Application of ethoxybenzone in the preparation of drugs for improving human early embryonic development arrest

Abstract

The present invention relates to the field of medicine and provides a method for preparing a drug for ameliorating early human embryonic developmental arrest. Specifically, the present invention innovatively reveals the key regulatory factors and mechanisms of cleavage-stage developmental arrest in human embryos, identifies H3K27ac as a potential therapeutic target, and designs an effective treatment for this target—ameliorating acetaminophen. Results demonstrate that ameliorating acetaminophen can effectively ameliorate embryonic developmental arrest, providing a reliable solution for improving the success rate of assisted reproductive technology.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of ethyphenidone in preparing a medicine for improving the developmental arrest of human early embryos. Background Art

[0002] The incidence of human infertility is 16.7%, and assisted reproductive technology is the most effective treatment. However, clinical practice has shown that the blastocyst formation rate after assisted reproductive technology is only around 45%, and 60% of morphologically normal 8-cell embryos fail to develop to the blastocyst stage. Currently, there is no effective technology to address this problem.

[0003] Assisted reproductive technology is an effective treatment for infertility. With the advent of sequential culture media, single-medium culture and co-culture methods have been replaced, allowing in vitro embryo culture time to be extended to a more stable and physiological blastocyst state. However, the human blastocyst formation rate is only around 45%, far lower than the blastocyst formation rate of mice and pigs (80%). Most of these embryos are arrested at the 8-cell stage, indicating that the culture conditions for early human embryos need to be optimized. However, the exact cause of the developmental arrest remains unknown.

[0004] Consistent with this, the primary human ZGA (zygotic genome activation) occurs at the 8-cell stage. Because ZGA coordinates with maternal mRNA degradation to complete the maternal-zygotic transition (MZT), preparing for subsequent lineage differentiation, the MZT regulatory mechanism may offer a breakthrough in uncovering the etiology of cleavage-stage developmental arrest in human embryos. However, the regulatory network underlying the human MZT remains unclear.

[0005] A 2022 study suggested that the SIRT (acetyltransferase sirtuin family) activators resveratrol or nicotinamide riboside can partially rescue the phenotype of cleavage-stage developmental arrest in human embryos. 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 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 Types II and Types III developmental arrest embryos by activating SIRT, which was 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 developmental arrest in early human embryos has not been fully elucidated. Existing technologies target embryos that have undergone developmental arrest and have been cultured in vitro for several additional days. Therefore, the pathological mechanisms targeted by the proposed treatment plans are relatively vague, which may limit the formulation of treatment plans and the treatment effects. Summary of the Invention

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

[0008] The first aspect of the present invention provides the use of oxaliplatin in the preparation of a drug for improving developmental arrest in early human embryos, wherein the embryo is a zygote on developmental day 0, 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 ethyphenidone is carried out by dissolving ethyphenidone in DMSO at a concentration of 1 M and adding the ethyphenidone to the culture medium of the zygote at a concentration of 0.02 uM.

[0010] In one embodiment, the effect of oxaliplatin on improving the developmental arrest of early human embryos is to reduce the acetylation level of embryos, increase the number of embryonic blastomeres, and increase the embryo 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 ethyphenidone is carried out by dissolving ethyphenidone in DMSO at a concentration of 1 M and adding the ethyphenidone to the culture medium of the zygote at a concentration of 0.02 uM.

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

[0014] This study innovatively reveals the key regulatory factors and mechanisms underlying cleavage-stage developmental arrest in human embryos, identifies potential therapeutic targets, and designs effective treatments targeting these targets, achieving breakthrough progress. This research will help address the issue of cleavage-stage developmental arrest in human embryos cultured in vitro 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 2: Human 3PN zygotes microinjected with siRNA to simultaneously knock down DPRX and ARGFX (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, 20 μm.

[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-treated group and the solvent control group (DMSO-treated group) (left); quantitative analysis of H3K27ac immunofluorescence intensity (right), Bars, 20 μm; Figure 4 B shows the developmental phenotypes of dKD embryos in the exifone drug-treated group and the solvent control group; Figure 4 C shows the developmental phenotypes of NC embryos in the exifone-treated group and the solvent control group. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention are described below with reference to 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: Embryos were cultured in G-1 plus medium (Cat. No.: Vitrolife, 10128) at 37°C and 6% CO2. On Day 3 (the third day of embryonic development), embryos were transferred to G-2 plus medium (Cat. No.: Vitrolife, 10132) for culture.

[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, degree of fragmentation, and whether compaction has occurred in the embryo are observed under a microscope to evaluate the quality of embryonic development.

[0024] First, embryos were obtained by intracytoplasmic sperm injection (ICSI) using oocytes and sperm donated by healthy volunteers. We performed single-cell, full-length transcriptome sequencing on 38 embryos from six consecutive stages, from zygote to blastocyst (days 1 to 6 of embryonic development), to map the transcriptional profiles of alleles in human preimplantation embryos. Pseudo-sequential analysis revealed that some morphologically normal 8-cell embryos exhibited a developmental arrest at the transcriptional level, with developmental trajectories significantly delayed compared to normal 8-cell embryos and closer to those of 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 developmentally arrested state 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 investigate whether DPRX and ARGFX are the key causes of developmental arrest in human cleavage-stage embryos, we collected a total of 95 discarded 3PN embryos (informed consent was signed by the donating patients) from the clinical diagnosis and treatment process of the Reproductive Medicine Center of Peking University Third Hospital. We divided them into a negative control group (49 embryos) and a dKD group (46 embryos). At the same time, we performed microinjections on both groups of human 3PN zygotes (developmental day 1). The negative control group was injected with only the solvent (labeled as siNC, control group), while the dKD group was injected with siRNA that could simultaneously knock down DPRX and ARGFX. 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 poor, with cleavage stage developmental arrest, specifically manifested by a significant decrease in the embryo compaction rate, a significant decrease in the number of cells, and an increase in the fragmentation rate on the fourth day of embryonic development (Day 4). 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 H3K27ac (acetylation of lysine 27 of histone H3) signal level in knockdown embryos was significantly increased compared with the control group, indicating that the H3K27ac deacetylation process that should occur during the ZGA stage is abnormal in dKD embryos, resulting in abnormal retention of H3K27ac.

[0028] Given that exifone activates HDAC1 and promotes H3K27ac deacetylation, we further verified whether reversing H3K27ac retention could rescue the cleavage-stage developmental arrest phenotype 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 negative control group (21 embryos). That is, the dKD group (52 embryos) was divided into a dKD exifone drug treatment group (n=25) and a dKD solvent control group (n=27). The negative control group (21 embryos) was divided into a negative control exifone drug treatment group (n=11) and a 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 medium at a concentration of 0.02 μM;

[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 increased in the two drug-treated groups ( Figure 4 B), which strongly suggests that abnormal H3K27ac retention is the direct cause of embryonic developmental arrest and that reversing H3K27ac retention can rescue the phenotype of developmental arrest in human cleavage-stage embryos.

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

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

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

Claims

1. Use of oxaliplatin in the preparation of a drug for improving infertility caused by human early embryonic development block, characterized in that: The embryo is a zygote on the 0th day of development. The developmental arrest refers to the embryo being in a developmental arrest state at the transcriptional level at the 8-cell stage. The human early embryonic developmental arrest is caused by abnormal retention of H3K27ac.

2. The use according to claim 1, characterized in that The administration method of the ethyphenidone is to dissolve the ethyphenidone 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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