Application of oxibenzophenone in preparation of medicine for improving human early embryonic block

Abstract

The invention relates to the field of medicines, and provides an application of oxibenzophenone in preparation of a medicine for improving early embryonic retardation of human beings. Specifically, the invention creatively discloses a key regulatory factor of human embryo cleavage stage development retardation and a specific mechanism thereof, provides a potential treatment target H3K27ac, and designs an effective treatment scheme, namely the epoxybenzophenone, aiming at the target, and a result shows that the epoxybenzophenone can effectively improve the embryonic development retardation and has a good treatment effect on the embryonic cleavage stage development retardation. And a reliable solution path is provided for improving the success rate of the assisted reproduction technology.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular, to the use of ethyphenone in preparing medicines for improving the blockage of early human embryonic development. Background Art

[0002] The probability of human infertility is 16.7%, and assisted reproductive technology is the most effective treatment method. However, clinical treatment has found that the blastocyst formation rate after assisted reproductive technology treatment is only about 45%, and 60% of 8-cell embryos with normal morphology fail to continue to develop to the blastocyst stage. There is still no effective technology to solve this problem.

[0003] Assisted reproductive technology is an effective method for treating infertility. With the emergence of sequential culture medium, single culture medium and co-culture methods have been replaced, and the in vitro embryo culture time can be extended to a more stable and physiological blastocyst. However, the human blastocyst formation rate is only about 45%, which is much lower than the blastocyst formation rate of mice and pigs (80%). Most of the embryos are blocked at the 8-cell stage, which indicates that the culture conditions of early human embryos need to be optimized. However, the exact cause of the developmental block remains unknown.

[0004] In line with this, the main ZGA (zygotic genome activation) in humans occurs at the 8-cell stage. Since ZGA and maternal mRNA degradation coordinate with each other to complete MZT (maternal-zygotic transition) and prepare for subsequent lineage differentiation, the MZT regulatory mechanism may be a breakthrough in revealing the cause of developmental arrest during the cleavage stage of human embryos. However, the regulatory network of human MZT is still unclear.

[0005] A 2022 study suggested that SIRT (acetyltransferase sirtuin family) activators resveratrol or nicotinamide riboside can partially rescue the phenotype of human embryonic cleavage stage developmental arrest. However, in this study, only a few of the human cleavage stage developmental arrest embryos treated with resveratrol or nicotinamide riboside redeveloped, and the redeveloped embryos were still unable to return to the normal developmental state and failed to successfully complete the subsequent developmental process. Some of the redeveloped embryos still had dysregulation of ribosome and protein translation, indicating that the phenotype of cleavage stage embryonic developmental arrest was not completely rescued. The effect of nicotinamide riboside treatment was similar to that described above. In addition, RNA sequencing results showed that resveratrol treatment improved the metabolic levels of TypesII and TypesIII developmental arrest embryos by activating SIRT, as manifested by increased levels of glycolysis and oxidative phosphorylation, but did not reach the metabolic levels of normal E4-E5 embryos.

[0006] Therefore, to date, the pathological mechanism of early human embryonic developmental arrest has not been fully elucidated. The existing technology targets embryos that have undergone developmental arrest and have been cultured in vitro for several additional days. Therefore, the pathological mechanism targeted by the proposed treatment options is relatively vague, which may limit the formulation of treatment options and the treatment effect. Summary of the invention

[0007] In order to fill the defects of the prior art, the present invention proposes for the first time to use ethoxybenzone to improve the developmental block of human early embryos. Specifically, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides the use of ethoxybenzone in the preparation of a drug for improving developmental arrest in early human embryos, wherein the embryo is a zygote on day 0 of development, and the developmental arrest refers to a developmental arrest state exhibited at the transcriptional level by the embryo at the 8-cell stage.

[0009] In one embodiment, the administration of exiphenone is carried out by dissolving exiphenone in DMSO at a concentration of 1 M and adding it to the culture medium of the zygotes at a concentration of 0.02 uM.

[0010] In one embodiment, the effect of ethoxybenzone on improving human early embryonic development arrest is to reduce the embryonic acetylation level, increase the number of embryonic blastomeres, and increase the embryonic compaction rate to improve the embryonic development phenotype.

[0011] The second aspect of the present invention provides the use of ethoxybenzone in the preparation of a drug for improving the development rate of human early embryos, wherein the embryo is an embryo that exhibits a developmental arrest state at the transcriptional level at the 8-cell stage.

