1. A blastula development potential evaluation method for a chromosome balance translocation patient is characterized in that the evaluation method is used for evaluating the blastula development potential based on mitochondrial DNA copy number and mainly comprises the following steps:

(1) blastocyst culture

For the infertility patients with balanced and translocated chromosome, performing embryo culture for 5-6 days to the stage of blastocyst according to the human assisted reproductive technology operation specification, taking 3-6 trophectoderm cells in the blastocyst by biopsy, and performing vitrification freezing and storing the blastocyst in liquid nitrogen;

(2) MCN assay

Performing single cell genome amplification on the biopsy cells by using an MALBAC method, and performing high-throughput DNA sequencing on an amplification product, wherein the sequencing amount is not less than 20 GB; analyzing chromosome euploidy of blastula and mitochondrial DNA copy number corresponding to a unit nuclear genome, namely MCN through sequencing data, wherein the MCN has a calculation formula of MCN = (autosomal mapping region multiplied by 2 multiplied by mitochondrial mapping reads)/(autosomal mapping reads multiplied by mitochondrial mapping region);

(3) selecting high development potential blastocyst for transplantation

Among the chromosome euploid blastocysts, the blastocyst with the MCN lower than 320.5 is preferably selected for unfreezing transplantation, and if no chromosome euploid blastocyst with the MCN lower than 320.5 exists, the chromosome euploid blastocyst with the lowest MCN is preferably selected for unfreezing transplantation.

Background

A balanced chromosomal translocation is one in which two chromosomes break and exchange non-centromere fragments to form two new derivative chromosomes called a reciprocal translocation, including reciprocal translocation between homologous and non-homologous chromosomes. Chromosomal cross-translocations, while causing a change in the location of the chromosomal segment, still preserve the total number of genes, are also known as chromosomal balance translocations. Chromosomal balance translocations (including roche translocations) are relatively common chromosomal abnormalities, which generally do not affect the phenotype of carriers, but gametes generated during meiosis are mostly abnormal, which in turn cause spontaneous abortion, dead fetuses or fetal malformations, and the like, for example, the detection rate of a chromosomal balance translocating patient in a habitual abortion couple is about 10 times higher than that of a general population. Infertility caused by balanced chromosomal translocation can be solved by means of third generation tube infant technology (Preimplantation Genetic Testing, abbreviated as PGT). Conventionally, insemination is completed by intracytoplasmic sperm microinjection technology, namely ICSI (intracytoplasmic sperm injection), fertilized eggs are cultured to a blastocyst stage, then blastocyst trophectoderm is subjected to biopsy, biopsy cells are taken to perform chromosome euploidy detection (based on technologies such as second-generation DNA sequencing), and blastocysts with chromosome euploidy and high morphological score are selected for transplantation. For the PGT-row patients with balanced chromosomal translocations, the quality assessment of the chromosomal euploid blastocysts is mainly based on the traditional morphological assessment method.

How to screen high-quality embryos for transplantation in the implementation process of an assisted reproduction technology is an important problem for embryologists to face, and because the quality of the transplanted embryos, namely the development potential of the embryos, directly influences the clinical outcome of assisted reproduction, a simple, reliable, accurate and safe method for evaluating the development potential of the embryos is very critical to construct. The embryo morphology evaluation method is an embryo quality evaluation method widely adopted clinically at present, is used for grading and selecting embryos according to embryo morphology at a specific time point, and has the characteristics of rapidness, simplicity, convenience, no wound, high efficiency and the like. The morphological scoring is mainly used for evaluating the development potential of the embryo according to the number and the form of pronuclei of the embryo, the number and the uniformity of blastomeres, the color and the cytoplasm form of the embryo, the states of zona pellucida and perivitelline space, the expansion state of blastocysts, the development of inner cell masses and trophoblasts and the like, and the existing morphological scoring standard mainly adopts the Istembull consensus, the Gardner scoring standard and related expert consensus. However, morphological evaluation of embryos is only based on morphological observation of embryos at specific development time points, and cannot obtain all information of embryo development; in addition, the method is influenced by subjective factors of observers, and the repeatability is poor; moreover, the quality of the embryo has no necessary correlation with the morphology, the morphological score cannot accurately and reliably reflect the development potential of the embryo, and the embryo with genetic material defects such as chromosome aneuploidy is difficult to effectively distinguish; furthermore, morphological scoring cannot subdivide the development potential of embryos with similar morphology, so that the evaluation of the development potential of embryos by simply applying an embryo morphological method has a defect.

