacement can give them a second chance to pregnancy without having to redo the cumbersome and expensive ART and PGD procedures.
”. It may be possible to make other "social selection" choices in the future that introduce socio-economic concerns.
Costs are substantial and insurance coverage may not be available.
PGD has the potential to screen for genetic issues unrelated to medical necessity
. The prospect of a “designer baby
” is closely related to the PGD technique.
By relying on the result of one cell from the multi-cell embryo, PGD operates under the assumption that this cell is representative of the remainder of the embryo. This may not be the case as the incidence of mosaicism is often relatively high. On occasion, PGD may result in a false negative result leading to the acceptance of an abnormal embryo, or in a false positive result leading to the deselection of a normal embryo.
ethics task force currently recommends using exclusion testing instead. Exclusion testing is based on a linkage analysis with polymorphic markers, in which the parental and grandparental origin of the chromosomes can be established. This way, only embryos are replaced that do not contain the chromosome derived from the affected grandparent, avoiding the need to detect the mutation itself.
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
and (clinical) genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
pre-implantation genetic diagnosis (PGD or PIGD) (also known as embryo screening) refers to procedures that are performed on embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
s prior to implantation, sometimes even on oocyte
Oocyte
An oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic...
s prior to fertilization
Fertilisation
Fertilisation is the fusion of gametes to produce a new organism. In animals, the process involves the fusion of an ovum with a sperm, which eventually leads to the development of an embryo...
. PGD is considered another way to prenatal diagnosis
Prenatal diagnosis
Prenatal diagnosis or prenatal screening is testing for diseases or conditions in a fetus or embryo before it is born. The aim is to detect birth defects such as neural tube defects, Down syndrome, chromosome abnormalities, genetic diseases and other conditions, such as spina bifida, cleft palate,...
. When used to screen for a specific genetic disease, its main advantage is that it avoids selective pregnancy termination
Abortion
Abortion is defined as the termination of pregnancy by the removal or expulsion from the uterus of a fetus or embryo prior to viability. An abortion can occur spontaneously, in which case it is usually called a miscarriage, or it can be purposely induced...
as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology
Assisted reproductive technology
Assisted reproductive technology is a general term referring to methods used to achieve pregnancy by artificial or partially artificial means. It is reproductive technology used primarily in infertility treatments. Some forms of ART are also used in fertile couples for genetic reasons...
, and requires in vitro fertilization
In vitro fertilisation
In vitro fertilisation is a process by which egg cells are fertilised by sperm outside the body: in vitro. IVF is a major treatment in infertility when other methods of assisted reproductive technology have failed...
(IVF) to obtain oocytes or embryos for evaluation.
The term pre-implantation genetic screening (PGS) is used to denote procedures that do not look for a specific disease but use PGD techniques to identify embryos at risk. PGD is a poorly chosen phrase because, in medicine, to "diagnose" means to identify an illness or determine its cause. An oocyte or early-stage embryo has no symptoms of disease. They are not ill. Rather, they may have a genetic condition that could lead to disease. To "screen" means to test for anatomical, physiological, or genetic conditions in the absence of symptoms of disease. So both PGD and PGS should be referred to as types of embryo screening.
The procedures may also be called preimplantation genetic profiling to adapt to the fact that they are sometimes used on oocytes or embryos prior to implantation for other reasons than diagnosis or screening.
Procedures performed on sex cells before fertilization may instead be referred to as methods of oocyte selection
Oocyte selection
Oocyte selection is a procedure that is performed prior to in vitro fertilization, in order to use oocytes with maximal chances of resulting in pregnancy. In contrast, embryo selection takes place after fertilization.-Techniques:...
or sperm selection, although the methods and aims partly overlap with PGD.
History
In 1967, Robert Edwards and David Gardner reported the successful sexing of rabbit blastocysts, setting the first steps towards PGD. It was not until the 1980s that human IVF was fully developed, which coincided with the breakthrough of the highly sensitive polymerase chain reactionPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) technology. Handyside and collaborators' first successful attempts at testing were in October 1989 with the first births in 1990 though the preliminary experiments had been published some years earlier. In these first cases, PCR was used for sex determination for patients carrying X-linked diseases.
PGD and society
As with all medical interventions associated with human reproduction, PGD raises strong, often conflicting opinions of social acceptability, particularly due to its eugenicEugenics
Eugenics is the "applied science or the bio-social movement which advocates the use of practices aimed at improving the genetic composition of a population", usually referring to human populations. The origins of the concept of eugenics began with certain interpretations of Mendelian inheritance,...
implications. For example, in Germany the use of PGD had been prohibited by the Embryo Protection Act of 1990 http://www.bmj.bund.de/files/-/1147/ESchG%20englisch.pdf, though this prohibition was relaxed somewhat in July of 2011 http://www.dw-world.de/dw/article/0,,15217136,00.html.
In other countries PGD is permitted in law but its operation is controlled by the state. In the UK, the use of PGD is controlled by the HFEA
Human Fertilisation and Embryology Authority
The Human Fertilisation and Embryology Authority is a statutory body in the United Kingdom that regulates and inspects all UK clinics providing in vitro fertilisation, artificial insemination and the storage of human eggs, sperm or embryos. It also regulates Human Embryo research...
