A method for assisted evaluation of a second biopsy in a preimplantation genetic test

Abstract

The present application relates to the assisted evaluation method of the second biopsy in the pre-implantation genetic testing. The blastocyst first biopsy process collects and cryopreserves the corresponding biopsy droplet; for the blastocyst with CNV detection failure or suspected abnormal CNV detection result in the first biopsy cell, the first biopsy droplet is thawed to complete genome amplification, CNV library construction, high-throughput sequencing and CNV analysis, and the second biopsy suggestion is given according to the detection result. If the biopsy droplet detects the abnormal aneuploidy or chimera clinically interpreted, it is suggested that the corresponding blastocyst has a high probability of chromosomal abnormality, and the second biopsy is not recommended; if the biopsy droplet does not detect the abnormal aneuploidy or chimera clinically interpreted, it is suggested that the corresponding blastocyst has a high probability of chromosomal normality, and the second biopsy is recommended. The present application proves that the first biopsy droplet contains detectable genetic material, and the consistency of the chromosomal ploidy detection result and the biopsy cell is more than 80%, which can assist the second biopsy decision and improve the decision efficiency and scientificity of PGT detection.

Description

Technical Field

[0001] This invention belongs to the field of gene testing, specifically relating to an auxiliary evaluation method for re-biopsy in preimplantation genetic testing of embryos. Background Technology

[0002] Preimplantation genetic testing (PGT) primarily involves biopsiing and genetically testing morphologically viable embryos obtained through in vitro fertilization and culture. Normal or non-pathogenic embryos are then selected for transfer, thus avoiding complications such as advanced maternal age infertility, adverse pregnancy history, unexplained recurrent miscarriages, unexplained recurrent implantation failures, and the birth of children with chromosomal and genetic defects. During PGT, a routine procedure involves microsurgical biopsy to obtain several trophoblastic ectoderm cells from the blastocyst. Single-cell genome amplification is then performed on the biopsied cells, followed by molecular biology techniques (next-generation DNA sequencing or nucleic acid microarray technology) to detect chromosomal aneuploidy and / or specific gene mutations. Embryonic chromosomal copy number variation (CNV) is fundamental to PGT. Significant CNV abnormalities in embryonic chromosomes can lead to adverse outcomes such as pregnancy failure, miscarriage, or birth defects. Selecting euploid embryos selected through PGT can minimize these risks.

[0003] In PGT testing, a certain percentage (1%-2%) of tests fail or produce suspected abnormal results. These failures are primarily due to issues with the sample itself (e.g., biopsy sample fragmentation), the procedure itself (e.g., improper biopsy tube loading), and the testing process itself. Given the high cost of PGT and the rarity of PGT blastocysts, a simple and effective re-evaluation of failed or suspected abnormal blastocysts is essential. However, relying on a re-biopsy for confirmation requires thawing the frozen blastocyst, refreezing it after the re-biopsy, and the added procedures may adversely affect the blastocyst's developmental potential. Furthermore, patients bear the significant financial burden of a re-biopsy (approximately 10,000-15,000 RMB per blastocyst). Currently, there is a lack of simple and effective auxiliary evaluation methods for PGT re-biopsy decisions, i.e., assessing the benefit of re-biopsy from a technical perspective. For example, if a high aneuploidy tendency in the blastocyst can be predicted, the likelihood of benefit from such a re-biopsy is low, making re-biopsy unnecessary. This requires additional information to support the decision.

[0004] PGT biopsies are performed on specialized biopsy droplets (prepared fresh before biopsy, approximately 3-5 μL, with an independent droplet for each blastocyst). Currently, blastocyst biopsies are routinely performed using laser, mechanical, or a combination of both methods. After the biopsy, the biopsy dishes are typically discarded as medical waste. During the biopsy process, due to the invasive opening in the trophectoderm, as well as the washing and temporary storage procedures, a significant amount of blastocyst fluid is released into the biopsy-related droplets. The inventors' previous research found that a significant amount of DNA can be detected in a large proportion of the biopsy-related droplets after blastocyst biopsy. Next-generation sequencing can determine the relevant genetic information, providing a basis for decision-making regarding repeat biopsies. Summary of the Invention

[0005] The purpose of this invention is to provide an auxiliary evaluation method for re-biopsy in preimplantation genetic testing of embryos.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for auxiliary evaluation of repeat biopsy in preimplantation genetic testing includes the following steps:

[0008] (1) Perform the first biopsy on blastocysts that meet the biopsy criteria in preimplantation genetic testing. Collect biopsy droplets of the corresponding blastocysts during the first biopsy. After marking the blastocysts with numbers, freeze the biopsy droplets in a sample preservation tube.