[0012] In one embodiment, the administration of exiphenone is carried out by dissolving exiphenone in DMSO at a concentration of 1 M and adding it to the culture medium of the zygotes at a concentration of 0.02 uM.

[0013] Compared with the prior art, the present invention has the following significant improvements:

[0014] The present invention innovatively reveals the key regulatory factors and specific mechanisms of developmental arrest during the cleavage stage of human embryos, proposes potential therapeutic targets, and designs effective treatment plans for these targets, achieving breakthrough progress. This will help solve the problem of developmental arrest during the cleavage stage of human in vitro cultured embryos and improve the success rate of assisted reproductive technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0016] Figure 1 The expression levels of DPRX and ARGFX were significantly downregulated in developmentally arrested 8 cells;

[0017] Figure 2 Figure 3 shows the embryonic development after microinjection of siRNA into human 3PN zygotes to knock down DPRX and ARGFX simultaneously (double knockdown, dKD). Figure 2 A shows the embryonic development phenotypes of the dKD group and the control group. Figure 2 B: The expression of genes related to zygotic genome activation was significantly downregulated in embryos of the dKD group. Figure 2 C shows abnormal degradation of maternal factors in embryos of the dKD group.

[0018] Figure 3 After microinjection of siRNA into human 3PN zygotes to simultaneously knock down DPRX and ARGFX (double knockdown, dKD), abnormal activation of acetylation occurred. Figure 3 A shows that the expression of deacetylase-related genes (HDAC1, HDAC2, SIRT1) is downregulated in dKD embryos; Figure 3 B H3K27ac immunofluorescence results of the group without simultaneous knockdown of DPRX and ARGFX (dKD) and the control group (siNC) (left); quantitative analysis of H3K27ac immunofluorescence intensity (right), Scale bars, 20μm.

[0019] Figure 4 The results of the drug-treated group and the solvent control group are compared. Figure 4 A is a representative H3K27ac immunofluorescence image of the exifone drug treatment group and the solvent control group (DMSO treatment group) (left); quantitative analysis of H3K27ac immunofluorescence intensity (right), Bars, 20μm; Figure 4 B is the developmental phenotype of dKD embryos in the exifone drug treatment group and the solvent control group; Figure 4 C shows the developmental phenotypes of NC embryos in the exifone drug-treated group and the solvent control group. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0021] Example

[0022] Embryonic development culture system: The embryos were cultured in G-1plus medium (Cat. No.: Vitrolife, 10128) at 37°C and 6% CO2, and transferred to G-2plus medium (Cat. No.: Vitrolife, 10132) for culture on Day 3 (the third day of embryonic development).

[0023] Embryo morphology evaluation indicators: According to the Chinese expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts, the number of blastomeres, the degree of fragmentation, and whether compaction has occurred in the embryo are observed under a microscope to evaluate the quality of embryo development.

[0024] First, we collected oocytes and sperm donated by healthy volunteers and obtained embryos through intracytoplasmic sperm injection (ICSI). We performed single-cell full-length transcriptome sequencing on 38 embryos from 6 consecutive stages, from zygote to blastocyst (day 1 to day 6 of embryonic development), and mapped the transcriptional profile of human preimplantation embryos. Pseudo-sequential analysis showed that some morphologically normal 8-cell embryos showed a developmental arrest at the transcriptional level, and their developmental trajectory was significantly delayed compared with normal 8-cell embryos, and closer to 4-cell embryos.

[0025] Next, we performed differentially expressed gene analysis on these developmentally arrested embryos and normal 8-cell stage embryos. The results showed that 1,325 of the upregulated genes in the developmentally arrested embryos were maternal factors, accounting for 24% of the maternal factors; 1,050 of the downregulated genes in the transcriptionally arrested embryos were major ZGA (zygotic genome activation) genes, accounting for 65% of the major ZGA genes, proving that MZT (maternal-zygotic transition) abnormalities exist in these developmentally arrested embryos. We define these embryos with normal morphology but actually in a state of developmental arrest as TAS embryos (transcriptionally arrested status). Further analysis found that the two key transcription factors that cause MZT abnormalities are DPRX and ARGFX, whose expression levels are significantly downregulated in the developmentally arrested 8-cells ( Figure 1 ).

