What Is Preimplantation Genetic Screening?

Preimplantation Genetic Screening (PGS ^[1] ) refers to the detection of chromosome numbers and structural abnormalities of early embryos before embryo implantation. By examining the structure and chromosomes of 23 embryos, Number, an early prenatal screening method to analyze embryos for abnormal genetic material. Therefore, normal embryos are selected for implantation in the uterus in order to obtain a normal pregnancy, improve the clinical pregnancy rate of patients, and reduce multiple pregnancy.

Preimplantation genetic screening

Right!

Preimplantation Genetic Screening, PGS ^[1]

Twenty years ago, the infertility rate among the people of childbearing age in China was only 3%, which is a relatively low level in the world. The 2009 China Infertility Summit Forum released the "Report on the Survey of the State of Infertility in China" shows that the number of infertility patients nationwide has exceeded 50 million, with the largest number of people aged 25 to 30, showing a trend of youth. On average, one in eight couples of childbearing age is facing fertility difficulties. The infertility rate has climbed to 12.5% -15%, which is close to the rate of 15% -20% in developed countries. The most serious thing is that the incidence is still rising. WHO experts predict that the infertility rate in China will rise to more than 20% in recent years.

WHO experts believe that the dual pressures of spirit and environment make infertility at a heavy "cost of life" a serious social problem. How to effectively help infertility patients to solve fertility problems has put forward higher requirements for society, hospitals and doctors.

Embryo abnormalities are one of the main reasons for the ultimate failure of in vitro fertilization. British researchers have developed a new technology that can screen embryo quality, which is expected to increase the success rate of IVF. ^[2]

The emergence of PGS is the result of the rapid development of assisted reproduction technology and molecular biology technology. The application of PGS technology has significant significance by selecting normal embryos.

(1) PGS can choose healthy embryos to improve the success rate of IVF and reduce abortion rate

Compared with the traditional morphology of embryos that rely on microscope technology to select embryos with high morphological grade for transplantation, PGS can directly analyze the genetic material of the embryo, accurately determine whether the embryo has chromosomal abnormalities, and screen out truly healthy embryos.

Data from clinical trials show that PGS can reduce the abortion rate of people with recurrent miscarriages receiving assisted reproductive therapy from 33.5% to 6.9% (Hodes-WertzB, 2012), while increasing the clinical pregnancy rate from 45.8% that depends on morphology to 70.9 % (Yang, 2012). The latest research results published by Martin D. Keltz et al. Show that PGS can significantly improve various indicators of IVF (see Table 1. Comparison of morphology and multiple indicators of embryos screened after PGS screening). PGS can significantly improve the indicators of the first and second generation of "IVF". It can fundamentally increase the pregnancy success rate of the first and second generation of "IVF", reduce the rate of spontaneous abortion, and improve the quality of pregnancy.

(B) to avoid multiple pregnancy, reduce the implementation of fetal reduction

Because the average success rate of IVF is 20-30%, in order to improve the success rate of one implantation, 2-3 embryos are usually implanted at the same time, so there are often multiple births. Multiple births are more risky than singletons, a danger that exists for both mothers and children. Authoritative surveys found that the cesarean section rate for twins was 78.45%, preterm births accounted for 47.07%, and comorbidities and complications accounted for 39.7%. Mothers of twins and multiples are more likely to develop pregnancy syndromes such as diabetes and hypertension during pregnancy, and the probability of postpartum hemorrhage is also higher, and premature birth is also more likely to occur. Most premature children will develop congenital lung disease, and the disease will be lifelong.

Therefore, in order to protect the safety of mothers and children, fetal reduction is required in principle for triplets and above. Fetal reduction surgery has a 10% chance of causing abortion of all embryos. Professor Cook, an internationally renowned expert in reproductive medicine, has always suggested that if the IVF method is adopted, it is better to accept only one fertilized egg transplant. He does not recommend IVF twins, because this method does not have much benefit to the mother and child. Although there are no mandatory rules in the United States, if an embryo is transferred to a woman and twins are finally born, then the doctor's transfer will usually be considered an unsuccessful transfer.

The use of PGS technology to select healthy embryos and increase the success rate of IVFs can avoid the harm caused by blindly transplanting multiple embryos to improve the success rate of pregnancy, resulting in multiple pregnancy, and the ethical and moral conflicts .

