CN112575068A - Method for evaluating embryonic development potential - Google Patents
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- 230000013020 embryo development Effects 0.000 title claims description 10
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 84
- 108020005196 Mitochondrial DNA Proteins 0.000 claims abstract description 60
- 239000012531 culture fluid Substances 0.000 claims abstract description 10
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Abstract
The invention relates to the technical field of reproductive medicine, in particular to a method for evaluating the development potential of an embryo, which comprises the following steps of obtaining a culture solution of the embryo; determining mitochondrial DNA copy number in the culture fluid; confirming the reference mitochondrial DNA copy number in the culture fluid; comparing the mitochondrial DNA copy number to the reference mitochondrial DNA copy number to obtain the relative amount of mitochondrial DNA. By using the culture solution of the embryo, the copy number of mitochondrial DNA in the culture solution is obtained, and the relative amount of mitochondria is used for evaluating the development potential of the embryo, the embryo does not need to be biopsied, and the evaluation process is noninvasive to the embryo.
Description
Technical Field
The invention relates to the technical field of reproductive medicine, in particular to a method for evaluating the development potential of an embryo, and particularly relates to a method for evaluating the development potential of the embryo by a non-invasive means.
Background
Embryo quality is always the most important factor affecting the success rate of Assisted Reproductive Technology (ART), but there is currently no method for effectively, objectively and comprehensively evaluating embryo quality.
Mitochondria are important organelles in cells, and in addition to providing more than 90% of energy for life activities, they are involved in regulating female reproductive processes, playing a key role in oocyte maturation, fertilization and early embryonic development. Since mitochondria have a set of independent genetic materials, namely mitochondrial DNA (mitochondrial DNA), which can be semi-autonomously replicated, transcribed and translated without completely depending on a group gene, many scholars have made relevant researches on the relationship between the copy number of the embryonic mtDNA and the embryo quality, the embryo development potential and the pregnancy outcome, but the research results are not the same, and most of them need to pass invasive research methods such as embryo biopsy technology.
However, embryo biopsy techniques used in mitochondrial DNA copy number detection can be traumatic to the embryo.
Disclosure of Invention
The invention aims to: aiming at the problem that the embryo biopsy technology causes trauma to the embryo in the prior art, the method for evaluating the development potential of the embryo is provided, and the method provides a new method for evaluating the development potential of the embryo by detecting the copy number of mitochondrial DNA in the embryo culture solution of the D3 cleavage apparatus and establishing the association between the copy number of the mitochondrial DNA and the morphological score.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for evaluating the development potential of embryo comprises the following steps,
obtaining a culture solution of the embryo;
determining mitochondrial DNA copy number in the culture fluid;
confirming the reference mitochondrial DNA copy number in the culture fluid;
comparing the mitochondrial DNA copy number to the reference mitochondrial DNA copy number to obtain the relative amount of mitochondrial DNA.
By using the culture solution of the embryo, the copy number of mitochondrial DNA in the culture solution is obtained, and the relative amount of mitochondria is used for evaluating the development potential of the embryo, the embryo does not need to be biopsied, and the evaluation process is noninvasive to the embryo.
Preferably, the embryo is a D3 embryo.
Preferably, the culture solution is obtained by a method,
single drops of the selected 2PN embryos are transferred into overnight balanced cleavage culture and are further cultured to D3, and the culture solution remaining after embryo transfer is collected according to the standard of 20 ul/drop.
Preferably, the relative amount of mitochondrial DNA is used to assess embryonic development potential.
Preferably, the method further comprises the step of performing morphological scoring on the embryo, and establishing the relation between the mitochondrial DNA copy number in the culture solution and the morphological scoring.
Preferably, the embryonic development potential comprises at least one of a gross evaluation of D3 embryo morphology and a rate of D3 cell development.
Preferably, the mitochondrial DNA copy number in the culture fluid is determined using digital PCR assays; the reference mitochondrial DNA copy number in the culture broth was determined using digital PCR detection.
Preferably, when using digital PCR detection, the amount of culture medium used is 20 ul.
Only 20ul of culture medium was required to accurately test mitochondrial DNA copy number using digital PCR.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the method for evaluating the development potential of the embryo selects the culture solution of the embryo in the D3 cleavage stage, detects the copy number of mitochondrial DNA in the culture solution by using a digital PCR technology, establishes association with morphological scoring by the non-invasive detection method, obtains a novel method for objectively and effectively judging the quality of the embryo in the cleavage stage, and provides a theoretical basis for screening high-quality embryo transplantation and improving the clinical pregnancy rate. Compared with the traditional embryo morphological scoring means, the metabonomics method can accurately and objectively reflect the development potential of the embryo; compared with other molecular biology means such as pre-implantation genetic diagnosis (PGD) and the like, the metabonomics method is safer and more comprehensive, and can avoid invasive trauma of embryo caused by biopsy and misdiagnosis caused by chimera. The function of the embryo mitochondria is directly related to the copy number of mtDNA and ATP activity thereof, the quality of the embryo with normal chromosome is evaluated by utilizing the copy number of the mitochondrial DNA, the ATP activity of a culture medium and the like, and the method has most advantages of a general metabonomics method and a PGD method.
