A brief history of 1 PGD/PGS technology development
As early as the late 1970s, when ART attracted worldwide attention, some scholars put forward the idea of screening abnormal embryos through this technology to reduce the risk of miscarriage. Subsequently, different scholars suggested that chromosome detection could be carried out by biopsy of polar body, 1~2 cell blastomere or several trophectodermal cells. 1990, British scholars conducted the research on the first X-linked recessive genetic disease PGD. After blastomere biopsy, the Y chromosome specific sequence was amplified by PCR for embryo sex selection and implanted into female embryos to obtain clinical pregnancy, which opened a new era of ART [1]. 1992 fluorescence in situ hybridization (FISH) is another sex selection scheme for monogenic diseases. X and Y chromosome specific probes can be used for sex selection, and X and Y chromosome aneuploidy can be detected [2]. 1995, based on the correlation between aneuploidy and female old age and abortion, polychromatic fish -PGS entered the PGD stage [3], which mainly screened aneuploidy of several chromosomes for elderly women, creating the PGS era and gaining popularity. However, the number of probes in FISH technology is limited, and a large number of (9~ 10) chromosomes can be detected simultaneously by fluorescence PCR, whole chromosome staining and other technologies, which makes people have a better choice [4]. Due to the research of sub-telomere probes, the method of mutual translocation of PGD has also begun to develop [5], which greatly reduces the risk of abortion in this group of people. PGD methods for various monogenic diseases have also made rapid progress during this period. 200 1 gene chip, 2002 comparative genome hybridization, 2003 single cell sequencing, qPCR and other comprehensive chromosome screening (CCS) technologies have entered people's field of vision. After 2004, the reports of gene chip -PGS began to increase, and after 20 10, it became the main PGD/PGS technology in most reproductive centers in the world [6]. Single nucleotide polymorphism (SNP) gene chip can rapidly and simply perform PGD on a variety of single gene diseases at the same time, and it is feasible to screen 23 pairs of chromosome aneuploidies at the same time [7]. In recent years, high-throughput sequencing technology may become the most potential PGD/PGS technology.
2 PGS put forward different indications.
The indications of PGD/PGS always adapt to the development of molecular genetics technology. Although the development of PGD/PGS is relatively short, the clinical transformation speed of various new molecular biology technologies in PGD/PGS field is very fast. At present, PGD/PGS has become an important part of art technology, which has greatly changed the face of ART, and turned ART technology from simply solving the problem of infertility into a favorable technology to optimize the pregnancy outcome, and may be applied to all ART people. 1990, PGD International Airport
Before the implementation, the International Working Group on Genetics was established to summarize and guide PGD's work around the world. 1997, the PGD working group of the European Society of Human Reproduction and Embryology (ESHRE) was established, and it was proposed to carry out PGS for high-risk groups, improve the success rate, make statistics every year and follow up the latest progress of PGD. In 2002, PGD International Group (PGDIS) was established, and in 2004, PGD Technical Guide [8] was published. This guide provides all-round guidance for PGD for the first time, and provides an international guidance scheme for establishing an efficient and orderly PGD center, but it does not give clear guidance for PGD/PGS indications. In 2005, the PGD working group of ESHRE further gave a more practical guideline for PGD/PGS inclusion on the basis of PGDIS guideline [9]. Indications are divided into two categories: recommended inclusion and not recommended inclusion. PGD suggests patients with confirmed diagnosis, severe teratogenesis or disability and high genetic possibility (possibility of chromosome rearrangement >); 10%; Monogenic diseases with a monogenic heritability of 25%~50%; Translocation chromosome problem; HLA matching and diseases treated by existing stem cells are included in genetically related indications. PGS recommended to be included in the registry system manager & gt2 times, and each center can decide the number of times according to the regulations of the region and the country; RIF> failed to transfer advanced embryos three times; Or the cumulative number of embryos failed to reach10; AMA & gt； 36 years old. However, women who may have a high risk of complications due to ovarian stimulation, follicular puncture, pregnancy, etc., are advised not to undergo ART, and their fertility is low, such as AMA women (over 40-45 years old) and their basic FSH value >; 15IU/L, body mass index >: 30kg/m2 and other diseases that are not suitable for ART are not recommended. Specifically, the number of antral follicles (AFC) is: at the age of 50, regardless of the age of women, the blastocyst formation rate decreases and the incidence of embryo aneuploidy increases significantly [17]. The influence of male factors on embryos still needs further study, and it is still inconclusive whether it is necessary to include remedial ICSI and elderly men in PGS.
