CN113834936A - Application of growth differentiation factor 9 in predicting embryonic development potential - Google Patents
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Abstract
The invention provides an application of a growth differentiation factor 9 in predicting embryonic development potential. Belongs to the technical field of assisted reproduction. To provide a safe and non-invasive indication of the potential for embryonic development. The method comprises the steps of obtaining an embryo culture solution, determining the absorbance of the embryo-free culture solution, determining the absorbance of a GDF9 standard solution and the concentration of a GDF9 standard solution as standard curves; and comparing the absorbance of the embryo culture solution with the standard curve to obtain the concentration of the GDF9 in the embryo culture solution, evaluating the development potential of the embryo by using the concentration of the GDF9, and not needing to carry out biopsy on the embryo, so that the method has the advantages of no wound, and can improve the survival rate of a single assisted reproductive treatment cycle, and has higher clinical significance.
Description
Technical Field
The invention belongs to the technical field of assisted reproduction, and particularly relates to application of a growth differentiation factor 9 in predicting embryonic development potential.
Background
With the progress of bioscience, Assisted Reproductive Technologies (ART) have been greatly developed in recent years, especially in the field of IVF laboratory technologies, but clinical outcomes still exist in unsatisfactory places. The us CDC publishes the latest 2017 full american ART data showing: for patients with the age of less than 35 years, the implantation rate of the transplanted embryo is only 37.1 percent; with the age increasing, the embryo implantation rate is also reduced, which is respectively: 27.5% (35-37 years old), 18.3% (38-40 years old), 9.7% (41-42), 4.1% (43-44), in the assisted pregnancy patients above 45 years old, the embryo is freshly implanted by 2.2%.
The current research shows that embryo selection is an important factor influencing embryo implantation, and the embryo implantation rate can be improved only by selecting the embryo with the most planting potential. How to screen out embryos with implantation potential for transplantation is the key for improving the success rate of ART and is a hotspot and difficulty in the reproductive field. The existing commonly used embryo selection technology is embryo morphology scoring, the quality of the embryo is mainly judged by evaluating the development speed, the size of blastomeres, fragments, vacuoles and the like of the embryo, and the method has the defects that the subjective factors of evaluation are large, the difference between different technicians and different laboratories is large, and the quality of the embryo cannot be quantified. In addition, the morphology of the embryo sometimes does not completely reflect the embryo's planting potential. Therefore, it is important to find a safe and non-invasive indication of embryo quality before early embryo transfer.
Disclosure of Invention
The invention aims to provide a safe and noninvasive marker of embryo development potential.
The invention provides an application of a growth differentiation factor 9 in prediction or auxiliary prediction of embryonic development potential.
The invention provides an application of a growth differentiation factor 9 in prediction or auxiliary prediction of embryo quality.
Further defined, the embryo is selected at the stage of the third day after fertilization.
Further defined, the detection method of the growth differentiation factor 9 is to detect by using a microplate reader.
Further defined, the method of culturing embryos is as follows: the fertilized embryos are placed into pre-balanced embryo culture drops in the cleavage stage for culture, the culture environment is 37 ℃, and the volume fraction of the culture environment is 5 percent of oxygen and the volume fraction of the culture environment is 6 percent of carbon dioxide.
Further defined, the pre-balanced cleavage stage embryo culture drop is formulated as an embryo culture solution G1-plus containing 5% HSA by mass volume fraction.
The invention provides a kit for predicting or assisting in predicting embryonic development potential, which comprises: a sample to be detected, a standard substance, a quality control substance and biotin.
Has the advantages that: the invention particularly relates to a method for predicting embryo development potential by detecting GDF9 level in human embryo in-vitro culture solution, wherein the GDF9 content in the embryo culture solution of the third day is obviously related to the embryo development potential. Where developmental potential refers to the likelihood of an embryo being implanted or developing into a fetus. The method does not need to carry out biopsy on the embryo, has the advantage of non-invasiveness, can improve the survival rate of a single assisted reproductive treatment cycle, shortens the treatment cycle of an infertility patient, and has higher clinical significance.
Drawings
FIG. 1 is a graph showing the relationship between embryo quality and GDF9 content in embryo culture fluid; A. GDF9 content in transplantable and non-transplantable embryo culture solutions; B. GDF9 content in high quality (good) embryo culture solution, medium quality (fair) embryo culture solution, and low quality (poor) embryo culture solution; the ordinate is the content of GDF9 and the abscissa is the group.