Drawings
FIG. 1 is a comparative graph showing the total evaluation of mtDNA copy number in different embryo culture solutions for the morphological evaluation of D3 embryo in test example 1 of the present invention.
FIG. 2 is a comparative graph showing mtDNA copy numbers in embryo culture solutions differing in the development rate of D3 in test example 2 of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
1. Embryo in cleavage stage and obtaining of culture solution thereof
Ovum is taken out after controlled hyperstimulation, fertilization is observed 17-20 hours after insemination, 2PN embryo is selected and transferred into overnight balanced cleavage culture (Quinns)
Clearage Medium SAGE 1026) single droplet culture was continued to D3,and the culture solution remaining after embryo transfer was collected according to the standard of 20 ul/drop.
2. Embryo morphology scoring
The scoring of D3 embryos was mainly performed according to the scoring standard of david k. gardner for embryos at the cleavage stage, improved according to the 2011 instein consensus content, and evaluated from the aspects of cleavage stage number, development speed, shape, uniformity, cytoplasm condition, fragment ratio, etc. (table 1), and abnormal phenomena such as abnormal fertilization, blastomere multinuclear embryos (MNB), and synovial Endoplasmic reticulum aggregation (SERa) were excluded. The D3 high-quality embryo can meet the above conditions, and the following three points are required to be reached on the development node: 1) d1 is 2PN embryo, 2) the number of D2 blastomeres is 3-5, 3) the number of D3 blastomeres is 7-9, and the score is more than or equal to 3.
TABLE 1D 3 embryo morphology scoring criteria at cleavage stage
| Scoring | Blastomere morphology | Cytoplasmic status | Fraction ratio |
| 4 is divided into | The blastomere is uniform and regular in shape | Clear cytoplasm without vacuole | Less than 5 percent |
| 3 points of | Slightly uneven blastomereSlightly irregular in shape | The cytoplasm may have granules or vacuoles | 6-20％ |
| 2 is divided into | Irregular blastomere and irregular shape | Severe cytoplasmic granules or vacuoles | 20-50％ |
| 1 minute (1) | Basically no identifiable blastomere | Complete granulation or vacuolation of the cytoplasm | Over 50 percent |
3 determination of mitochondrial DNA copy number of embryo culture fluid in cleavage stage
3.1, DNA concentration was determined by Nanodrop. Design and synthesis of primers: the primer sequence is designed and synthesized by Chengdu Zhike Biotechnology limited company,
upstream (ND1-F) 5'-CCCTAAAACCCGCCACATCT-3',
downstream (ND1-R) 5'-GAGCGATGGTGAGAGCTAAGGT-3'.
Adopts a digital PCR (digital PCR) method and utilizes Bio-RadQX200TMThe droplet-based digital PCR system detects mitochondrial DNA copy number. PCR systems were formulated as in table 2:
TABLE 2 digital PCR formulation system
3.2 mixing the samples evenly, transferring 20 mul of reaction system to a special intermediate plate of a micro-droplet generating plate, and ensuring no bubble in the transferring process; adding 60 mul of special oil into the oil hole of the wanted micro-droplet generating plate, sleeving a leather sleeve, and putting the micro-droplet generating plate on a machine; gently pipette 35 μ l of the produced droplets into a dedicated 96well without creating air bubbles; and (3) sealing the membrane by using a membrane sealing machine, then, loading a digital PCR instrument, wherein the reaction conditions are shown in table 3, and after the reaction is finished, putting the embryo into a microdroplet reader to read data.
TABLE 3 digital PCR reaction condition table
The reference mitochondrial DNA copy number was determined by obtaining D3 embryo morphology scores for multiple D3 embryo broth embryos and mitochondrial DNA copy number in the embryos by the methods described above. A correlation table was established between mitochondrial DNA copy number in culture and the morphological score of the D3 embryos. The determined copy number of mitochondrial DNA in the culture embryo is the reference copy number of mitochondrial DNA, which is used as a reference for the embryo to be tested later.
By the method, after the mitochondrial DNA copy number of the embryo to be evaluated is determined, the mitochondrial DNA copy number of the culture solution is compared with the reference mitochondrial DNA copy number to obtain the relative quantity of the mitochondrial DNA copy number, and the relative quantity can be used for evaluating the development potential of the embryo to be evaluated.
Test example 1
General evaluation of embryo morphology of D3 comparison of mtDNA copy number in different embryo culture solutions
The digital PCR detection result shows that under the condition that the female age, BMI, basic endocrine conditions, HCG daily internal membrane thickness and other conditions have no significant difference, the copy numbers of mtDNA in different embryo culture solutions of D3 embryo morphological total evaluation are compared, the standard deviation (mean plus or minus SD) is that the average copy number of mtDNA in a high-quality embryo group is (1.30 plus or minus 1.99) copy/ul, the average copy number is obviously higher than that in a non-high-quality embryo group (0.91 plus or minus 0.94), and the difference has statistical significance (P <0.05) (Table 4).
