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Year : 2019  |  Volume : 3  |  Issue : 3  |  Page : 129-132

Application and challenge of preimplantation genetic testing in reproductive medicine

Department of Reproductive Medicine, Institute of Embryo-Fetal Original Adult Disease, School of Medicine, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University, Shanghai 200030, China

Date of Submission22-Aug-2019
Date of Web Publication27-Sep-2019

Correspondence Address:
He-Feng Huang
School of Medicine, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University, Shanghai 200030
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.268163

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How to cite this article:
Liu XL, Xu CM, Huang HF. Application and challenge of preimplantation genetic testing in reproductive medicine. Reprod Dev Med 2019;3:129-32

How to cite this URL:
Liu XL, Xu CM, Huang HF. Application and challenge of preimplantation genetic testing in reproductive medicine. Reprod Dev Med [serial online] 2019 [cited 2020 Aug 13];3:129-32. Available from: http://www.repdevmed.org/text.asp?2019/3/3/129/268163

Prof. He-Feng Huang, Doctoral Supervisor and Chief Physician, is the chair professor of Shanghai Jiao Tong University, a distinguished professor of the Zhejiang University, an academician of the Chinese Academy of Sciences, an academician of the Third World Academy of Sciences, and an honorary academician of the Royal College of Obstetricians and Gynaecologists. She is currently the dean of the International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, China; the dean of the Embryonic Disease Research Institute; the dean of Shanghai Key Laboratory of Embryonic Disease; and the vice-president of Savaid Medical School, University of Chinese Academy of Sciences. Prof. Huang has also undertaken the “863” and “the 12th five-year plan” and the National Key Research and Development Plan. She serves on the editorial board of several SCI journals, such as Endocrinology.

There has been a very rapid development in the area of preimplantation genetic testing (PGT) since the first baby applied PGT technology in the world was born in the last 30 years. As an alternative treatment of prenatal diagnosis, the transfer of the fetus with normal testing result has benefited many families. PGT is mainly classified into PGT for aneuploidies (PGT-A), PGT for chromosomal structural rearrangement (PGT-SR), and PGT for monogenic/single-gene defects (PGT-M). Here, we reviewed the application, development, limitations, and challenges of the current PGT in reproductive medicine.

  Application and Challenge in Biopsy Top

Embryo biopsy is a critical step in the assessment of the obtained genetic material during PGT, and its influences on the development potential of the fetus and health of the offspring have always attracted attention. The time and manner in which to perform the biopsy remain controversial. According to the sampling time, the biopsy is classified into polar body biopsy (PBB) of the oocytes, blastomere biopsy (BB) of the embryo in the cleavage stage, and trophectoderm biopsy (TB) in the blastocyst stage. PBB involves minimal damage to the embryo and has obtained ethical clearance. Sequential biopsy of the first and the second polar bodies can predict maternal chromosomal euploidy and the site of disease-related, single-nucleotide polymorphism (SNP). As only maternal genetic information can be detected, up to 40% of all chromosomal abnormalities are missed when PBB is used to screen aneuploidy.[1] BB was once widely applied in the clinical setting because it eliminated the need to wait for the embryo to reach the blastocyst stage. However, disadvantages include severe damage to the embryo, few cells available for detection, low accuracy, high mosaicism rate, and wide existence of chromosome instability in the embryo during the cleavage stage. A multicentric large-cohort study conducted at the International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, China, has reported that [2] loss of blastomere in the frozen-thawed, cleavage-stage embryos will lead to reduced implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate. In addition, there is a difference in the biopsy results of the same embryo between the cleavage stage and the blastocyst stage.[1] The possible explanations are laboratory error or mechanism for aneuploid cells to self-correct or self-remove during embryonic development. If embryonic self-correction is a common phenomenon during early embryonic development, biopsy before the self-correction will result in wastage of embryos. In the early 2000, preimplantation genetic screening (PGS) 1.0 based on fluorescence in situ hybridization (FISH) and BB, now called PGT-A 1.0, was implemented, until Mastenbroek et al. indicated that the pregnancy rate could decrease adversely, leading to the overturn of effectiveness of PGS.[3] The possible interpretations of its ineffectiveness includes excessive indications, the ability to detect limited number of chromosomes, mosaic status of embryos and insufficient experience of technicians.[4] After the introduction of comprehensive chromosome screening and TB, PGS 2.0 was started to be reused in clinical practice. Currently, TB has gradually replaced BB. Biopsy of the trophoblast cells results in limited damage to the embryos, and multiple cells can be detected each time with more reliable diagnosis. However, mosaic embryos also ensue. The mosaicism rate of the TB samples is 20%–40%, and most embryos have euploid inner cell mass (ICM). Although the mosaicism rate detected using TB is relatively lower, whether the karyotype of trophoblast cells could reflect the extent of karyotype of ICM has not been confirmed. It is noteworthy that some noninvasive detection methods provide a new direction for embryo detection. Magli et al.[5] found that DNA in the blastocoelic fluid is highly fragmented, which is presumed as the apoptotic products of aneuploid cells. It proves the existence of self-correction in early embryo and its scavenging process of abnormal cells. Thus, scientists believe that the embryonic quality can be evaluated based on the results of blastocoelic fluid amplification. Above all, the potential damage caused by the performance of embryonic biopsy on fetus needs further exploration. The optimal biopsy method should be selected as per the disease type (chromosomal or genetic diseases) and the molecular mechanism of the disease (meiosis or mitosis). Meanwhile, a noninvasive detection technique is an important development tendency for PGT.