FIG. 2 shows GDF9 content in culture media corresponding to implanted and non-implanted embryos; the ordinate is the content of GDF9 and the abscissa is the group.
FIG. 3 shows GDF9 content in culture solution corresponding to live embryos and embryos not obtained; the ordinate is the content of GDF9 and the abscissa is the group.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
A commercial embryo culture solution G1-plus (Vitroffe, Sweden).
Sources of embryo culture solution: ovum is taken out after controlled superovulation, fertilization situation is observed 17-20 hours after insemination, 2PN embryo is selected to be transferred into a single liquid drop (G1+ 5% HSA) of embryo culture solution in a cleavage period balanced overnight to be continuously cultured for the third day, and residual culture solution after embryo transplantation is collected according to the standard of 10 mu L/drop.
Live embryo culture solution: after controlled hyperstimulation, ova are taken out, fertilization conditions are observed 17-20 hours after insemination, 2PN embryos are selected to be transferred into overnight balanced embryo culture solution in the cleavage stage (G1+ 5% HSA, 30 mu L/drop) and are continuously cultured for the third day, the rest culture solution after embryo transplantation is collected according to the standard of 10 mu L/drop, and only in the later stage identification, the embryos are embryos which can successfully produce live fetuses after embryo transplantation.
Implanted embryo culture solution: taking out ovum after controlled superovulation, observing fertilization situation 17-20 hours after insemination, selecting single liquid drop of 2PN embryo transferred into overnight balanced embryo culture solution in cleavage stage (G1+ 5% HSA) to continue culturing to the third day, and collecting the residual culture solution after embryo transplantation according to the standard of 10 mul/drop, wherein the embryo is the embryo which can successfully produce live fetus after embryo transplantation in later stage identification.
Example 1.
1. Obtaining a culture solution of embryos: the fertilized embryos are put into pre-balanced embryo culture drops (G1+ 5% HSA recombinant human serum albumin) in the cleavage stage for continuous culture under the culture environment of 37 ℃, 5% of oxygen and 6% of carbon dioxide, the embryo evaluation is carried out according to the traditional morphology scores including cleavage speed, fragmentation degree, blastomere uniformity, the presence of single or multiple blastomeres and the like, the third day of transplantable embryos are defined as embryos with the number of blastomeres not less than 6, fragments less than 30% and no obvious morphological abnormality, and good-quality embryo culture solution and poor-quality embryo culture solution are obtained.
2. Detecting the content of GDF9 (growth factor 9) in the embryo culture solution with good quality and the embryo culture solution with poor quality: determining the absorbance of the embryo culture solution with good quality and the embryo culture solution with poor quality; determining the absorbance of the culture solution without the embryo, the absorbance of the GDF9 standard solution and the concentration of the GDF9 standard solution as standard curves; comparing the embryo culture solution absorbance with the standard curve to obtain the concentration of GDF9 in the embryo culture solution, and using the concentration of GDF9 to evaluate the embryo development potential.
3. The results are shown in fig. 1, the content of GDF9 in the culture solution of the third day is significantly related to the quality of the embryo, wherein the content of GDF9 in the culture solution corresponding to the embryo with better embryo quality is significantly lower.
3. Detecting the content of GDF9 in the implanted embryo culture solution and the non-implanted embryo culture solution: determining the absorbance of the embryo culture solution with good quality and the embryo culture solution with poor quality; determining the absorbance of the culture solution without the embryo, the absorbance of the GDF9 standard solution and the concentration of the GDF9 standard solution as standard curves; comparing the embryo culture solution absorbance with the standard curve to obtain the concentration of GDF9 in the embryo culture solution, and using the concentration of GDF9 to evaluate the embryo development potential.
The results are shown in fig. 2, the GDF9 content in the culture solution of the third day is significantly related to the implantation rate, wherein the culture solution corresponding to the implanted embryo has significantly lower GDF9 content.
4. Detecting the GDF9 content in the live embryo culture solution and the embryo culture solution without live birth: determining the absorbance of the embryo culture solution with good quality and the embryo culture solution with poor quality; determining the absorbance of the culture solution without the embryo, the absorbance of the GDF9 standard solution and the concentration of the GDF9 standard solution as standard curves; comparing the embryo culture solution absorbance with the standard curve to obtain the concentration of GDF9 in the embryo culture solution, and using the concentration of GDF9 to evaluate the embryo development potential.
