Application of in-vitro regenerated platelets to treatment of premature ovarian insufficiency

Abstract

The invention is in the field of medical formulations. Specifically, the invention provides a pharmaceutical composition, the pharmaceutical composition comprises in-vitro regenerated platelets and / or a culture supernatant of the in-vitro regenerated platelets, and the in-vitro regenerated platelets have the following characteristics: the FGF level is significantly increased and / or the VEGF level is significantly increased; the pharmaceutical composition is used for promoting ovarian regeneration, improving ovarian endocrine function, improving ovarian reserve function, and / or improving mouse fertility outcome.

Description

Technical Field

[0001] The present invention belongs to the field of medical preparations, and specifically relates to the application of in vitro regenerated platelets in the treatment of premature ovarian insufficiency. Background Art

[0002] Premature ovarian insufficiency (POI) refers to a clinical syndrome in which ovarian function declines in women before the age of 40, mainly manifested as menstrual abnormalities (amenorrhea, oligomenorrhea), follicle stimulating hormone (FSH) > 25 U / L, and fluctuating decline in estrogen levels.

[0003] The incidence of POI is 1% - 4%, and the global analysis of the incidence of POI shows 3.7%. Common causes of POI include genetic factors, immune factors, infectious factors, iatrogenic factors, and environmental factors, etc. However, the causes of more than half of POI patients are still unclear, which is idiopathic POI.

[0004] During the long-term diagnosis and treatment process, experts found that POI presents a progressive course. The domestic guidelines put forward the concept of "subclinical POI", which refers to the FSH level between 15 - 25 U / L. This part of patients belongs to the high-risk population for progressing to POI. The diagnostic criteria for POI are two elevations in FSH levels (> 25 IU / L) within 4 weeks. When the FSH level exceeds 40 IU / L twice, it progresses to premature ovarian failure (POF), indicating that POI has progressed to the late stage, which will not only affect sexual health, cause infertility, but also trigger long-term complications such as cardiovascular diseases, osteoporosis, and neurocognitive disorders, and even premature death.

[0005] Since the cause of POI is still unclear, there is still a lack of effective specific treatment methods so far. The current main measure is symptomatic treatment, and the treatment principle is: adjusting menstruation, relieving symptoms, improving the quality of life, solving fertility problems, reducing long-term health problems and long-term complications.

[0006] Therefore, there is an urgent need to develop POI treatment drugs with excellent effects, good stability, relatively clear components, and wide sources, and there is an urgent need to develop efficient and stable POI treatment means. Summary of the Invention

[0007] The present invention provides an in vitro regenerated platelet and its application in the treatment of premature ovarian insufficiency.

[0008] In a first aspect of the present invention, there is provided a pharmaceutical composition for treating premature ovarian insufficiency in a subject, the pharmaceutical composition comprising in vitro regenerated platelets and / or the culture supernatant of the platelets, wherein the in vitro regenerated platelets have the following characteristics: a significantly increased FGF level and / or a significantly increased VEGF level.

[0009] In another preferred embodiment, the "significantly increased FGF level" means that the FGF level X1 of the platelets is compared with the FGF level X0 of human platelets, and X1 / X0 ≥ 1.1, preferably X1 / X0 ≥ 1.3, more preferably X1 / X0 ≥ 1.5, and most preferably X1 / X0 ≥ 2.

[0010] In another preferred embodiment, the "significantly increased VEGF level" means that the VEGF level Y1 of the platelets is compared with the VEGF level Y0 of human platelets, and Y1 / Y0 ≥ 1.1, preferably Y1 / Y0 ≥ 1.3, more preferably Y1 / Y0 ≥ 1.5, and most preferably Y1 / Y0 ≥ 2.

[0011] In another preferred embodiment, the TGF-β level, EGF level, BDNF level, FGF level, VEGF level, PDGF-AB level, PDGF-BB level, and / or IGF level of the in vitro regenerated platelets show no significant change compared with human platelets.

[0012] In another preferred embodiment, the "no significant change" means no statistically significant difference.

[0013] In another preferred embodiment, the content of the in vitro regenerated platelets in the pharmaceutical composition is 0.2 - 3E9 / mL, preferably 1E9 / mL, and most preferably 2E9 / mL.