  Application and Challenge in the Use of Detection and Analysis Techniques Top

After PGS 1.0, PGT-A detection high throughput technologies, for example, next-generation sequencing (NGS) and array CGH (aCGH). NGS is widely applied in PGT-SR for patients with chromosome inversion, balanced translocation, unbalanced translocation, or Robertsonian translocation. NGS can detect microcopy number variation and mosaic. Detection of mosaic by using high-resolution NGS (hr-NGS) of Illumina VeriSeq PGS as a representative is more sensitive and accurate than that using aCGH. The detection rate of mosaic is 20%–80% among the embryos; however, it is still difficult to differentiate between low proportional mosaic and background noise. No uniform standard for the evaluation of TB mosaicism is available. A very strict scoring standard may classify the mosaic embryo with development potential as an abnormal embryo, leading to wastage. Otherwise, it will result in the transplantation of abnormal mosaic embryos, giving adverse reproductive outcomes.

Allele dropout (ADO), preferential amplification of allele, failure in amplification, and DNA contamination are important factors that influence the accurate diagnosis of PGT-M. ADO indicates the loss of one of the two alleles during PCR amplification. When the pathogenicity genes (especially complex hybrid disease genes) are directly sequenced, the occurrence of high-frequency ADO considerably influences the diagnosis. In particular, during the PGT-M process, because of the limited cell number of biopsies and low DNA concentration, the whole genetic amplification (WGA) greatly increases the possibility of ADO occurrence. Although WGA based on multiple displacement amplification possesses high fidelity and amplification efficiency, the occurrence of ADO is inevitable. Thus, the establishment of a detection method that can discover potential ADO is an important way to avoid PGT-M misdiagnosis.

Currently, whether the embryo carries pathogenic mutations is indirectly detected by pathogenic genes and bilateral interlocking polymorphic markers simultaneously, such as SNP or short tandem repeat (STR), namely preimplantation genetic haplotyping (PGH), which reduces the possibility of misdiagnosis caused by high ADO rate. The involved STR or SNP site in the detection is often designed for different monogenic diseases or families. Thus, the detection is of high cost and time-consuming. Although the development of new generation of sequencing technique reduces the difficulty and cost of seeking the SNP site, experienced technicians are still needed to perform artificial screening and provide informative SNP. Moreover, the current strategy is targeted capture sequencing, rather than SNP haplotyping based on the whole genome; therefore, the quantity of detectable monogenic disease remains limited.