(http://www.hfea.gov.uk) - the UK regulator for fertility treatment and embryo research. The HFEA only permits the use of PGD where the clinic concerned has a licence from the HFEA and sets out the rules for this licensing in its Code of Practice (http://cop.hfea.gov.uk/cop). Each clinic, and each medical condition, requires a separate application where the HFEA check the suitability of the genetic test proposed and the staff skills and facilities of the clinic. Only then can PGD be used for a patient.
Indications and applications
PGD can potentially be used to select embryos to be without a genetic disorder, to have increased chances of successful pregnancy, to match a sibling in HLA type in order to be a donor, to have less cancer predisposition, and for sex selectionSex selection
Sex selection is the attempt to control the sex of the offspring to achieve a desired sex. It can be accomplished in several ways, both pre- and post-implantation of an embryo, as well as at birth...
.
Monogenic disorders
PGD is available for a large number of monogenic disorders, that is, a condition is due to a single gene only, (autosomal recessive, autosomal dominant or X-linked disorders) or a chromosomal structural aberration (such as a balanced translocationChromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...
). PGD helps these couples identify embryos carrying a genetic disease or a chromosome abnormality, thus avoiding diseased offspring. The most frequently diagnosed autosomal recessive disorders are cystic fibrosis
Cystic fibrosis
Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...
, Beta-thalassemia
Thalassemia
Thalassemia is an inherited autosomal recessive blood disease that originated in the Mediterranean region. In thalassemia the genetic defect, which could be either mutation or deletion, results in reduced rate of synthesis or no synthesis of one of the globin chains that make up hemoglobin...
, sickle cell disease and spinal muscular atrophy
Spinal muscular atrophy
Spinal Muscular Atrophy is a neuromuscular disease characterized by degeneration of motor neurons, resulting in progressive muscular atrophy and weakness. The clinical spectrum of SMA ranges from early infant death to normal adult life with only mild weakness...
type 1. The most common dominant diseases are myotonic dystrophy
Myotonic dystrophy
Myotonic dystrophy is a chronic, slowly progressing, highly variable inherited multisystemic disease. It is characterized by wasting of the muscles , cataracts, heart conduction defects, endocrine changes, and myotonia. Myotonic dystrophy can occur in patients of any age...
, Huntington's disease
Huntington's disease
Huntington's disease, chorea, or disorder , is a neurodegenerative genetic disorder that affects muscle coordination and leads to cognitive decline and dementia. It typically becomes noticeable in middle age. HD is the most common genetic cause of abnormal involuntary writhing movements called chorea...
and Charcot-Marie-Tooth disease
Charcot-Marie-Tooth disease
Charcot–Marie–Tooth disease- , known also as Morbus Charcot-Marie-Tooth, Charcot-Marie-Tooth neuropathy, hereditary motor and sensory neuropathy , hereditary sensorimotor neuropathy , or peroneal muscular atrophy, is an inherited disorder of nerves that takes different forms...
; and in the case of the X-linked diseases, most of the cycles are performed for fragile X syndrome
Fragile X syndrome
Fragile X syndrome , Martin–Bell syndrome, or Escalante's syndrome , is a genetic syndrome that is the most commonly known single-gene cause of autism and the most common inherited cause of intellectual disability...
, haemophilia A
Haemophilia A
Haemophilia A is a deficiency in clotting factor VIII.Haemophilia A is inherited as an X-linked recessive trait, and thus occurs in males and in homozygous females. However, mild haemophilia A has been described in heterozygous females, presumably due to extremely unfavourable lyonization...
and Duchenne muscular dystrophy
Duchenne muscular dystrophy
Duchenne muscular dystrophy is a recessive X-linked form of muscular dystrophy, which results in muscle degeneration, difficulty walking, breathing, and death. The incidence is 1 in 3,000 boys. Females and males are affected, though females are rarely affected and are more often carriers...
. Though it is quite infrequent, some centers report PGD for mitochondrial disorders or two indications simultaneously.
PGD is also now being performed in a disease called Hereditary multiple exostoses
Hereditary multiple exostoses
Hereditary multiple exostoses is a rare medical condition in which multiple bony spurs or lumps develop on the bones of a child...
(MHE / MO / HME)
In addition, there are infertile couples who carry an inherited condition and who opt for PGD as it can be easily combined with their IVF treatment.
Pregnancy chances
Preimplantation genetic profiling (PGP) has been suggested to be applied as a method of assisted reproductive technologyAssisted reproductive technology
Assisted reproductive technology is a general term referring to methods used to achieve pregnancy by artificial or partially artificial means. It is reproductive technology used primarily in infertility treatments. Some forms of ART are also used in fertile couples for genetic reasons...
to perform embryo selection of an embryo that appears to have the greatest chances for successful pregnancy. However, as the results of PGP rely on the assessment of a single cell, PGP has inherent limitations as the tested cell may not be representative of the embryo because of mosaicism
Mosaic (genetics)
In genetic medicine, a mosaic or mosaicism denotes the presence of two populations of cells with different genotypes in one individual who has developed from a single fertilized egg...