[0014] In another preferred embodiment, the form of the composition is a liquid preparation.

[0015] In another preferred embodiment, the culture system of the in vitro regenerated platelets includes a first-stage medium, a second-stage medium, a third-stage medium, and / or a fourth-stage medium. Wherein, the first-stage medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021. The second-stage medium includes StemSpan™-ACF Erythroid expansion medium, BMP4, VGEF, bFGF. The third-stage medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA. The fourth-stage culture medium includes a basal medium, TPO, SCF, and PFHM-II.

[0016] In another preferred example, the method for preparing in vitro regenerated platelets includes the following steps: (S1) Culturing and expanding ESC / iPSC cells with a stem cell culture medium; (S2) Performing mesoderm differentiation culture on the cells obtained in step (S1) with the first-stage culture medium; (S3) Performing hemogenic endothelium and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S2) with the second-stage culture medium; (S4) Continuing to culture the cells obtained in step (S3) with the third-stage culture medium to obtain megakaryocytes; (S5) Continuing to culture the cells obtained in step (S4) with the fourth-stage culture medium to obtain platelets.

[0017] In the second aspect of the present invention, there is provided the use of the pharmaceutical composition described in the first aspect of the present invention for preparing a kit for treating premature ovarian insufficiency in a subject.

[0018] In the third aspect of the present invention, there is provided a kit for treating premature ovarian insufficiency in a subject, the kit comprising: (i) A detection reagent for premature ovarian insufficiency; and (ii) The pharmaceutical composition described in the first aspect of the present invention.

[0019] In another preferred example, the kit further includes an instruction manual, and the instruction manual guides the kit for treating premature ovarian insufficiency in a subject.

[0020] In the fourth aspect of the present invention, there is provided the use of in vitro regenerated platelets for preparing a pharmaceutical composition, and the pharmaceutical composition is used for one or more of the following uses: (Z1) Treating premature ovarian insufficiency in a subject; (Z2) Promoting ovarian regeneration in a subject; (Z3) Improving the estrous cycle of a subject; (Z4) Improving the sex hormone level of a subject; (Z5) Improving the ovarian reserve function of a subject; (Z6) Improving the fertility outcome of a subject.

[0021] In another preferred example, the subject is a subclinical premature ovarian insufficiency subject; a clinical premature ovarian insufficiency subject; a premature ovarian failure subject; a spontaneous premature ovarian insufficiency subject; an iatrogenic premature ovarian insufficiency subject; an idiopathic premature ovarian insufficiency subject; an immune premature ovarian insufficiency subject; a congenital ovarian hypoplasia syndrome subject (Turner syndrome).

[0022] In another preferred example, the in vitro regenerated platelets are platelets differentiated from human pluripotent stem cells ESC / iPSC cells in vitro.

[0023] In another preferred example, the in vitro regenerated platelets are platelets differentiated from hematopoietic stem cells HSPCs in vitro.

[0024] In another preferred example, the HSPCs are HSPCs derived from peripheral blood and / or HSPCs derived from umbilical cord blood.

[0025] In another preferred example, the hematopoietic stem cells include hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells or combinations thereof.

[0026] In another preferred example, the hematopoietic stem cells are CD34 + cells.

[0027] In another preferred example, the in vitro regenerated platelets are platelets differentiated from megakaryocytes MK and / or megakaryocyte progenitors MKP in vitro.

[0028] In another preferred example, the in vitro differentiation system includes a first-stage medium, a second-stage medium, a third-stage medium and / or a fourth-stage medium, wherein, the first-stage medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021; the second-stage medium includes StemSpan™-ACF Erythroid expansion medium, BMP4, VGEF, bFGF; the third-stage medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA; the fourth-stage medium includes a basal medium, TPO, SCF, PFHM-II.

[0029] In another preferred example, the first-stage medium includes STEMdiff™ APEL™2 medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 25 ng / mL of activin A, 1.5 uM of CHIR-99021, 1% PS.

[0030] In another preferred example, the second-stage culture medium comprises StemSpan™-ACF Erythroid expansion culture medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, and 1% PS.

[0031] In another preferred example, the third-stage culture medium comprises STEMdiff™ APEL™2 culture medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 25 ng / mL of Flt3, 10 ng / mL of IL3, 10 ng / mL of IL6, 5% of PFHM-II, 100 μg / mL of PVA, and 1% PS.

