JP2022538763A - Biomarkers and their uses - Google Patents

Abstract

The present invention uses mtDNA point mutation rate as a biomarker of egg quality and reduces mtDNA point mutation rate in the preparation of medicaments for improving egg quality or treating or preventing female infertility. provide the use of agents for [Selection figure] None

Description

The present disclosure relates to the field of bioengineering, in particular biomarkers and their uses. More particularly, the present disclosure relates to the use of mitochondrial deoxyribonucleic acid (mtDNA) point mutation rate as a biomarker for egg quality, kits, use of reagents in the manufacture of kits, pharmaceutical compositions, reagents in the manufacture of medicaments , methods for screening drugs, and systems for determining egg quality, efficacy of drugs for treating female infertility, or prognosis for patients with female infertility.

Age is one of the important factors affecting a woman's fertility. A woman's fertility usually peaks at age 24 and declines after age 30, rarely becoming pregnant after age 50. In recent years, the phenomenon of infertility and low fertility has been on the rise, and rapid changes in the environment are considered to be the most likely explanation.

Therefore, at this stage, it is of great importance to find new biomarkers specifically indicative of egg quality for the prevention and treatment of infertility.

The present disclosure is based on the findings and knowledge of the inventors in solving the following problems and facts.

The present inventors analyzed clinical reproductive data and found that the oocyte blastocyst formation rate, which is an index of egg quality, was significantly lower in older women than in younger women. It indicates that the quality is significantly lower than that of young women. On the other hand, the present inventors used high-throughput next-generation sequencing to detect mtDNA mutations in young female ova and old female ova discarded by intracytoplasmic sperm injection (ICSI) in assisted reproductive medicine. We found that more mitochondrial DNA point mutations were significantly accumulated in oocytes from older versus younger women. We further analyzed the frequencies of these mtDNA point mutations and found that the mutation frequencies of these mtDNA point mutations were mainly concentrated in the region of (0.002, 0.005). These experimental results indicate that mtDNA point mutation rates, especially low-frequency mtDNA point mutation rates, can be used as markers of oocyte aging.

Thus, in a first aspect, the disclosure in some embodiments provides for the use of mtDNA point mutation rate as a biomarker of egg quality. Through experiments, the inventors found that more mtDNA point mutations were significantly accumulated in low quality oocytes versus high quality oocytes. Therefore, mtDNA point mutation rate can be used as a biomarker of egg quality. In addition, "egg quality" means a high or low quality of ova indicated by the ovum blastocyst formation rate, and the higher the ovum blastocyst formation rate, the higher the ovum quality. The lower the rate, the lower the egg quality. Furthermore, "point mutation rate" means the number of point mutations occurring in the mitochondrial genome obtained after sequencing the mitochondrial genome of the tested egg. We found that the higher the point mutation rate, i.e., the higher the number of point mutations, the lower the egg quality, while the lower the point mutation rate, i.e., the lower the number of point mutations, the lower the egg quality. found high. In some embodiments of the present disclosure, the inventors have found through experimentation that more mtDNA point mutations with a low frequency of 0.005 or less but greater than 0.002 are low for high quality eggs. It was found that it was significantly accumulated in quality oocytes. Therefore, a low frequency mtDNA point mutation rate below 0.005 but above 0.002 is more accurate to use as a biomarker of egg quality.

In a second aspect, the present disclosure in embodiments provides kits for determining egg quality, efficacy of a pharmaceutical for treating female infertility, or prognosis for female infertility patients. According to embodiments of the present disclosure, kits include reagents used to detect mtDNA point mutation rates. As mentioned above, mtDNA point mutation rate can be used as a biomarker of egg quality. Thus, kits containing reagents used to detect mtDNA point mutation rates according to embodiments of the present disclosure may be used to determine the quality of eggs, the efficacy of pharmaceuticals for treating female infertility, or the efficacy of female infertility patients. It can be used to determine prognosis. It should be noted that "female infertility" should be understood broadly and means not only women who are unable to conceive or give birth, but also women who have difficulty becoming pregnant or giving birth.

According to embodiments of the present disclosure, the kit may further comprise at least one of the following additional technical features.

