OVARIAN RESERVE BIOMARKER AND USE THEREOF

Abstract

According to a composition and a kit for diagnosing ovarian reserve, and a method of providing information for diagnosing ovarian reserve by using the same, according to an aspect, the development of follicles may be predicted early. In addition, by predicting not only the number of oocytes but also the quality of oocytes, a comprehensive diagnosis of ovarian reserve may be made.

Claims

1. A composition for diagnosing ovarian reserve, comprising an agent capable of measuring expression levels of miR-145-5p or miR-425-5p.

2. The composition of claim 1, wherein the ovarian reserve indicates whether or not there is activation of primordial follicles, production of ovarian hormones, or ovarian hypofunction.

3. The composition of claim 1, wherein the agent is a nucleic acid comprising a polynucleotide identical to or complementary to the nucleic acid sequence of the miRNA.

4. The composition of claim 3, wherein the nucleic acid is a primer or a probe.

5. The composition of claim 1, wherein the miRNA stimulates a bone morphogenetic protein (BMP) signaling pathway.

6. A kit for diagnosing ovarian reserve, comprising an agent capable of measuring expression levels of miR-145-5p or miR-425-5p.

7. A method of detecting a marker to provide information for diagnosing ovarian reserve, comprising: measuring an expression level of miR-145-5p or miR-425-5p in a subject suspected of having ovarian hypofunction; and comparing the measured expression level with an expression level of a gene of a normal control group.

8. The method of claim 7, wherein the ovarian reserve indicates whether or not there is activation of primordial follicles, production of ovarian hormones, or ovarian hypofunction.

9. The method of claim 7, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, sheep, a cow, a goat, or a pig.

10. The method of claim 7, wherein the measuring comprises incubating a nucleic acid comprising a polynucleotide identical to or complementary to the nucleic acid sequence of the gene.

11. The method of claim 7, wherein the measuring is performed by electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, microarray, northern blotting, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCT), real-time PCR, or a combination thereof.

12. The method of claim 8, further comprising, determining that the subject has high ovarian reserve or high activation of ovarian function, when the measured expression level of the miR-145-5p gene is decreased compared to the measured expression level in the normal control group; or when the measured expression level of the miR-425-5p gene is increased compared to the measured expression level in the normal control group.

13. The method of claim 8, further comprising, determining that the subject has a low risk of early ovarian failure, when the measured expression level of the miR-145-5p gene is decreased compared to the measured expression level in the normal control group, or when the measured expression level of the miR-425-5p gene is increased compared to the measured expression level in the normal control group.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0057] FIG. 1 shows a schematic diagram of a plan of stem cell treatment for functional recovery of aged ovaries.

[0058] FIGS. 2A and 2B show images for analyzing implantability in the ovary of human placenta-derived mesenchymal stem cells (hPD-MSCs) injected into aged mouse models.

[0059] FIG. 3A shows a graph showing numbers of primary follicles after stem cell treatment; FIG. 3B is a graph showing E2 expression after stem cell treatment; and FIG. 3C is a graph showing changes of a marker of early follicle development after stem cell treatment.

[0060] FIGS. 4A and 4B show graphs and tables for analyzing circulating miRNAs related to early follicle development, based on miRNA-Seq.

[0061] FIG. 5A shows a table showing the target of miR-145-5p; FIG. 5B shows a graph identifying changes in protein expression due to a change in miRNA expression; FIG. 5C shows a table showing the miR-425-5p target; and FIG. 5D shows an image for analyzing an effect of selected miRNA on the bone morphogenetic protein (BMP) signaling pathway related to activation of primordial follicles.

[0062] FIG. 6 is a schematic diagram showing the principle by which changes in the expression of miR-145 and miR-425 by stem cell treatment stimulate the BMP signaling pathway and the activation of primordial follicles.

MODE OF DISCLOSURE

[0063] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these examples.

Example 1. Selection and Identification of Biomarkers for Ovarian Reserve or Premature Ovarian Failure

1. Stem Cell Treatment for Restoring Function of Aged Ovaries

[0064] As shown in FIG. 1, 5×10.sup.5 human placenta-derived mesenchymal stem cells were injected through the tail vein of naturally aged 52-week-old female mice three times at 10-day intervals, and groups at week 1, week 2, week 3, and week 5 were defined as stem cell treatment experimental groups (hereinafter referred to as “experimental groups”), and a group to which the same amount of PBS was injected under the same conditions was defined as a control group, and the groups were analyzed for comparison.

