Introduction The development and enhancement of in vitro fertilization IVF over the last 35 years has resulted in dramatic improvements in the treatment of infertility.
At present, IVF provides both the most successful and often the most cost effective approach to the care of most infertile couples [1][2].
In the short time since the first IVF birth in [3], our greater understanding of embryo development had allowed for the development of new technologies which can be implemented to enhance embryo selection. The primary goal is to distinguishing those embryos that are reproductively competent and are capable of producing a healthy child from those that cannot. The drive to select healthy embryos and avoid failed pregnancy attempts, miscarriages, and the need for pregnancy termination led to the first applications of preimplantation genetic diagnosis PGD in [4].
The concept of PGD is not a new one. John Rock predicted that human IVF, gender selection, and gestational carriers would be utilized in reproductive science [5]. He stated that one day science will allow for parents to obtain sons or daughters "according to specification", foreshadowing the ability to screen out detrimental disease states with PGD.
The first description of PGD came years later published by the great Dr. Robert Edwards and Dr. Richard Gardner in a Nature manuscript. In it he described the use of PGD for sexing of rabbit blastocysts [6].
Further work in animal models continued, including Marilyn Monk's work in demonstrating PGD in a murine model for Lesch-Nyan syndrome [7]. The development and implementation of several techniques in human embryology were instrumental in the application of PGD in human reproduction. First, Leeanda Wilton pioneered the cleavage stage biopsy in Then, in , two additional approaches to obtaining genetic material from embryos were described with Yuri Verlinsky describing polar body biopsy and Audrey Muggleton-Harris describing trophectoderm biopsy.
The first applications for PGD came in testing monogenic disorders and sex-linked disorders. This was made possible by Elana Kontogianni's work in which showed PCR for the Y chromosome was possible from a blastomere. Focusing on X-chromosome linked diseases, amplification and detection of Y-chromosome specific repeat sequences allowed for selection of embryos that were female and thus not at risk of carrying the disease.
These early approaches gave way to technologies that allowed for the detection of gene mutations on autosomes and sex chromosomes enabling clinicians to select embryos that do not harbor the mutation for embryo transfer. The success of PGD to predict embryos which did not have genetic disease led to attempts to apply the technology more widely as a selective tool to all embryos in a particular cohort and identify those embryos with normal chromosome complements and thus a higher chance of success on a per cycle basis [8—16].
This practice became known as preimplantation genetic screening PGS. A prominent goal in this case was to decrease the reliance on high embryo transfer order to achieve high success rates. This is an important given that the practice vastly increases the prevalence of multiple gestations, which are associated with high maternal and neonatal morbidity as compared to singletons [12].
The contribution of embryonic aneuploidy to the inefficiency of human reproduction is well established [17—19] and it seemed intuitive that assessment of the ploidy status of each embryo within the developing cohort would allow selection of only euploid embryos and would ultimately improve IVF outcomes [20]. While this premise was always valid, early attempts at embryonic aneuploidy screening were suboptimal [21,22]. The early techniques entailed molecular analysis that lacked sufficient precision to be clinically meaningful.
More recently, application of newer and more powerful molecular technologies have overcome some of the early limits and produced meaningful improvements in clinical outcomes.
Applications for Genetic Testing of Embryos The impetus to develop PGD in the clinical realm was to identify only unaffected children prior to implantation and thus eliminate the need for pregnancy termination after a diagnosis was made at a later time in the pregnancy. Mastenbroek's group, in , published a paper of a multicentric, randomized, controlled trial RCT where they compared three cycles of IVF with and without Preimplantation Genetic Screening PGS in women in the age group of 35—41 years.
The concept of better pregnancy outcome using this technology finally picked up when Munne and other scientists demonstrated its benefits. New terminology was developed to differentiate between aneuploidy screening and detection of monogenic disorders.
Considering the difficulties in the use of FISH technology, several groups perfected the long learning curve for PGT which included perfecting embryo biopsy techniques without harming the embryo and genetic diagnosis using different molecular techniques. Different groups studied the effect of day 3 cleavage-stage biopsy and day 5 blastocyst biopsy on embryo implantation and live birth outcomes.