The primary function of mitochondria is to produce ATP to supply energy, and it is the center of energy supply for the cell and is closely related to the metabolism and growth of the embryo. Mitochondria play a crucial role in embryonic development and maintenance of embryonic metabolism through their critical cellular functions. These functions include: maintenance of ion homeostasis, amino acid metabolism, glycolysis, fatty acid metabolism, signal transduction, and apoptosis regulation, among others. Mitochondria are the most numerous organelles in oocytes and embryos, not only provide energy for maturation and fertilization of oocytes and subsequent embryonic development, but also are closely related to life processes such as oocyte activation, calcium oscillation, apoptosis program initiation and the like, and mitochondria in embryos are all derived from oocytes. Impairment of mitochondrial function is also closely related to oocyte aging, fertilization disorders, and decreased embryo quality. Mature oocytes are the highest Mitochondrial content of human cells, in numbers of hundreds of thousands to over a million, which is associated with a tremendous energy requirement in oocyte fertilization and early embryo development, with 1 copy of Mitochondrial DNA (mitochonddrial DNA, mtDNA) in each mitochondrion. The fertilized egg mtDNA will be diluted and regulated continuously as the embryo develops and cell divides, eventually resulting in a specific mtDNA low copy state in the embryo cell. Embryos with good developmental potential must not leave a reasonable mitochondrial copy, however the correlation between mtDNA copy number in embryos and developmental potential of embryos has not been elucidated.

Disclosure of Invention

The invention provides a chromosome balance translocation patient blastocyst development potential evaluation method based on mitochondrial DNA copy number, which can accurately and effectively screen blastocysts with high development potential for transplantation. The method of the invention comprises the following main steps:

(1) blastocyst culture

For the infertility patients with balanced and translocated chromosome, performing embryo culture for 5-6 days to the stage of blastocyst according to the human assisted reproductive technology operation specification, taking 3-6 trophectoderm cells in the blastocyst by biopsy, and performing vitrification freezing and storing the blastocyst in liquid nitrogen;

(2) MCN assay

Performing single cell genome amplification on the biopsy cells by using an MALBAC method, and performing high-throughput DNA sequencing on an amplification product, wherein the sequencing amount is not less than 20 GB; analyzing chromosome euploidy of blastocysts and mitochondrial DNA copy number corresponding to a unit nuclear genome, namely MCN (MCN is (autosomal mapping region x 2 x mitochondrial mapping reads)/(autosomal mapping reads x mitochondrial mapping region);

(3) selecting high development potential blastocyst for transplantation

Among the chromosome euploid blastocysts, the blastocyst with the MCN lower than 320.5 is preferably selected for unfreezing transplantation, and if no chromosome euploid blastocyst with the MCN lower than 320.5 exists, the chromosome euploid blastocyst with the lowest MCN is preferably selected for unfreezing transplantation.

The method for evaluating the blastocyst development potential of the chromosome balance translocation patient based on the copy number of the mitochondrial DNA is beneficial to screening the blastocyst with the most development potential for transplantation, so that the chromosome balance translocation patient carrying out PGT realizes pregnancy to the greatest extent and the fastest extent, the cost of the patient is not increased, and the clinical resources and the cost can be effectively saved.

Drawings

FIG. 1 is a graph of correlation analysis of blastocyst MCN and female age.

FIG. 2 is a graph of association analysis of blastocyst MCN and its chromosomal euploidy.

FIG. 3 is a graph of correlation analysis of blastocyst MCN and morphological quality, wherein A is a comparative analysis of the difference in MCN between two groups of high quality blastocysts and non-high quality blastocysts in all blastocysts, B is a comparative analysis of the difference in MCN between two groups of high quality blastocysts and non-high quality blastocysts in euploid blastocysts, C is a comparative analysis of the difference in MCN between two groups of high ICM quality and low ICM quality in all blastocysts, and D is a comparative analysis of the difference in MCN between two groups of high TE quality and low TE quality in all blastocysts.

FIG. 4 is a correlation analysis of chromosomal euploid blastocysts MCN and their clinical pregnancies and the corresponding ROC curves.