[0032] In another preferred example, the fourth-stage culture medium comprises STEMdiff™ APEL™2 culture medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 5% of PFHM-II, and 1% PS.

[0033] In another preferred example, treating premature ovarian insufficiency in a subject includes preventing premature ovarian insufficiency in the subject, improving the symptoms of premature ovarian insufficiency in the subject, maintaining the degree of premature ovarian insufficiency in the subject, slowing down the rate of deterioration of the symptoms of premature ovarian insufficiency in the subject, treating premature ovarian insufficiency in the subject, or a combination thereof.

[0034] In another preferred example, promoting ovarian regeneration in a subject means that the ovarian weight A1 of the subject after administering the pharmaceutical composition is compared with the ovarian weight A0 of the subject before administering the pharmaceutical composition, and A1 / A0 ≥ 1.05, more preferably, A1 / A0 ≥ 1.10.

[0035] In another preferred example, A1 - A0 ≥ 0.005% of the subject's mass, preferably ≥ 0.01% of the subject's mass.

[0036] In another preferred example, improving the estrous cycle of a subject means that after administering the pharmaceutical composition, the estrus period of the subject is significantly increased and / or the diestrus period of the subject is significantly decreased.

[0037] In another preferred example, the significant increase in the estrus period of the subject means that the estrus period B1 of the subject after administering the pharmaceutical composition is compared with the estrus period B0 of the subject before administering the pharmaceutical composition, and B1 / B0 ≥ 1.2, more preferably, B1 / B0 ≥ 1.5, most preferably, B1 / B0 ≥ 2.

[0038] In another preferred example, the significant reduction in the dioestrus of the subject means that the dioestrus C1 of the subject after administering the pharmaceutical composition is compared with the dioestrus C0 of the subject before administering the pharmaceutical composition, and C1 / C0 ≤ 0.9, more preferably, C1 / C0 ≤ 0.8, and most preferably, C1 / C0 ≤ 0.6.

[0039] In another preferred example, the improvement of the sex hormone level of the subject includes: improving the AMH level of the subject, improving the estrogen level of the subject, improving the FSH level of the subject, improving the LH level of the subject, or a combination thereof.

[0040] In another preferred example, the improvement of the AMH level of the subject means that the AMH level D1 of the subject after administering the pharmaceutical composition is compared with the AMH level D0 of the subject before administering the pharmaceutical composition, and D1 / D0 ≥ 1.2, more preferably, D1 / D0 ≥ 1.5, and most preferably, D1 / D0 ≥ 2.

[0041] In another preferred example, the improvement of the estrogen level of the subject means that the estrogen level E1 of the subject after administering the pharmaceutical composition is compared with the estrogen level E0 of the subject before administering the pharmaceutical composition, and E1 / E0 ≥ 1.1, more preferably, E1 / E0 ≥ 1.3, and most preferably, E1 / E0 ≥ 1.5.

[0042] In another preferred example, the improvement of the FSH level of the subject means that the FSH level F1 of the subject after administering the pharmaceutical composition is compared with the FSH level F0 of the subject before administering the pharmaceutical composition, and F1 / F0 ≤ 0.9, more preferably, F1 / F0 ≤ 0.8, and most preferably, F1 / F0 ≤ 0.6.

[0043] In another preferred example, the improvement of the LH level of the subject means that the LH level G1 of the subject after administering the pharmaceutical composition is compared with the LH level G0 of the subject before administering the pharmaceutical composition, and G1 / G0 ≤ 0.95, more preferably, G1 / G0 ≤ 0.9, and most preferably, G1 / G0 ≤ 0.85.

[0044] In another preferred example, the improvement of the ovarian reserve function of the subject means that the total number of follicles of the subject increases significantly and / or the number of growing follicles increases significantly.

[0045] In another preferred example, the significant increase in the total number of follicles of the subject means that the total number of follicles H1 of the subject after administering the pharmaceutical composition is compared with the total number of follicles H0 of the subject before administering the pharmaceutical composition, and H1 / H0 ≥ 1.3, more preferably, H1 / H0 ≥ 1.5, and most preferably, H1 / H0 ≥ 2.