[0009] (2) For blastocysts in which CNV detection of the first biopsy cells failed or CNV detection results were suspected to be abnormal, the first biopsy droplets corresponding to the blastocyst frozen in step (1) were thawed.

[0010] (3) The thawed biopsy droplets in step (2) were subjected to genomic DNA amplification, CNV sequencing library construction and purification in sequence. The concentration of the purified library was measured, and then high-throughput sequencing and CNV analysis were performed to obtain the CNV detection results of the biopsy droplets.

[0011] (4) Based on the CNV detection results of the biopsy droplets obtained in step (3), provide auxiliary assessment suggestions on whether the corresponding blastocyst should be biopsied again.

[0012] In step (1), the collection process of the biopsy droplets is as follows: After the first biopsy of the blastocyst is completed, for the biopsy operation droplets used for blastocyst biopsy operation and the washing droplets used for washing the blastocysts and biopsy cells after biopsy, as well as the temporary storage droplets used for temporarily storing the blastocysts to be frozen after biopsy, the embryo culture oil in the biopsy operation dish and the temporary storage dish is first removed with a pipette, and then the biopsy operation droplets, washing droplets and temporary storage droplets of the corresponding blastocysts are completely aspirated one by one and combined. The combined liquid is the biopsy droplet, which is transferred to the sample preservation tube and labeled with the blastocyst number to complete the cryopreservation.

[0013] In step (2), the CNV detection failure indicates that there is no valid detection data and CNV interpretation cannot be completed, including: single-cell genome amplification failure, high-throughput sequencing library construction failure, sequencing detection failure, and data analysis failure.

[0014] In step (3), the interpretation criteria for CNV analysis include: reporting abnormal numbers of the entire chromosome; reporting ≥10Mb chromosome segments with deletions or duplications; performing quantitative analysis of mosaicism on ≥10Mb chromosome abnormalities, which can technically detect mosaicism ratios as low as 20%; and making clinical interpretations based on the mosaicism ratio: a mosaicism ratio <30% is judged as clinical euploidy, 30%-70% is judged as clinical mosaicism, and >70% is judged as clinical aneuploidy.

[0015] In step (4), the specific criteria for the auxiliary assessment recommendations are as follows: if the biopsy droplet detects aneuploidy or mosaicism that is clinically interpreted as abnormal, it indicates that the corresponding blastocyst chromosome has a high probability of abnormality and a second biopsy is not recommended; if the biopsy droplet does not detect aneuploidy or mosaicism that is clinically interpreted as abnormal, it indicates that the corresponding blastocyst chromosome has a high probability of normality and a second biopsy is recommended.

[0016] The absence of clinically interpreted aneuploidy or mosaicism includes one of the following situations: the biopsy droplet CNV detection result is a clear neuploidy; the biopsy droplet CNV detection result shows a chromosomal variation with a mosaic ratio of <30%; or no valid CNV detection result is obtained.

[0017] The failure to obtain valid CNV detection results includes: the concentration of the CNV purification library in the biopsy droplets is less than twice the average concentration of the negative control, making high-throughput sequencing impossible; or although high-throughput sequencing is completed, no CNV analysis results that can be clinically interpreted are obtained.

[0018] If the assessment recommends a second biopsy, the blastocyst will be thawed using commercial thawing solution. After the blastocyst cavity has recovered, biopsy cells will be obtained again for CNV testing following the initial biopsy procedure.

[0019] The above-mentioned auxiliary assessment methods are applied in the decision support for blastocyst re-biopsy in preimplantation genetic testing.

[0020] The application of biopsy droplets collected during the first biopsy of blastocysts in preimplantation genetic testing in the preparation of a test reagent for auxiliary evaluation of re-biopsy. The collection process of the biopsy droplets is as follows: After the first biopsy of blastocysts, for the biopsy operation droplets used for blastocyst biopsy operation in the biopsy operation dish, the washing droplets used for washing the blastocysts and biopsy cells after biopsy, and the temporary storage droplets used for temporarily storing the blastocysts to be frozen after biopsy, the embryo culture oil in the biopsy operation dish and the temporary storage dish is first removed with a pipette. Then, the biopsy operation droplets, washing droplets and temporary storage droplets of the corresponding blastocyst are completely aspirated one by one and combined. The combined liquid is the biopsy droplet. It is transferred to a sample preservation tube, labeled with the blastocyst number, and then cryopreserved.

