- Email: [email protected]
Preimplantation genetic diagnosis for monogenic diseases
Accepted Manuscript Preimplantation genetic diagnosis for monogenic diseases Vivian Chi Yan Lee, Judy FC. Chow, William Shu Biu Yeung, Pak Chung Ho
PII:
S1521-6934(17)30055-X
DOI:
10.1016/j.bpobgyn.2017.04.001
Reference:
YBEOG 1707
To appear in:
Best Practice & Research Clinical Obstetrics & Gynaecology
Received Date: 20 February 2017 Revised Date:
5 April 2017
Accepted Date: 7 April 2017
Please cite this article as: Lee VCY, Chow JF, Yeung WSB, Ho PC, Preimplantation genetic diagnosis for monogenic diseases, Best Practice & Research Clinical Obstetrics & Gynaecology (2017), doi: 10.1016/j.bpobgyn.2017.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Preimplantation genetic diagnosis for monogenic diseases Vivian Chi Yan Lee1*, Judy FC Chow2, William Shu Biu Yeung2, Pak Chung Ho3 Affiliations: 1Department
of Obstetrics & Gynaecology, Queen Mary Hospital, Hong Kong,
2Department
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Hong Kong SAR.
of Obstetrics and Gynaecology, the University of Hong Kong, Hong
Kong, Hong Kong SAR.
of Reproductive Medicine and Shenzhen Key Laboratory of Fertility
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3Centre
* corresponding author: Dr Vivian Lee
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Regulation, The Hong Kong University – Shenzhen Hospital, Shenzhen, China
Address: 5th floor, block K, Queen Mary Hospital, Pok Fu Lam, Hong Kong.
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Telephone: 825-22553111
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Email address: [email protected]
ACCEPTED MANUSCRIPT Abstract Preimplantation genetic diagnosis (PGD) was firstly report in 1990. Thereafter, more and more indications for PGD, including monogenic diseases (MGD) and translocations, are available nowadays and the list of indications of PGD is
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expanding from early onset and serious conditions to late onset diseases.
Polymerase chain reaction (PCR) has been used for PGD for MGD, while newer
techniques emerge, including karyomapping and next generation sequencing, in
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recent decade. Limitations of various methods for PGD were discussed in this
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review.
Keywords
Preimplantation genetic diagnosis (PGD); monogenic diseases; polymerase chain
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reaction (PCR); next generation sequencing (NGS).
ACCEPTED MANUSCRIPT Background
The first baby delivered after sex selection by genetic test on embryo for Xlinked diseases in a process known as preimplantation genetic diagnosis (PGD),
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was reported in 1990 (1). In the report, two pairs of twin baby girls without
severe X-linked diseases were born. Soon after, the first baby by PGD for another monogenic disease (MGD), cystic fibrosis, was born free from the parental
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mutant alleles (2). The aim of PGD is to provide an alternative reproductive option for couples at risks of giving birth to offspring with severe genetic
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diseases of childhood-onset, so as to avoid the physical and psychological trauma related to abortions when an affected pregnancy is diagnosed after spontaneous
Indications of PGD
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pregnancy.
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Potentially fatal genetic conditions are common indications for PGD (http://guide.hfea.gov.uk/pgd/). Cystic fibrosis is the most common potentially
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fatal autosomal recessive disease in the Caucasian population. The offspring of couples both carrying heterozygous pathogenic mutations would have a quarter (25%) chance of having this debilitating condition primarily affecting lung. The disease has no cure and requires multidisciplinary medical care. The affected individuals would have shortened life expectancy.. For example in the Chinese population, thalassaemia is the commonest autosomal recessive disease, especially in Southeastern part of China. The babies with major thalassaemia (i.e. homozygous for deletion of alpha-globin genes in alpha-thalassaemia or
ACCEPTED MANUSCRIPT heterozygous mutations in beta-globin genes in beta-thalassaemia) are either non-compatible with life, in haemoglobin Bart’s hydrops fetalis syndrome, or blood transfusion dependent from early infancy in Cooley’s anaemia. PGD is a reproductive option for couples carrying thalassaemia traits (3-5) In addition to
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childhood onset genetic diseases, the indications of PGD have been extended to adult-onset diseases (6). Huntington’s disease (HD) and spinocerebellar ataxia
are good examples. Patients with these mutations or trinucleotide repeats in the
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disease range will develop these debilitating diseases later in their lives, most
likely in adulthood with no cure. Mutations with incomplete penetrance are also
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indications for PGD. Mutations of tumour suppressor genes, BRCA-1 and 2 are good examples and both can cause cancer-predisposing syndrome (6). Patients with mutation of the BRCA1 or BRCA2 gene will experience substantially
origins.
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elevated risks of cancers of breast, ovarian, colonic, pancreatic and prostatic
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There are some ethical concerns regarding the use of PGD in these adult onset diseases or conditions with incomplete penetrance due to the ‘tradition’ to offer
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PGD to early onset conditions because their phenotypes show up early. In conditions with incomplete penetrance, there is still a possibility, though may be very slim, that the individuals carrying the mutations may not develop the genetic conditions. However, there is no consensus on the line drawn to demarcate what conditions are serious enough or what age onset is eligible for PGD. Individuals carrying the mutations suffer from psychological burden to various extent worrying about development of the full blown diseases later in their lives. One author concluded that it may be preferable to allow testing for
ACCEPTED MANUSCRIPT every detectable genetic condition according to the dictates of parental choice as the sole justification required for embryo selection rather than restricting PGD to serious conditions (7).
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The list of indications of PGD is expanding. The pace of discovery of novel rare disease causing genes by whole exome sequencing using next generation
sequencing (NGS) has rapidly increased in the past 3-5 years (8, 9). More and
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more genetic mutations for congenital syndromes or inherited diseases are
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found, and all these mutations can potentially become indications for PGD.
As said before, the aim of PGD is to avoid vertical transmission of the pathogenic gene mutation to next generation. In some occasions, the at-risk individuals may not be prepared to know whether they carry the pathogenic mutation or not and
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they are not ready to do the pre-symptomatic tests. Take HD as an example. The at-risk individuals may have strong family history of HD and understand clearly
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that there is no cure. This bad news may be too burdensome and they are not prepared to face it yet, but would like to avoid the possibilities of vertical
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transmission. PGD with exclusion test can be one option (10). Embryos with the at-risk haplotype would be discarded. This approach can avoid disclosure of the status of the at-risk individual against their wills and at the same time, prevents the vertical transmission to the next generation. However, using this approach, there are concerns of unnecessary invasive procedures for the couple, in case they do not carry the genetic mutations, and embryo wastage with disposal of the unaffected embryos. Therefore, the approach is not allowed even in some developed countries (11).