[0046] In another preferred example, the significant increase in the number of growing follicles in the subject means that the number of growing follicles J1 in the subject after administering the pharmaceutical composition is compared with the number of growing follicles J0 in the subject before administering the pharmaceutical composition, and J1 / J0 ≥ 1.3. More preferably, J1 / J0 ≥ 1.5. Most preferably, J1 / J0 ≥ 2.

[0047] In another preferred example, the improvement of the reproductive outcome of the subject includes: a significant increase in the number of ovulations of the subject, a significant decrease in the number of abnormal eggs, a significant increase in the number of MII-phase eggs, and a significant increase in the number of two-cell embryos.

[0048] In another preferred example, the significant increase in the number of ovulations of the subject means that the number of ovulations K1 in the subject after administering the pharmaceutical composition is compared with the number of ovulations K0 in the subject before administering the pharmaceutical composition, and K1 / K0 ≥ 1.3. More preferably, K1 / K0 ≥ 1.5. Most preferably, K1 / K0 ≥ 2.

[0049] In another preferred example, the significant decrease in the number of abnormal eggs means that the number of abnormal eggs L1 in the subject after administering the pharmaceutical composition is compared with the number of abnormal eggs L0 in the subject before administering the pharmaceutical composition, and L1 / L0 ≤ 0.2. More preferably, L1 / L0 ≤ 0.1. Most preferably, L1 / L0 ≤ 0.05.

[0050] In another preferred example, the significant decrease in the number of abnormal eggs includes that the number of abnormal eggs L1 in the subject after administering the pharmaceutical composition is compared with the number of abnormal eggs L0 in the subject before administering the pharmaceutical composition, and L1 / L0 = 0.

[0051] In another preferred example, the significant increase in the number of MII-phase eggs in the subject means that the number of MII-phase eggs M1 in the subject after administering the pharmaceutical composition is compared with the number of MII-phase eggs M0 in the subject before administering the pharmaceutical composition, and M1 / M0 ≥ 2. More preferably, M1 / M0 ≥ 3. Most preferably, M1 / M0 ≥ 4.

[0052] In another preferred example, the significant increase in the number of two-cell embryos in the subject means that the number of two-cell embryos N1 in the subject after administering the pharmaceutical composition is compared with the number of two-cell embryos N0 in the subject before administering the pharmaceutical composition, and N1 / N0 ≥ 1.5. More preferably, N1 / N0 ≥ 2. Most preferably, N1 / N0 ≥ 3.

[0053] In another preferred example, the culture system for in vitro regenerated platelets includes a first-stage culture medium, a second-stage culture medium, a third-stage culture medium, and / or a fourth-stage culture medium. Among them, the first-stage culture medium includes a basal medium, BMP4, VEGF, activin A, and CHIR-99021. The second-stage culture medium includes StemSpan™-ACF Erythroid expansion medium, BMP4, VGEF, and bFGF; The third-stage culture medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA; The fourth-stage culture medium includes a basal medium, TPO, SCF, and PFHM-II.

[0054] In another preferred example, the basal medium includes E8 medium, complete medium, StemSpan TM SFEM hematopoietic cell medium, StemPro-34, STEMdiff™ APEL™2 medium, and / or BPEL.

[0055] In another preferred example, the basal medium is STEMdiff™ APEL™2 medium.

[0056] In another preferred example, the first-stage culture medium includes STEMdiff™ APEL™2 medium, 20 - 40 ng / mL of BMP4, 40 - 60 ng / mL of VEGF, 20 - 30 ng / mL of activin A, and 1 - 3 μM of CHIR-99021.

[0057] In another preferred example, the second-stage culture medium includes StemSpan™-ACF Erythroid expansion medium, 20 - 4 ng / mL of BMP4, 40 - 60 ng / mL of VEGF, and 40 - 60 ng / mL of bFGF.

[0058] In another preferred example, the third-stage culture medium includes STEMdiff ™ APEL ™ 2 medium, 40 - 60 ng / mL of TPO, 40 - 60 ng / mL of SCF, 20 - 30 ng / mL of Flt3, 5 - 15 ng / mL of IL3, 5 - 15 ng / mL of IL6, 1% - 10% of PFHM-II, and 50 - 200 μg / mL of PVA.