[0021] The significant advantages of this invention are:

[0022] This invention innovatively utilizes biopsy droplets, which are clinical waste in preimplantation genetic testing (PGS), as genetic analysis samples. This represents a novel sample and analysis method, unseen in previous reports. Based on this sample, a re-biopsy-assisted evaluation method can provide a scientific basis for re-biopsy decisions in blastocysts where initial biopsy cell detection failed in PGS, effectively avoiding meaningless re-biopsy and reducing clinical operational costs. This method collects frozen biopsy droplets without additional manipulation of the blastocyst; detection can be completed simply by thawing the droplets, reducing damage to the blastocyst caused by repeated manipulation and improving its preservation and utilization safety. Furthermore, the method relies on existing conventional PGS equipment and operating systems, eliminating the need for specialized equipment. It is simple to operate, easy to clinically promote, and can improve the overall decision-making efficiency of PGS. Attached Figure Description

[0023] Figure 1 : Schematic diagram of the biopsy droplet collection process.

[0024] Figure 2 CNV sequencing results of 6 typical samples.

[0025] Figure 3 CNV sequencing results of re-biopsy cell samples and droplet samples from 5 blastocysts that failed the initial biopsy cell test. Detailed Implementation

[0026] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0027] Example 1:

[0028] Patients who underwent PGT assisted reproduction at the Reproductive Medicine Center of Fujian Provincial Maternal and Child Health Hospital starting in 2024 were selected. The procedures included ovulation induction, oocyte retrieval, sperm optimization, in vitro fertilization, embryo culture and observation, blastocyst biopsy, and genetic testing. Biopsy droplets from the blastocysts were collected for genetic analysis, and basic and clinical information of the patients was also collected and analyzed.

[0029] 1. Materials and Methods

[0030] 1.1 In vitro fertilization, embryo culture and biopsy procedures

[0031] After degranulation of oocytes, mature oocytes at the mid-incubation stage (MII) are retrieved and fertilized via ICSI (micromanipulation system: eppendorf, Germany; oocyte holding needle and injection needle: Cooper, USA). Most in vitro embryo culture uses sequential culture, with a few using one-step culture. Sequential culture is divided into cleavage-stage embryo culture (days 1-3, D1-D3) and blastocyst-stage embryo culture (days 4-6, D4-D6). Cleavage-stage embryo culture uses G1-PLUS medium (Vitrolife, Sweden), and blastocyst-stage embryo culture uses G2-PLUS medium (Vitrolife, Sweden). Both stages of embryo culture are performed in a benchtop incubator (COOK, Australia). One-step culture uses G-TL medium (Vitrolife, Sweden) in a GERI Timelapse incubator (Genea, Australia). All embryos are cultured at 37°C, with a CO2 concentration of 6% (v / v) and an O2 concentration of 5% (v / v).

[0032] Blastocyst formation was observed on days 5 and 6 (approximately 116 and 140 hours post-fertilization, respectively), and scored using the Gardner scoring system. Blastocysts scoring 3BC or 3CB or higher were selected for biopsy. Blastocyst biopsy preparation dishes were prepared on the day of biopsy: several 3-5 μL biopsy preparation and washing droplets were prepared using overnight equilibrated GM-PLUS medium (Vitrolife, Sweden). Both droplets were completely covered with sufficient embryo culture oil (Vitrolife, Sweden) (oil layer thickness approximately 2-3 mm), and incubated in a COOK incubator (culture conditions: 37℃, CO2 concentration 6% (V / V), O2 concentration 5% (V / V)) for at least half an hour. In addition, the temporary storage dish was prepared the day before the biopsy: the temporary storage droplets (about 10 μL / droplet) were prepared with G2-PLUS culture medium (vitrolife, Sweden), and a sufficient amount of embryo culture oil (vitrolife, Sweden) was added to completely cover the droplets (oil layer thickness about 2-3 mm), and the dish was placed in a COOK incubator (culture conditions: 37°C, CO2 concentration 6% (V / V), O2 concentration 5% (V / V)) for overnight equilibration.