New methods were introduced for the detection of aneuploidy of all chromosomes within 24—48 hours. In , Scott's group published their clinical trial showing that the biopsy of cleavage-stage embryos significantly impaired implantation potential; however, trophectoderm biopsy of blastocyst did not have any negative effect on implantation [ Figure 4 ]. Dahdouh et al. They concluded that PGS with the use of CCS technology increases clinical and sustained IRs, thus improving embryo selection particularly in patients with normal ovarian reserve [ Figure 6 ].
Day 3 versus day 5 biopsy. Implantation rates following a randomized paired analysis of the effects of cleavage-stage and blastocyst-stage biopsies on embryo reproductive potential.
A similar paired analysis demonstrated that the developmental potential of embryos undergoing trophectoderm biopsy at the blastocyst stage was equivalent to the nonbiopsied control sibling. Day 5, day 6, and day 7 biopsies should be included for preimplantation genetic testing analysis. Meta-analysis of randomized controlled trials on preimplantation genetic screening with comprehensive chromosome screening versus routine care. Forman et al. Overall implantation rate increases in comprehensive chromosome screening with eSET cases independent of age.
The current indications for PGT include repeated implantation failures, repeated pregnancy loss, advanced maternal and paternal age, male factor infertility, and genetic disorders in the parents including mosaicism of sex chromosomes, structural rearrangements, and monogenic genetic diseases. Scott et al. Chromosomal aneuploidies are one of the major causes of infertility and maternal age-related reduced fertility potential.
Euploid embryo transfer results in highest pregnancy rates and live birth rates reducing miscarriage risk independent of maternal age.
However, PGT has its limitations under certain circumstances. Furthermore, experienced laboratory personnel in IVF and genetics are important for a patient's success. One of the new modalities for enhancing success is the evaluation of the mitochondrial DNA mtDNA content of the embryo.
Victor et al. Embryonic chromosomal mosaicism is a condition in which more than one cell line is present, where one has a normal chromosomal constituent and others have abnormalities in chromosome number. It is assumed that mosaicism has adverse effects to the implantation and development of the embryo.
Kushnir et al. However, there was no significant difference in the on-going pregnancy rates or miscarriage rates among mosaic embryo transfers at any threshold of aneuploidy, and the degree of trophectoderm mosaicism was a poor predictor of on-going pregnancy and miscarriage.
In , Alan Handyside with his group described the concept of karyomapping. It is genome-wide parental haplotyping using high-density SNP genotyping.
Here, a linkage-based diagnosis is carried out for any single-gene defect. By knowing the genotyping of the parents and a close relative of known disease status, generally a previously affected child, this technology eliminates the need for customized test development.
Karyomapping identifies informative loci for each of the four parental haplotypes across each chromosome and maps the inheritance of these haplotypes and the position of any crossovers in the proband as well as in the preimplantation embryos.
Thus, it identifies the embryo-carrying normal chromosome copies. As embryo biopsy is an invasive procedure, efforts are being made to find different embryonic samples which do not require embryo biopsy.
One of the novel approaches is the use of noninvasive PGS. Palini et al. Our team reported the first live births in India for a Robertsonian[ 62 ] and reciprocal translocation,[ 63 ] inversion with a cryptic translocation picked up on pre-PGT-A workup[ 64 ] and pregnancy after PGT for a complex translocation.
We have also successfully carried out PGT-M for the first time, in India, for genetic disorders such as Duchenne muscular dystrophy, neurofibromatosis, sickle cell anemia, Leigh syndrome, retinoblastoma, hereditary inclusion body myopathy, cardiac disorders, and carriers of BRCA1.
We also have several pregnancies in couples carrying mutations for beta thalassemia. From the above review, we can conclude that PGT is a major diagnostic tool to prevent transmission of any known genetic disorder.
It also helps in populations which are at high risk of having babies with certain genetic aberrations. PGT reduces the trauma of multiple failed IVF cycles, early miscarriages, and helps in cases of advanced maternal age to prevent the birth of a syndromic child. PGT-M protects the child from inherited monogenic disorders. With the concept of savior sibling, PGT-M is useful in some of the hematological disorders to cure an affected child.