The results are shown in fig. 3, the GDF9 content in the culture fluid of the third day is significantly correlated with live birth, wherein the culture fluid corresponding to embryos obtained from live birth has significantly lower GDF9 content.
5. GDF9 in the culture fluid of the third day can be used as a marker for non-invasive assessment of the potential for embryo development. During embryo screening, GDF9 content in an embryo culture solution can be sequenced to serve as an auxiliary noninvasive evaluation method.
Example 2A kit for predicting or aiding in the prediction of the potential of embryonic development
Firstly, the components of the kit: detection samples, standard substances, quality control substances and biotin, concentrated washing solution A, GDF-9 experiment buffer solution, biotin conjugate and TMB substrate solution.
II, GDF9 detection method:
1. equilibrating all reagents and samples to room temperature;
2. dissolving standard products B-F (respectively containing 48pg, 222pg, 775pg, 2550pg and 5800pg of recombinant human GDF9 protein) and a quality control product I, II (respectively containing 400pg of recombinant human GDF9 protein 240-containing and 4302pg of recombinant human GDF 9-containing protein 2868-containing) by 1mL of deionized water respectively, dissolving, whirling and uniformly mixing;
3. diluting the concentrated washing solution A (phosphate buffer solution containing nonionic detergent) with deionized water by 25 times;
4. diluting the sample to a proper multiple with a fresh blank culture solution (embryo culture solution in the cleavage stage);
5. marking the microporous strip;
6. respectively adding 50 mu L of calibrator, quality control material and sample into corresponding micropores;
7. add 50. mu.L of GDF-9 assay buffer (phosphate buffer with bovine serum albumin) to each well;
8. incubating for 3 hours at room temperature with shaking on a microplate orbital shaker (600-;
9. the biotin conjugate (protein-based buffer containing the detection antibody biotin complex) was diluted 50-fold 20-30 minutes before the incubation was completed. Accurately sucking 220 mu L of concentrated solution into 11mL of diluted solution by the whole plate operation;
10. washing the plate for 5 times;
11. add 100. mu.L of diluted biotin conjugate per well;
12. incubating with shaking on a microplate orbital shaker (600-;
13. washing the plate for 5 times;
14. add 100. mu.L of streptavidin conjugate per well (ready-to-use);
15. incubation with shaking on a microplate orbital shaker (600-;
16. washing the plate for 5 times;
17. 100 μ L of TMB substrate was added to each well. Attention is paid to avoiding direct sunlight;
18. incubation with shaking on a microplate orbital shaker (600-;
19. add 100. mu.L of stop buffer to each well. Reading with microplate reader at 450nm wavelength within 20 min.
Example 3A method for predicting embryonic development potential for non-therapeutic and diagnostic purposes
1. Selecting a plurality of embryo culture solutions to be implanted into the same person, wherein the source and the period of the embryo culture solutions are the same as those in the embodiment 1;
2. the kit of example 2 is used to detect the content of the growth factor 9 in the embryo culture solution in step 1, and the embryo with the lowest content of the growth factor-9 is selected as the embryo with the highest development potential.
Claims (7)
1. The application of the growth differentiation factor 9 in predicting or assisting in predicting the development potential of embryos.
2. The application of the growth differentiation factor 9 in predicting or assisting in predicting the quality of the embryo.
3. Use according to claim 1 or 2, wherein the embryo is selected at a time period of three days after fertilization.
4. The use according to claim 1 or 2, wherein the growth differentiation factor 9 is detected by a microplate reader.
5. Use according to claim 1 or 2, wherein the method of culturing embryos is as follows: the fertilized embryos are placed into pre-balanced embryo culture drops in the cleavage stage for culture, the culture environment is 37 ℃, and the volume fraction of the culture environment is 5 percent of oxygen and the volume fraction of the culture environment is 6 percent of carbon dioxide.
6. The use of claim 5, wherein the pre-equilibrated embryo culture drop at the cleavage stage is formulated as embryo culture broth G1-plus containing 5% by volume of HSA.
7. A kit for predicting or aiding in the prediction of embryonic development potential, the kit comprising: a sample to be detected, a standard substance, a quality control substance and biotin.
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徐清华等: "单卵母细胞对应的卵丘颗粒细胞中生长分化因子-9和骨形成蛋白-15的表达水平与胚胎发育潜能的研究", 中国优生与遗传杂志, no. 09, pages 120 - 127 * |
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