[0033] Blastocyst biopsy employs a laser-mechanical approach: after the blastocyst has fully expanded, a laser perforator (Hamilton, USA) is used to create a hole in the zona pellucida region, avoiding the inner cell mass. A small number of trophectoderm cells are aspirated and gently pulled using a biopsy needle, while the laser continuously cuts the intercellular junctions during the pulling process, ultimately yielding 3-7 biopsy cells. Each biopsy droplet is used for the biopsy of only one blastocyst, and the biopsy time for each blastocyst typically does not exceed 10 minutes.

[0034] 1.2 Biopsy droplet collection and genome amplification library construction

[0035] The relevant procedures for sample collection and pretreatment are as follows: Figure 1 .

[0036] After the blastocysts are biopsied in the biopsy procedure droplets within the biopsy procedure dish, the biopsied blastocysts and biopsy cells are transferred to the corresponding washing droplets in the biopsy procedure dish for washing. The biopsied blastocysts are then placed in the temporary storage droplets in the temporary storage dish for freezing, while the biopsy cells are loaded into sample preservation tubes (Econ Medical Systems, Suzhou, China) for subsequent genetic testing. During the biopsy process, the corresponding blastocyst number must be clearly marked on the bottom of both the biopsy procedure dish and the temporary storage dish. After the biopsy, the embryo culture oil in the biopsy procedure dish and the temporary storage dish is first removed using a pipette. For each biopsied blastocyst, its corresponding biopsy procedure droplet, washing droplet, and temporary storage droplet are completely aspirated. These three droplets are collected and combined to obtain the biopsy-related droplet (also known as the biopsy droplet), which is then stored in a sample preservation tube and frozen. The blastocyst number is marked on the sample preservation tube.

[0037] For biopsy cells and biopsy droplets, genomic DNA amplification (MALBAC method, i.e., multiple annealing circular amplification technology) and CNV sequencing library construction were performed using the ChromInst kit (Econ Medical Systems, Suzhou, China) according to the manufacturer's instructions. CNV library purification was performed using the ChromInst kit's accompanying CMPure magnetic beads according to the manufacturer's instructions, and the concentration of the purified library was determined using a Qubit 3.0 (Thermo Fisher Scientific, USA). An equal volume of blank mixed droplets with the same composition as the biopsy droplets served as a negative control, and a known quality control (Econ Medical Systems, Suzhou, China) served as a positive control.

[0038] 1.3 High-throughput sequencing and CNV analysis

[0039] Next-generation sequencing (NGS) technology was used, and the submitted samples were sequenced by Decoding Biomedical Technology Co., Ltd. (Shanghai, China). The quality control standards were as follows: the concentration of the purified CNV library from the biopsy cells must be ≥5.0 ng / µL. The sequencing platform was BGI Genomics' DNBSEQ-T7, employing a PE150 paired-end sequencing strategy, with an effective sequencing data volume of no less than 2.0M reads.

[0040] After sequencing data was processed, CNV analysis was performed using ChromGo software (version V1.9.1, Yikang Medical Co., Ltd., Suzhou, China) according to standard procedures. The report included: Chromosomal aneuploidy: reporting abnormalities in the number of entire chromosomes; Chromosomal segment deletion / duplication: abnormal segments of ≥4Mb were detected in biopsy cell samples, and abnormal segments of ≥10Mb were detected in biopsy droplet samples; Mosaic analysis: quantitative analysis of mosaicism was performed for chromosomal abnormalities of ≥10Mb (including aneuploidy of entire chromosomes and chromosomal segment deletion / duplication); Technically, this detection platform can detect mosaicism as low as 20%; Clinically, the detected mosaicism is graded according to industry consensus: mosaicism <30% is considered euploidy (clinically considered normal), 30%~70% is considered mosaicism (clinically defined), and >70% is considered aneuploidy (clinically considered abnormal).

[0041] 1.4 Statistical Analysis Methods

[0042] Data analysis was performed using SPSS 26.0 statistical software. For quantitative data, those conforming to a normal distribution and having passed the homogeneity of variance test were expressed as mean ± standard deviation. Categorical data were expressed as proportions or percentages (%). Depending on the nature of the quantitative data, independent samples t-tests were used for comparisons between groups; alternatively, quantitative data were categorized and then performed together with categorical data for descriptive statistical analysis, and chi-square tests or Fisher's exact test were used to compare differences between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0043] 2 Results

[0044] 2.1 CNV library detection results of biopsy droplets

[0045] One hundred and fifty-five PGT-assisted reproductive couples were randomly enrolled in 115 PGT oocyte retrieval cycles. Of these, 97.14% (102 / 105) had secondary infertility, and 85.22% (98 / 115) and 13.04% (15 / 115) of the patients received GnRH-A antagonist and GnRH-A ultra-long protocols, respectively, for ovulation induction. A total of 318 biopsy-available blastocysts were involved, and all were subjected to chromosomal CNV (chromosomal variability) detection using biopsy cells. 313 blastocysts yielded valid results on the first attempt; 5 blastocysts failed on the first attempt but successfully obtained CNV results after re-biopsy and testing. Biopsy droplets from all blastocysts were collected for genome amplification and CNV library construction.