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Another indication of PGD is for tissue typing. For diseases, like betathalassaemia, cord blood or bone marrow transplantation is a curative treatment option for an affected child and alleviates the need for regular blood transfusion
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and the complications of iron overload. However, the possibility of finding an
unrelated donor compatible for bone marrow donation is slim. The purpose of performing PGD for beta-thalassaemia couples together with HLA typing is to
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select disease-free embryos with HLA that matches the affected child for the
transplantation. Successful implantation and birth of the transferred embryos
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would allow collection of HLA-matched cord blood stem cells for transplantation. These savior children can offer an opportunity to cure the affected siblings of the family and the family can have another healthy member without the severe diseases (12-15). However, this raises ethical concern (16) and the National
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Health Service (NHS) of England does not include the condition in their clinical
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commission policy (17).
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Diagnostic methods
The method of choice for diagnosis of MGD in PGD is usually polymerase chain reaction (PCR) (18). PCR can be tailor-made for particular mutations or changes in the deoxyribonucleic acid (DNA) sequences. However, PCR on single cells encounters a number of challenges. First, amplification efficiency of single cell samples is generally lower than that of PCR for genomic DNA in routine genetic testing (19). The lower amplification efficiency can be attributed to the collection of samples and the PCR procedure itself. The collection of biopsy samples is
ACCEPTED MANUSCRIPT operator-dependent and cell loss may occur during manipulation of single cells or tubing process. Spontaneous cell lysis, and biopsy of anucleated fragment or degenerated cells are other causes of reduced amplification efficiency. Use of different cell lysis protocol would pose some variation in the amplification
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efficiency (19).
The timing of biopsy also affects the amplification efficiency. In a polar body
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biopsy, collection of both the first and the second polar bodies is preferred, while in a cleavage-stage biopsy, collection of one cell instead of two cells is preferred
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because available evidences strongly suggest that the latter severely jeopardizes implantation rate and pregnancy rate (20, 21). Evidences also show that the removal of one blastomere from 6-8 cell cleavage-stage embryos (about 10-20% of cell mass) does not detrimentally affect the development of the biopsied
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embryos into good quality blastocysts (22-25). With the current techniques, results of the genetic tests can be available within 2 days. Therefore, the
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biopsied Day 3 embryos are usually cultured to Day 5 or 6 and the resulting good quality blastocysts with normal genetic results are replaced in the same cycles.
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For trophectoderm biopsy, 3 to 5 cells out of 100-150 cells in a blastocyst (about 2-5% of cell mass) (29) are collected, and the risk of amplification failure is lower than that with single cells (26-28). Other than amplification efficiency, the improved implantation rate after trophectoderm biopsy was clearly illustrated by a paired randomised trial in 2013. The sustained implantation rate was not affected by trophectoderm biopsy, while it was reduced by 39% after blastomere biopsy (30). Although the clinical outcome is better with trophectoderm biopsy, time is usually insufficient to complete the genetic test within the tight schedule
ACCEPTED MANUSCRIPT of a PGD cycle. Therefore, except in rare circumstances, the biopsied blastocysts require vitrification right after the biopsy, and the genetically normal blastocysts are cryopreserved to be replaced in subsequent frozen-thawed embryo transfer (FET) cycles. A good vitrification programme is a prerequisite for a successful
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PGD program using trophectoderm biopsy. The pregnancy rate is good after warming of the vitrified biopsied blastocysts. A pregnancy rate of 73% was
reported in one retrospective analysis (31) and many studies showed similar
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efficacy of vitrification for both non-biopsied or biopsied blastocysts (32, 33).
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Another challenge of single cell PCR is allele dropout (ADO) phenomenon, which can greatly increase the misdiagnosis rate (18). ADO occurs in around 10-20% of cases (34). When it occurs, only one of the two alleles is amplified by PCR. In PGD for autosomal recessive genetic diseases, ADO can cause problem in
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differentiating the carrier from the affected embryos. In autosomal dominant or X-linked genetic diseases, ADO can also cause misdiagnosis between normal and
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affected embryos. This is the reason why incorporation of linked markers is recommended to identify ADO. The linked markers could be either
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microsatellite markers or single nucleotide polymorphism (SNP) markers, and ideally, they should be intragenic (18). Using multiplex PCR, both the mutation and the polymorphic markers are amplified together to enhance the diagnostic accuracy.
Only a tiny amount of DNA (about 5-10 pg/ml) can be obtained from a single cell (29). A large number of amplification cycles are needed before a mutation can be detected. The amplification may also amplify contaminants in the samples
ACCEPTED MANUSCRIPT causing misdiagnosis. Contamination can be from genomic DNA of the technical operators or patients, either from cumulus cells or sperm sticking on the zona pellucida, or even from carry-over contaminations from PCR products amplified previously (35). Intra-cytoplasmic sperm injection (ICSI) can reduce the
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contamination from zona-bound sperm or from cumulus cells, and ICSI becomes one of the requirements for PGD using PCR nowadays. Strict aseptic protocols in PGD laboratory, separation of the pre- and post-PCR rooms and dedicated
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equipment and reagents for different procedures can further reduce the chances
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of contamination (19, 35).
There has been much advancement in recent decades for PGD, which includes use of comprehensive chromosomal screening by array comparative genomic hybridisation (aCGH), single nucleotide polyporphism array (SNP array) and
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next generation sequencing (NGS) for aneuploidy screening. aCGH was established as a commercially available platform. It involves a relatively
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straightforward and rapid protocol for PCR library-based whole-genome amplification, DNA labelling, hybridization and array scanning. These procedures
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can be completed within the tight schedule of PGD, and are used worldwide (36, 37). However, aCGH can only detect copy number variants including duplications or deletions, but cannot detect mutations of monogenic diseases. The use of SNP array is mainly limited in laboratories in USA, due to high cost of the required equipment, length and complexity of its protocol and need of extensive interpretation of the results. Compared with aCGH, SNP array has some advantages, like higher resolution with the average spacing of about 5kb and possibility of identification of origin of genetic aberrations according to the
ACCEPTED MANUSCRIPT genotype information of parents (37). The combined use of parental SNP genotypes and straightforward Mendelian genetic analysis enables the generation of a karyomap for each chromosome or chromosome segment inherited by each embryo for diagnosis of MGD or chromosomal abnormalities,
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which is the basic principle of PGD by karyomapping (38). Although
karyomaping is commercially available and theoretically applicable to all MGDs without need of prior optimization of the assay for the disease concerned, the
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cost of the platform is higher than other platforms and the requirement for
genetic information from an affected individual or embryos as reference make
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the platform not commonly used for PGD of MGD.