[0059] In another preferred example, the fourth-stage culture medium includes STEMdiff™ APEL™2 medium, 40 - 60 ng / mL of TPO, 40 - 60 ng / mL of SCF, and 1% - 10% of PFHM-II.

[0060] In another preferred example, the culture medium includes 0.5% - 3% of PS.

[0061] In another preferred example, the first-stage culture medium comprises STEMdiff™ APEL™2 medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 25 ng / mL of activin A, 1.5 μM of CHIR-99021, and 1% PS.

[0062] In another preferred example, the second-stage culture medium comprises StemSpan™-ACF Erythroid expansion medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, and 1% PS.

[0063] In another preferred example, the third-stage culture medium comprises STEMdiff™ APEL™2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 25 ng / mL of Flt3, 10 ng / mL of IL3, 10 ng / mL of IL6, 5% of PFHM-II, 100 μg / mL of PVA, and 1% PS.

[0064] In another preferred example, the fourth-stage culture medium comprises STEMdiff™ APEL™2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 5% of PFHM-II, and 1% PS.

[0065] In another preferred example, the culture system comprises M1 medium, and the medium contains StemSpan™ SFEMII medium, IL-3, IL-6, SCF, TPO, and FLt.

[0066] In another preferred example, the culture system comprises M2 medium, and the medium contains IMDM medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, IL-6, SCF, and TPO.

[0067] In another preferred example, the M1 medium contains StemSpan™ SFEM II medium, 5 - 50 ng / ml of IL-3, 5 - 50 ng / ml of IL-6, 5 - 50 ng / ml of SCF, 5 - 50 ng / ml of TPO, and 5 - 50 ng / ml of FLt.

[0068] In another preferred example, the M2 medium contains IMDM medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, 5 - 50 ng / ml of IL-6, 5 - 50 ng / ml of SCF, and 5 - 50 ng / ml of TPO.

[0069] In another preferred example, the culture system comprises a stem cell culture medium.

[0070] In another preferred example, the stem cell culture medium is mTeSR1 medium.

[0071] In another preferred example, the method for preparing in vitro regenerated platelets comprises the following steps: (S1) Culturing and expanding ESC / iPSC cells with a stem cell culture medium; (S2) Performing mesoderm differentiation culture on the cells obtained in step (S1) with the first-stage culture medium; (S3) Performing hemogenic endothelium and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S2) with the second-stage culture medium; (S4) Continuing to culture the cells obtained in step (S3) with the third-stage culture medium to obtain megakaryocytes; (S5) Continuing to culture the cells obtained in step (S4) with the fourth-stage culture medium to obtain platelets.

[0072] In another preferred example, the stem cell culture medium comprises: mTesR1 medium containing Y27632 or mTesR1 medium not containing Y27632.

[0073] In another preferred example, step (S1) comprises: culturing ESC / iPSC cells with mTesR1 medium containing Y27632 for 12 - 24 h; and then continuing to culture with mTesR1 medium not containing Y27632 until the confluence reaches 70% - 80%.

[0074] In another preferred example, the culture time of step (S2) is 2 days.

[0075] In another preferred example, the culture time of step (S3) is 5 days.

[0076] In another preferred example, the culture time of step (S4) is 7 days.

[0077] In another preferred example, the culture time of step (S5) is 5 - 8 days, preferably 7 days, and most preferably 6 days.

[0078] In another preferred example, the method for preparing in vitro regenerated platelets comprises the following steps: (Q1) Culturing hematopoietic stem cells with the M1 medium to obtain megakaryocyte progenitor cells; (Q2) Continuing to culture the megakaryocyte progenitor cells obtained in step (Q1) with the M2 medium to obtain platelets.

[0079] In another preferred example, the hematopoietic stem cells are CD34 + cells.

[0080] In another preferred example, the culture time of step (Q1) is 7 days.

[0081] In another preferred example, the culture time in step (Q2) is 10 - 12 days.

[0082] In another preferred example, the in vitro regenerated platelets have the following characteristics: a significantly increased FGF level and / or a significantly increased VEGF level.

[0083] In another preferred example, "a significantly increased FGF level" means that the FGF level X1 of the platelets compared with the FGF level X0 of human platelets, X1 / X0 ≥ 1.1, preferably, X1 / X0 ≥ 1.3, more preferably, X1 / X0 ≥ 1.5, most preferably, X1 / X0 ≥ 2.