However, this technology should be used judiciously, and its pitfalls should be understood. National Center for Biotechnology Information , U. J Hum Reprod Sci. Author information Copyright and License information Disclaimer. Address for correspondence: Dr. Deshmukh Marg, Mumbai - , Maharashtra, India. E-mail: moc. This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.
This article has been cited by other articles in PMC. Abstract Preimplantation genetic testing PGT is an early form of prenatal genetic diagnosis where abnormal embryos are identified, thereby allowing transfer of genetically normal embryos.
K EYWORDS: Assisted reproductive technology , preimplantation genetic diagnosis , preimplantation genetic screening , preimplantation genetic testing , fluorescence in situ hybridization , array comparative genomic hybridization , next-generation sequencing. Open in a separate window. Figure 1. Figure 2. Figure 3. In spite of these pioneering attempts, why did PGD not become popular? Figure 4. Figure 5. Figure 6. Figure 7. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
Heape W. Preliminary note on the transplantation and growth of mammalian ova within a uterine foster-mother. Sexing of live rabbit blastocysts. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Birth after the reimplantation of a human embryo.
Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Penetration of the zona-free hamster egg by human sperm. Fertil Steril. The conclusion was that 24 chromosome screening after trophectoderm biopsy and eSET produced as high a pregnancy rate as the transfer of two untested embryos without encountering the increased risk for twin gestations.
Structural chromosome rearrangements include reciprocal and Robertsonian translocations and inversions. Individuals who carry balanced chromosome translocations or inversions generally have no clinical findings related to the translocation, but will produce high rates of abnormal gametes after meiotic segregation.
The involved chromosomes will orient at the metaphase plate in a quadrivalent pattern and will segregate to the two daughter cells in one of 30 or so segregation patterns. Scriven and colleagues [ ] published a method for the evaluation of segregation in translocations involving FISH using commercially available specific centromeric and sub-telomeric probes. Although this technique could identify embryos that were balanced for the involved chromosomes, it was subject to the same technical limitations described above for aneuploidy testing utilizing FISH.
In addition, no information was provided for chromosomes not involved in the translocation, which could produce aneuploidy and decreased reproductive potential. Fiorentino et al. This was applied in 27 PGD cycles where 18 couples achieved a clinical pregnancy. Shortly thereafter, mCGH or aCGH was applied after WGA, and this analysis allowed not only aneuploidy evaluation of the translocated chromosomes, but also detected age-related aneuploidy in other chromosomes not involved in the translocation.
In the report of Alfarawati et al. Treff et al. Of normally developing blastocysts, Overall, including arrested embryos, Less than one quarter of the embryos produced in these cycles therefore were truly euploid and capable of producing a healthy pregnancy.
The reproductive outcome in couples carrying a translocation is likely dependent on the shape of the quadrivalent and subsequent modes of segregation of the specific translocation and the risk of producing a viable abnormal gestation. Scriven et al. Rapid advances in DNA sequencing technology have made it possible to generate very large amounts of sequence data with the use of high-throughput NGS and bioinformatics tools. On the flow cell surface, hundreds of thousands of the fragments are sequenced in parallel reactions involving the successive addition of fluorescent nucleotides and ultra-high resolution imaging of the successively added base.
The results are compared to a reference genome using a bioinformatics algorithm, and the protocol is repeated until a sufficient read depth is obtained by the sequencing of other fragments from the same genomic region.
The use of barcodes allows multiple samples from different analyses to be sequenced simultaneously in the same flow cell, allowing efficiency of cost.
For PGD of single gene disorders using NGS, sequencing of the region containing the mutation until sufficient read depth is accomplished to be confident that the base calling is straightforward.
Methods are now being developed to enrich WGA samples for specific genomic regions, where single gene mutations are present to increase the read depth of these sequences [ ]. Treff and colleagues [ ] evaluated NGS-based PGD for single gene disorders in six couples at-risk of transmitting either autosomal recessive disease Walker Warburg syndrome, cystic fibrosis, familial dysautonomia , dominant disease neurofibromatosis 1 or X-linked hypophosphatemic rickets to their children.