.
A systematic review and meta-analysis of existing randomized controlled trial
Randomized controlled trial
A randomized controlled trial is a type of scientific experiment - a form of clinical trial - most commonly used in testing the safety and efficacy or effectiveness of healthcare services or health technologies A randomized controlled trial (RCT) is a type of scientific experiment - a form of...
s came to the result that there is no evidence of a beneficial effect of PGP as measured by live birth rate. On the contrary, for women of advanced maternal age, PGP significantly lowers the live birth rate. Technical drawbacks, such as the invasiveness of the biopsy, and chromosomal mosaicism are the major underlying factors for inefficacy of PGP.
When used for women of advanced maternal age and for patients with repetitive IVF failure, PGP is mainly carried out as a screening for detection of chromosomal abnormalities such as aneuploidy
Aneuploidy
Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders . Some cancer cells also have abnormal numbers of chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate...
, reciprocal and Robertsonian translocations, and few cases for other abnormalities such as chromosomal inversion
Chromosomal inversion
An inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end. An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself. Inversions are of two types: paracentric and pericentric.Paracentric inversions do not include the...
s or deletions. The principle behind it is that, since it is known that numerical chromosomal abnormalities explain most of the cases of pregnancy loss, and a large proportion of the human embryos are aneuploid, the selective replacement of euploid embryos should increase the chances of a successful IVF treatment. Comprehensive chromosome analysis methods include array-comparative genomic hybridization (aCGH), quantitative PCR and SNP microarrays. Combined with single blastomere biopsy on day-3 embryos, aCGH is very robust with 2.9% of tested embryos with no results, and associated with low error rates (1.9%).
In addition to screening for specific abnormalities, techniques are in development that can avail for up to full genome sequencing
Full genome sequencing
Full genome sequencing , also known as whole genome sequencing , complete genome sequencing, or entire genome sequencing, is a laboratory process that determines the complete DNA sequence of an organism's genome at a single time...
, from which genetic profiling can score the DNA patterns by comparing with ones that have previously been found among embryos in successful or unsuccessful pregnancies.
HLA matching
Human leukocyte antigenHuman leukocyte antigen
The human leukocyte antigen system is the name of the major histocompatibility complex in humans. The super locus contains a large number of genes related to immune system function in humans. This group of genes resides on chromosome 6, and encodes cell-surface antigen-presenting proteins and...
(HLA) typing of embryos, so that the child's HLA matches a sick sibling, availing for cord-blood stem cell donation. The child is in this sense a "savior sibling
Savior sibling
A savior sibling is a child who is born to provide an organ or cell transplant to a sibling that is affected with a fatal disease, such as cancer or Fanconi anemia, that can best be treated by hematopoietic stem cell transplantation....
" for the recipient child. HLA typing has meanwhile become an important PGD indication in those countries where the law permits it. The HLA matching can be combined with the diagnosis for monogenic diseases such as Fanconi anaemia or b-thalassemia in those cases where the ailing sibling is affected with this disease, or it may be exceptionally performed on its own for cases such as children with leukaemia. The main ethical argument against is the possible exploitation of the child, although some authors maintain that the Kantian imperative is not breached since the future donor child will not only be a donor but also a loved individual within the family.
Cancer predisposition
A more recent application of PGD is to diagnose late-onset diseases and (cancer) predisposition syndromes. Since affected individuals remain healthy until the onset of the disease, frequently in the fourth decade of life, there is debate on whether or not PGD is appropriate in these cases. Considerations include the high probability of developing the disorders and the potential for cures. For example, in predisposition syndromes, such as BRCA1 mutations which predispose the individual to breast cancer, the outcomes are unclear. Although PGD is often regarded as an early form of prenatal diagnosis, the nature of the requests for PGD often differs from those of prenatal diagnosis requests made when the mother is already pregnant. Some of the widely accepted indications for PGD would not be acceptable for prenatal diagnosis.Sex discernment
Preimplantation genetic diagnosis provides a method of prenatal sex discernmentPrenatal sex discernment
Prenatal sex discernment is the prenatal testing for discerning the sex of a fetus before birth.-Methods:Prenatal sex discernment can be performed by preimplantation genetic diagnosis before conception....
even before implantation, and may therefore be termed preimplantation sex discernment. Potential applications of preimplantation sex discernment include:
- A complement to specific gene testing for monogenic disorders, which can be very useful for genetic diseases whose presentation is linked to the sexSex linkageSex linkage is the phenotypic expression of an allele related to the chromosomal sex of the individual. This mode of inheritance is in contrast to the inheritance of traits on autosomal chromosomes, where both sexes have the same probability of inheritance...
, such as, for example, X-linked diseases. - Ability to prepare for any sex-dependent aspects of parenting.
- Sex selectionSex selectionSex selection is the attempt to control the sex of the offspring to achieve a desired sex. It can be accomplished in several ways, both pre- and post-implantation of an embryo, as well as at birth...