NGS is one kind of massively parallel sequencing, which greatly reduce the cost of sequencing of human genome (39). NGS-based preimplantation genetic
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aneuploidy testing has been validated in various centres (40-43). However, the resolution of the current NGS-based preimplantation genetic screening is not
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good enough for detection of genetic mutation in the majority of MGDs. There was one report using targeted NGS-based PGD for MGD (44). This method can
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offer diagnosis of both MGD and aneuploidy in one test and the cost calculated by the authors in their centres is comparable to other platforms used routinely. However, there is no other report following the publication, probably due to difficulty and cost in designing primer sequences for individual MGD and need of bioinformatics experts in interpretation of data for every single MGD. Further studies and modifications of the method are needed before its routine clinical application.
ACCEPTED MANUSCRIPT Research agendas 1.
Use of NGS for simultaneous diagnosis of MGD and aneuploidy screening;
2.
Direct comparison of different platforms of PGD for MGD, including PCR,
cost effectiveness.
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Results of PGD for monogenic diseases
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karyomapping and NGS is needed, especially on their diagnostic accuracy and
The aim of PGD is to avoid delivery of an affected baby with severe genetic
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diseases. Development of an affected pregnancy or delivery of an affected baby may not be necessarily arisen from misdiagnosis. Transfer of a wrong embryo due to human error is another possibility, which can be reduced by strict laboratory protocol (45, 46). There are other sources of errors in PGD, like
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sample mislabelling and misidentification, and unprotected sexual intercourse during treatment cycles, especially in vitrified-warmed embryo transfer cycles,
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producing in-vivo developed embryos that skip genetic test with unknown genotype (18, 47). As mentioned above, complete removal of all cumulus cells
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and use of ICSI can reduce the contamination from maternal and paternal cells (45). Adequate pre-PGD PCR validation and meticulous design of PCR primers, together with some physical strategies like separation of pre-, PCR, and post-PCR laboratories and biopsy laboratories can further improve the efficiency of PCR and reduce the chance of cross contamination and risks of human errors (18).
There were 12 adverse misdiagnoses reported in the ESHRE PGD consortium 2005, which probably under-estimated the actual misdiagnosis rate (48). One
ACCEPTED MANUSCRIPT paper looked deeply into the causes and correlations with the misdiagnosis of PGD cases by reanalyses of 940 untransferred embryos (49). 93.7% of embryos were correctly classified at the time of PGD, with a sensitivity of 99.2% and a specificity of 80.9%. The diagnostic accuracy was significantly higher with two-
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cells than one-cell biopsies, but was not affected by morphology of the embryos. However, the sensitivity was significantly higher if multiplex instead of
singleplex protocols were used and in embryos with good morphology than
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those with poor morphology. The use of multiplex protocol on one-cell is as
robust as those on two-cells regarding the false negative rate (49). Two-cells
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biopsy in cleavage-stage embryos is associated with adverse effect on both the implantation rate and the pregnancy rate (21).
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Long term effects of PGD
The long term effects of embryo biopsy on early human embryos have been
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studied. There were a few studies on babies born after PGD up to 2-years old on early development parameters like biometric and health outcome, mental and
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psychomotor developments. All showed outcomes similar to that of ICSI babies or naturally conceived babies and ICSI babies together (50, 51). The biggest studies on 581 children born after blastomere biopsy revealed similar birthweights and major malformation rate at birth or 2 months of age in comparison with ICSI babies. However, significantly more perinatal deaths were seen in postPGD/PGS multiple pregnancies than ICSI multiple pregnancies, while no such difference was observed in singleton pregnancies after PGD/PGS (52). Another follow-up study compared babies delivered either after single blastocyst transfer
ACCEPTED MANUSCRIPT after trophectoderm biopsy and aneuploidy screening with those after double blastocyst transfer of untested embryos, the results of which echoed with that of previous studies. The multiple pregnancy rate was significantly lower in the single embryo transfer group, 1.6% compared with 47%, while the adverse
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neonatal outcomes including preterm deliveries, low birthweight and neonatal intensive care unit admission were all significantly higher in the double
blastocyst transfer group (53). Therefore, papers published thereafter focused
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on singleton pregnancies after PGD (54, 55). The longer follow-up study on the children born after PGD up to 5 to 6 years of age showed comparable cognitive
Future developments
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and psychosocial development in children born after ICSI or conceived naturally.
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The success rate after PGD was reported to be similar to the conventional assisted reproductive technology, in-vitro fertilisation (IVF). According to the
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recent ESHRE PGD consortium data collection, the clinical pregnancy rate and
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delivery rate per oocyte retrieval was 28% and 24% after PCR for MGD (46).
In PGD cycles for MGD, embryo biopsy is necessary and the biopsied genetic materials can go for aneuploidy screening in the same setting. There are several studies showing beneficial effects of PGD with concurrent aneuploidy screening. In one study on MGD, about 50% of embryos diagnosed to be transferrable after PGD for MGD were aneuploid, which might result in implantation failure, miscarriages or abnormal pregnancies (56). Patients with concurrent preimplantation genetic screening (PGS) had higher live birth rate (59.4% vs
ACCEPTED MANUSCRIPT 37.5%) and half the abortion rate (40% vs 20%), though the difference did not reached statistical significance. Another study on the use of PGS in cases with or without human leukocyte antigen typing, the pregnancy rate was significantly improved from 45.4% to 68.4% and there was a 3-fold reduction of spontaneous
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abortion rate from 15% to 5.5% after PGS (57). Although these are all
retrospective studies, using the same biopsied genetic materials for PGD to do
on either the patients or the embryos.
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aneuploidy screening seems to be logical as it does not require extra procedures
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There are several limitations of aneuploidy screening that the couple must be counselled on before proceeding to PGD with PGS treatment cycles. One important message, which must be delivered clearly to the couple undergoing these treatments, is that the chance of not having any genetically transferrable
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embryos after both tests is high, and only 25.6% of embryos are transferrable (56). Another limitation is due to mosaicism of early human embryos.