Aneuploidy testing was performed by qPCR. The genetic disease results were compared with Taqman allelic discrimination assays or PGD results from an outside reference laboratory. In all cases, the NGS results were perfectly consistent with those of the other two methodologies. To determine chromosome copy number by NGS, the shotgun sequencing-chromosome mapping protocol originally developed by Fan et al.
In this protocol, WGA embryo biopsy material is randomly fragmented, and the sequencing of 33—36 base pairs is carried out to allow the mapping of the fragment to the chromosome of origin. The number of fragments that map to a particular chromosome should be proportional to the copy number of that chromosome, with trisomic or monosomic chromosomes having more or less fragments, respectively.
Yin and colleagues [ ] studied trophectoderm biopsy samples from 38 donated blastocysts from 16 IVF cycles by both NGS and SNP microarray, with qPCR being used to define any inconsistencies between the two protocols.
High throughput sequencing was performed using an Illumina HiSeq sequencer. An average of 9. Results showed that 26 of the embryos The euploid embryos were correctly identified by NGS and SNP array, and consistent abnormalities were identified in six uniformly aneuploid embryos. This group Li et al. Uniformly euploid embryos were identified in Combining both aneuploidy testing and genetic disease diagnosis, Wells and colleagues [ ] have reported the births of children to couples at-risk for cystic fibrosis or mitochondrial DNA defects after PGD.
NGS using the Ion Torrent platform was performed on trophectoderm biopsy samples after MDA WGA to diagnose chromosome aneuploidy, as well as direct sequencing of the mutations present in the family. It was noted that the high throughput of the NGS system allowed simultaneous genetic analysis of up to embryos, which could significantly reduce the cost of PGD to two-thirds of the current cost using aCGH.
In addition, the pre-workup for single gene disorder PGD would no longer be needed, adding additional savings. A very significant question to be answered at this time is what will PGD look like in five to 10 years?
As molecular technologies continue to evolve and allow us to accumulate huge amounts of sequence data, we need to decide what will be the role of PGD in the future.
It is not clear, however, that universal PGD makes sense in terms of the extra cost for patients and the small number of qualified laboratories currently able to perform the procedure. It has also been suggested that additional genetic screening be added to current PGD aneuploidy testing. In this regard, we will probably follow the experience of classical prenatal diagnosis, which is now, and will be in the future, most frequently performed by cell-free testing of fetal DNA in the maternal plasma.
It would be possible to do multiplex enrichment for these genomic abnormalities, which could be detected by NGS sequencing, likely in a protocol with embryo vitrification, data analysis and subsequent frozen embryo transfer. The use of PGD for severe genetic disorders has always been non-controversial, and chromosome aneuploidy testing is only currently beginning to be more widely accepted as data accumulates from RCTs showing improved pregnancy and delivery rates.
Other uses of PGD have been viewed more skeptically, such as HLA matching, adult onset genetic disorders, conditions with variable penetrance and non-medical sex selection. As our understanding of genomic information improves, new prenatal tests will be developed, which are likely to be DNA sequence-based, and many could be adapted for prenatal or preimplantation diagnosis. It is not clear, however, whether PGD is the appropriate clinical setting to introduce new genetic testing strategies that have not yet been validated in the prenatal diagnostic clinic.
While the use of PGD to prevent the transmission of known serious genetic disease in a family is considered appropriate, the situation becomes less clear as more extensive genetic screening of embryos by technologies, such as whole exome or whole genome screening, become possible in the context of PGD. Questions arise regarding the moral obligation of the parents to perform extensive genetic testing on their embryos, simply because it is possible, as well as the obligation of health professionals to provide such testing.
Preliminary discussions regarding the future use of PGD and an ethical framework for its application have been carried out with panels of experts in the field [ , , ].