[0065] Human placenta-derived mesenchymal stem cells injected into aged mice were stained with PKH67 to confirm whether the stem cells were settled in the ovary, as shown in FIG. 2A. PKH67 staining was performed immediately before cell injection using PKH67 Green Fluorescent Cell Linker Kits (Sigma-Aldrich). The extracted ovaries were washed with Dulbecco's phosphate-buffered saline (DPBS) and then fixed with 4% paraformaldehyde. After making a frozen block at the optimal cutting temperature (OCT), frozen sections with a thickness of 12 μm were prepared. The prepared frozen sections were fixed and counter stained with 4′,6-diamidino-2-phenylindole (DAPI) and then, the presence of human placenta-derived mesenchymal stem cells in the aged ovary was identified by using a confocal microscope.

[0066] As a result, as shown in FIG. 2B, it was confirmed that the human placenta-derived mesenchymal stem cells stained with PKH67 were settled in the ovary from week 1 after injection, and were also observed to be settled around the follicle. Also, human placenta-derived mesenchymal stem cells could be observed in the samples of the mice of the experimental group at week 5 after injection.

[0067] The above results indicate that when stem cells are injected through the tail vein of aged mice three times at 10-day intervals, the injected cells are well settled in the ovaries.

[0068] Next, to analyze efficacy of the human placenta-derived mesenchymal stem cells settled in the aged ovary, the number of follicles and the levels of hormones in the blood were analyzed.

[0069] The ovaries, extracted to identify the number of follicles, were fixed by using 4% paraformaldehyde, and then paraffin blocks were prepared. The entire ovary was cut to a thickness of 7 μm to make paraffin sections. In this regard, the tissue sections were sequentially attached to polarized slides. The slides of the 10th slide number were selected and subjected to deparaffinization and fixation, followed by hematoxylin and eosin (H&E) staining. The stained slides were analyzed for the number of follicles according to the stage of follicle development (primordial follicle, primary follicle, secondary follicle, and antral follicle) using an optical microscope. Classification of follicles according to developmental stages is as follows. A primordial follicle is in a form in which several flattened granulosa cells surround an oocyte, a primary follicle is a follicle in which granulosa cells are transformed to cuboidal granulosa cells as the primordial follicle starts to grow. Secondary follicles are follicles into which primary follicles have grown, and are follicles in which granulosa cells have increased to two layers or more, while theca cells begin to appear outside the granulosa cells. The number of granulosa cells continues to increase, and when the cells grow beyond a certain level, an antrum is formed in the follicle, which is called an antral follicle.

[0070] E2 and AMH were measured in the serum. After fasting the mice for 12 hours, breathing anesthesia was performed with CO.sub.2 gas, blood was collected from the abdominal aorta by using a syringe, and a serum separation tube (polyethylene tube) was used for various blood biochemical tests. Serum was separated from the collected blood using a centrifuge within 40 minutes after blood collection. For measurement of hormones, the serum was separated by 300 μl and stored at −20° C. E2 concentration in the serum was measured by using Elecsys® Estradiol III (Roche Diagnostics GmbH), and the AMH concentration was measured by using Elecsys® AMH immunoassay (Roche Diagnostics GmbH). Values of both hormones were measured by using the cobas 6000 system (Roche Diagnostics GmbH), and a concentration of each hormone in the serum was calculated by using the standard curve of the standard substance.

[0071] As a result, as shown in FIG. 3A, after repeated injection of human placenta-derived mesenchymal stem cells three times, it was observed that the number of primary follicles more than doubled in the week-2 and week-3 groups.

[0072] In addition, as a result of comparing E2 produced in the ovaries in the experimental group and the control group, as shown in FIG. 3B, E2 was observed to be higher in the blood of mice injected with stem cells. In addition, as shown in FIG. 3C, it was also observed that the concentration of AMH, which is a marker of early follicle development, was significantly increased in the week-2 and week-3 groups after stem cell injection.

[0073] The above results indicate that the human placenta-derived mesenchymal stem cells settled in the aged ovary activate the dormant primordial follicles. That is, when human placenta-derived mesenchymal stem cells settle in the aged ovary, the stem cells were confirmed to improve ovarian reserve by increasing development of early follicles, and a phenotype of enhanced ovarian function was observed through production of ovarian hormones.

2. Selection of Circulating miRNA Biomarkers

[0074] miRNA-Seq was performed, in order to select circulating miRNAs related to early follicle development. More specifically, based on the data of Example 1, it was confirmed that the number of primary follicles in the plasma of the mice group at week 2 after stem cell injection sharply increased, and based on this, the plasma of the mice in the experimental group at week 2 after injection was collected and miRNA-Seg was performed.

[0075] Circulating miRNAs were isolated from serum of the animals at week 2 after injection of human placenta-derived mesenchymal stem cells using a miRNeasy serum/plasma kit (Qiagen). eBiogen Co., Ltd. was requested to perform the miRNA-seq. Briefly, concentration of circulating miRNA was measured using a trace spectrophotometer (ND 2000; Nano Drop), and then adjusted to 200 ng, before applying the miRNA to the experiment. Library construction was performed by using a multiple small RNA library prep kit (NEB), and miRNA-seq was performed by using IIlumina NextSeq500 (IIlumina) and Illumina SE75 (IIlumina). Thereafter, using Excel-based miRNA-seq data analysis (ExDEGA) tool provided by eBiogen, circulating miRNAs which are expressed significantly (p<0.05) differently by two-fold or more compared to the control group were selected.