According to embodiments of the present disclosure, the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. As mentioned above, low frequency mtDNA point mutation rates below 0.005 but above 0.002 are more accurate for use as biomarkers of egg quality. Thus, according to embodiments of the present disclosure, kits containing reagents used to detect mtDNA point mutation rates of 0.005 or less, but greater than 0.002 mutation frequency are used to detect egg quality, female infertility It is more accurate for use in determining the efficacy of drugs to treat disease, or the prognosis of female infertility patients.

According to embodiments of the present disclosure, the reagents include at least one selected from primers and probes that specifically detect mtDNA point mutation rates. In some embodiments of the disclosure, the primers have nucleotide sequences set forth in SEQ ID NOs: 1-8. The inventors have found that the reagent has higher precision and sensitivity in detecting mtDNA point mutation rate.

In a third aspect, the present disclosure in embodiments provides use of a reagent for detecting mtDNA point mutation rate in the manufacture of a kit, said kit comprising: egg quality, pharmaceutical for treating female infertility or to determine the prognosis of female infertility patients. As mentioned above, mtDNA point mutation rate can be used as a biomarker of egg quality. Thus, kits prepared with reagents used to detect mtDNA point mutation rates can be used to determine egg quality, the efficacy of pharmaceuticals for treating female infertility, or the prognosis of female infertility patients. can be used for

In embodiments of the present disclosure, the uses described above may further include at least one of the following additional technical features.

According to embodiments of the present disclosure, the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. As mentioned above, low frequency mtDNA point mutation rates below 0.005 but above 0.002 are more accurate for use as biomarkers of egg quality. Thus, according to embodiments of the present disclosure, kits prepared with reagents used to detect mtDNA point mutation rates of 0.005 or less, but greater than 0.002 mutation frequency, include: oocyte quality, It is more accurate for use in determining the efficacy of pharmaceuticals for treating female infertility or the prognosis of female infertility patients.

According to embodiments of the present disclosure, the reagents include at least one selected from primers and probes that specifically detect mtDNA point mutation rates. In some embodiments of the disclosure, the primers have nucleotide sequences set forth in SEQ ID NOs: 1-8. The inventors have found that the reagent has higher precision and sensitivity in detecting mtDNA point mutation rates.

In a fourth aspect, the disclosure in embodiments provides kits. According to embodiments of the present disclosure, the kit includes reagents for reducing mtDNA point mutation rate, and the kit is used to improve egg quality. As mentioned above, the mtDNA point mutation rate can be used as a biomarker of egg quality, the higher the point mutation rate, i.e., the higher the number of point mutations, the lower the egg quality, whereas the higher the point mutation rate, the lower the egg quality. The lower the rate, ie the lower the number of point mutations, the higher the egg quality. Therefore, egg quality can be improved by reducing the mtDNA point mutation rate. Thus, kits containing reagents used to reduce mtDNA point mutation rates according to embodiments of the present disclosure can be used to improve egg quality for scientific research and experiments.

In a fifth aspect, the present disclosure in embodiments provides pharmaceutical compositions for improving egg quality or treating or preventing female infertility. According to embodiments of the present disclosure, the pharmaceutical composition comprises a reagent for reducing mtDNA point mutation rate. As mentioned above, the mtDNA point mutation rate can be used as a biomarker of egg quality, the higher the point mutation rate, i.e., the higher the number of point mutations, the lower the egg quality, whereas the higher the point mutation rate, the lower the egg quality. The lower the rate, ie the lower the number of point mutations, the higher the egg quality. Therefore, by reducing the mtDNA point mutation rate, egg quality can be improved, or female infertility can be treated or prevented. Thus, according to embodiments of the present disclosure, pharmaceutical compositions comprising reagents for reducing mtDNA point mutation rates can be used to improve egg quality or treat or prevent female infertility. .

According to embodiments of the present disclosure, the aforementioned pharmaceutical composition may further comprise at least one of the following additional technical features.

According to embodiments of the present disclosure, the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. As mentioned above, low frequency mtDNA point mutation rates below 0.005 but above 0.002 are more accurate for use as biomarkers of egg quality. Therefore, a pharmaceutical composition comprising a reagent capable of reducing the mtDNA point mutation rate of 0.005 or less but greater than 0.002 mutation frequency will more effectively improve egg quality or female infertility. disease can be treated or prevented.