. A 2006 survey http://www.dnapolicy.org/resources/PGDSurveyReportFertilityandSterilitySeptember2006withcoverpages.pdf found that 42 per cent of clinics that offer PGD have provided it for sex selection for non-medical reasons. Nearly half of these clinics perform it only for “family balancing”, which is where a couple with two or more children of one sex desire a child of the other, but half do not restrict sex selection to family balancing. In India, this practice has been used to select only male embryos although this practice is illegal . Opinions on whether sex selection for non-medical reasons is ethically acceptable differ widely, as exemplified by the fact that the ESHRE Task Force could not formulate a uniform recommendation.
Minor disabilities
A 2006 survey reveals that PGD has occasionally been used to select an embryo for the presence of a particular disease or disability, such as deafness, in order that the child would share that characteristic with the parents.http://www.newscientist.com/blog/shortsharpscience/2006/09/designer-deafness.htmlTechnical aspects
PGD is a form of genetic diagnosis performed prior to implantation. This implies that the patient’s oocytes should be fertilized in vitro and the embryos kept in culture until the diagnosis is established. It is also necessary to perform a biopsy on these embryos in order to obtain material on which to perform the diagnosis. The diagnosis itself can be carried out using several techniques, depending on the nature of the studied condition. Generally, PCR-based methods are used for monogenic disorders and FISH for chromosomal abnormalities and for sexing those cases in which no PCR protocol is available for an X-linked disease. These techniques need to be adapted to be performed on blastomeres and need to be thoroughly tested on single-cell models prior to clinical use. Finally, after embryo replacement, surplus good quality unaffected embryos can be cryopreserved, to be thawed and transferred back in a next cycle.Obtaining embryos for preimplantation genetic diagnosis
Currently, all PGD embryos are obtained by assisted reproductive technologyAssisted reproductive technology
Assisted reproductive technology is a general term referring to methods used to achieve pregnancy by artificial or partially artificial means. It is reproductive technology used primarily in infertility treatments. Some forms of ART are also used in fertile couples for genetic reasons...
, although the use of natural cycles and in vivo fertilization followed by uterine lavage was attempted in the past and is now largely abandoned. In order to obtain a large group of oocytes, the patients undergo controlled ovarian stimulation (COH). COH is carried out either in an agonist protocol, using gonadotrophin-releasing hormone (GnRH) analogues for pituitary desensitisation, combined with human menopausal gonadotrophins (hMG) or recombinant follicle stimulating hormone (FSH), or an antagonist protocol using recombinant FSH combined with a GnRH antagonist according to clinical assessment of the patient’s profile (age, body mass index (BMI), endocrine parameters). hCG is administered when at least three follicles of more than 17 mm mean diameter are seen at transvaginal ultrasound scan. Transvaginal ultrasound-guided oocyte retrieval is scheduled 36 hours after hCG administration. Luteal phase supplementation consists of daily intravaginal administration of 600 µg of natural micronized progesterone.
Oocytes are carefully denudated from the cumulus cells, as these cells can be a source of contamination during the PGD if PCR-based technology is used. In the majority of the reported cycles, intracytoplasmic sperm injection
Intracytoplasmic sperm injection
Intracytoplasmic sperm injection is an in vitro fertilization procedure in which a single sperm is injected directly into an egg.-Indications:...
(ICSI) is used instead of IVF. The main reasons are to prevent contamination with residual sperm adhered to the zona pellucida and to avoid unexpected fertilization failure. The ICSI procedure is carried out on mature metaphase-II oocytes and fertilization is assessed 16–18 hours after. The embryo development is further evaluated every day prior to biopsy and until transfer to the woman’s uterus. During the cleavage stage, embryo evaluation is performed daily on the basis of the number, size, cell-shape and fragmentation rate of the blastomeres. On day 4, embryos were scored in function of their degree of compaction and blastocysts were evaluated according to the quality of the throphectoderm and inner cell mass, and their degree of expansion.
Biopsy procedures
As PGD can be performed on cells from different developmental stages, the biopsy procedures vary accordingly. Theoretically, the biopsy can be performed at all preimplantation stages, but only three have been suggested: on unfertilised and fertilised oocytes (for polar bodies, PBs), on day three cleavage-stage embryos (for blastomeres) and on blastocysts (for trophectoderm cells).The biopsy procedure always involves two steps: the opening of the zona pellucida
Zona pellucida
The zona pellucida is a glycoprotein membrane surrounding the plasma membrane of an oocyte. It is a vital constitutive part of the latter, external but of essential importance to it...
and the removal of the cell(s). There are different approaches to both steps, including mechanical, chemical (Tyrode’s acidic solution) and laser technology for the breaching of the zona pellucida, extrusion or aspiration for the removal of PBs and blastomeres, and herniation of the trophectoderm cells.
Polar body biopsy
The first and second polar bodyPolar body
A polar body is a cell structure found inside an ovum. Both animal and plant ova possess it. It is also known as a polar cell.Asymmetrical cell division leads to the production of polar bodies during oogenesis. To conserve nutrients, the majority of cytoplasm is segregated into the secondary...
of the oocyte are extruded at the time of the conclusion of the meiotic division
Meiosis
Meiosis is a special type of cell division necessary for sexual reproduction. The cells produced by meiosis are gametes or spores. The animals' gametes are called sperm and egg cells....