[0076] Circulating miRNA was isolated from 200 μl of serum by using an miRNeasy serum/plasma kit (Qiagen), and during the isolation process, cel-miR-39 mimic (1.6×10.sup.8 copies/μl; Qiagen) was added and used as an external spike-in control. The extracted circulating miRNA was reverse transcribed using a HB_I RT Reaction kit (Heimbiotek). All quantitative real-time RT-PCR analyses were performed by using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and a Nucleic mix II kit. The PCR reactant was made by using HB miR Multi assay kit system I, and PCR was performed under the reaction cycle conditions of 15 minutes at 95° C., followed by 40 cycles of 10 seconds at 95° C., and single fluorescence measurement for 40 seconds at 60° C. The primers used in the experiment were purchased from Heimbiotek, and the measured miRNA expression level was corrected by the expression level of cel-miR-39.

[0077] As a result, as shown in FIG. 4A, various circulating miRNAs that changed after stem cell injection into aged mice could be identified, and among them, underexpressed miR-145-5p and overexpressed miR-425-5p compared to the control group were selected in the serum of the groups injected with stem cells.

[0078] Next, quantitative real-time RT-PCR was performed to verify expression of the selected circulating miRNA. As a result, as shown in FIG. 4B, it was confirmed that expression of miRNA-145-5p was decreased at week 2 and week 3 after injection of human placenta-derived mesenchymal stem cells, and expression of miRNA-425-5p was increased.

3. Confirmation of Stimulation of BMP Signaling Pathway of Selected Biomarkers

[0079] In order to confirm the subgenes of the circulating miRNA selected in Example 1 above, the results were analyzed using various methods of bioinformatics (computational algorithms) such as miRWalk, TargetScan, miRmap, and miRanda.

[0080] As a result, as shown in FIG. 5A, miR-145-5p was found to target genes belonging to the TGFβ superfamily (Acvr1b, Acvr2a, Bmpr2, Tgfbr2, Smad1, Smad3). The pathways of the TGFβ superfamily is largely divided into the TGFβ signaling pathway and the BMP signaling pathway, and it was confirmed that the miR-145-5p targets both of these pathways.

[0081] Changes in the expression of sub-proteins due to changes in the expression of the selected circulating miRNAs were confirmed in the ovarian tissue by using Western blotting. Proteins were extracted from the ovarian tissue using a lysis buffer and then electrophoresed on 10% or 12% SDS-polyacrylamide gel. The extracted proteins were transferred to a polyvinyldene difluoride membrane (PVDF membrane), and then reacted with a TBS-T buffer solution containing 5% skim milk for 1 hour. After reacting the proteins with primary antibodies overnight at 4° C., secondary antibodies attached to horseradish peroxidases (HRP) were reacted with the proteins for 1 hour at room temperature, and then protein expression was confirmed using the enhanced chemiluminescence (ECL) method.

[0082] As shown in FIG. 5B, as a result of confirming change of the protein expression by Western blot, in the ovaries of the groups at week 2, week 3, and week 5 after stem cell injection that underexpress miR-145-5p, expression of proteins related to the BMP signaling pathway (Acvr2a, Bmpr2, Smad1, p-Smad1/5) was confirmed to be higher than that of the control group.

[0083] In addition, as shown in FIG. 5D, it was confirmed that miR-425-5p targets Grem2, a BMP antagonist, and as a result of confirming the change in protein expression in the ovaries, it was observed that protein expression was lower in the week-2 and week-3 groups after stem cell injection compared to the control group.

[0084] The above results indicate that the expression of circulating miRNAs present in the blood is changed by human placenta-derived mesenchymal stem cells injected into aged mice, and the change in their expression stimulates the BMP signaling pathway in the ovary.

[0085] As a result, as shown in FIG. 6, the stimulation of the BMP pathway suggests that the dormant primordial follicles in the ovary are activated due to the activation of the BMP signaling pathway in the ovary. When follicle development from the stage of the primordial follicle to the primary follicle resumed, the follicle growth proceeded vigorously, and as a result, production and secretion of ovarian hormones also increased, suggesting that the ovarian function of the aged mice was improved.

[0086] Therefore, changes in the expression of miR-145-5p and miR-425-5p present in the blood may be used as a marker of follicle development to predict ovarian reserve of women. In addition, changes in the expression of miR-145-5p and miR-425-5p present in the blood may predict ovarian function, such as hormone production, and be used as a marker for diagnosis of early ovarian failure.