In a sixth aspect, the disclosure in embodiments provides use of a reagent for reducing mtDNA point mutation rate in the manufacture of a kit for improving egg quality. As mentioned above, the mtDNA point mutation rate can be used as a biomarker of egg quality, the higher the point mutation rate, i.e., the higher the number of point mutations, the lower the egg quality, whereas the higher the point mutation rate, the lower the egg quality. The lower the rate, ie the lower the number of point mutations, the higher the egg quality. Therefore, egg quality can be improved by reducing the mtDNA point mutation rate. Thus, kits prepared with reagents used to reduce mtDNA point mutation rates can be used to improve egg quality for scientific research and experiments.

In a seventh aspect, the present disclosure in embodiments provides use of a reagent for reducing mtDNA point mutation rate in the manufacture of a medicament for improving egg quality or treating or preventing female infertility. do. As mentioned above, the mtDNA point mutation rate can be used as a biomarker of egg quality, the higher the point mutation rate, i.e., the higher the number of point mutations, the lower the egg quality, whereas the higher the point mutation rate, the lower the egg quality. The lower the rate, ie the lower the number of point mutations, the higher the egg quality. Therefore, reducing the rate of mtDNA point mutations can treat or prevent female infertility, which can improve egg quality. Thus, a medicament prepared with a reagent for reducing mtDNA point mutation rate can be used to improve egg quality or to treat or prevent female infertility.

According to embodiments of the present disclosure, the uses described above may further include at least one of the following additional technical features.

According to embodiments of the present disclosure, the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. As mentioned above, low frequency mtDNA point mutation rates below 0.005 but above 0.002 are more accurate for use as biomarkers of egg quality. Therefore, a medicament prepared with a reagent that can reduce the mtDNA point mutation rate of 0.005 or less but greater than 0.002 mutation frequency will more effectively improve egg quality or reduce female infertility. can be treated or prevented.

In an eighth aspect, the present disclosure in embodiments provides methods for screening drugs used to improve egg quality or treat or prevent female infertility. According to an embodiment of the present disclosure, the method comprises contacting an egg derived from a female patient with poor egg quality or infertility with a candidate drug; and comparing, wherein a decrease in the mtDNA point mutation rate of the egg after said contacting relative to before said contacting is an indication that said drug candidate is a drug of interest. As mentioned above, mtDNA point mutation rate can be used as a biomarker of egg quality. Thus, methods according to embodiments of the present disclosure can effectively screen for drugs that can improve egg quality or treat or prevent female infertility. Egg quality is measured according to the common knowledge of those skilled in the art or according to common standards. For example, an egg with a significantly lower blastocyst formation rate than normal standards is a poor quality egg.

According to embodiments of the present disclosure, the aforementioned method may further include at least one of the following additional technical features.

According to embodiments of the present disclosure, the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. As mentioned above, low frequency mtDNA point mutation rates below 0.005 but above 0.002 are more accurate for use as biomarkers of egg quality. Therefore, the methods according to embodiments of the present disclosure can more effectively and accurately screen drugs that can improve egg quality or treat or prevent female infertility.

In a ninth aspect, the present disclosure in embodiments provides a system for determining egg quality, efficacy of a pharmaceutical for treating female infertility, or prognosis for female infertility patients. According to an embodiment of the present disclosure, referring to FIG. 4, the system includes an acquisition device 100 configured to acquire mtDNA point mutation rates of a test sample (eg, an egg); , a determination device 200 configured to determine egg quality, efficacy of a drug for treating female infertility, or prognosis of a female infertility patient based on the mtDNA point mutation rate of the test sample; include.

According to embodiments of the present disclosure, the system described above may further include at least one of the following additional technical features.

According to embodiments of the present disclosure, the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002.

According to embodiments of the present disclosure, a higher mtDNA point mutation rate in a test sample than in a normal sample is indicative of poor oocyte quality in the test sample; A reduction in the mtDNA point mutation rate of a test sample of is indicative of increased efficacy of said pharmaceutical agent in treating female infertility; Comparability with normal samples is indicative of a good prognosis for female infertility patients.