[0046] The results showed that the average concentration of the CNV purified library in the blank mixed droplets (i.e., negative control) was 0.246 ng / ul (3 parallel results); among the 318 biopsy droplet samples corresponding to 318 blastocysts, 50.63% (161 / 318) of the biopsy droplet samples had a CNV purified library concentration (CSL, Concentration of Sequencing Library) ≥ 0.5 ng / ul (approximately twice the average CSL concentration of the negative control), and among these 161 samples, 51.55% (83 / 161) had a CSL ≥ 1.0 ng / ul. Next-generation sequencing (NGS) was performed on the CNV libraries of the 83 biopsy droplet samples with CSL ≥ 1.0 ng / ul, and valid CNV analysis results were obtained in 92.77% (77 / 83) of the samples. Figure 2 Table 1 shows the results for six representative samples.

[0047] In addition, NGS was performed on 20 blastocyst biopsy droplet samples with CSL < 1.0 ng / μL, corresponding euploid biopsy cells, and completed embryo transfer. This group included 12 samples with 0.5 ng / μL ≤ CSL < 1.0 ng / μL and 8 samples with 0.35 ng / μL ≤ CSL < 0.5 ng / μL, with an overall effective CNV detection rate of 70% (14 / 20). Among the 6 samples for which no effective CNV results were obtained, 4 samples had CSL below 0.5 ng / μL. Stratified by concentration, the effective CNV detection rate was 83.33% (10 / 12) for samples with 0.5 ng / μL ≤ CSL < 1.0 ng / μL and 50% (4 / 8) for samples with 0.35 ng / μL ≤ CSL < 0.5 ng / μL. Negative controls consistently failed to yield valid CNV analysis results, while positive controls consistently showed the expected results (+21, i.e., trisomy 21, consistent with theoretical results). This indicates that blastocyst biopsy droplets contain detectable genetic material, with approximately 50% of biopsy droplet samples having a CSL exceeding 0.5 ng / μL, suggesting the potential for detecting chromosomal CNVs.

[0048] Table 1. Consistency of blastocyst biopsy cells and their corresponding biopsy droplets with CNV (6 typical samples)

[0049]

[0050] 2.2 Analysis of factors affecting nucleic acid content in biopsy droplet samples

[0051] Using a CSL concentration of 0.5 ng / uL in the biopsy droplets (approximately twice the average CSL of the negative control) as the cutoff line, the differences between the high CSL group (CSL≥0.5 ng / uL, abbreviated as HCSL, corresponding to 161 blastocysts) and the low CSL group (CSL<0.5 ng / uL, abbreviated as LCSL, corresponding to 157 blastocysts) were analyzed. The analytical indicators included female age, male age, female infertility duration, male semen quality, total GN, GN days, number of oocytes retrieved, number of mature oocytes at MII, number of high-quality embryos at D3, number of blastocysts formed, number of high-quality blastocysts, blastocyst age (D5 or D6), total blastocyst score, ICM (inner cell) score, TE (trophectoderm) score, biopsy operator, and CNV detection results of biopsy cells (based on PGT detection). The results are shown in Table 2.

[0052] The CSL of biopsy droplets was significantly correlated only with blastocyst age and CNV detection results in biopsy cells. Compared with D5 blastocysts, D6 blastocyst biopsy droplets had higher nucleic acid content (P=0.018); compared with euploid blastocysts, biopsy droplets of chromosomally abnormal (aneuploid or mosaic) blastocysts had higher nucleic acid content (P=0.001).