, normally the first polar body is noted after ovulation
Ovulation
Ovulation is the process in a female's menstrual cycle by which a mature ovarian follicle ruptures and discharges an ovum . Ovulation also occurs in the estrous cycle of other female mammals, which differs in many fundamental ways from the menstrual cycle...
, and the second polar body after fertilization
Fertilisation
Fertilisation is the fusion of gametes to produce a new organism. In animals, the process involves the fusion of an ovum with a sperm, which eventually leads to the development of an embryo...
. PB biopsy is used mainly by two PGD groups in the USA and by groups in countries where cleavage-stage embryo selection is banned. They have been used for diagnosing translocations and monogenic disorders of maternal origin, as well as for PGS.
The first PB is removed from the unfertilised oocyte, and the second PB from the zygote, shortly after fertilization. The main advantage of the use of PBs in PGD is that they are not necessary for successful fertilisation or normal embryonic development, thus ensuring no deleterious effect for the embryo. One of the disadvantages of PB biopsy is that it only provides information about the maternal contribution to the embryo, which is why cases of autosomal dominant and X-linked disorders that are maternally transmitted can be diagnosed, and autosomal recessive disorders can only partially be diagnosed. Another drawback is the increased risk of diagnostic error, for instance due to the degradation of the genetic material or events of recombination that lead to heterozygous first PBs. It is generally agreed that it is best to analyse both PBs in order to minimize the risk of misdiagnosis. This can be achieved by sequential biopsy, necessary if monogenic diseases are diagnosed, to be able to differentiate the first from the second PB, or simultaneous biopsy if FISH is to be performed.
In Germany, where the legislation bans the selection of preimplantation embryos, PB analysis is the only possible method to perform PGD. The biopsy and analysis of the first and second PBs can be completed before syngamy, which is the moment from which the zygote is considered an embryo and becomes protected by the law.
Cleavage-stage biopsy (Blastomere biopsy)
Cleavage-stage biopsy is generally performed the morning of day three post-fertilization, when normally developing embryos reach the eight-cell stage. The biopsy is usually performed on embryos with less than 50% of anucleated fragments and at an 8-cell or later stage of development. A hole is made in the zona pellucida and one or two blastomeres containing a nucleus are gently aspirated or extruded through the opening.The main advantage of cleavage-stage biopsy over PB analysis is that the genetic input of both parents can be studied. On the other hand, cleavage-stage embryos are found to have a high rate of chromosomal mosaicism, putting into question whether the results obtained on one or two blastomeres will be representative for the rest of the embryo. It is for this reason that some programs utilize a combination of PB biopsy and blastomere biopsy. Furthermore, cleavage-stage biopsy, as in the case of PB biopsy, yields a very limited amount of tissue for diagnosis, necessitating the development of single-cell PCR and FISH
Fish
Fish are a paraphyletic group of organisms that consist of all gill-bearing aquatic vertebrate animals that lack limbs with digits. Included in this definition are the living hagfish, lampreys, and cartilaginous and bony fish, as well as various extinct related groups...
techniques.
Although theoretically PB biopsy and blastocyst biopsy are less harmful than cleavage-stage biopsy, this is still the prevalent method. It is used in approximately 94% of the PGD cycles reported to the ESHRE PGD Consortium. The main reasons are that it allows for a safer and more complete diagnosis than PB biopsy and still leaves enough time to finish the diagnosis before the embryos must be replaced in the patient’s uterus, unlike blastocyst biopsy.
Of all cleavage-stages, it is generally agreed that the optimal moment for biopsy is at the eight-cell stage. It is diagnostically safer than the PB biopsy and, unlike blastocyst biopsy, it allows for the diagnosis of the embryos before day 5. In this stage, the cells are still totipotent and the embryos are not yet compacting. Although it has been shown that up to a quarter of a human embryo can be removed without disrupting its development, it still remains to be studied whether the biopsy of one or two cells correlates with the ability of the embryo to further develop, implant and grow into a full term pregnancy.
Blastocyst biopsy
In an attempt to overcome the difficulties related to single-cell techniques, it has been suggested to biopsy embryos at the blastocyst stage, providing a larger amount of starting material for diagnosis. It has been shown that if more than two cells are present in the same sample tube, the main technical problems of single-cell PCR or FISH would virtually disappear. On the other hand, as in the case of cleavage-stage biopsy, the chromosomal differences between the inner cell mass and the trophectoderm (TE) can reduce the accuracy of diagnosis, although this mosaicism has been reported to be lower than in cleavage-stage embryos.TE biopsy has been shown to be successful in animal models such as rabbits, mice and primates. These studies show that the removal of some TE cells is not detrimental to the further in vivo development of the embryo.
Human blastocyst-stage biopsy for PGD is performed by making a hole in the ZP on day three of in vitro culture. This allows the developing TE to protrude after blastulation, facilitating the biopsy. On day five post-fertilization, approximately five cells are excised from the TE using a glass needle or laser energy, leaving the embryo largely intact and without loss of inner cell mass. After diagnosis, the embryos can be replaced during the same cycle, or cryopreserved and transferred in a subsequent cycle.