3 PGS indications dispute
RSA, RIF, AMA and severe male factor are also the main medical indications of PGS at present. The original idea of PGS is to reduce the risk of implantation failure, abortion, induced labor and birth defects caused by chromosomal abnormalities. Old age is one of the factors leading to poor pregnancy outcome in women, and it is also the primary indication of PGS. However, the practice of some ART centers shows that PGS can reduce the risk of chromosome abnormality, but it can not improve the pregnancy outcome and reduce the live birth rate. There are two important prospective cohort studies on the influence of PGS on AMA women, both of which show [18- 19]. Compared with the control group, the number of transplantable embryos, the rate of persistent pregnancy and the live birth rate in PGS group were significantly reduced. Once these two articles were published, they caused great controversy, shook the confidence of the international community in PGS, and directly led to a sharp decline in the number of PGS cycles after 2007 [20]. At the same time, the randomized case-control or retrospective analysis of RSA, RIF and male factors does not support the role of PGS in improving pregnancy rate [2 1-23].
Although the negative evaluation of PGS continues, most scholars still believe that PGS has a positive impact on pregnancy outcome, mainly in the following aspects: ① Although the live birth rate has not been significantly improved, the abortion rate and multiple births rate have decreased significantly; ② PGS in ②AMA women greatly reduces the risk of Down syndrome, and PGS is more economical for women over 40 years old [24]; ③PGS can reduce the emotional trauma caused by repeated abortion in elderly women and reduce the risk of abortion in the second trimester; ④ Some people can still see the obvious improvement of pregnancy outcome.
Before 2007, most ART-PGS used blastomere biopsy and limited chromosome FISH (PGS# 1). Most scholars think that the limitations of this technology may lead to differences in clinical effects of PGS. Biopsy of blastomeres has high limitations: ① it may have a great influence on embryo development potential; ② Nuclear fragmentation and dissolution due to biopsy or fixation may lead to inaccurate results; ③ There are many chimeras in early embryos, and the error rate of blastomeres may be high. The sustained pregnancy rate increased after PGS polar body biopsy in fish, which also suggested the possible influence of blastomere biopsy on embryo development potential. However, FISH technology has many limitations: ① it is difficult to interpret fish signals; ②FISH probe cannot cover all chromosomes; ③ The mechanism of aneuploidy is different in people with different indications, which leads to the difference of FISH-PGS results [25]. After an in-depth analysis of the results published in the New England Journal by Mastenbroek et al. [26], some scholars think that the team lacks experience, and the final conclusion may be inaccurate due to the wrong biopsy technology, cell fixation technology, FISH technology, FISH report and patient selection bias. Based on the limitations of blastomere biopsy and FISH technology, the international community began to advocate changing the biopsy method, conducting CCS at the same time, and terminating FISH-PGS[27-28].