Detailed Description

The invention is further illustrated below with reference to specific examples.

Example 1

Selecting a chromosome balance translocation patient assisted by PGT (PGT) Preimplantation Genetic Testing in Fujian province, taking in 246 blasts in 94 PGT cycles, wherein the average age of female patients is 29.27 +/-4.15 years (20-40 years), and meanwhile collecting blast culture information, clinical information and pregnancy results (transplants) of the patients, wherein the basic conditions of 10 chromosome balance translocation patients and blasts thereof are shown in Table 1.

Table 110 cases of basic information on chromosome balance translocation patients and blastocyst culture test information

Note: x, Y representative chromosome, t denotes balanced translocation, rob denotes roche translocation; the number before the decimal point of the blastocyst number represents the number of the patient, and the number after the decimal point represents the number of the blastocyst in the same patient; d5 and D6 represent day 5 and day 6 blastocysts, respectively, blastocyst score follows Gardner score standard, mitochondrial copy number is MCN, NGS represents second generation DNA sequencing, and NGS result shows chromosome variation; the diagnosis result is determined based on the detection result of the chromosomal NGS, and the normal representation is a chromosome-balanced blastocyst, and the abnormal representation is a chromosome-unbalanced blastocyst.

The operations of oocyte collection, semen treatment, ICSI, blastocyst culture, blastocyst trophoblast cell biopsy, blastocyst freeze-thaw transplantation and the like are all carried out according to relevant specifications (Huangguoning, et al, assisted reproductive laboratory technology, people health Press, 2014, and the like), and all biopsies are obtained by a laser method at the blastocyst stage. The relevant studies were fully informed by patient consent and were subject to ethical approval by the scientific ethical committee of the department of gynaecology and health care of the Fujian province (ethical batch No.: 2015066). The chromosome euploidy and the mitochondrial copy number of the biopsy blastocyst are determined and analyzed by adopting a second-generation high-throughput DNA sequencing technology, and finally the correlation analysis of the same development potential is carried out by combining the blastocyst culture and clinical pregnancy information. The main operational and analytical link information is as follows:

(1) blastocyst culture, biopsy and cryopreservation

Insemination was achieved by intracytoplasmic sperm microinjection, ICSI (intracytoplasmic sperm injection) technique, followed by tabletop incubators (K-MINC-1000 incubator, COOK, Australia) and a culture system G1/G2 (Vitroffe, Sweden) in 6% CO₂And culturing the embryos for 5-6 days at 37 ℃ to a blastocyst stage, evaluating the quality of the blastocysts by adopting a Gardner scoring standard, and selecting the blastocysts with the score of more than 3 BB; then, performing biopsy on the trophectoderm by adopting laser, and taking 3-6 blastocyst trophectoderm biopsy cells; the biopsied blastocysts were then vitrified frozen (using cryogens from the company gabonese, japan) and stored in liquid nitrogen.

(2) Single cell genome amplification sequencing

Biopsy cells were subjected to Whole Genome Amplification (WGA) using the MALBAC (multiple annealing and partitioning-based Amplification cycles, i.e., multiple annealing circular cycle Amplification technique) method, followed by NGS (next-generation sequencing) high-throughput gene sequencing using the HiSeq-2500 platform (Illumina, usa) according to standard procedures, with no less than 200 million effective reads per sample. Single cell genome amplification was performed using model YK001B kit from Yikang corporation, and sequencing Library construction was performed using Next Ultra DNA Library Prep kit (NEB corporation, USA) according to the protocol.

(3) Blastocyst thawing and transplantation

After the chromosome or gene of the biopsy cell is detected to be normal, the corresponding blastocyst is unfrozen (by adopting unfreezing liquid of Valencia company in Spain) and transplanted. Among the chromosome euploid (i.e., chromosome balance) blastocysts, those with high morphological scores are preferably selected for thawing. Thawed blastocysts are transplanted on day 5 or 6 after natural or artificial ovulation. The endometrium of the patient is prepared by selecting the proper scheme, the thickness of the endometrium is not less than 6mm, and each patient is transplanted with one blastocyst at a time. The intrauterine gestational sac with heartbeat observed by ultrasonic after 40 days of blastocyst transplantation is judged as clinical pregnancy, and all pregnant patients need to adopt amniocentesis for birth control to determine the fetal chromosome karyotype.