There are two drawbacks to this approach, due to the stage at which it is performed. First, only approximately half of the preimplantation embryos reach the blastocyst stage. This can restrict the number of blastocysts available for biopsy, limiting in some cases the success of the PGD. Mc Arthur and coworkers report that 21% of the started PGD cycles had no embryo suitable for TE biopsy. This figure is approximately four times higher than the average presented by the ESHRE PGD consortium data, where PB and cleavage-stage biopsy are the predominant reported methods. On the other hand, delaying the biopsy to this late stage of development limits the time to perform the genetic diagnosis, making it difficult to redo a second round of PCR or to rehybridize FISH probes before the embryos should be transferred back to the patient.
Cumulus cell sampling
Sampling of cumulus cells can be performed in addition to a sampling of polar bodies or cells from the embryo. Because of the molecular interactions between cumulus cells and the oocyte, gene expression profiling of cumulus cells can be performed to estimate oocyte quality and the efficiency of an ovarian hyperstimulation protocol, and may indirectly predict aneuploidyAneuploidy
Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders . Some cancer cells also have abnormal numbers of chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate...
, embryo development and pregnancy outcomes.
Genetic analysis techniques
Fluorescent in situ hybridizationFluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
(FISH) and Polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) are the two commonly used, first-generation technologies in PGD. Other approaches have been proposed or are currently in development (such as whole genome amplification and comparative genomic hybridization
Comparative genomic hybridization
Comparative genomic hybridization or Chromosomal Microarray Analysis is a molecular-cytogenetic method for the analysis of copy number changes in the DNA content of a given subject's DNA and often in tumor cells....
) . PCR is generally used to diagnose monogenic disorders and FISH is used for the detection of chromosomal abnormalities (for instance, aneuploidy
Aneuploidy
Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders . Some cancer cells also have abnormal numbers of chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate...
screening or chromosomal translocations). Over the past few years, various advancements in PGD testing have allowed for an improvement in the comprehensiveness and accuracy of results available depending on the technology used. Recently a method was developed allowing to fix metaphase plates from single blastomeres. This technique in conjunction with FISH, m-FISH can produce more reliable results, since analysis is done on whole metaphase plates
FISH
FISH is the most commonly applied method to determine the chromosomal constitution of an embryo. In contrast to karyotyping, it can be used on interphase chromosomes, so that it can be used on PBs, blastomeres and TE samples. The cells are fixated on glass microscope slides and hybridised with DNA probes. Each of these probes are specific for part of a chromosome, and are labelled with a fluorochrome. Currently, a large panel of probes are available for different segments of all chromosomes, but the limited number of different fluorochromes confines the number of signals that can be analysed simultaneously.The type and number of probes that are used on a sample depends on the indication. For sex determination (used for instance when a PCR protocol for a given X-linked disorder is not available), probes for the X and Y chromosomes are applied along with probes for one or more of the autosomes as an internal FISH control. More probes can be added to check for aneuploidies, particularly those that could give raise to a viable pregnancy (such as a trisomy 21). The use of probes for chromosomes X, Y, 13, 14, 15, 16, 18, 21 and 22 has the potential of detecting 70% of the aneuploidies found in spontaneous abortions.
In order to be able to analyse more chromosomes on the same sample, up to three consecutive rounds of FISH can be carried out. In the case of chromosome rearrangements, specific combinations of probes have to be chosen that flank the region of interest. The FISH technique is considered to have an error rate between 5 and 10%.
The main problem of the use of FISH to study the chromosomal constitution of embryos is the elevated mosaicism rate observed at the human preimplantation stage. A meta-analysis of more than 800 embryos came to the result that approximately 75% of preimplantation embryos are mosaic, of which approximately 60% are diploid–aneuploid mosaic and approximately 15% aneuploid mosaic. Li and co-workers found that 40% of the embryos diagnosed as aneuploid on day 3 turned out to have a euploid inner cell mass at day 6. Staessen and collaborators found that 17.5% of the embryos diagnosed as abnormal during PGS, and subjected to post-PGD reanalysis, were found to also contain normal cells, and 8.4% were found grossly normal. As a consequence, it has been questioned whether the one or two cells studied from an embryo are actually representative of the complete embryo, and whether viable embryos are not being discarded due to the limitations of the technique.
PCR
Kary MullisKary Mullis
Kary Banks Mullis is a Nobel Prize winning American biochemist, author, and lecturer. In recognition of his improvement of the polymerase chain reaction technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and earned the Japan Prize in the same year. The process was first...
conceived PCR
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
in 1985 as an in vitro simplified reproduction of the in vivo process of DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
. Taking advantage of the chemical properties of DNA and the availability of thermostable DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
s, PCR allows for the enrichment of a DNA sample for a certain sequence. PCR provides the possibility to obtain a large quantity of copies of a particular stretch of the genome, making further analysis possible. It is a highly sensitive and specific technology, which makes it suitable for all kinds of genetic diagnosis, including PGD. Currently, many different variations exist on the PCR itself, as well as on the different methods for the posterior analysis of the PCR products.