[0053] Table 2. Analysis of factors affecting CSL concentration in biopsy droplets (N=318)

[0054]

[0055] 2.3 CNV Analysis of Biopsy Cells and Biopsy Droplets

[0056] For the aforementioned 91 biopsy droplet samples that yielded valid CNV analysis results, their CNV detection results were compared with the CNV results of the corresponding blastocyst biopsy cells. Three types of results were observed (Table 1 and...). Figure 2 Without considering fragment size and chimerism ratio: Category I is completely consistent, accounting for 49.45% (45 / 91), that is, the CNV results of the biopsy droplet and the biopsy cell are completely consistent, including both being euploid (such as blastocyst B1) or having the same chromosomal CNV abnormality (such as blastocyst B2); Category II is partially consistent, accounting for 18.68% (17 / 91), that is, both have the same chromosomal CNV abnormality, and each is accompanied by additional chromosomal CNV abnormalities (such as blastocysts B3 and B4); Category III is completely inconsistent, accounting for 31.87% (29 / 91), including one being euploid and the other having a chromosomal CNV abnormality (such as blastocyst B5), or both having completely different chromosomal CNV abnormalities (such as blastocyst B6).

[0057] If chromosome ploidy is used as the sole criterion, discrepancies are only considered when one of the biopsy cell and biopsy droplet results is euploid and the other is aneuploid (a total of 17 blastocysts, such as B5). All other cases are considered consistent. Based on this, the chromosome ploidy consistency between the biopsy droplet and biopsy cell results was 81.32% (74 / 91) in the 91 blastocysts.

[0058] Example 2: Auxiliary evaluation method for repeat biopsy in preimplantation genetic testing of embryos

[0059] Example 1 confirmed that detectable genetic material exists in blastocyst biopsy droplets. Blastocysts with chromosomal abnormalities detected by PGT release more genetic material into the biopsy droplets, and the consistency between the chromosome ploidy detection results of the biopsy droplets and biopsy cells exceeds 80%. Based on this, this example constructs an auxiliary evaluation method for re-biopsy in PGT detection and verifies the effectiveness of the method through experiments.

[0060] Five blastocysts from Example 1 that failed initial cell biopsy were selected. These blastocysts were thawed using commercially available thawing solution (Kato, Japan) according to the manufacturer's instructions. After the blastocyst cavity recovered, a second biopsy was performed. The procedure for the second biopsy was the same as the first biopsy. CNV detection was also performed on the biopsy cells obtained from the second biopsy. Simultaneously, the corresponding biopsy droplets were collected after the second biopsy, and relevant tests were performed according to the detection standards for the droplets from the first biopsy. The test results showed that the CSL levels of the collected second biopsy droplets were all at extremely low levels (<0.35 ng / uL), making subsequent sequencing analysis difficult.

[0061] Of the five blastocysts that failed initial biopsy cell detection, four had high CSL values ​​(>1.0 ng / uL), while the remaining one had a relatively low CSL value but still exceeded 0.5 ng / uL. Sequencing analysis was performed on the initial biopsy droplets of all five blastocysts (Table 3). Figure 3 Of these, two blastocysts (B26 and B61) showed euploidy in their initial biopsy droplet CNV analysis, while blastocyst B2 showed aneuploidy (+4, +16). The initial biopsy droplet CNV results for these three blastocysts were consistent with the CNV results from subsequent biopsies. For blastocyst B4, the CNV results from subsequent biopsies were -7, -16, and -21, which were largely consistent with the initial biopsy droplet results [-7 (~69%), del (11)(p15.5p14.3)(~25.50Mb, ~45%), -16 (~62%), -21 (~54%)]. Furthermore, the chromosome ploidy results from both were consistent, both being aneuploid. In addition, sequencing of blastocyst B107 failed due to the low concentration of the CNV purification library in the first biopsy droplet, resulting in no analyzable CNV detection results. This situation is consistent with the euploidy results of the second biopsy cell detection.

[0062] Table 3. Consistency of CNV between repeat biopsy cells and the initial biopsy droplet.

[0063]