(4) mtDNA copy number assay

And (3) utilizing NGS data analysis to calculate the mtDNA content of each sequencing sample, and adopting the mtDNA copy number corresponding to the unit nuclear genome to characterize and analyze, namely the ratio of mtDNA mapping reads number to autosomal reads number. The calculation formula of mtDNA copy number per nuclear genome (MCN) for a unit nuclear genome is MCN ═ autosomal mapped region × 2 × number of mitochondria mapped reads)/(autosomal mapped reads number × number of mitochondria mapped region. For example, assuming that the sequencing results in 100 reads, wherein 90 reads correspond to autosomes, the normal reads length is 100 units long, 5 reads correspond to mitochondria, and the average reads length is 1 unit long, then MCN ═ 100 × 2 × 5)/(90 × 1) ═ 11.1, which means that the copy number of mitochondria corresponding to each nuclear genome is 11.1, and each cell only contains 1 nuclear genome, thus representing the average copy number of mitochondria in the cell.

(5) Statistical analysis

The relevant parameters are expressed by mean value plus or minus standard deviation, the difference of female patient age, chromosome euploidy (balance), blastocyst overall morphological quality, ICM score and TE score is compared by independent t test, and the difference of pregnancy outcome is analyzed by chi-square test. A P value of <0.05 indicates that the difference is statistically significant, and P <0.01 indicates that the difference is significant. The ROC curve is drawn by using the social science statistics software package (SPSS 23.0).

(6) Analysis of results

For PGT patients with balanced chromosomal translocations, among the currently known major influencing factors of copy number of blastocyst mitochondria, due to the differences of age, blastocyst chromosome ploidy and blastocyst quality of different patients, the major influencing factors do not have large influence on the copy number of mitochondria, so that the study is worthy, because the important influencing factors determine whether the copy number of mitochondria can be used as a blastocyst development potential evaluation index alone, or else, the comprehensive evaluation is combined with other factors.

(i) Blastocyst MCN independent of female patient age

Mitochondria are closely related to the physiological status of the human body, and since 94 PGT patients are different in age, which does not affect the clarity of the mitochondrial copy number of their blastocysts, the relationship between blastocyst MCN and the age of female patients was first evaluated. 203 of the 246 blastocysts were from younger female patients (mean age 27.78 years, age group: 20-33 years) and the remaining 43 blastocysts were derived from medically advanced women (mean age 35.63 years, age group: 34-40 years), and the MCNs of the blastocysts were analyzed in comparison between these two groups of patients, with no statistically significant difference (p 0.77) as shown in the results of fig. 1. Among the 246 blastocysts, 96 full-length blastocysts were found, and further, the results of fig. 1 showed no statistically significant difference in MCN between the aged and aged groups (79 and 17) as to the difference between the full-length blastocysts analyzed (p ═ 0.83). Blastocyst MCN is therefore independent of the age of the female patient.

(ii) Blastocyst MCN and chromosomal ploidy independence thereof

The association between blastocyst MCN and its chromosomal ploidy was then analyzed. 96 of 246 blastocysts were examined by NGS as chromosomally euploid blastocysts and another 150 blastocysts as chromosomally aneuploid embryos, and the difference in blastocyst MCN between these two groups was analyzed by comparison, and the results in fig. 2 showed no statistically significant difference (p ═ 0.75). Blastocyst MCN is therefore independent of its chromosomal euploidy.

(iii) Blastocyst MCN and morphological quality thereof

The association between blastocyst MCN and its morphological quality score was further analyzed. The selection of the transplanted blastocysts is mainly based on morphological criteria, and the morphological grading criteria of the blastocysts are based on the size and hatching degree of the blastocyst cavity, and are mainly based on the Gardner scoring system. Wherein the mass of ICM (inner cell mass) is classified into A grade (dense cells), B grade (loose aggregation of cells) and C grade (few cells); the quality of TE (Trophectoderm) is classified into class A (multiple cells forming a cohesive layer), class B (few cells forming a loose epithelium), and class C (fewer large cells). Blastula higher than 3BB (including 3 BB) are superior blastula, and blastula not higher than 3BB are non-superior blastula.