A recently developed Karyomapping technique is the preimplantation detection method based on linkage analysis for a wide range of monogenic diseases. Compared with direct mutation detection and targeted haplotype analysis, Karyomapping has many advantages, such as easy operation, good flexibility, and accurate results. No corresponding detection protocol is required for specific patients or diseases.[6] In addition, the SNP site identified using Karyomapping detection covers the whole genome, and chromosome mutation detection can be conducted simultaneously, including aneuploid and large-fragment chromosome deletions. However, conditions such as no proband, new mutation, genital system mosaicism, or lack of critical disease family member will influence the construction of the haplotype. If PGT-M is performed at this time, the parental haplotype information should be obtained via haplotype detection in the sperm and/or PPB under the precondition of confirmed pathogenic mutations. When the husband and wife are close relatives, the homology of the bilateral genetic markers of the pathogenic gene is high, affecting the screening of specific DNA markers and limiting the application of the PGH technique.

Confirmation of the pathogenic gene and pathogenicity of the genetic variant is a precondition for PGT-M application. With the popularization of the gene detection technique, more gene mutation sites are being detected. The pathogenicity analysis of the site becomes an important part of genetic counseling before PGT-M detection. Owing to the lack of necessary evidence, based on the guidelines for the categories of sequence variants recommended by the American College of Medical Genetics and Genomics (ACMG),[7] some rare variants that are not included in the disease database are classified as variants of uncertain significance (VUS). These variants will not be classified as pathogenic or benign to be the evidence to rule in or rule out PGT until the performance of genetic function experiments or update of the database and the emergence of literature support. A long wait time and uncertainty of database update is a big challenge for patients with VUS.

  Selection of Embryo Transfer Top

Mosaic embryos include cells with ≥2 karyotypes, mostly derived from meiosis or mitotic error in the early embryonic development stage, which is the distinct result of PGT-A except for euploid and aneuploid embryos. Some research have proved some mechanisms including abnormal apoptosis, normal cell trending to distribute ICM and correction of chromosomal abnormality at the time of mitosis which lead to self-correction, and selection of the development of mosaic embryo before being transferred.[8] A study on a live birth after transferring mosaic embryo indicated the development potential of mosaic embryo;[9] therefore, there is rethinking about the clinical management strategy for mosaic embryos. Several trials have proved that compared with euploid embryos, the transplantation rate of mosaic embryo is lower and the abortion rate is higher. Mosaicism may appear in the two-cell stage, and the proportions of embryonic mosaicism that develop into the cleavage stage and blastocysts stage are 15%–90% and 15%–30%, respectively.[10] The occurrence of embryonic mosaicism is not related to the age of the women; however, the mechanism and its potential influence are unclear. Thus, for PGT-A detection in women with advanced age and recurrent miscarriages or repeatedly failed implantations, normal euploid embryos should be selected as the priority for embryonic transplant. If there is no other available embryo, patients should be informed about the risks of potential abortion or fetal abnormality before mosaic embryo transplant.

It is widely known that embryo mosaicism occurs in the cleavage stage and the blastocyst stage. Owing to the ethical and legal restrictions as well as limited studies being conducted on the subject, current investigations on the mosaicism mainly focus on description without verification of function. Therefore, it is still a challenge to confirm the incidence of mosaicism and the actual proportion and quantify embryonic capability and development potential. In principle, when there are no euploid embryos available, patients should be completely informed about the risk during consultation. Mosaic embryo transplantation can be considered after obtaining informed consent from the patients. The Preimplantation Genetic Diagnosis International Society and the Congress on Controversies in Preconception, Preimplantation and Prenatal Genetic Diagnosis suggest that when using a hr-NGS platform to detect 5–10 cells in the blastocyst, an embryo with abnormal cells >70% cannot be transferred; avoid the transfer of embryo with mosaic trisomy 13, 18, 21, 22 that could lead to live birth, uniparental disomy-related mosaic trisomy 14, 15, and intrauterine growth retardation-related mosaic trisomy 2, 7, 16.