1 is a bar graph showing egg blastocyst formation rates between older and younger women, according to embodiments of the present disclosure; FIG. FIG. 2 is a bar graph showing the number of point mutations in the mitochondrial genome between eggs obtained from aged and young women, respectively, according to embodiments of the present disclosure. FIG. 3 is a bar graph showing the frequency of point mutations in the mitochondrial genome between eggs obtained from aged and young women, respectively, according to embodiments of the present disclosure. FIG. 4 is a block diagram illustrating a system for determining egg quality, efficacy of pharmaceuticals for treating female infertility, or prognosis for female infertility patients, according to an embodiment of the present disclosure;

DETAILED DESCRIPTION OF EMBODIMENTS Embodiments of the present disclosure will now be described in detail. The embodiments described herein with reference to the drawings are illustrative, exemplary, and used to provide an overview of the disclosure. The embodiments are not to be construed as limiting the disclosure.

Embodiment 1
The experimental method was as follows. The present inventors divided the female patients into a young group (30 years old or younger) and an old group (38 years old or older) according to age from collaborators who underwent intracytoplasmic sperm injection (ICSI). of clinical data were analyzed. The oocyte blastocyst formation rates of 157 female patients in the younger group and 103 female patients in the older group who underwent ICSI from May 2017 to March 2018 were systematically analyzed. The experimental results are shown in FIG.

FIG. 1 shows the oocyte blastocyst formation rate for 157 female patients in the younger group and 103 female patients in the older group who underwent ICSI, according to embodiments of the present disclosure. The experimental results showed that the oocyte blastocyst formation rate of the young group was higher than that of the old group, which indicated that the quality of the oocytes of the old female patients was lower than that of the young female patients.

Embodiment 2
The experimental method was as follows. We collected discarded immature oocytes that underwent ICSI from collaborators, 9 from young female patients and 10 from older female patients. 1-4 discarded immature oocytes from each patient, containing 20 μL cell lysis buffer, composed of 17.75 μL H ₂ O, 2 μL 10×KOD buffer, and 0.25 μL proteinase K placed in a centrifuge tube. The lysis procedure was as follows: 55°C for 45 min; 94°C for 5 min; hold at 4°C. 5 μL of egg lysate from each patient was then subjected to amplification of the mitochondrial genome by PCR, using four pairs of primers to generate the 3561-9794 region, 9795-14567 region, and 14562-14562 region of the mitochondrial genome, respectively. The 139 region and the 115-3560 region were amplified. The four regions covered the entire length of the mitochondrial genome. Table 1 shows the primers used. The PCR procedure was as follows: (i) denaturation at 95°C for 5 minutes; (ii) denaturation at 94°C for 30 seconds, annealing at 57°C for 45 seconds, and extension at 68°C (extension time was , was calculated according to the length of the fragment, i.e., 1000 bp per minute.The extension times were 6 min 30 sec for the 3561-9794 region, 5 min for the 9795-14567 region, 2 min for the 14562-139 region, and 115-115 min. 3 min 30 sec at 3560 area.) Step (ii) was set to 40 cycles; (iii) extension at 68°C for 10 min; and (iv) hold at 4°C. The four PCR-amplified fragments were subjected to 1% agarose gel electrophoresis followed by gel extraction. Four fragments were pooled for high-throughput next-generation sequencing to analyze the mutation rate of the mitochondrial genome. Experimental results are shown in FIG.
Table 1: Primers for amplifying small amounts of egg mtDNA

FIG. 2 shows the number of point mutations in the mitochondrial genome between eggs from 9 young female patients and 10 elderly female patients in an embodiment of the present disclosure. Experimental results showed that the number of point mutations in the mitochondrial genome of eggs obtained from elderly female patients was significantly higher than that of young female patients.

Embodiment 3
The experimental method was as follows. We further analyzed the results shown in Figure 2 for the mutation frequency of point mutations in the mitochondrial genome. The experimental results are shown in FIG.

FIG. 3 shows mutation frequencies of point mutations in the mitochondrial genome between eggs from 9 young female patients and 10 elderly female patients, in an embodiment of the present disclosure. The experimental results show that the mutation frequencies of point mutations in the mitochondrial genome of eggs obtained from elderly patients are mainly located between (0.002, 0.005), and the frequencies of eggs obtained from elderly female patients are within the above range. The number of certain point mutations was shown to be significantly higher than in younger female patients.