When using PCR in PGD, one is faced with a problem that is inexistent in routine genetic analysis: the minute amounts of available genomic DNA. As PGD is performed on single cells, PCR has to be adapted and pushed to its physical limits, and use the minimum amount of template possible: one strand. This implies a long process of fine-tuning of the PCR conditions and a susceptibility to all the problems of conventional PCR, but several degrees intensified. The high number of needed PCR cycles and the limited amount of template makes single-cell PCR very sensitive to contamination. Another problem specific to single-cell PCR is the allele drop out (ADO) phenomenon. It consists of the random non-amplification of one of the alleles present in a heterozygous sample. ADO seriously compromises the reliability of PGD as a heterozygous embryo could be diagnosed as affected or unaffected depending on which allele would fail to amplify. This is particularly concerning in PGD for autosomal dominant disorders, where ADO of the affected allele could lead to the transfer of an affected embryo.
Establishing a diagnosis
The establishment of a diagnosis in PGD is not always straightforward. The criteria used for choosing the embryos to be replaced after FISH or PCR results are not equal in all centres.In the case of FISH, in some centres only embryos are replaced that are found to be chromosomally normal (that is, showing two signals for the gonosomes and the analysed autosomes) after the analysis of one or two blastomeres, and when two blastomeres are analysed, the results should be concordant. Other centres argue that embryos diagnosed as monosomic could be transferred, because the false monosomy (i.e. loss of one FISH signal in a normal dipoloid cell) is the most frequently occurring misdiagnosis. In these cases, there is no risk for an aneuploid pregnancy, and normal diploid embryos are not lost for transfer because of a FISH error. Moreover, it has been shown that embryos diagnosed as monosomic on day 3 (except for chromosomes X and 21), never develop to blastocyst, which correlates with the fact that these monosomies are never observed in ongoing pregnancies.
Diagnosis and misdiagnosis in PGD using PCR have been mathematically modelled in the work of Navidi and Arnheim and of Lewis and collaborators. The most important conclusion of these publications is that for the efficient and accurate diagnosis of an embryo, two genotypes are required. This can be based on a linked marker and disease genotypes from a single cell or on marker/disease genotypes of two cells. An interesting aspect explored in these papers is the detailed study of all possible combinations of alleles that may appear in the PCR results for a particular embryo. The authors indicate that some of the genotypes that can be obtained during diagnosis may not be concordant with the expected pattern of linked marker genotypes, but are still providing sufficient confidence about the unaffected genotype of the embryo. Although these models are reassuring, they are based on a theoretical model, and generally the diagnosis is established on a more conservative basis, aiming to avoid the possibility of misdiagnosis. When unexpected alleles appear during the analysis of a cell, depending on the genotype observed, it is considered that either an abnormal cell has been analysed or that contamination has occurred, and that no diagnosis can be established. A case in which the abnormality of the analysed cell can be clearly identified is when, using a multiplex PCR for linked markers, only the alleles of one of the parents are found in the sample. In this case, the cell can be considered as carrying a monosomy for the chromosome on which the markers are located, or, possibly, as haploid. The appearance of a single allele that indicates an affected genotype is considered sufficient to diagnose the embryo as affected, and embryos that have been diagnosed with a complete unaffected genotype are preferred for replacement. Although this policy may lead to a lower number of unaffected embryos suitable for transfer, it is considered preferable to the possibility of a misdiagnosis.
Preimplantation genetic haplotyping
Preimplantation genetic haplotypingPreimplantation Genetic Haplotyping
Preimplantation Genetic Haplotyping is a clinical method of preimplantation genetic diagnosis . PGH was first developed in 2006 at London's Guy's Hospital and greatly advances PGD by using DNA fingerprinting rather than identifying the actual genetic signature .- Scope :Compared with previous PGD...
(PGH) is a new clinical method of Preimplantation genetic diagnosis (PGD). PGH was first developed in 2006 at London's Guy's Hospital and greatly advances PGD by using DNA fingerprinting rather than identifying the actual genetic signature (such as point mutations).
Embryo transfer and cryopreservation of surplus embryos
Embryo transferEmbryo transfer
Embryo transfer refers to a step in the process of assisted reproduction in which embryos are placed into the uterus of a female with the intent to establish a pregnancy...
is usually performed on day three or day five post-fertilization, the timing depending on the techniques used for PGD and the standard procedures of the IVF
In vitro fertilisation
In vitro fertilisation is a process by which egg cells are fertilised by sperm outside the body: in vitro. IVF is a major treatment in infertility when other methods of assisted reproductive technology have failed...
centre where it is performed.
With the introduction in Europe of the single-embryo transfer policy, which aims at the reduction of the incidence of multiple pregnancies after ART, usually one embryo or early blastocyst is replaced in the uterus. Serum hCG is determined at day 12. If a pregnancy is established, an ultrasound examination at 7 weeks is performed to confirm the presence of a fetal heartbeat. Couples are generally advised to undergo PND
Prenatal diagnosis
Prenatal diagnosis or prenatal screening is testing for diseases or conditions in a fetus or embryo before it is born. The aim is to detect birth defects such as neural tube defects, Down syndrome, chromosome abnormalities, genetic diseases and other conditions, such as spina bifida, cleft palate,...
because of the, albeit low, risk of misdiagnosis.