  Ethical Conflicts Top

Previous prenatal diagnosis does not aim at delayed genetic diseases, such as adult polycystic kidney disease, and Huntington chorea. With the rapid development of PGT-relevant techniques and improvement in the health consciousness of individuals, the indications have extended from chromosomal diseases and monogenic disease to delayed genetic disorder and PGT for human leukocyte antigen (PGT-HLA). The former mainly includes cancer susceptibility diseases (such as breast cancer and ovarian cancer), heart diseases, and neurodegenerative diseases. With the extension of clinical applications, PGT will inevitably raise more ethical controversies. Although the development of PFT in diseases with a nonearly onset or non-100% onset remains controversial, more and more patients consider PGT detection as a priority. Thus, during genetic consulting, patients should be completely informed about the clinic phenotype, disease outcome, susceptible risk, early prevention, and PGT selection. Moreover, some blood diseases or immunodeficiency diseases, such as thalassemia and high IgM syndrome, could go through HLA match at the same time with the routine PGT-M detection, which will provide stem cell donor for the fetus. However, embryos obtained from normal PGT-M detection result are few, leading to a relatively low probability of embryos being matched with HLA (in theory, the probability of obtaining an HLA match is 3/16 for recessive hereditary disease and 1/8 for dominant genetic disease). If these patients are at advanced age, high risk of abnormal aneuploidy in the embryo should be conveyed during the consultation. Moreover, some ethical issues such as whether a substandard embryo or savior fetus should be materialized still need further discussion. If secondary findings are not conveyed to the patient before whole-exome sequencing, some ethical issues will be caused. The ACMG states that 59 genetic mutations can lead to severe diseases, such as tumor, cardiovascular diseases, whereas other genetic mutations could be intervened with medical treatment. As a secondary finding in implementing PGT, the American Society for Reproductive Medicine notes that patients should be informed about the baby's sex, but doctors should not consider it as an influencing factor in the reproductive decision.

  Conclusion Top

PGT is a revolutionary development for human reproductive health. High-throughput technology to obtain genome information of the embryo and selection of the most appropriate embryo to transplant into the mother greatly improves the possibility of having healthy offspring in families with genetic diseases and provides a new way to treat patients with specific genetic diseases. The rapid development of PGT will definitely bring new opportunities and challenges. Doctors must make a reproductive decision following the principle of “Reproductive freedom, Do not harm,” which underlies the well-being brought to humans by PGT.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Wu YT, Li C, Zhu YM, Zou SH, Wu QF, Wang LP, et al. Outcomes of neonates born following transfers of frozen-thawed cleavage-stage embryos with blastomere loss: A prospective, multicenter, cohort study. BMC Med 2018;16:96. doi: 10.1186/s12916-018-1077-8.  Back to cited text no. 2
Mastenbroek S, Twisk M, van Echten-Arends J, Sikkema-Raddatz B, Korevaar JC, Verhoeve HR, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007;357:9-17. doi: 10.1056/NEJMoa067744.  Back to cited text no. 3
Brezina PR, Anchan R, Kearns WG. Preimplantation genetic testing for aneuploidy: What technology should you use and what are the differences? J Assist Reprod Genet 2016;33:823-32. doi: 10.1007/s10815-016-0740-2.  Back to cited text no. 4
Magli MC, Albanese C, Crippa A, Tabanelli C, Ferraretti AP, Gianaroli L. Deoxyribonucleic acid detection in blastocoelic fluid: A new predictor of embryo ploidy and viable pregnancy. Fertil Steril 2019;111:77-85. doi: 10.1016/j.fertnstert.2018.09.016.  Back to cited text no. 5
Handyside AH, Harton GL, Mariani B, Thornhill AR, Affara N, Shaw MA, et al. Karyomapping: A universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes. J Med Genet 2010;47:651-8. doi: 10.1136/jmg.2009.069971.  Back to cited text no. 6
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24. doi: 10.1038/gim.2015.30.  Back to cited text no. 7
McCollin A, Swann RL, Summers MC, Handyside AH, Ottolini CS. Abnormal cleavage and developmental arrest of human preimplantation embryos in vitro. Eur J Med Genet 2019. pii: S1769-7212(19)30129-6. doi: 10.1016/j.ejmg.2019.04.008.  Back to cited text no. 8
Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N Engl J Med 2015;373:2089-90. doi: 10.1056/NEJMc1500421.  Back to cited text no. 9
Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK, et al. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update 2014;20:571-81. doi: 10.1093/humupd/dmu016.  Back to cited text no. 10


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