Table 1. Comparison of morphology and PGS-selected indicators after embryo transfer *

	Transplantation rate	Clinical pregnancy rate	Ongoing pregnancy rate	Multiple pregnancy rate	Miscarriage
IVF (-PGS)	19.15%	43.91-45.8%	32.49-41.7%	34.38%	26.01-33.5%
IVF (+ PGS)	45-52.63%	55-70.9%	61.54-92%	8.33%	6.9-11.11%

* Data source BrookeHodes-Wertz, JamieGrifo, etal. (2012). Fertility and Sterility, 98: 675-680. ZhihongYang, JiaenLiu, etal. (2012). MolecularCytogenetics, 5: 24MartinD.Keltz, MarioVega, etal. (2013). JAssistReprodGene , 30: 1333--1339.

In 1990, the British Handyside successfully used the polymerase chain reaction (PCR) technology to analyze the pregnancy and childbirth after embryonic sex screening of sex-linked disease carriers by blastomeres, completed the first case of PGS in the world, and pioneered the prenatal screening. Check the new era. In the 1990s, the pre-implantation screening technology developed rapidly.

In 1994, Monne used fluorescence in situ hybridization (fluorescentinsituhybridization (FISH)) technology to successfully screen chromosome aneuploidies and embryo sex before implantation. Since then, multiplex PCR, fluorescent PCR, multicolor FISH and other technologies have been developed.

In 1998, FISH was applied to PGS with balanced chromosomal translocation. By choosing normal and balanced gametes or embryos, PGS can significantly reduce repeated spontaneous abortions due to chromosomal translocations. In the same year, commercially available five-color probes that can simultaneously screen the five chromosomes of 13, 18, 21, X, and Y were also used to screen aneuploidy of eggs and embryos in older women in PGS.

Interphasenuclerconversion technology, comparative genomic hybridization (CGH), and whole genome amplification (WGA) technologies have been successively used in PGS since 1999, which has further promoted the research and application of this technology.

Since 2010, high-throughput sequencing has begun to develop rapidly. Sequences of hundreds of thousands to millions of DNA molecules can be sequenced at once, and a detailed and comprehensive analysis of the genome of an embryo has become possible. Into new territory.

The research enthusiasm for PGS technology at home and abroad has never stopped. At present, the challenges faced by PGS technology are mainly the following two points:

(1) How to safely and effectively obtain the genetic material of the embryo for testing.

PGS biopsies include:

(1) gametes such as sperm or eggs;

(2) The blastomere of the cleavage stage embryo;

(3) Blastocysts nourish ectodermal cells. Each type of genetic material has its advantages and disadvantages.

l gamete. The use of gametes for screening before fertilization is currently rare. The key to this method is how to complete the genetic analysis of sperm or eggs without affecting their fertility. At present, eggs are often used for PGS. The genetic analysis of the first or second polar body is mainly used to indirectly infer whether the egg is normal or not. However, the use of polar body analysis to infer the egg's genome or its chromosome structure and number does not fully reflect the true situation of egg genetic composition. And the polar body can only reflect the genetic laws of the mother, but not the genetic laws of the father.

l blastomere. Blastocyst biopsy is the main method for obtaining PGS. Generally, the blastomeres of 6 to 10 cell stages are selected to be cultured until the third day. The cells in this stage have the potential of totipotent differentiation. Taking out 1 or 2 cells will not affect the embryo development. The blastomere carries a full set of genomes that the parents passed on to the embryo, allowing for a more comprehensive examination.

Blastocyst cells. Blastocyst biopsy is another approach for PGS screening. In this method, fertilized eggs are cultured in vitro to the fifth day, and 5-10 cells are drawn from the blastocyst stage embryo trophectoderm by micromanipulation for genetic screening. The advantage of using blastocyst stage embryo cells is that there are no debris and degraded cells, while cleavage stage embryos often have debris and degraded cells. Because it does not affect the inner cell mass, it does not affect embryo development. However, due to the limitation of culture technology, more than 40% of the embryos cannot develop well to the blastocyst stage in vitro. Therefore, blastocyst stage cells cannot be obtained for PGS. Although taking a certain number of blastocyst stage cells will not affect the normal development of the embryo, the genetic composition of endoblasts and trophectoderm cells is not exactly the same. Therefore, PGS with trophoderm cells may cause misdiagnosis. It is better to use several cells for screening than single cells for screening, which can reduce the rate of misdiagnosis.

(2) How to overcome the influence of extremely low sample size on the accuracy and effectiveness of screening.