It is not unusual that after the PGD, there are more embryos suitable for transferring back to the woman than necessary. For the couples undergoing PGD, those embryos are very valuable, as the couple's current cycle may not lead to an ongoing pregnancy. Embryo cryopreservation
Embryo cryopreservation
Cryopreservation of embryos is the process of preserving an embryo at sub-zero temperatures, generally at an embryogenesis stage corresponding to pre-implantation, that is, from fertilisation to the blastocyst stage.-Indications:...
and later thawing and replacement can give them a second chance to pregnancy without having to redo the cumbersome and expensive ART and PGD procedures.
Ethical issues
PGD has raised ethical issues, although this approach could reduce reliance on fetal deselection during pregnancy. The technique can be used to determine the sex of the embryo, and thus can be used to select embryos of one sex in preference of the other in the context of “family balancingSex selection
Sex selection is the attempt to control the sex of the offspring to achieve a desired sex. It can be accomplished in several ways, both pre- and post-implantation of an embryo, as well as at birth...
”. It may be possible to make other "social selection" choices in the future that introduce socio-economic concerns.
Costs are substantial and insurance coverage may not be available.
PGD has the potential to screen for genetic issues unrelated to medical necessity
Medical necessity
Medical necessity is a United States legal doctrine, related to activities which may be justified as reasonable, necessary, and/or appropriate, based on evidence-based clinical standards of care. Other countries may have medical doctrines or legal rules covering broadly similar grounds...
. The prospect of a “designer baby
Designer baby
The colloquial term "designer baby" refers to a baby whose genetic makeup has been artificially selected by genetic engineering combined with in vitro fertilisation to ensure the presence or absence of particular genes or characteristics. The term is derived by comparison with "designer clothing"...
” is closely related to the PGD technique.
By relying on the result of one cell from the multi-cell embryo, PGD operates under the assumption that this cell is representative of the remainder of the embryo. This may not be the case as the incidence of mosaicism is often relatively high. On occasion, PGD may result in a false negative result leading to the acceptance of an abnormal embryo, or in a false positive result leading to the deselection of a normal embryo.
Non-disclosure
Another problematic case is the cases of desired non-disclosure of PGD results for some genetic disorders that may not yet be apparent in a parent, such as Huntington disease. It is applied when patients do not wish to know their carrier status but want to ensure that they have offspring free of the disease. This procedure can place practitioners in questionable ethical situations, e.g. when no healthy, unaffected embryos are available for transfer and a mock transfer has to be carried out so that the patient does not suspect that he/she is a carrier. The ESHREEuropean Society of Human Reproduction and Embryology
The European Society of Human Reproduction and Embryology was founded in 1985 by Robert Edwards and J. Cohen , who felt that the study and research in the field of reproduction needed to be encouraged and recognized.- Aims :...
ethics task force currently recommends using exclusion testing instead. Exclusion testing is based on a linkage analysis with polymorphic markers, in which the parental and grandparental origin of the chromosomes can be established. This way, only embryos are replaced that do not contain the chromosome derived from the affected grandparent, avoiding the need to detect the mutation itself.
Religious objections
Some religious organizations disapprove of this procedure. The Roman Catholic Church, for example, takes the position that it involves the destruction of human life. and besides that, opposes the necessary in vitro fertilization of eggs as contrary to Aristotelian principles of nature.Information on clinic websites
In a study of 135 IVF clinics, 88% had websites, 70% mentioned PGD and 27% of the latter were university- or hospital-based and 63% were private clinics. Sites mentioning PGD also mentioned uses and benefits of PGD far more than the associated risks. Of the sites mentioning PGD, 76% described testing for single-gene diseases, but only 35% mentioned risks of missing target diagnoses, and only 18% mentioned risks for loss of the embryo. 14% described PGD as new or controversial. Private clinics were more likely than other programs to list certain PGD risks like for example diagnostic error, or note that PGD was new or controversial, reference sources of PGD information, provide accuracy rates of genetic testing of embryos, and offer gender selection for social reasons.External links
- Preimplantation genetic diagnosis and sex selection- How does it work in the UK?
- In Focus "Preimplantation Genetic Diagnosis: scientific, legal and moral aspects" (German Reference Centre for Ethics in the Life Sciences )
- Fertility Trends Make Headline News from Coastal Fertility Medical Center
- Huntington's Disease and PGD
- List of diseases screened in the UK licensed by the HFEA
- Screening Embryos for Disease by Joe Palca @ NPR.org
- Preimplantation genetic diagnosis and sex selection
- Religious views on PGD
- List of some diseases that can be screened for - Australia
- Preimplantation Genetic Diagnosis images. Polar body and blastomere biopsy images. Normal and abnormal FISH images.
- Saving Henry by Laurie Strongin, a non-fiction account of Strongin's pioneering, yet failed attempts at using PGD to save the life of her son Henry
- HDBuzz article on PGD, prenatal diagnosis, exclusion testing and non-disclosure PGD for people at risk of Huntington's disease