[0084] In another preferred example, "a significantly increased VEGF level" means that the VEGF level Y1 of the platelets compared with the VEGF level Y0 of human platelets, Y1 / Y0 ≥ 1.1, preferably, Y1 / Y0 ≥ 1.3, more preferably, Y1 / Y0 ≥ 1.5, most preferably, Y1 / Y0 ≥ 2.

[0085] In a fifth aspect of the present invention, there is provided an in vitro regenerated platelet for treating premature ovarian insufficiency in a subject, the in vitro regenerated platelet having the following characteristics: a significantly increased FGF level and / or a significantly increased VEGF level.

[0086] In another preferred example, "a significantly increased FGF level" means that the FGF level X1 of the platelets compared with the FGF level X0 of human platelets, X1 / X0 ≥ 1.1, preferably, X1 / X0 ≥ 1.3, more preferably, X1 / X0 ≥ 1.5, most preferably, X1 / X0 ≥ 2.

[0087] In another preferred example, "a significantly increased VEGF level" means that the VEGF level Y1 of the platelets compared with the VEGF level Y0 of human platelets, Y1 / Y0 ≥ 1.1, preferably, Y1 / Y0 ≥ 1.3, more preferably, Y1 / Y0 ≥ 1.5, most preferably, Y1 / Y0 ≥ 2.

[0088] In another preferred example, the culture system of the in vitro regenerated platelets includes a first-stage culture medium, a second-stage culture medium, a third-stage culture medium, and / or a fourth-stage culture medium. Wherein, the first-stage culture medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021; The second-stage culture medium includes StemSpan™-ACF Erythroid expansion medium, BMP4, VGEF, bFGF; The third-stage culture medium comprises a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA; The fourth-stage culture medium comprises a basal medium, TPO, SCF, and PFHM-II.

[0089] In another preferred example, the method for preparing in vitro regenerated platelets comprises the following steps: (S1) Culturing and expanding ESC / iPSC cells with a stem cell culture medium; (S2) Performing mesoderm differentiation culture on the cells obtained in step (S1) with the first-stage culture medium; (S3) Performing hemogenic endothelium and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S2) with the second-stage culture medium; (S4) Continuing to culture the cells obtained in step (S3) with the third-stage culture medium to obtain megakaryocytes; (S5) Continuing to culture the cells obtained in step (S4) with the fourth-stage culture medium to obtain platelets.

[0090] In the sixth aspect of the present invention, there is provided a method for treating premature ovarian insufficiency, the method comprising administering to a subject the in vitro regenerated platelets described in the fourth aspect of the present invention, the pharmaceutical composition described in the first aspect of the present invention, or the kit described in the second aspect of the present invention.

[0091] In another preferred example, the subject is a human.

[0092] In another preferred example, the administered dose is 0.2E9 - 6E9 per ovary, preferably 1E9 - 4E9 per ovary.

[0093] In another preferred example, the subject is a non-human mammal.

[0094] In another preferred example, the subject is a mouse.

[0095] In another preferred example, the administered dose is 0.1E7 - 5E7 per ovary, preferably 0.5E7 - 2E7 per ovary.

[0096] In another preferred example, the administration cycle is ≥21 days, preferably ≥28 days.

[0097] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0098] Figure 1 showed the ( Figure 1 A) body weight changes of each group of mice during the adaptive feeding period to the end point of sample collection and ( Figure 1 B) ovarian weights at the end point (n = 6).

[0099] Figure 2 showed the estrous cycle changes of each group of mice (n = 6).

[0100] Figure 3 showed the serum hormone levels of each group of mice. Specifically, it showed the ( Figure 3 A) AMH, ( Figure 3 B) E2, ( Figure 3 C) FSH, ( Figure 3 D) LH contents in the serum of mice at the end of the first estrous cycle; and the ( Figure 3 E) AMH, ( Figure 3 F) E2, ( Figure 3 G) FSH, ( Figure 3 H) LH contents in the serum of mice at the end of the fourth estrous cycle; biological n = 3, technical replicates n = 3.