The proposal of 4 PGS2.0
The differences between PGS#2(2.0) and Fish -PGS(PGS# 1) are as follows: ① blastocyst trophectoderm biopsy or polar body biopsy was taken on the 5th/6th day (D5/D6) instead of the 3rd day (D3); ② Use gene chip or other technologies for CCS and abandon FISH technology [29-30]. In 20 10, the PGD working group of ESHRE called for evaluating the clinical effect of PGS2.0 [3 1], which received a lot of support, and then the report that CCS was beneficial to ART's pregnancy outcome exploded: blastomere biopsy+comparative genomic hybridization gene chip (aCGH) significantly improved the pregnancy rate and reduced the multiple pregnancy and abortion rate [32]; ACGH is more accurate than FISH, and the implantation rate and pregnancy rate are improved [33]; Compared with PGS alone, regular monitoring combined with aCGH-PGS significantly increased the pregnancy rate [34-35]; CCS combined with blastocyst transfer can significantly improve the pregnancy rate, persistent pregnancy rate and live birth rate, reduce the abortion rate [36] and reduce the risk of multiple births. The clinical efficacy of AMA (female aged 40-43) and RSA population is also satisfactory [37-38]. American PGS big data analysis 20 1 1—20 12 shows that the live birth rate per cycle and the live birth rate per transplantation cycle of 37-year-old women after PGS are significantly higher than those of the control group [39]; Some small prospective studies show that CCS can significantly improve the implantation rate and pregnancy rate of women with poor prognosis.
The main indications of PGS2.0 are the same as those published by es hre PGD Working Group in 2005, but male factors are added: abnormal sperm routine examination; Sperm genome decline
Subtraction (SGD) includes chromosome breakage, chromatin dispersion and the increase of aneuploidy rate. These men can give birth through ICSI, but the probability of abnormal embryos may increase, and the indications for PGS should be increased. Specifically: ① Abnormal semen indexes need ICSI to assist pregnancy. For PGS [severe oligozoospermia (
In 20 15 years, some IVF centers reported that non-selective PGS for all IVF patients significantly improved the pregnancy rate [4 1], and began the debate on whether to conduct PGS for all IVF patients. In view of the rapid development of high-throughput sequencing technology and the proposal of non-invasive PGS using free DNA in embryo culture medium [42], it is possible to screen all embryos quickly and accurately in the future.
Limitations of 5 PGS2.0
Although PGS2.0 is a good method to screen embryos and improve pregnancy outcome, its limitations are obvious: ① Biopsy may bring embryo damage, decreased implantation potential, epigenetic changes and possible long-term effects in adulthood, so it is urgent to develop less invasive or even non-invasive detection methods to reduce possible effects; ② At present, PGS analysis results need more than 24 hours, so frozen embryos need to be transplanted at a suitable time. Although the existing research shows that the pregnancy outcome of fresh embryos and frozen embryos is similar [43], the potential safety hazard of embryo freezing on embryo development has always been the focus and hot spot in the field of assisted reproduction. With the continuous development of technology and the shortening of PGS reporting time, fresh blastocyst transplantation after PGS2.0 may become the direction of future efforts; ③ The existing PGS2.0 detection technologies are all based on embryonic cell DNA amplified from the whole genome, and the accuracy of analysis depends on the integrity of the amplified products. However, the amplification bias can not be eliminated, which may lead to false positive and false negative. Therefore, the currently defined PGS is only used to screen aneuploidy. With the development of technology, can we expand the screening scope of PGS and increase the screening of small fragment duplication deletion? ④PGS2.0 still can't predict the outcome of chimera embryos, and PGS2.0 may have a high recognition degree for chimera embryos. Because chimera embryos may develop into normal individuals or miscarriages, chimeras account for about 2.88% of all early abnormal karyotype abortions [44], so it is difficult to define the pregnancy outcome of chimera embryos, and the pre-transplant consultation of chimera embryos has become a difficult point at present, which needs further study.
6 PGS2.0 potential customers
At present, the focus of PGS debate has shifted from whether PGS is beneficial to clinic to discussing the necessity of expanding the indications of PGS. Although there is still lack of evidence from multicenter prospective randomized controlled studies [45], it is still necessary to re-understand and evaluate PGS. Undeniably, after 20 10 years, most ART centers in the world have applied blastocyst biopsy +PGS(PGS2.0)+FET as a routine means to the main indications (AMA, RIF, RSA and male factors), with the main purpose of reducing the risk of abortion and improving the success rate. PGS2.0 has greatly changed the artistic surface.
Appearance may become a routine item for all patients in the future reproductive center.