Preimplantation genetic diagnosis (PGD) is the analysis of the genetic material of gametes or embryos before embryo implantation and the selection of embryos without genetic abnormalities for transfer. Preimplantation genetic screening (PGS) is the selection of chromosomally haploid embryos for transfer, thereby increasing the success rate of in vitro fertilization-embryo transfer (IVF-ET). The world’s first PGD baby was born in 1990, and by 2004, more than 1,000 normal PGD-diagnosed babies had been born, and by 2010, approximately 10,000 PGD- and PGS-diagnosed babies will be born worldwide.
1. Indications for PGD and PGS
(1) Indications for PGD
Monogenic genetic diseases and sex-linked genetic diseases: The first case of PGD in the world was diagnosed with X-linked recessive genetic diseases, and Handyside et al. selected female embryos for transfer by testing the sex of the embryos and obtained a successful pregnancy. Since then, PGD has diagnosed many genetic diseases, such as cystic fibrosis, thalassemia, spinal muscular atrophy, Huntington’s disease, etc. In 1999, the First Hospital of Sun Yat-sen University reported the first case of PGD in mainland China. monogenic genetic disease PGD is the genetic detection of disease-causing genes and the selection of embryos without genetic mutations for transfer to avoid offspring morbidity; and for sex-linked genetic diseases, it is also In the case of sex-linked genetic disorders, the possibility of avoiding the disease in the offspring can be achieved by identifying the sex of the embryo. However, in the case of X-linked recessive disorders, if male embryos are selected for transfer, although the offspring will not develop the disease, theoretically 50% of the male normal embryos will be discarded and 50% of the female carrier embryos will be retained.
(2) Chromosomal disorders: Chromosomal disorders can be divided into chromosomal number abnormalities and structural abnormalities: for example, 47,XXX (Creutzfeldt-Jakob disease) and Turner syndrome (45,XO) are chromosomal number abnormalities; while reciprocal translocations, Robertson translocations and chromosomal inversions are structural abnormalities. Reciprocal translocation refers to the rejoining of two chromosomes after breaking and exchanging each other without a mitotic break, with an incidence of about 1/6×106 in the population. robertsonian translocation breaks occur at two proximal mitotic chromosomes at the mitotic break, and after the break the mitotic grains of the two long-arm chromosomes fuse with each other to form a derived chromosome, with an incidence of about 1/1000. because there is no important genetic material missing, reciprocal translocation and robertsonian translocation Also known as balanced translocations, intelligence and phenotype are usually normal, but in reproduction, the production of unbalanced gametes can lead to reduced fertility, recurrent miscarriages, and malformed children. Fertility problems in this group can be solved by selecting normal or balanced embryos for transfer through PGD.
Fluorescence in situ hybridization (FISH) technology has been used within the field of PGD for chromosomal disorders for more than a decade. Although the number of diagnosed chromosome streaks is very limited by having to select probes and design experimental protocols according to different chromosomal disorders, FISH is easy to perform and has solved fertility problems for many couples with chromosomal disorders. In recent years, with the development of molecular biology technology, more and more PGDs for chromosomal disorders have adopted gene chips or second-generation sequencing technologies. These new technologies can detect not only the problematic chromosomes, but also all other chromosomes, providing more genetic information.
(3) Human leukocyte antigen (HLA) matching: For some families with children with hematologic disorders, PGD allows for the transfer of embryos with HLA selection of the same matching type as the affected child to save an existing child with a hematologic disorder. However, the PGD baby is born because it is a “life-saving baby”, while other embryos are discarded because they have no “life-saving function”, which is ethically controversial.
(4) Mitochondrial diseases: Approximately 15% of mitochondrial or oxidative phosphorylation diseases are caused by maternally inherited mitochondrial DNA (mtDNA) mutations. Therefore, the risk of disease in the offspring can be reduced by selecting embryos with mtDNA mutation rates below the pathogenesis threshold through PGD.
Indications for PGS: PGS is an embryo screening method aimed at improving pregnancy and live birth rates. PGS was first reported by Munné et al. in 1993 for embryos with abnormal chromosome numbers, and since then, the application has increased each year, with the European Collaboration on Human Reproduction and Embryology ( ESHRE) PGD reporting 3551 cycles of PGS in 2009.
The main indications for PGS include unexplained recurrent fertility failure, unexplained recurrent miscarriage, and advanced age of the female partner. Although the use of PGS is still controversial, most scholars advocate the abandonment of FISH for PGS, and many studies have shown that PGS can increase the rate of fertilization, decrease the rate of spontaneous abortion, reduce aneuploid pregnancies, and improve the success rate of assisted reproduction techniques; however, further confirmation is needed in multicenter studies with large samples.