In addition, extensive genomic sequencing will undoubtedly reveal unintended findings that may be clinically significant, or alternatively, variants of unknown clinical significance may be identified. This underscores the need for carefully obtaining the consent of couples for PGD along with both pre- and post-test genetic counseling to make sure the results are correctly interpreted and clearly explained. PGD was initially performed nearly 25 years ago as an alternative for the prenatal diagnosis of single gene disorders in ongoing intrauterine gestations with the potential interruption of affected pregnancies.
In the second phase of PGD development, cycles were performed for the detection of aneuploidy to improve the outcome of IVF in patients with translocations, advanced reproductive age, recurrent IVF failure or recurrent pregnancy loss. While there has been the general acceptance of PGD for genetic disorders, there has been skepticism of the benefit of PGD for chromosome aneuploidy, specifically the lack in multiple RCTs of a statistically significant improvement in the live birth rate with PGD, as compared to IVF without testing.
This has also been achieved with a change in the tissue biopsied from Day 3 blastomeres to Day 5 trophectoderm or polar bodies, which causes less harm to the embryo and, in the case of trophectoderm biopsy, provides a multi-cellular sample, which can mitigate the mosaicism common in cleavage stage embryos, as well as lower the risk of ADO.
Initial reports have shown improved implantation and live birth rates with lower miscarriage rates in IVF when chromosomal PGD is performed. The identification and transfer of euploid embryos appears to blunt the effect of advancing maternal age and is the ideal method to select embryos for use in eSET to reduce the incidence of multiple gestations with IVF.
With the increasing use of NGS in PGD, both single gene disorder and chromosomal testing can be performed simultaneously on the same sequencing platform without the need for the pre-test workup of single gene disorders.
In the near future, NGS is likely to be used to identify embryos with microdeletion syndromes or common pathologic copy number variations. New developments in sequencing technology and bioinformatics will likely allow even more sequence information to be rapidly generated from embryos.
The objectives of such testing and the role that PGD should play in IVF will need to be further defined and validated, or the technology could be used for the identification of non-medical traits, such as stature, memory, hair and eye color or athletic ability. National Center for Biotechnology Information , U. Journal List J Clin Med v. J Clin Med. Published online Mar Harvey J. Stern 1, 2. Author information Article notes Copyright and License information Disclaimer.
This article has been cited by other articles in PMC. Abstract Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos.
Keywords: preimplantation genetic diagnosis, chromosomal microarray, embryo biopsy, next generation sequencing, inherited genetic disorders. Introduction Preimplantation genetic diagnosis PGD is a form of prenatal diagnosis that is performed on early embryos created by in vitro fertilization IVF.
Open in a separate window. Table 2 Results of a survey of in vitro fertilization IVF centers regarding the indications for which PGD was offered in their clinic. Tissue Biopsy for PGD Decisions regarding the optimal time to perform PGD biopsy and testing involve careful consideration of multiple factors, including: 1 When in embryologic development is the abnormality first identifiable? Polar Body Biopsy The first and second polar bodies are by-products of the meiosis of the egg, and their removal is believed to be less harmful to the embryo than either blastomere or trophectoderm biopsy, which, by their nature, reduce the embryonic cell number [ 23 ].
Blastocyst Trophectoderm Biopsy The human blastocyst contains approximately cells distributed between the inner cell mass, which will develop into the fetus proper, and the surrounding trophectoderm cells, which will become the placenta and fetal membranes. Figure 1. Age-Related Chromosome Aneuploidy The most common use of PGD involves the analysis of embryos for chromosome aneuploidy arising from either meiotic mal-segregation in oocytes or less commonly sperm or mitotic abnormalities of embryos after syngamy, which results in mosaicism; defined as the presence of two or more cytogenetically defined cell lines in the same embryo.
Figure 2. Structural Chromosome Rearrangements Structural chromosome rearrangements include reciprocal and Robertsonian translocations and inversions. Conclusions PGD was initially performed nearly 25 years ago as an alternative for the prenatal diagnosis of single gene disorders in ongoing intrauterine gestations with the potential interruption of affected pregnancies. Conflicts of Interest The author declares no conflicts of interest.
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