1 Brief history of the development of PGD/ PGS technology
As early as the late 1970s, when ART attracted worldwide attention, some scholars proposed the idea of screening abnormal embryos to reduce the risk of miscarriage through this technology. Subsequently, different scholars proposed that chromosome detection could be performed by biopsy of polar body, 1~2-cell cleavage bulb cells or several trophoblast ectodermal cells. 1990, British scholars carried out the first case of PGD for X-linked recessive genetic disease, and after the cleavage bulb biopsy, the Y chromosome specific sequences were amplified through the PCR method for the sex selection of the embryo, and female embryos were implanted to obtain clinical pregnancies, thus opening up a new era in ART[1]. 1992, dual-color ART was introduced in the world. In 1992, two-color fluorescent in situ hybridization (FISH) was developed as another option for sex selection in monogenic diseases, applying X and Y chromosome-specific probes for sex selection and detecting X and Y chromosome aneuploidy [2]. 1995, based on the correlation of aneuploidy with advanced age and miscarriage, multicolor FISH was developed for sex selection. In 1995, based on the correlation between aneuploidy and female advanced age and miscarriage, multicolor FISH-PGS was introduced into the PGD arena [3], which mainly screens several chromosomal aneuploidies in women of advanced age, creating the era of PGS, and has been popularized. However, the number of probes of FISH technology is limited, and techniques such as fluorescent PCR and whole chromosome staining can simultaneously perform PGS detection on a larger number (9~10) of chromosomes, which gives people a better choice [4]. Mutual translocation PGD methods have also begun to be developed as a result of the study of subtelomeric probes [5], greatly reducing the risk of miscarriage in this population. Various PGD methods for single-gene disorders also progressed rapidly during this period. gene chip, comparative genomic hybridization in 2001, single-cell sequencing, qPCR and other comprehensive chromosomescreening (CCS) techniques came into the limelight in 2003. gene chip-PGS reports began to increase in 2004, and the number of reports of gene chip-PGS began to rise. After 2004, the reports of PGS began to increase, and became the main PGD/ PGS technology in most of the international fertility centers after 2010 [6]. Single nucleotide polymorphism (SNP) gene chips can quickly and easily perform PGD for a variety of single-gene diseases at the same time, and at the same time feasible to screen 23 pairs of chromosomes for aneuploidy [7]. In recent years, high-throughput sequencing may become the most promising PGD/ PGS technology.
2 Different indications for PGS are proposed
The indications for PGD/PGS have always been in line with the development of molecular genetic technologies. Although the development time of PGD/PGS is relatively short, but in the field of PGD/PGS a variety of new molecular biology technology clinical transformation speed is very fast, at present PGD/PGS has become an important part of the ART technology, greatly changed the face of ART, so that the ART technology from the simple solution to the problem of infertility to become the optimization of the pregnancy outcome of the favorable technology, and may be applied to all the ART population. In 1990, the International Working Group on Preimplantation Genetics was established to summarize and guide the global work on PGD. 1997, the European Society of Human Reproduction and Embryology (ESHRE) PGD Working Group was established, proposing a new approach for high-risk pregnancies. In 1997, the PGD Working Group of the European Society of Human Reproduction and Embryology (ESHRE) was established, proposing PGS for high-risk groups to improve the success rate, and conducting annual statistics to follow up the latest progress of PGD. 2002, the PGD International Study Group (PGDIS) was established, and the PGD Technical Guidelines were published in 2004 [8], which for the first time combined with the Guidelines for the Establishment of ART centers and the Guidelines for the PCR/FISH experiments, and provided guidance for the establishment of an efficient and orderly PGD system. In 2005, the PGD Working Group of the ESHRE provided further guidelines for the inclusion of PGD/PGS on the basis of the PGDIS guideline, which is more practical [9]. The indications are divided into two categories: those recommended for inclusion and those not recommended for inclusion. PGD is recommended to include single-gene disorders with a clear diagnosis, severe teratogenicity or disability, and a high likelihood of inheritance (chromosomal rearrangement probability >10%; monogenic inheritance ratio of 25% to 50%); translocation chromosome problems; and disorders with an HLA mismatch for which there is already an available stem cell therapy in the genetically related indications. RSA> 2 times, the number of times may be determined by each center according to regional and national regulations; RIF> 3 transfers with high-level embryo failure; or multiple transfers with a cumulative total of 10 embryo failures; AMA> 36 years of age. However, ART is not recommended for any woman who may be at high risk of complications due to ovarian stimulation, follicular puncture, pregnancy, low fertility such as women with AMA (age 40-45 years or older), basal FSH value >15IU/L, body mass index >30kg/m2 and other diseases that make ART unsuitable for inclusion, specifically, sinus follicle count ( Specifically, sinus follicle count (AFC) number <7, and poor embryo quality were not considered. This version of the guideline did not include male factor infertility as an indication for PGS, and the inclusion of male factor infertility as an indication for PGS is more controversial.In 2006, there is still an international preference for severe male factor infertility as an indication for PGS [10], and it is practiced in multicenters, the main reason is that severe male factor can lead to an elevated chance of chromosomal abnormalities of the embryo [11]: embryonic primary chromosomal abnormalities such as trisomy, trisomy and monosomy and sex chromosome abnormalities; PGS results of embryos from patients with obstructive and non-obstructive azoospermia show aneuploidy rates higher than 50% [12]; spermatozoa from patients with severe oligozoospermia have a significantly higher rate of aneuploidy compared to controls [13]; intracytoplasmic sperm injection (ICSI)/testis is not recommended for patients with severe oligozoospermia [14]; the use of ICSI/testis is not recommended for patients with severe oligozoospermia [15]; the use of ICSI is not recommended for patients with severe oligozoospermia [16]. ICSI/testicularspermextraction (TESE)-PGS resulted in significantly improved pregnancy outcomes [14]; high prevalence of chromosomal abnormalities in embryos after remedial ICSI, and biopsy and PGS were effective in improving pregnancy outcomes [15-16]; whether male age is associated with the occurrence of aneuploidy in embryos has not been conclusively demonstrated, but one However, an embryo-based study showed that older men (>50 years old, independent of female age) had a lower rate of blastocyst formation and a significantly higher incidence of embryonic aneuploidy [17]. The effect of male factors on embryos still needs further study, and the need to include remedial ICSI and older men in PGS is inconclusive.