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Chromosome mosaicism poses a great challenge on genetic counselling. There is no consensus on the outcome of transfer of mosaic embryos, with one
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prospective study showing 8 out of 18 of these mosaic embryos implanted resulted in 6 healthy livebirths and 2 miscarriages (58). The recommended strategy up-to-date is to prioritise euploid blastocysts to be transferred before these mosaic blastocysts (59). So far, there is no randomised trial looking at the use of PGS in PGD setting, which is urgently needed.
Another direction is to perform non-invasive PGD. One way to go is to use blastocoelic fluid, which is sampled before vitrification of blastocysts (60). This
ACCEPTED MANUSCRIPT technique was used in a few studies showing its efficacy in sex determination using NGS (61, 62). Apart from the blastocoelic fluid, spent medium after embryo culture is another source of embryonic DNA. The possibility has been shown by different investigators (63, 64). However, before routine use of these non-
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invasive techniques, there are still many problems that we need to tackle (65).
Currently, the origin of DNA in these non-invasive techniques has not been fully evaluated and there are several potential sources of contamination that may
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contribute to the genetic material detected in the culture medium. In the
majority of studies, there is no 100% guarantee of detection of DNA in the
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blastocoelic fluid or culture medium samples (65). Further studies must be performed before the routine use of these techniques.
Research agendas
Concurrent use of aneuploidy screening with PGD for MGD.
2.
Use of non-invasive PGD with blastocoelic fluid or culture medium.
3.
More studies on the long term effect of PGD should be performed.
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Conclusion
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PGD is an alternative to those couples with high risks of transmitting genetic mutations causing severe medical conditions for avoiding abortion of an affected pregnancy. The technique of PGD evolves quickly and new techniques hopefully can reduce the cost and improve the diagnostic accuracy of the test. Proper genetic counselling is needed before PGD.
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Conflict of interest
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All authors do not have any conflict of interest.
Practical points:
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1. The indications for PGD is still emerging, expanding from early onset severe life-threatening conditions to late onset conditions. 2. The diagnostic methods keep changing and evolving. Newer methods may provide shorter turnaround time frame and lower cost in the future.
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3. Strict laboratory protocol can reduce the chance of misdiagnosis.
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4. The use of preimplantation aneuploidy screening in PGD for monogenic diseases may improve the efficacy of the treatment with a reduction of miscarriage rate and chance of carrying aneuploid pregnancies.
ACCEPTED MANUSCRIPT Important reference 1, 7, 30, 58, 59 Reference:
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31. Schoolcraft WB, Treff NR, Stevens JM, Ferry K, Katz-Jaffe M, Scott RT. Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and singlenucleotide polymorphism microarray–based comprehensive chromosome screening in infertile patients. Fertility and sterility. 2011;96(3):638-40. 32. Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertility and sterility. 2012;98(5):1138-46. e1. 33. Capalbo A, Treff NR, Cimadomo D, Tao X, Upham K, Ubaldi FM, et al. Comparison of array comparative genomic hybridization and quantitative realtime PCR-based aneuploidy screening of blastocyst biopsies. European Journal of Human Genetics. 2015;23(7):901-6. 34. Gil LAL, Lucena C, Lucena E. Methods in preimplantation genetic diagnosis. Reproductive BioMedicine Online. 2001;2(1):20-31. 35. Sermon K. Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist’s view. Human Reproduction Update. 2002;8(1):11-20. 36. Fiorentino F, Spizzichino L, Bono S, Biricik A, Kokkali G, Rienzi L, et al. PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization. Human Reproduction. 2011;26(7):1925-35. 37. Handyside AH. PGD and aneuploidy screening for 24 chromosomes by genome-wide SNP analysis: seeing the wood and the trees. Reproductive biomedicine online. 2011;23(6):686-91. 38. Handyside AH, Harton GL, Mariani B, Thornhill AR, Affara N, Shaw M-A, et al. Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes. Journal of medical genetics. 2010;47:651-8. 39. Martín J, Cervero A, Mir P, Martinez JAC, Pellicer A, Simón C. The impact of next-generation sequencing technology on preimplantation genetic diagnosis and screening. Fertility and sterility. 2013;99(4):1054-61. e3. 40. Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, Cottone G, et al. Development and validation of a next-generation sequencing–based protocol for 24-chromosome aneuploidy screening of embryos. Fertility and sterility. 2014;101(5):1375-82. e2. 41. Fiorentino F, Bono S, Biricik A, Nuccitelli A, Cotroneo E, Cottone G, et al. Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles. Human Reproduction. 2014;29(12):2802-13. 42. Kung A, Munné S, Bankowski B, Coates A, Wells D. Validation of nextgeneration sequencing for comprehensive chromosome screening of embryos. Reproductive biomedicine online. 2015;31(6):760-9. 43. Zheng H, Jin H, Liu L, Liu J, Wang W-H. Application of next-generation sequencing for 24-chromosome aneuploidy screening of human preimplantation embryos. Molecular cytogenetics. 2015;8:38(1-9). 44. Treff NR, Fedick A, Tao X, Devkota B, Taylor D, Scott Jr RT. Evaluation of targeted next-generation sequencing–based preimplantation genetic diagnosis of monogenic disease. Fertility and sterility. 2013;99(5):1377-84. e6. 45. Wilton L, Thornhill A, Traeger-Synodinos J, Sermon KD, Harper JC. The causes of misdiagnosis and adverse outcomes in PGD. Human reproduction. 2009;24(5):1221-8.