2. Preimplantation diagnosis and screening sampling
It is an important step in PGD and PGS to ensure that the cells obtained are suitable for genetic diagnosis, but also to minimize the impact of sampling on embryo development.
Sources of material for PGD
(1) Polar bodies: Polar bodies are by-products of the oocyte and have less impact on embryo development. The polar bodies can provide a relatively long genetic diagnosis and facilitate the transfer of embryos in fresh cycles. However, the use of polar bodies can only infer genetic information from the mother.
(2) Schizonts: Single-cell genetic analysis using schizonts from oogenesis embryos allows simultaneous analysis of genetic information from both parents and has been the predominant source of sampling in previous years of PGD work. However, due to the limited availability of cells for diagnosis and the high percentage of chimerism in oogenesis embryos, the proportion of split-ball sampling is gradually decreasing.
(3) Blastocyst retrieval: The percentage of blastocyst stage chimerism is significantly lower than that of oogenesis embryos, and the trophectoderm retrieval of blastocyst not only increases the number of cells obtained and improves the accuracy of genetic diagnosis, but also causes relatively little damage to the embryo, and in recent years, more and more PGD and PGS cycles have adopted the blastocyst stage retrieval technique. Of course, the increasing maturity of blastocyst culture technology, vitrification freeze-thaw technology, and the development of laser instruments also provide the technical guarantee for the wide application of blastocyst retrieval.
PGD retrieval methods
Embryo retrieval is an important step in the PGD process. The success of retrieval directly affects the final diagnosis of PGD, and it is an invasive procedure for the embryos. The process of retrieval includes two processes: zona pellucida punching and cell retrieval. The hyaline zone perforation includes chemical, mechanical and laser methods. The chemical method is rarely used due to the potential damage of Tyrode’s acid to the embryo; the mechanical method is relatively less damaging to the embryo, but is technically difficult and takes a long time to perform in vitro, and is gradually replaced by the laser method.
The laser method of zona pellucida perforation refers to the local zona pellucida melting and volatilization by the laser energy generated by the laser generator, and the size of the zona pellucida perforation can be controlled by adjusting the laser energy, action time and number of pulses.
3.Genetic diagnosis technology
(1) Application of PCR technology in PGD: Polymerase chain reaction (PCR) uses a pair of oligonucleotide chains as primers to guide DNA polymerase to carry out DNA synthesis on two complementary chains between primer recognition sites, after three steps of template denaturation, annealing and extension for one cycle, the product of each cycle can be used as a template for the next cycle, multiple cycles can make specific DNA fragments in the number of The product of each cycle can be used as a template for the next cycle. The first PGD case in the world was performed by PCR.
Since the diagnostic material for PGD and PGS is one or a few cells with minimal DNA content, it is prone to amplification failure or amplification bias, which severely limits the development of PGD and PGS. Although many scholars have developed PCR-derived techniques, such as nested PCR, multiplex PCR, and fluorescent quantitative PCR, which have broadened the scope of PGD diagnosis to some extent, they are still very limited.
In recent years, whole ge?nome amplification (WGA), a technique for non-selective amplification of the entire genomic sequence, has been rapidly developed, leading the way in the field of PGD and PGS. WGA is a technique for non-selective amplification of the entire genomic sequence, which aims to substantially increase the total amount of DNA without sequence predisposition. The WGA techniques that are currently used in the field of PGD and PGS are: De Minimis Oligonucleotide Primer PCR (DOP-PCR), Pre-amplification Primer Extension Reaction PCR (PEP-PCR), Multiplexed Strand Displacement Amplification (MDA) and Multiple Annealing Loop Amplification (MALBAC).
Gene microarray detection or second-generation sequencing using WGA products is currently the most widely used method within the field of PGD and PGS, as will be described in detail later. The single-cell PCR technique is not only demanding on the laboratory, but also has some problems, such as amplification failure, contamination, and allelic decapping.
(2) Application of FISH technique in PGD and PGS: FISH is the in situ hybridization of specific DNA probes labeled with fluorochromes and specific chromosomal sequences to be measured in tissue cells under certain conditions, and the hybridization signal is displayed and observed under a fluorescent microscope for diagnosis. fISH is mainly applied in PGD for the diagnosis of sex-linked diseases and chromosomal diseases. Although the FISH technique is relatively simple and does not require DNA amplification, the number of chromosomes that can be detected by FISH is limited (up to 10~12), different probes need to be selected according to the chromosomes to be detected, and sometimes signal judgment is difficult. In recent years, FISH technology has been gradually replaced by gene chip technology and next generation sequencing (NGS) technology.
(3) Application of gene microarray technology in PGD and PGS: Gene microarray technology can detect polymorphisms or mutations in thousands of known genes simultaneously. Currently, the main clinical applications of gene chips include comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP). CGH and SNP techniques can detect more subtle chromosomal changes, such as >3Mbp repeats and deletions.
detected. The combination of single-cell whole-genome amplification and microarray technologies allows the detection of chromosomal conditions in single cells or microcells. Therefore, CGH and SNP microarrays have been widely used in the field of PGS and PGD of chromosomal diseases.