3 Controversy over the indications of PGS
RSA, RIF, AMA, and severe male factor are the main medical indications for PGS, which was initially envisioned as a way to reduce the risk of implantation failure, miscarriage, induced abortion, and birth defects due to chromosomal abnormality, and advanced age is one of the factors that contribute to the poorer outcome of pregnancy in females, and it is also the first indication for PGS, but it has been shown in some ART centers. A subset of ART centers have shown that PGS may reduce the risk of some chromosomal abnormalities, but does not improve pregnancy outcomes and reduces the live birth rate. Two important prospective cohort studies on the effect of PGS in women with AMA have shown [18-19] that the number of transferable embryos was significantly lower in the PGS group compared with the control group, and that the rate of sustained pregnancies was significantly lower and the live birth rate was significantly lower. The publication of these two articles caused great controversy and shook the confidence of the international community in PGS, which directly led to the sharp decline in the number of PGS cycles after 2007 [20]. At the same time, randomized case-control or retrospective analyses of RSA, RIF, and male factor indication did not support the effectiveness of PGS in improving pregnancy rates [21-23].
While the negative evaluation of PGS continues, most scholars still believe that PGS has a positive impact on pregnancy outcomes, mainly focusing on the following aspects: (1) although there is no significant increase in the rate of live births, the rate of miscarriages, the rate of multiple births decreased significantly; (2) PGS in AMA women greatly reduces the risk of Down's syndrome, > PGS in women aged 40 years is more economical [24]; (3) PGS can reduce the risk of Down's syndrome in women aged 40 years [24]; (4) PGS is more cost-effective in women aged 40 years. PGS can reduce the emotional trauma caused by repeated miscarriages and reduce the risk of mid-trimester miscarriages in women of advanced age;④ Significant improvements in pregnancy outcomes can still be seen in some populations.
Since most ART-PGS prior to 2007 used cleavage sphere cell biopsy + limited chromosome FISH (PGS#1), most scholars believe that the limitations of this technique may have contributed to the variations in clinical outcomes of PGS. Ovoid biopsy has high limitations: (1) it may have a greater impact on the developmental potential of the embryo; (2) the results may be inaccurate due to the fragmentation and lysis of nuclei caused by biopsy or fixation; (3) chimerism is frequent in early embryos, and the ovoid results may have a high error rate. The increase in the pregnancy rate after FISH-PGS of polar body biopsy also suggests that the cleavage ball biopsy may have an effect on the developmental potential of embryos. The limitations of FISH technology are many: (1) the interpretation of FISH signals is difficult; (2) the FISH probe cannot cover all chromosomes; (3) the mechanism of aneuploidy may be different in different indicative populations, leading to differences in the results of FISH-PGS [25]. After an in-depth analysis of the results published in the New England Journal by Mastenbroek et al [26], some scholars believed that the team was inexperienced, and that the biopsy technique, cell fixation technique, FISH technique, FISH reporting errors, and patient selection bias might have caused the inaccurate final conclusions. Based on the limitations of the oocyte biopsy and FISH technology, the international community has begun to advocate changing the biopsy method, performing CCS at the same time, and terminating FISH-PGS [27-28].