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46. De Rycke M, Belva F, Goossens V, Moutou C, SenGupta SB, TraegerSynodinos J, et al. ESHRE PGD Consortium data collection XIII: cycles from January to December 2010 with pregnancy follow-up to October 2011. Human Reproduction. 2015;30(8):1763-89. 47. Bettio D, Capalbo A, Albani E, Rienzi L, Achille V, Venci A, et al. 45,X product of conception after preimplantation genetic diagnosis and euploid embryo transfer: evidence of a spontaneous conception confirmed by DNA fingerprinting. Reproductive biology and endocrinology : RB&E. 2016;14(1):55. 48. Harper J, Wilton L, Traeger-Synodinos J, Goossens V, Moutou C, SenGupta S, et al. The ESHRE PGD Consortium: 10 years of data collection. Human reproduction update. 2012;18(3):234-47. 49. Dreesen J, Destouni A, Kourlaba G, Degn B, Mette WC, Carvalho F, et al. Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study. European Journal of Human Genetics. 2014;22(8):1012-8. 50. Nekkebroeck J, Bonduelle M, Desmyttere S, Van den Broeck W, PonjaertKristoffersen I. Mental and psychomotor development of 2-year-old children born after preimplantation genetic diagnosis/screening. Human Reproduction. 2008;23(7):1560-6. 51. Desmyttere S, Bonduelle M, Nekkebroeck J, Roelants M, Liebaers I, De Schepper J. Growth and health outcome of 102 2-year-old children conceived after preimplantation genetic diagnosis or screening. Early human development. 2009;85(12):755-9. 52. Liebaers I, Desmyttere S, Verpoest W, De Rycke M, Staessen C, Sermon K, et al. Report on a consecutive series of 581 children born after blastomere biopsy for preimplantation genetic diagnosis. Hum Reprod. 2010;25(1):275-82. 53. Forman EJ, Hong KH, Franasiak JM, Scott RT. Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates. American journal of obstetrics and gynecology. 2014;210(2):157. e1-. e6. 54. Winter C, Van Acker F, Bonduelle M, Desmyttere S, De Schrijver F, Nekkebroeck J. Cognitive and psychomotor development of 5-to 6-year-old singletons born after PGD: a prospective case–controlled matched study. Human Reproduction. 2014;29(9):1968-77. 55. Winter C, Van Acker F, Bonduelle M, Desmyttere S, Nekkebroeck J. Psychosocial development of full term singletons, born after preimplantation genetic diagnosis (PGD) at preschool age and family functioning: a prospective case-controlled study and multi-informant approach. Human Reproduction. 2015;30(5):1122-36. 56. Goldman KN, Nazem T, Berkeley A, Palter S, Grifo JA. Preimplantation Genetic Diagnosis (PGD) for Monogenic Disorders: the Value of Concurrent Aneuploidy Screening. Journal of Genetic Counseling. 2016;25:1327-37. 57. Rechitsky S, Pakhalchuk T, San Ramos G, Goodman A, Zlatopolsky Z, Kuliev A. First systematic experience of preimplantation genetic diagnosis for single-gene disorders, and/or preimplantation human leukocyte antigen typing, combined with 24-chromosome aneuploidy testing. Fertility and sterility. 2015;103(2):503-12.
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58. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. New England Journal of Medicine. 2015;373(21):2089-90. 59. http://www.pgdis.org/docs/newsletter_071816.html 60. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid. Reproductive biomedicine online. 2013;26(6):603-10. 61. Galluzzi L, Palini S, Stefani SD, Andreoni F, Primiterra M, Diotallevi A, et al. Extracellular embryo genomic DNA and its potential for genotyping applications. Future Science OA. 2015;1(4). 62. Herrera C, Morikawa MI, Castex CB, Pinto M, Ortega N, Fanti T, et al. 317 sex determination of equine embryos by PCR using blastocoele fluid. Reproduction, Fertility and Development. 2015;27(1):247-8. 63. Liu W, Liu J, Du H, Ling J, Sun X, Chen D. Non-Invasive Pre-Implantation Aneuploidy Screening and Diagnosis of Beta Thalassemia IVSII654 Mutation using Spent Embryo Culture Medium. Annals of Medicine. 2016:110,DOI:.1080/07853890.2016.1254816. 64. Xu J, Fang R, Chen L, Chen D, Xiao J-P, Yang W, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proceedings of the National Academy of Sciences. 2016;113(42):11907-12. 65. Hammond ER, Shelling AN, Cree LM. Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use. Human Reproduction. 2016;31(8):1653-61.
ACCEPTED MANUSCRIPT Highlights
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Various indications for PGD are available. The indications for PGD have expanded from early onset serious diseases to the more controversial late onset diseases. Limitations of PGD should be addressed properly so as to reduce the chance of misdiagnosis. Technical errors can be reduced by strict laboratory strategies.
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PII:
S1521-6934(17)30055-X
DOI:
10.1016/j.bpobgyn.2017.04.001
Reference:
YBEOG 1707
To appear in:
Best Practice & Research Clinical Obstetrics & Gynaecology
Received Date: 20 February 2017 Revised Date:
5 April 2017
Accepted Date: 7 April 2017
Please cite this article as: Lee VCY, Chow JF, Yeung WSB, Ho PC, Preimplantation genetic diagnosis for monogenic diseases, Best Practice & Research Clinical Obstetrics & Gynaecology (2017), doi: 10.1016/j.bpobgyn.2017.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preimplantation genetic diagnosis for monogenic diseases Vivian Chi Yan Lee1*, Judy FC Chow2, William Shu Biu Yeung2, Pak Chung Ho3 Affiliations: 1Department
of Obstetrics & Gynaecology, Queen Mary Hospital, Hong Kong,
2Department
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Hong Kong SAR.
of Obstetrics and Gynaecology, the University of Hong Kong, Hong
Kong, Hong Kong SAR.
of Reproductive Medicine and Shenzhen Key Laboratory of Fertility
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3Centre
* corresponding author: Dr Vivian Lee
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Regulation, The Hong Kong University – Shenzhen Hospital, Shenzhen, China
Address: 5th floor, block K, Queen Mary Hospital, Pok Fu Lam, Hong Kong.
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Telephone: 825-22553111
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Email address: [email protected]
ACCEPTED MANUSCRIPT Abstract Preimplantation genetic diagnosis (PGD) was firstly report in 1990. Thereafter, more and more indications for PGD, including monogenic diseases (MGD) and translocations, are available nowadays and the list of indications of PGD is
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expanding from early onset and serious conditions to late onset diseases.
Polymerase chain reaction (PCR) has been used for PGD for MGD, while newer
techniques emerge, including karyomapping and next generation sequencing, in
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recent decade. Limitations of various methods for PGD were discussed in this
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review.
Keywords
Preimplantation genetic diagnosis (PGD); monogenic diseases; polymerase chain
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reaction (PCR); next generation sequencing (NGS).
ACCEPTED MANUSCRIPT Background
The first baby delivered after sex selection by genetic test on embryo for Xlinked diseases in a process known as preimplantation genetic diagnosis (PGD),
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was reported in 1990 (1). In the report, two pairs of twin baby girls without
severe X-linked diseases were born. Soon after, the first baby by PGD for another monogenic disease (MGD), cystic fibrosis, was born free from the parental
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mutant alleles (2). The aim of PGD is to provide an alternative reproductive option for couples at risks of giving birth to offspring with severe genetic
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diseases of childhood-onset, so as to avoid the physical and psychological trauma related to abortions when an affected pregnancy is diagnosed after spontaneous
Indications of PGD
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pregnancy.