(4) Application of NGS technology in PGD and PGS: NGS is relative to traditional Sanger sequencing, which has changed the scale of sequencing, and its technical feature is that it no longer distinguishes a single template, but turns the template into a “library”, including all the templates that need to be sequenced, also based on the template The complementary strand is formed by sequence synthesis or hybridization, and each base is identified by a fluorescent marker introduced during the extension of the complementary strand. Since 2008, the cost of whole genome sequencing has declined exponentially, which is an important condition for NGS to be able to enter clinical applications for PGD and PGS.
Speaking of NGS, we also need to mention an important term: sequencing depth. Sequencing depth refers to the ratio of the total number of bases (bp) obtained from sequencing to the size of the genome, which is commonly understood as the number of times the genome has been sequenced, and is one of the indicators for evaluating the sequencing volume. There is a positive correlation between sequencing depth and genome coverage, and the error rate or false positive results from sequencing decreases as the sequencing depth increases.
NGS for PGD and PGS is also divided into 2 types: one is to perform WGA on the sampled cells first, followed by NGS, which is also the more common way. The other is that the sampled embryonic cells are not subjected to WGA, but rather a large number of specific fragments are amplified using mimic PCR techniques, and then these amplified fragments are subjected to NGS.
At the ESHRE annual meeting in 2013, Wells et al. reported a successful case of NGS-PGS pregnancy; in the same year, UW-Gene and Xiangya Hospital also reported a successful case of PGS with NGS detection of blastocysts. In 2013, Treff et al. performed PGD in 6 couples (2 with cystic fibrosis, 1 with Walker-Warburg syndrome, 1 with familial vegetative dysfunction, 1 with X-linked hypophosphatasia rickets, and 1 with neurofibroma). cases) were diagnosed with PGD, and genetic diagnostic techniques were used in other laboratories, qPCR and NGS techniques, respectively. The results showed that the diagnostic results of NGS were completely consistent with the other two methods. 2014 saw the birth of the world’s first and second MALBAC amplification sequenced PGD infant at the Reproductive Center of Peking University Hospital, with the diagnosis of hereditary multiple osteochondroma and X-linked hypohidrotic ectodermal dysplasia, respectively.
The application of NGS technology in the field of PGD and PGS is gradually developing, and the greatest advantage of NGS technology is also that it can detect not only aneuploidy in embryos, but also monogenic diseases. This is something that other technologies cannot reach yet.
4. Ethical considerations
Although PGD and PGS can avoid abortions or induced abortions associated with conventional prenatal diagnosis, there are still many issues that deserve attention in the actual clinical application of this technology. PGD and PGS are performed by invasive manipulation of embryos to obtain a final diagnosis, which requires long-term follow-up of a large sample of offspring. In addition, there are many controversies regarding the abandonment of embryos during PGD. For example, in sex screening for X-linked recessive disorders, half of the healthy male embryos will be discarded and half of the female carrier embryos will be retained; and for autosomal recessive disorders, it has been controversial whether heterozygous embryos will be transferred.
There has also been controversy about some indications for PGD, such as susceptibility analysis for some familial tumors (e.g., familial colon polyposis, breast cancer, etc.), HLA selection to save children with pre-existing blood disorders, etc.
It is important to note that genetic counseling prior to PGD is very important, and ESHRE analysis of the data they collected on PGD in 2009 showed that the average pregnancy rate for PGD was only 23.04%. Therefore, physicians should be objective in their delivery of the PGD process and results, rather than overstating its successful outcome, which will be more favored by patients. The diagnosis of PGD should not end with the transfer of the embryo into the mother, but should continue with the follow-up until the prenatal diagnosis and even for the later birth of the baby. follow-up would also significantly improve patient satisfaction.
In summary, single-cell genetic diagnostic techniques are changing rapidly and leading the field of PGD and PGS to advance rapidly. If the birth of Louis Brown in 1978 opened a new chapter in assisted reproduction, the rapid development of single-cell genetic diagnostics has brought new hope to many couples at high risk for genetic disorders. In recent years, both Europe and the United States have formulated technical guidelines or recommendations for PGD and PGS accordingly, and China’s Health and Family Planning Commission has also issued the Notice on the Pilot Clinical Application of High-Throughput Genetic Sequencing for Preimplantation Embryo Genetic Diagnosis in Assisted Reproduction Institutes, and relevant departments have also started to develop detailed technical specifications. With the continuous development and improvement of single-cell genetic diagnosis technology, it is believed that more and more couples with high risk of genetic diseases will benefit from it.