4 The proposal of PGS2.0
The difference between PGS#2 (2.0) and FISH-PGS (PGS#1): ① the blastocyst trophectoderm biopsy or polar body biopsy on day 5/day 6 (D5/D6), and no longer the cleavage sphere biopsy on day 3 (D3); ② the use of gene microarray or other technologies for CCS, and the abandonment of the FISH technology [29-30]. In 2010, the ESHRE PGD working group called for an evaluation of the clinical effectiveness of PGS 2.0 [31], which received much support, and reports of the benefits of CCS for ART pregnancy outcomes exploded: cleavage bulb biopsy+comparative genomic hybridization gene chip (aCGH) significantly increased pregnancy rates and reduced multiple pregnancies and miscarriages [32]; aCGH was more accurate than FISH, and implantation rates and miscarriage rates were higher than in FISH [33]; aCGH was more accurate than FISH, and implantation rates and miscarriage rates were higher than in FISH [34]; aCGH was more effective than FISH [35]; and aCGH was more effective than FISH, and implantation rates, and miscarriage rates were higher than FISH. The accuracy of aCGH was higher than that of FISH, and the implantation and pregnancy rates were increased [33]; time-lapsemonitoring combined with aCGH-PGS significantly increased the pregnancy rate compared with PGS alone [34-35]; CCS combined with blastocyst transfer significantly increased the pregnancy rate, the sustained pregnancy rate, the live-birth rate, and lowered the miscarriage rate [36], and reduced the risk of multiple births; and AMA (for women aged 40-43 years old), RSA population also have satisfactory clinical results [37-38]. The analysis of 2011-2012 PGS data in the United States showed that > the live birth rate per cycle and the live birth rate per transplantation cycle after PGS in women aged 37 years were significantly higher than those in the control group [39]; and some small prospective studies showed that [40], CCS significantly increased the implantation rate and the pregnancy rate of women with poorer prognosis.
The main indications for PGS2.0 are the same as those published by the PGD Working Group of the ESHRE in 2005, but with the addition of male factors: abnormalities in routine sperm examination; sperm genomedecay (SGD), including increased chromosome fragmentation, dispersed chromatin, and increased aneuploidy. These men can have children by ICSI, but the chance of embryo abnormality may increase, so PGS should be added to the indications. Specifically: ① semen index abnormalities need ICSI fertilization for PGS [severe sperm reduction (<5×106/mL); obstructive or non-obstructive azoospermia]; ② percutaneous TESE-ICSI. secondary indications are: previous episodic chromosomal aneuploidies miscarriage; poor embryo quality; selective single-embryo transfer.
In 2015, some IVF centers reported that unselective PGS for all IVF patients significantly improved pregnancy rates [41], starting a debate on whether to perform PGS for all IVF patients. Given the rapid development of high-throughput sequencing technology and the proposal of noninvasive PGS using free DNA from embryo culture fluid [42], it is possible that minimally damaging but rapid and accurate chromosome screening of all embryos will be possible in the future.
5 Limitations of PGS2.0
While PGS2.0 is currently a better method for selecting embryos and improving pregnancy outcomes, its limitations are obvious: (1) biopsy may result in embryo damage, decreased implantation potential, epigenetic changes, and possible long-term effects in adulthood, and there is an urgent need for the development of less damaging or even noninvasive tests to minimize the possible effects; (2) the analysis of PGS results requires the use of a non-invasive test. At present, the time required for PGS result analysis is more than 24h, and embryo freezing is needed to select the appropriate time for transfer, although existing studies show that fresh and frozen embryos have similar pregnancy outcomes [43], however, the safety hazards of embryo freezing on embryo development have been the focus of the research and hotspot in the field of assisted reproduction, with the continuous development of technology to shorten the time of the PGS report, PGS2.0 after the transfer of fresh blastocysts may become the future of PGS. The transfer of fresh blastocysts after PGS2.0 may become the direction of future efforts; ③ Existing PGS2.0 detection technology is based on the analysis of genome-wide amplification of embryonic cell DNA, the accuracy of its analysis depends on the integrity of the amplification product, however, amplification bias can not be eliminated, which may lead to false positives and false negatives, therefore, the current definition of PGS is only a screening for aneuploidy, with the advancement of technology, is it possible to With the advancement of technology, is it possible to expand the screening scope of PGS to include screening for small segment duplication deletions? PGS2.0 is still unable to predict the outcome of chimeric embryos, and the recognition of chimeras by PGS2.0 may be higher, because chimeric embryos may develop into normal individuals or miscarry, and chimeras account for about 2.88% of all early anomalous karyotypic miscarriages [44], so it is difficult to define the outcome of pregnancies in chimeric embryos, and pre-implantation counseling of chimeric embryos has become a difficult problem, and further research is needed. Further research is needed.
6 Prospects of PGS2.0
The current debate on PGS has shifted from whether PGS is clinically beneficial to the necessity of expanding the indications for PGS. Although there is still a lack of evidence from multicenter prospective randomized controlled studies [45], and there is still a need to re-conceptualize and reevaluate PGS, it is undeniable that most ART centers in the international community have used blastocyst biopsy + PGS (PGS2.0) + FET as a routine tool for the main indications of this population (AMA, RIF, RSA, and male factor) since 2010, with the main aim of reducing the risk of miscarriage in this population, and the main purpose is to reduce the risk of abortion in this population. PGS2.0 has dramatically changed the face of ART and may become a routine program for all patients in fertility centers in the future.
The use of PGS2.0+FET as a routine tool in the primary indicative population (AMA, RIF, RSA, male factor) is aimed at reducing the risk of miscarriage and improving the success rate of the population, and has shown good results.