Throughout this specification, "an embodiment," "some embodiments," "one embodiment," "another example," "an example," "a particular example," or "some The phrase "example" means that a particular feature, structure, material, or property described in connection with an embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, “in some embodiments,” “in one embodiment,” “in an embodiment,” “in another example,” “in examples,” “in certain examples,” or “in some examples.” Phrases such as"in various places throughout this specification are not necessarily all referring to the same embodiment or example of the disclosure. Moreover, the particular features, structures, materials, or properties may be combined in any suitable manner in one or more embodiments or examples. Furthermore, a person skilled in the art can combine different embodiments or implementations and features of different embodiments or implementations without contradicting each other.

Although illustrative embodiments have been shown and described, such embodiments cannot be construed as limiting the present disclosure and alterations, substitutions, and modifications may be made to the embodiments within the scope of the present disclosure. can be done.

100: Acquisition device 200: Determination device

Claims (12)

1. The use of mitochondrial deoxyribonucleic acid (mtDNA) point mutation rate as a biomarker of egg quality, comprising:
Optionally, use characterized in that the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. A kit for determining egg quality, the efficacy of a drug for treating female infertility, or the prognosis of a female infertility patient, comprising reagents used to detect mtDNA point mutation rates. A kit characterized by: the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002;
optionally, the reagent includes at least one selected from primers and probes that specifically detect the mtDNA point mutation rate;
Optionally, the kit of claim 2, wherein said primers have nucleotide sequences set forth in SEQ ID NOS: 1-8. Use of a reagent for detecting mtDNA point mutation rate in the manufacture of a kit, wherein the kit determines egg quality, the efficacy of a pharmaceutical for treating female infertility, or the prognosis of female infertility patients A use characterized in that it is used to the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002;
optionally, the reagent includes at least one selected from primers and probes that specifically detect the mtDNA point mutation rate;
Optionally, the use according to claim 4, wherein said primers have the nucleotide sequences shown in SEQ ID NOS: 1-8. A kit comprising reagents for reducing mtDNA point mutation rate, characterized in that the kit is used to improve egg quality. 1. A pharmaceutical composition for improving egg quality or treating or preventing female infertility, comprising a reagent for reducing mtDNA point mutation rate,
Optionally, a pharmaceutical composition characterized in that the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. Use of reagents for reducing mtDNA point mutation rates in the manufacture of kits for improving egg quality. Use of a reagent for reducing mtDNA point mutation rate in the manufacture of a medicament for improving egg quality or treating or preventing female infertility, comprising:
Optionally, use characterized in that the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. A method for screening a drug used to improve egg quality or to treat or prevent female infertility, comprising:
contacting an egg from a female patient with poor egg quality or infertility with a drug candidate;
comparing the mtDNA point mutation rate of said egg before and after said contacting;
A decrease in the mtDNA point mutation rate of the egg after the contact compared to before the contact is an indicator that the candidate drug is a target drug,
Optionally, the method characterized in that the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. 1. A system for determining egg quality, the efficacy of a pharmaceutical for treating female infertility, or the prognosis of a female infertility patient, comprising:
an acquisition device configured to acquire the mtDNA point mutation rate of a test sample;
connected to the acquisition device to determine the quality of the egg, the efficacy of the pharmaceutical for treating the female infertility, or the prognosis of the female infertility patient based on the mtDNA point mutation rate of the test sample; and a determination device configured to
Optionally, the system characterized in that the mutation frequency of mtDNA point mutations is less than or equal to 0.005 but greater than 0.002. a higher mtDNA point mutation rate in the test sample than in a normal sample is indicative of poor egg quality in the test sample; or
a reduction in the mtDNA point mutation rate of the test sample after treatment with the pharmaceutical agent relative to prior to treatment with the pharmaceutical agent is indicative of increased efficacy of the pharmaceutical agent in treating female infertility; or
Comparability of the mtDNA point mutation rate of the test sample to the normal sample after treatment with the pharmaceutical agent relative to prior to treatment with the pharmaceutical agent is indicative of a favorable prognosis for the female infertility patient. 12. The system of claim 11.

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