TABLE 4 Total assessment of embryo morphology Table of D3
As shown in FIG. 1, the morphology of the embryos evaluated as good quality and normal development speed generally has higher mtDNA copy number, and in the embryo at the cleavage stage, the higher the mtDNA copy number is, the stronger the ability of the embryos to develop into blastula and implantation is.
Test example 2
Comparison of mtDNA copy number in embryo culture solutions with different D3 development rates
The digital PCR detection result shows that under the condition that the female age, BMI, basic endocrine conditions, HCG daily membrane thickness and other conditions have no significant difference, the standard deviation (mean plus or minus SD) of the copy numbers of the mtDNA of the embryo culture solution of different groups of D3 cell development speeds is that the copy/ul of the normal speed group (1.28 plus or minus 1.86) is obviously higher than that of the abnormal speed group (0.78 plus or minus 0.71), and the difference has statistical significance (P <0.05) (Table 5).
TABLE 5 embryo Condition Table for different development speeds of D3
As shown in FIG. 2, the embryo with normal development speed has higher mtDNA copy number, and the higher the mtDNA copy number is, the stronger the ability of the embryo to develop into blastocyst and to be implanted is.
Test example 3
Comparison of mtDNA copy number in embryo culture scored differently
The digital PCR assay results showed that there was no statistical difference (P >0.05) between the high (1.13 ± 1.76) and low (1.15 ± 1.15) groupings of copies/ul of mtDNA in embryo culture solutions with different D3 scores compared to no significant difference in age, BMI, basal endocrine conditions, HCG day film thickness, etc., in women (table 6).
TABLE 6 embryo Condition Table with different D3 scores
As can be seen from the test results of test examples 1-3, mtDNA copy number has a statistically significant correlation with the overall evaluation of D3 embryo morphology and the development rate of D3 cells, and thus the development potential of the embryo can be evaluated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Chengdu university of traditional Chinese medicine
<120> a method for evaluating the developmental potential of embryos
<141> 2020-12-18
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccctaaaacc cgccacatct 20
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gagcgatggt gagagctaag gt 22
Claims (8)
1. A method for assessing the developmental potential of an embryo comprising the steps of,
obtaining a culture solution of the embryo;
determining mitochondrial DNA copy number in the culture fluid;
confirming the reference mitochondrial DNA copy number in the culture fluid;
comparing the mitochondrial DNA copy number to the reference mitochondrial DNA copy number to obtain the relative amount of mitochondrial DNA.
2. The method of assessing embryo developmental potential of claim 1 wherein the embryo is a D3 embryo.
3. The method of evaluating the developmental potential of an embryo according to claim 1, wherein the culture fluid is obtained by,
single drops of the selected 2PN embryos are transferred into overnight balanced cleavage culture and are further cultured to D3, and the culture solution remaining after embryo transfer is collected according to the standard of 20 ul/drop.
4. The method of assessing embryonic development potential of claim 1, wherein the relative amount of mitochondrial DNA is used to assess embryonic development potential.
5. The method of claim 1, further comprising morphologically scoring the embryo and correlating mitochondrial DNA copy number in culture to the morphological score.
6. The method of assessing embryo development potential of claim 1, wherein said embryo development potential comprises at least one of a gross evaluation of D3 embryo morphology and D3 cell development rate.
7. The method of assessing embryonic development potential of claim 1, wherein the number of mitochondrial DNA copies in the culture fluid is determined using digital PCR assays; the reference mitochondrial DNA copy number in the culture broth was determined using digital PCR detection.
8. The method of claim 7, wherein the amount of culture medium used is 20ul as determined by digital PCR.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312349A (en) * | 2015-10-16 | 2019-02-05 | 酷博尔外科器械有限公司 | The method of the quantitative and determining embryo quality of mitochondrial DNA |
| CN111172259A (en) * | 2020-03-09 | 2020-05-19 | 云南省第一人民医院 | Embryo chromosome detection method for blastomere period of embryo culture solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312349A (en) * | 2015-10-16 | 2019-02-05 | 酷博尔外科器械有限公司 | The method of the quantitative and determining embryo quality of mitochondrial DNA |
| CN111172259A (en) * | 2020-03-09 | 2020-05-19 | 云南省第一人民医院 | Embryo chromosome detection method for blastomere period of embryo culture solution |
Non-Patent Citations (2)
| Title |
|---|
| S. STIGLIANI等: "Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome", MOLECULAR HUMAN REPRODUCTION, vol. 20, no. 12, pages 1238 - 1246, XP055365979, DOI: 10.1093/molehr/gau086 * |
| SARA STIGLIANI等: "Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study", MOLECULAR HUMAN REPRODUCTION, vol. 25, no. 9, pages 527 - 537 * |
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