The differences in MCN between the two groups of high-quality blastocysts and non-high-quality blastocysts (fig. 3A, P ═ 0.22), between the two groups of high-quality blastocysts and non-high-quality blastocysts (fig. 3B, P ═ 0.90), between the two groups of high ICM quality and low ICM quality (fig. 3C, P ═ 0.60), and between the two groups of high TE quality and low TE quality (fig. 3D, P ═ 0.08), were analyzed for comparison in all blastocysts, respectively, and the results showed that there were no statistically significant differences between the groups (fig. 3). As can be seen, blastocyst MCN is independent of its morphological quality.

(iv) MCN can be used to predict blastocyst development potential

From the foregoing analysis, blastocyst MCN is independent of female patient age, chromosomal euploidy, and embryo morphological quality. On this basis, the association between blastocyst MCN and clinical pregnancy rate was further analyzed. Only chromosomal euploid (i.e., chromosomal balance) blastocysts are currently transplanted clinically. A total of 57 chromosome euploid blastocysts out of 246 were transplanted during the study period, of which 36 were successfully pregnant and 21 were not.

The difference between the blastocyst MCNs of the clinical pregnancy group and the non-pregnancy group was analyzed by comparison, and the results showed (fig. 4) that there was a very significant statistical difference in MCNs between the two groups (p ═ 0.01). The MCN of the blastocyst in the clinical pregnancy group was lower than that in the non-clinical pregnancy group (199.6vs 322.4, p <0.05), and there was a significant statistical difference between the two. The results indicate that clinical pregnancy is associated with MCN of the transplanted blastocyst. To explore the predictive effect of blastocyst MCN on clinical pregnancy rate, blastocyst pregnancy scores were analyzed using a receiver operating characteristic curve (ROC). An ROC curve is drawn by taking the false positive rate (specificity) as an abscissa and the true positive rate (sensitivity) as an ordinate, the area under the ROC curve (the area under the ROC curve, namely AUC) is 0.63, and the specificity is 92% (FIG. 4); further analysis gave an optimum cut-off value of 320.5 for MCN for clinical and non-clinical pregnancies. The implantation blastocyst is selected with the cut-off value 320.5 of MCN as the threshold value, the clinical pregnancy rate of the blastocyst below the cut-off value is as high as 70 percent and is obviously higher than 60 percent depending on the morphological quality score (the average pregnancy data of the blastocyst in recent years in the center).

Example 2

In order to verify the reliability and accuracy of the assessment method for blastocyst development potential of the chromosome balance translocation patients based on mitochondrial DNA copy number constructed in example 1, 6 chromosome balance translocation patients (numbers #1- #6) subjected to assisted reproductive gene detection are further included, and blastocysts with balanced chromosomes (namely chromosome euploidy) are selected by performing blastocyst culture, TE biopsy, biopsy cell genome amplification and sequencing analysis. The 6 patients were randomly divided into two groups according to the difference of the evaluation method of blastocyst development potential, the experimental group was based on the MCN method of the present invention, the control group was based on the evaluation method of the conventional morphology, the best blastocyst was selected for transplantation according to the respective method and followed up to the clinical pregnancy results, and the results are detailed in table 2.

TABLE 2 comparative analysis of two blastocyst development potential evaluation methods

Among them, patients #1- #3 successfully achieved clinical pregnancy by selecting blastocysts with MCN less than 320.5 (but not all blastocysts with the highest morphological score) for transplantation according to the MCN method provided by the present invention. Patients #4- #6 selected the best blastocyst for transplantation based on traditional morphological methods, and only patient #5 with MCN less than 320.5 successfully achieved clinical pregnancy, although the morphological evaluation of the blastocyst transplanted by this patient was general, only 5CB was non-quality blastocyst. The remaining two patients (#4 and #6) had good quality blastocysts transplanted, but had MCNs 572 and 469, respectively, greater than 320.5, and did not achieve clinical pregnancy. Therefore, the evaluation method for blastocyst development potential of the chromosome balance translocation patient based on the copy number of the mitochondrial DNA is superior to the traditional blastocyst evaluation method based on morphology, and the blastocyst with high development potential can be accurately and effectively screened out for transplantation, so that the chromosome balance translocation patient carrying out PGT realizes pregnancy to the greatest extent and the fastest extent, the cost of the patient is not increased, and the clinical resources and the cost can be effectively saved.