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Potentially fatal genetic conditions are common indications for PGD (http://guide.hfea.gov.uk/pgd/). Cystic fibrosis is the most common potentially
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fatal autosomal recessive disease in the Caucasian population. The offspring of couples both carrying heterozygous pathogenic mutations would have a quarter (25%) chance of having this debilitating condition primarily affecting lung. The disease has no cure and requires multidisciplinary medical care. The affected individuals would have shortened life expectancy.. For example in the Chinese population, thalassaemia is the commonest autosomal recessive disease, especially in Southeastern part of China. The babies with major thalassaemia (i.e. homozygous for deletion of alpha-globin genes in alpha-thalassaemia or
ACCEPTED MANUSCRIPT heterozygous mutations in beta-globin genes in beta-thalassaemia) are either non-compatible with life, in haemoglobin Bart’s hydrops fetalis syndrome, or blood transfusion dependent from early infancy in Cooley’s anaemia. PGD is a reproductive option for couples carrying thalassaemia traits (3-5) In addition to
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childhood onset genetic diseases, the indications of PGD have been extended to adult-onset diseases (6). Huntington’s disease (HD) and spinocerebellar ataxia
are good examples. Patients with these mutations or trinucleotide repeats in the
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disease range will develop these debilitating diseases later in their lives, most
likely in adulthood with no cure. Mutations with incomplete penetrance are also
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indications for PGD. Mutations of tumour suppressor genes, BRCA-1 and 2 are good examples and both can cause cancer-predisposing syndrome (6). Patients with mutation of the BRCA1 or BRCA2 gene will experience substantially
origins.
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elevated risks of cancers of breast, ovarian, colonic, pancreatic and prostatic
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There are some ethical concerns regarding the use of PGD in these adult onset diseases or conditions with incomplete penetrance due to the ‘tradition’ to offer
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PGD to early onset conditions because their phenotypes show up early. In conditions with incomplete penetrance, there is still a possibility, though may be very slim, that the individuals carrying the mutations may not develop the genetic conditions. However, there is no consensus on the line drawn to demarcate what conditions are serious enough or what age onset is eligible for PGD. Individuals carrying the mutations suffer from psychological burden to various extent worrying about development of the full blown diseases later in their lives. One author concluded that it may be preferable to allow testing for
ACCEPTED MANUSCRIPT every detectable genetic condition according to the dictates of parental choice as the sole justification required for embryo selection rather than restricting PGD to serious conditions (7).
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The list of indications of PGD is expanding. The pace of discovery of novel rare disease causing genes by whole exome sequencing using next generation
sequencing (NGS) has rapidly increased in the past 3-5 years (8, 9). More and
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more genetic mutations for congenital syndromes or inherited diseases are
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found, and all these mutations can potentially become indications for PGD.
As said before, the aim of PGD is to avoid vertical transmission of the pathogenic gene mutation to next generation. In some occasions, the at-risk individuals may not be prepared to know whether they carry the pathogenic mutation or not and
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they are not ready to do the pre-symptomatic tests. Take HD as an example. The at-risk individuals may have strong family history of HD and understand clearly
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that there is no cure. This bad news may be too burdensome and they are not prepared to face it yet, but would like to avoid the possibilities of vertical
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transmission. PGD with exclusion test can be one option (10). Embryos with the at-risk haplotype would be discarded. This approach can avoid disclosure of the status of the at-risk individual against their wills and at the same time, prevents the vertical transmission to the next generation. However, using this approach, there are concerns of unnecessary invasive procedures for the couple, in case they do not carry the genetic mutations, and embryo wastage with disposal of the unaffected embryos. Therefore, the approach is not allowed even in some developed countries (11).
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Another indication of PGD is for tissue typing. For diseases, like betathalassaemia, cord blood or bone marrow transplantation is a curative treatment option for an affected child and alleviates the need for regular blood transfusion
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and the complications of iron overload. However, the possibility of finding an
unrelated donor compatible for bone marrow donation is slim. The purpose of performing PGD for beta-thalassaemia couples together with HLA typing is to
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select disease-free embryos with HLA that matches the affected child for the
transplantation. Successful implantation and birth of the transferred embryos
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would allow collection of HLA-matched cord blood stem cells for transplantation. These savior children can offer an opportunity to cure the affected siblings of the family and the family can have another healthy member without the severe diseases (12-15). However, this raises ethical concern (16) and the National
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Health Service (NHS) of England does not include the condition in their clinical
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commission policy (17).
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Diagnostic methods
The method of choice for diagnosis of MGD in PGD is usually polymerase chain reaction (PCR) (18). PCR can be tailor-made for particular mutations or changes in the deoxyribonucleic acid (DNA) sequences. However, PCR on single cells encounters a number of challenges. First, amplification efficiency of single cell samples is generally lower than that of PCR for genomic DNA in routine genetic testing (19). The lower amplification efficiency can be attributed to the collection of samples and the PCR procedure itself. The collection of biopsy samples is
ACCEPTED MANUSCRIPT operator-dependent and cell loss may occur during manipulation of single cells or tubing process. Spontaneous cell lysis, and biopsy of anucleated fragment or degenerated cells are other causes of reduced amplification efficiency. Use of different cell lysis protocol would pose some variation in the amplification
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efficiency (19).
The timing of biopsy also affects the amplification efficiency. In a polar body
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biopsy, collection of both the first and the second polar bodies is preferred, while in a cleavage-stage biopsy, collection of one cell instead of two cells is preferred
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because available evidences strongly suggest that the latter severely jeopardizes implantation rate and pregnancy rate (20, 21). Evidences also show that the removal of one blastomere from 6-8 cell cleavage-stage embryos (about 10-20% of cell mass) does not detrimentally affect the development of the biopsied
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embryos into good quality blastocysts (22-25). With the current techniques, results of the genetic tests can be available within 2 days. Therefore, the
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biopsied Day 3 embryos are usually cultured to Day 5 or 6 and the resulting good quality blastocysts with normal genetic results are replaced in the same cycles.