[0064] In summary, among the five blastocysts that failed initial biopsy, the consistency of chromosome ploidy detection results between the initial biopsy droplets and the re-biopsy cells reached 80% (4 / 5), comparable to the overall chromosome ploidy consistency of 81.32% (74 / 91) in the aforementioned study. This result further confirms that the detection of CNV abnormalities in biopsy droplets indicates a high probability of chromosomal abnormalities in the corresponding blastocyst. Therefore, CNV detection results from biopsy droplets are expected to provide auxiliary basis for the decision on re-biopsy of blastocysts. The specific evaluation strategy is as follows: when CNV detection of biopsy cells fails or the CNV detection result is suspected to be abnormal, a re-biopsy evaluation is carried out in conjunction with the CNV detection results of the initial biopsy droplets of the corresponding blastocyst. The specific judgment criteria and basis are as follows: ① Re-biopsy is not recommended: if the CNV sequencing analysis results of the initial biopsy droplet sample detect chromosomal variations that are clinically interpreted as abnormal, including a. clear aneuploidy (abnormal number of chromosomes); b. clinically abnormal mosaicism with a mosaicism ratio ≥30%. This result strongly suggests that the blastocyst itself has a very high probability of chromosomal abnormalities. Even if a repeat biopsy yields a valid diagnosis, it is still highly likely to be abnormal, and the patient's chances of obtaining a transferable embryo are low. The clinical benefit of a repeat biopsy is limited. Furthermore, to avoid unnecessary secondary manipulation of the embryo, a repeat biopsy is not recommended. ② Recommendation for a repeat biopsy: If the initial biopsy droplet sample did not detect clinically interpreted chromosomal abnormalities, or if a valid CNV analysis result could not be obtained, this result suggests that the blastocyst is relatively likely to be chromosomally normal (euploid), or there is no valid evidence to support a clear chromosomal abnormality. In this case, a repeat biopsy to clarify the diagnosis has a high probability of yielding a clinically interpreted normal diagnosis, allowing the patient to still have the opportunity to use the embryo. Therefore, a repeat biopsy is recommended to clarify the diagnosis. Note: 1. No clinically interpretable chromosomal abnormalities were detected, including biopsy droplet CNV sequencing analysis results showing definite euploidy, or detection of chromosomal variations with a mosaicism rate of <30% (clinically determined to be euploidy); 2. Unable to obtain effective CNV analysis results, including two situations: first, the content of free DNA in the biopsy droplets is extremely low, resulting in the CNV purified library concentration being less than twice the average concentration of the negative control, making high-throughput sequencing impossible; second, although high-throughput sequencing was completed, no clinically interpretable CNV analysis results were obtained due to data quality issues.

[0065] Taking the five blastocysts re-biopsied in Table 3 as examples, the CNV sequencing results of the initial biopsy droplets of blastocysts B2 and B4 showed aneuploidy, indicating a high probability of chromosomal abnormalities, thus re-biopsy was not recommended. Subsequent re-biopsy results also confirmed that both were blastocysts with chromosomal abnormalities. The CNV sequencing results of the initial biopsy droplets of blastocysts B26 and B61 showed euploidy, while no chromosomal abnormalities were detected in the initial biopsy droplet of blastocyst B107, indicating a low probability of chromosomal abnormalities in these three blastocysts. Subsequent re-biopsy results also confirmed that all three were euploid blastocysts. These results indicate that CNV detection results based on the initial biopsy droplets can provide a reliable basis for the decision to re-biopsy blastocysts.

[0066] To further validate the effectiveness of this assessment method, a larger sample size was collected from relevant biopsy droplet samples. Three blastocysts (YZ1-YZ3) whose initial biopsy cell detection failed were selected. One blastocyst (YZ1) had an initial biopsy droplet CSL < 0.35 ng / μL, and no valid CNV results were detected after sequencing. The other two blastocysts had initial biopsy droplet CSL > 0.5 ng / μL. NGS sequencing was performed according to the aforementioned procedure. The CNV results of the re-biopsy cells and the initial biopsy droplets are shown in Table 4. The results show that the re-biopsy auxiliary assessment suggestions based on the initial biopsy droplet CNV detection results are consistent with the CNV detection results of the re-biopsy cells.

[0067] Table 4. Validation of PGT rebiopsy-assisted evaluation method

[0068]

[0069] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for auxiliary evaluation of re-biopsy in preimplantation genetic testing, characterized in that: Includes the following steps: (1) Perform the first biopsy on blastocysts that meet the biopsy criteria in preimplantation genetic testing. Collect biopsy droplets of the corresponding blastocysts during the first biopsy. After marking the blastocysts with numbers, freeze the biopsy droplets in a sample preservation tube. (2) For blastocysts in which CNV detection of the first biopsy cells failed or CNV detection results were suspected to be abnormal, the first biopsy droplets corresponding to the blastocyst frozen in step (1) were thawed. (3) The thawed biopsy droplets in step (2) were subjected to genomic DNA amplification, CNV sequencing library construction and purification in sequence. The concentration of the purified library was measured, and then high-throughput sequencing and CNV analysis were performed to obtain the CNV detection results of the biopsy droplets. (4) Based on the CNV detection results of the biopsy droplets obtained in step (3), provide auxiliary assessment suggestions on whether the corresponding blastocyst should be biopsied again.