[0026] To explore whether DPRX and ARGFX are the key causes of developmental arrest of human cleavage-stage embryos, we collected a total of 95 discarded 3PN embryos (informed consent had been signed by the donating patients) during clinical diagnosis and treatment at the Reproductive Medicine Center of Peking University Third Hospital and divided them into a negative control group (49 embryos) and a dKD group (46 embryos). At the same time, both groups of human 3PN zygotes (developmental day 1) were microinjected. The negative control group was only injected with the solvent (labeled as siNC, control group), while the dKD group was injected with siRNA that could simultaneously knock down DPRX and ARGFX, and the developmental phenotypes of the two groups of 3PN embryos were observed.

[0027] The results showed that the development quality of 3PN embryos in the dKD group was significantly low, and there was a developmental block in the cleavage stage. Specifically, on the fourth day of embryonic development (Day 4), the embryo compaction rate decreased significantly, the number of cells decreased significantly, and the fragmentation rate increased ( Figure 2 A). Single-cell RNA sequencing analysis of the two groups of 3PN embryos revealed that ZGA-related gene activation was inhibited and maternal degradation was abnormal in dKD embryos, indicating that their MZT process was abnormal ( Figure 2 BC). Further analysis revealed that the expression of deacetylase-related genes (HDAC1, HDAC2, SIRT1) was downregulated ( Figure 3 A). Immunofluorescence staining showed ( Figure 3 B) The signal level of H3K27ac (acetylation of lysine 27 of histone H3) in the knockdown embryos was significantly higher than that in the control group, indicating that the H3K27ac deacetylation process that should have occurred during the ZGA stage was abnormal in the dKD embryos, and H3K27ac was abnormally retained.

[0028] Given that exifone can activate HDAC1 and promote H3K27ac deacetylation, we further verified whether reversing H3K27ac retention could rescue the phenotype of cleavage stage developmental arrest in dKD embryos. Based on this, we further set up exifone drug treatment groups and solvent control groups for the dKD group (52 embryos) and the negative control group (21 embryos), that is, the dKD group (52 embryos) was divided into dKD exifone drug treatment group (n=25) and dKD solvent control group (n=27). The negative control group (21 embryos) was divided into negative control exifone drug treatment group (n=11) and negative control solvent control group (n=10).

[0029] The dosage is:

[0030] Drug treatment group: exifone (Cat. No.: TCI America, H17830) was dissolved in DMSO (Cat. No.: Sigma-Aldrich, D2650) at a concentration of 1 M and stored at -20°C; it was added to G-1plus or G-2plus culture medium at a concentration of 0.02 uM;

[0031] Solvent control group: an equal amount of DMSO solvent was added to the culture medium.

[0032] The results showed that compared with the two solvent control groups, the acetylation levels of embryos in the two drug-treated groups were significantly reduced ( Figure 4 A). At the same time, the number of embryonic blastomeres and the compaction rate of the two drug-treated groups increased ( Figure 4 B), which strongly suggests that abnormal retention of H3K27ac is the direct cause of embryonic developmental arrest and that reversing H3K27ac retention can rescue the phenotype of human cleavage-stage embryonic developmental arrest.

[0033] Considering that some of the normally developed embryos collected above have normal morphology but are actually in a state of developmental arrest, we further applied the study to these NC embryos that have not undergone double gene knockdown but may actually be in a state of developmental arrest. Figure 4 C, which shows the same results as the double gene knockdown 3PN embryos.

[0034] In summary, we used human 3PN embryos for research and found that exifone drug treatment can significantly increase the number of embryonic blastomeres and improve the embryonic development phenotype, proving that reversing the abnormal retention of H3K27ac can effectively improve the human embryonic development rate.

[0035] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The use of ethoxybenzone in the preparation of a drug for improving human early embryonic development block, characterized in that: The embryo is a zygote on the 0th day of development, and the developmental arrest refers to the embryo being in a developmental arrest state at the transcriptional level at the 8-cell stage.

2. The use according to claim 1, characterized in that The administration method of the ethyphenone is to dissolve ethyphenone in DMSO at a concentration of 1 M and add it to the culture medium of the zygotes at a concentration of 0.02 uM.

3. The use according to claim 1, characterized in that: The effect of ethoxybenzone on improving early human embryonic development block is to reduce the level of embryonic acetylation, increase the number of embryonic blastomeres, and increase the embryonic compaction rate to improve the embryonic development phenotype.

4. The embryo is an embryo that exhibits a developmental arrest state at the transcriptional level at the 8-cell stage.

5. The use according to claim 1, characterized in that The administration method of the ethyphenone is to dissolve ethyphenone in DMSO at a concentration of 1 M and add it to the culture medium of the zygotes at a concentration of 0.02 uM.

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