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For trophectoderm biopsy, 3 to 5 cells out of 100-150 cells in a blastocyst (about 2-5% of cell mass) (29) are collected, and the risk of amplification failure is lower than that with single cells (26-28). Other than amplification efficiency, the improved implantation rate after trophectoderm biopsy was clearly illustrated by a paired randomised trial in 2013. The sustained implantation rate was not affected by trophectoderm biopsy, while it was reduced by 39% after blastomere biopsy (30). Although the clinical outcome is better with trophectoderm biopsy, time is usually insufficient to complete the genetic test within the tight schedule
ACCEPTED MANUSCRIPT of a PGD cycle. Therefore, except in rare circumstances, the biopsied blastocysts require vitrification right after the biopsy, and the genetically normal blastocysts are cryopreserved to be replaced in subsequent frozen-thawed embryo transfer (FET) cycles. A good vitrification programme is a prerequisite for a successful
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PGD program using trophectoderm biopsy. The pregnancy rate is good after warming of the vitrified biopsied blastocysts. A pregnancy rate of 73% was
reported in one retrospective analysis (31) and many studies showed similar
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efficacy of vitrification for both non-biopsied or biopsied blastocysts (32, 33).
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Another challenge of single cell PCR is allele dropout (ADO) phenomenon, which can greatly increase the misdiagnosis rate (18). ADO occurs in around 10-20% of cases (34). When it occurs, only one of the two alleles is amplified by PCR. In PGD for autosomal recessive genetic diseases, ADO can cause problem in
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differentiating the carrier from the affected embryos. In autosomal dominant or X-linked genetic diseases, ADO can also cause misdiagnosis between normal and
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affected embryos. This is the reason why incorporation of linked markers is recommended to identify ADO. The linked markers could be either
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microsatellite markers or single nucleotide polymorphism (SNP) markers, and ideally, they should be intragenic (18). Using multiplex PCR, both the mutation and the polymorphic markers are amplified together to enhance the diagnostic accuracy.
Only a tiny amount of DNA (about 5-10 pg/ml) can be obtained from a single cell (29). A large number of amplification cycles are needed before a mutation can be detected. The amplification may also amplify contaminants in the samples
ACCEPTED MANUSCRIPT causing misdiagnosis. Contamination can be from genomic DNA of the technical operators or patients, either from cumulus cells or sperm sticking on the zona pellucida, or even from carry-over contaminations from PCR products amplified previously (35). Intra-cytoplasmic sperm injection (ICSI) can reduce the
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contamination from zona-bound sperm or from cumulus cells, and ICSI becomes one of the requirements for PGD using PCR nowadays. Strict aseptic protocols in PGD laboratory, separation of the pre- and post-PCR rooms and dedicated
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equipment and reagents for different procedures can further reduce the chances
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of contamination (19, 35).
There has been much advancement in recent decades for PGD, which includes use of comprehensive chromosomal screening by array comparative genomic hybridisation (aCGH), single nucleotide polyporphism array (SNP array) and
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next generation sequencing (NGS) for aneuploidy screening. aCGH was established as a commercially available platform. It involves a relatively
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straightforward and rapid protocol for PCR library-based whole-genome amplification, DNA labelling, hybridization and array scanning. These procedures
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can be completed within the tight schedule of PGD, and are used worldwide (36, 37). However, aCGH can only detect copy number variants including duplications or deletions, but cannot detect mutations of monogenic diseases. The use of SNP array is mainly limited in laboratories in USA, due to high cost of the required equipment, length and complexity of its protocol and need of extensive interpretation of the results. Compared with aCGH, SNP array has some advantages, like higher resolution with the average spacing of about 5kb and possibility of identification of origin of genetic aberrations according to the
ACCEPTED MANUSCRIPT genotype information of parents (37). The combined use of parental SNP genotypes and straightforward Mendelian genetic analysis enables the generation of a karyomap for each chromosome or chromosome segment inherited by each embryo for diagnosis of MGD or chromosomal abnormalities,
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which is the basic principle of PGD by karyomapping (38). Although
karyomaping is commercially available and theoretically applicable to all MGDs without need of prior optimization of the assay for the disease concerned, the
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cost of the platform is higher than other platforms and the requirement for
genetic information from an affected individual or embryos as reference make
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the platform not commonly used for PGD of MGD.
NGS is one kind of massively parallel sequencing, which greatly reduce the cost of sequencing of human genome (39). NGS-based preimplantation genetic
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aneuploidy testing has been validated in various centres (40-43). However, the resolution of the current NGS-based preimplantation genetic screening is not
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good enough for detection of genetic mutation in the majority of MGDs. There was one report using targeted NGS-based PGD for MGD (44). This method can
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offer diagnosis of both MGD and aneuploidy in one test and the cost calculated by the authors in their centres is comparable to other platforms used routinely. However, there is no other report following the publication, probably due to difficulty and cost in designing primer sequences for individual MGD and need of bioinformatics experts in interpretation of data for every single MGD. Further studies and modifications of the method are needed before its routine clinical application.
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Use of NGS for simultaneous diagnosis of MGD and aneuploidy screening;
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Direct comparison of different platforms of PGD for MGD, including PCR,
cost effectiveness.
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Results of PGD for monogenic diseases
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karyomapping and NGS is needed, especially on their diagnostic accuracy and
The aim of PGD is to avoid delivery of an affected baby with severe genetic
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diseases. Development of an affected pregnancy or delivery of an affected baby may not be necessarily arisen from misdiagnosis. Transfer of a wrong embryo due to human error is another possibility, which can be reduced by strict laboratory protocol (45, 46). There are other sources of errors in PGD, like
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sample mislabelling and misidentification, and unprotected sexual intercourse during treatment cycles, especially in vitrified-warmed embryo transfer cycles,
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producing in-vivo developed embryos that skip genetic test with unknown genotype (18, 47). As mentioned above, complete removal of all cumulus cells
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and use of ICSI can reduce the contamination from maternal and paternal cells (45). Adequate pre-PGD PCR validation and meticulous design of PCR primers, together with some physical strategies like separation of pre-, PCR, and post-PCR laboratories and biopsy laboratories can further improve the efficiency of PCR and reduce the chance of cross contamination and risks of human errors (18).