2. The auxiliary evaluation method according to claim 1, characterized in that: In step (1), the collection process of the biopsy droplets is as follows: After the first biopsy of the blastocyst is completed, for the biopsy operation droplets used for blastocyst biopsy operation and the washing droplets used for washing the blastocysts and biopsy cells after biopsy, as well as the temporary storage droplets used for temporarily storing the blastocysts to be frozen after biopsy, the embryo culture oil in the biopsy operation dish and the temporary storage dish is first removed with a pipette, and then the biopsy operation droplets, washing droplets and temporary storage droplets of the corresponding blastocysts are completely aspirated one by one and combined. The combined liquid is the biopsy droplet, which is transferred to the sample preservation tube and labeled with the blastocyst number to complete the cryopreservation.

3. The auxiliary evaluation method according to claim 1, characterized in that: In step (2), the CNV detection failure indicates that there is no valid detection data and CNV interpretation cannot be completed, including: single-cell genome amplification failure, high-throughput sequencing library construction failure, sequencing detection failure, and data analysis failure.

4. The auxiliary evaluation method according to claim 1, characterized in that: In step (3), the interpretation criteria for CNV analysis include: reporting abnormal numbers of the entire chromosome; reporting ≥10Mb chromosome segments with deletions or duplications; performing quantitative analysis of mosaicism on ≥10Mb chromosome abnormalities, which can technically detect mosaicism ratios as low as 20%; and making clinical interpretations based on the mosaicism ratio: a mosaicism ratio <30% is judged as clinical euploidy, 30%-70% is judged as clinical mosaicism, and >70% is judged as clinical aneuploidy.

5. The auxiliary evaluation method according to claim 1, characterized in that: In step (4), the specific criteria for the auxiliary assessment recommendations are as follows: if the biopsy droplet detects aneuploidy or mosaicism that is clinically interpreted as abnormal, it indicates that the corresponding blastocyst chromosome has a high probability of abnormality, and it is not recommended to perform a second biopsy; if the biopsy droplet does not detect aneuploidy or mosaicism that is clinically interpreted as abnormal, it indicates that the corresponding blastocyst chromosome has a high probability of normality, and it is recommended to perform a second biopsy.

6. The auxiliary evaluation method according to claim 5, characterized in that: The absence of clinically interpreted aneuploidy or mosaicism includes one of the following situations: the biopsy droplet CNV detection result is a clear euploidy; the biopsy droplet CNV detection result shows a chromosomal variation with a mosaic ratio of <30%; or no valid CNV detection result is obtained.

7. The auxiliary evaluation method according to claim 6, characterized in that: The failure to obtain valid CNV detection results includes: the concentration of the CNV purification library in the biopsy droplets is less than twice the average concentration of the negative control, making high-throughput sequencing impossible; or although high-throughput sequencing is completed, no CNV analysis results that can be clinically interpreted are obtained.

8. The auxiliary evaluation method according to claim 1, characterized in that: If the assessment recommends a second biopsy, the blastocyst will be thawed using commercial thawing solution. After the blastocyst cavity has recovered, biopsy cells will be obtained again for CNV testing following the initial biopsy procedure.

9. The application of the auxiliary evaluation method according to any one of claims 1 to 8 in assisting decision-making for blastocyst re-biopsy in preimplantation genetic testing.

10. The application of biopsy droplets collected from the first biopsy of a blastocyst in preimplantation genetic testing in the preparation of a testing reagent for auxiliary evaluation by re-biopsy, characterized in that: The collection process of the biopsy droplets is as follows: After the first biopsy of the blastocysts, for the biopsy operation droplets placed in the biopsy operation dish for the blastocyst biopsy operation, the washing droplets used to wash the blastocysts and biopsy cells after the biopsy, and the temporary storage droplets placed in the temporary storage dish for temporarily storing the blastocysts to be frozen after the biopsy, the embryo culture oil in the biopsy operation dish and the temporary storage dish is first removed with a pipette. Then, the biopsy operation droplets, washing droplets and temporary storage droplets of the corresponding blastocysts are completely aspirated one by one and combined. The combined liquid is the biopsy droplet. It is transferred to the sample preservation tube and labeled with the blastocyst number to complete the cryopreservation.

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