Because only a very small number of cells can be taken (the minimum is only one or two), how to accurately detect the low sample volume after taking the cells is an urgent problem. The current methods are:

Fluorescence in situ hybridization FISH

Chromosome analysis generally uses fluorescence in situ hybridization (FISH). The DNA probes were labeled with fluorescent dyes of different colors, and after hybridizing with different chromosomes of the cells fixed on the glass slide, the hybridized portion showed fluorescence of different colors under a fluorescence microscope. In order to screen for chromosomal abnormalities. FISH is still the main method for screening for chromosomal diseases. It is mainly used for screening aneuploidy of chromosomes, especially for abnormal numbers of 13, 18, 21, X and Y chromosomes.

However, the current problem of FISH technology is that it cannot detect all chromosomes at once. Generally, each blastomere cell can only label 5 chromosomes, which takes about 5 hours. Up to 13 chromosomes can only be detected by three rounds of FISH, and as the number of nuclear degeneration increases, the hybridization efficiency of the probe decreases. Therefore, in the PGS for aneuploidy screening of embryos, a full set of 23 chromosomes cannot be screened simultaneously, and true karyotype analysis cannot be performed. It has been reported that at least about 20% of aneuploidies are missed with FISH ^[3]

Step 1: Drug stimulates ovary superovulation

Women's natural menstrual cycle discharges only one mature egg at a time, only one embryo can be formed after fertilization, and the pregnancy rate for transferring one embryo is very low. In order to obtain multiple embryos that can be used for testing and transplantation, ovarian superovulation needs to be stimulated with hormone drugs.

Step 2: Collect eggs from the ovary

After the use of superovulation drugs, ultrasound and hormonal levels monitor egg maturity. When the eggs are mature, collect the eggs. Sperm were collected at the same time.

Step 3: Two ways to complete fertilization

l In vitro fertilization (IVF): Place eggs and sperm in the same culture dish and co-cultivate them for natural fertilization.

l Intracytoplasmic sperm microinjection (ICSI): When sperm quality is poor, natural fertilization cannot be performed, and ICSI needs to be used to complete fertilization.

Step 4: In vitro fertilization

After the fertilization is completed, it is necessary to monitor whether each fertilized egg is successfully fertilized, and the fertilized egg after the fertilization is cultured in vitro.

Step 5: Embryo biopsy

One blastomere cell was selected from the blastomere cultured for three days in vitro, or several blastocyst trophoblast cells were selected from a blastocyst cultured for five days in a culture dish.

Step 6: PGS detects embryo cells

The above-selected embryonic cells were tested for PGS.

Step 7: Embryo transfer

Select 1-2 normal embryos tested by PGS to be transferred into the woman's uterus, and freeze the remaining normal embryos. If the first embryo transfer fails, it can be used for subsequent transfers.

Step 8: Confirming pregnancy

Two weeks after embryo transfer, pregnancy is confirmed by urine test or blood test. IVF-pregnant pregnant women are monitored in the same way as naturally-pregnant pregnant women.

Senior pregnant women (age 35 years)

Pregnant women with a history of repeated spontaneous abortions (3 spontaneous abortions)

Pregnant women who fail repeated embryo implantation (failure 3 times)

Couples with children with chromosomal abnormalities

Couples with abnormal chromosome number and structure

With PGS, routine prenatal examinations cannot be ignored. Because there are more than 4,000 kinds of genetic diseases all over the world, currently PGS can only detect abnormalities in the structure and number of 23 pairs of chromosomes in embryos, and cannot cover all diseases. Because PGS materials are limited, only a certain number of blastomere or blastocyst stage cells are taken. Although the normal development of the embryo will not be affected, the genetic composition of the taken cells and the cell clusters that leave them to develop is not exactly the same. Chimeric disease may have inconsistent screening results. In addition, the cause of chromosomal diseases is unknown, and there is no way to prevent it. Although a healthy embryo has been selected, the fetus may have abnormal changes in chromosomes at any stage of life development after embryo transfer due to maternal reasons and environmental factors. Therefore, after successful pregnancy with PGS, pregnant women still need to undergo routine prenatal examinations.

PGS is not a substitute for prenatal screening. If the routine prenatal examination reveals abnormalities in the fetus, or the pregnant woman has an indication for prenatal screening, it is strongly recommended that pregnant women choose prenatal screening methods such as amniocentesis for confirmation.