There were 12 adverse misdiagnoses reported in the ESHRE PGD consortium 2005, which probably under-estimated the actual misdiagnosis rate (48). One
ACCEPTED MANUSCRIPT paper looked deeply into the causes and correlations with the misdiagnosis of PGD cases by reanalyses of 940 untransferred embryos (49). 93.7% of embryos were correctly classified at the time of PGD, with a sensitivity of 99.2% and a specificity of 80.9%. The diagnostic accuracy was significantly higher with two-
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cells than one-cell biopsies, but was not affected by morphology of the embryos. However, the sensitivity was significantly higher if multiplex instead of
singleplex protocols were used and in embryos with good morphology than
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those with poor morphology. The use of multiplex protocol on one-cell is as
robust as those on two-cells regarding the false negative rate (49). Two-cells
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biopsy in cleavage-stage embryos is associated with adverse effect on both the implantation rate and the pregnancy rate (21).
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Long term effects of PGD
The long term effects of embryo biopsy on early human embryos have been
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studied. There were a few studies on babies born after PGD up to 2-years old on early development parameters like biometric and health outcome, mental and
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psychomotor developments. All showed outcomes similar to that of ICSI babies or naturally conceived babies and ICSI babies together (50, 51). The biggest studies on 581 children born after blastomere biopsy revealed similar birthweights and major malformation rate at birth or 2 months of age in comparison with ICSI babies. However, significantly more perinatal deaths were seen in postPGD/PGS multiple pregnancies than ICSI multiple pregnancies, while no such difference was observed in singleton pregnancies after PGD/PGS (52). Another follow-up study compared babies delivered either after single blastocyst transfer
ACCEPTED MANUSCRIPT after trophectoderm biopsy and aneuploidy screening with those after double blastocyst transfer of untested embryos, the results of which echoed with that of previous studies. The multiple pregnancy rate was significantly lower in the single embryo transfer group, 1.6% compared with 47%, while the adverse
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neonatal outcomes including preterm deliveries, low birthweight and neonatal intensive care unit admission were all significantly higher in the double
blastocyst transfer group (53). Therefore, papers published thereafter focused
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on singleton pregnancies after PGD (54, 55). The longer follow-up study on the children born after PGD up to 5 to 6 years of age showed comparable cognitive
Future developments
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and psychosocial development in children born after ICSI or conceived naturally.
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The success rate after PGD was reported to be similar to the conventional assisted reproductive technology, in-vitro fertilisation (IVF). According to the
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recent ESHRE PGD consortium data collection, the clinical pregnancy rate and
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delivery rate per oocyte retrieval was 28% and 24% after PCR for MGD (46).
In PGD cycles for MGD, embryo biopsy is necessary and the biopsied genetic materials can go for aneuploidy screening in the same setting. There are several studies showing beneficial effects of PGD with concurrent aneuploidy screening. In one study on MGD, about 50% of embryos diagnosed to be transferrable after PGD for MGD were aneuploid, which might result in implantation failure, miscarriages or abnormal pregnancies (56). Patients with concurrent preimplantation genetic screening (PGS) had higher live birth rate (59.4% vs
ACCEPTED MANUSCRIPT 37.5%) and half the abortion rate (40% vs 20%), though the difference did not reached statistical significance. Another study on the use of PGS in cases with or without human leukocyte antigen typing, the pregnancy rate was significantly improved from 45.4% to 68.4% and there was a 3-fold reduction of spontaneous
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abortion rate from 15% to 5.5% after PGS (57). Although these are all
retrospective studies, using the same biopsied genetic materials for PGD to do
on either the patients or the embryos.
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aneuploidy screening seems to be logical as it does not require extra procedures
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There are several limitations of aneuploidy screening that the couple must be counselled on before proceeding to PGD with PGS treatment cycles. One important message, which must be delivered clearly to the couple undergoing these treatments, is that the chance of not having any genetically transferrable
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embryos after both tests is high, and only 25.6% of embryos are transferrable (56). Another limitation is due to mosaicism of early human embryos.
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Chromosome mosaicism poses a great challenge on genetic counselling. There is no consensus on the outcome of transfer of mosaic embryos, with one
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prospective study showing 8 out of 18 of these mosaic embryos implanted resulted in 6 healthy livebirths and 2 miscarriages (58). The recommended strategy up-to-date is to prioritise euploid blastocysts to be transferred before these mosaic blastocysts (59). So far, there is no randomised trial looking at the use of PGS in PGD setting, which is urgently needed.
Another direction is to perform non-invasive PGD. One way to go is to use blastocoelic fluid, which is sampled before vitrification of blastocysts (60). This
ACCEPTED MANUSCRIPT technique was used in a few studies showing its efficacy in sex determination using NGS (61, 62). Apart from the blastocoelic fluid, spent medium after embryo culture is another source of embryonic DNA. The possibility has been shown by different investigators (63, 64). However, before routine use of these non-
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invasive techniques, there are still many problems that we need to tackle (65).
Currently, the origin of DNA in these non-invasive techniques has not been fully evaluated and there are several potential sources of contamination that may
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contribute to the genetic material detected in the culture medium. In the
majority of studies, there is no 100% guarantee of detection of DNA in the
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blastocoelic fluid or culture medium samples (65). Further studies must be performed before the routine use of these techniques.
Research agendas
Concurrent use of aneuploidy screening with PGD for MGD.
2.
Use of non-invasive PGD with blastocoelic fluid or culture medium.
3.
More studies on the long term effect of PGD should be performed.
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Conclusion
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PGD is an alternative to those couples with high risks of transmitting genetic mutations causing severe medical conditions for avoiding abortion of an affected pregnancy. The technique of PGD evolves quickly and new techniques hopefully can reduce the cost and improve the diagnostic accuracy of the test. Proper genetic counselling is needed before PGD.
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Conflict of interest
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All authors do not have any conflict of interest.
Practical points:
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1. The indications for PGD is still emerging, expanding from early onset severe life-threatening conditions to late onset conditions. 2. The diagnostic methods keep changing and evolving. Newer methods may provide shorter turnaround time frame and lower cost in the future.
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3. Strict laboratory protocol can reduce the chance of misdiagnosis.
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4. The use of preimplantation aneuploidy screening in PGD for monogenic diseases may improve the efficacy of the treatment with a reduction of miscarriage rate and chance of carrying aneuploid pregnancies.
ACCEPTED MANUSCRIPT Important reference 1, 7, 30, 58, 59 Reference:
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ACCEPTED MANUSCRIPT Highlights
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Various indications for PGD are available. The indications for PGD have expanded from early onset serious diseases to the more controversial late onset diseases. Limitations of PGD should be addressed properly so as to reduce the chance of misdiagnosis. Technical errors can be reduced by strict laboratory strategies.
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