[0101] Figure 4 showed the gross ovarian pathology and follicle count of mice. Figure 4 A showed the scanned images of mouse ovarian tissue sections stained with HE; Figure 4 B showed the total follicle (including primordial follicles, primary follicles, secondary follicles, antral follicles and atretic follicles) count, n = 3; Figure 4 C showed the growing follicle (including primary follicles, secondary follicles and antral follicles) count, n = 3.

[0102] Figure 5 showed the ovulation situation and two-cell embryos of mice. Figure 5 A showed the oocyte morphology of each group of mice, the morphology of MII-stage eggs and two-cell embryos; Figure 5 B showed the total ovulation number of each group of mice, n = 3; Figure 5 C showed the number of MII-stage eggs of each group of mice, n = 3; Figure 5 D showed the two-cell embryo count of each group of mice, n = 3.

[0103] Figure 6 showed that the platelets of the present invention counteracted POI ovarian cell apoptosis by releasing cytokines. Detailed implementation manners

[0104] After extensive and in-depth research, the inventor of the present invention unexpectedly discovered for the first time an in vitro differentiated platelet, which significantly improved the development and function of POI ovaries in terms of ovarian volume, texture, follicular development, hormone levels, and fertility outcomes. At the same time, it also has the advantages of clear composition, high safety, and simple preparation. Meanwhile, the present invention constructed an in vitro cell model and observed the significant anti-apoptotic effect of the platelets of the present invention by treating with cytokines released by PLT. The present invention was completed on this basis.

[0105] Term To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0106] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0107] As used herein, the terms "comprising," "including," and "containing" may be used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of."

[0108] Where a numerical range is provided, it should be understood, unless the context clearly dictates otherwise, that each intermediate integer value of that value, each tenth of each intermediate integer value, each other intermediate value between the upper and lower limits of that range, and any other intermediate value within the specified range are included within the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also covered by the present invention, subject to any explicit exclusions within the specified range. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0109] As used herein, the terms "in vitro regeneration" and "in vitro differentiation" may be used interchangeably and both refer to the in vitro differentiation of hematopoietic stem and progenitor cells HSPCs, megakaryocytes MK, and / or megakaryocyte progenitors MKP into platelets.

[0110] As used herein, the levels of TGF-β, EGF, BDNF, FGF, VEGF, PDGF-AB, PDGF-BB, and / or IGF in the in vitro regenerated platelets having no significant change compared with those of human platelets means that, compared with human platelets, the levels of one or more cytokines in TGF-β, EGF, BDNF, FGF, VEGF, PDGF-AB, PDGF-BB, and IGF in the in vitro regenerated platelets increase, but the increase does not exceed 1.05-fold; or decrease, but the decrease is not less than 95%; generally, there is no statistically significant difference. For example, when the TGF-β level P1 of the in vitro regenerated platelets is compared with the TGF-β level P0 of human platelets, 0.95 ≤ P1 / P0 ≤ 1.05, preferably 0.97 ≤ P1 / P0 ≤ 1.03, and most preferably 0.99 ≤ P1 / P0 ≤ 1.01.

[0111] Premature ovarian insufficiency (POI) Premature ovarian insufficiency (POI) refers to a clinical syndrome in which a woman shows ovarian function decline before the age of 40, mainly manifested as menstrual abnormalities (amenorrhea, oligomenorrhea), follicle-stimulating hormone (FSH) > 25 U / L, and fluctuating decline in estrogen levels.

[0112] In the long-term diagnosis and treatment process, POI presents a progressive course. "Subclinical POI" refers to an FSH level of 15 - 25 U / L, and these patients belong to the high-risk population progressing to POI. The diagnostic criteria for POI are two elevations in FSH levels (> 25 IU / L) within 4 weeks. When the FSH level exceeds 40 IU / L twice, it progresses to premature ovarian failure (POF), indicating that POI has progressed to the late stage, which will not only affect sexual health, lead to infertility, but also cause long-term complications such as cardiovascular diseases, osteoporosis, and neurocognitive disorders, and even premature death.

[0113] In the present invention, the "premature ovarian insufficiency" includes "subclinical POI", "primary and secondary POI", and "premature ovarian failure".

[0114] Symptoms and common treatment methods of POI The current treatment of POI includes mental health management, hormone therapy, fertility treatment, treatment and health management of other complications, etc. Usually, it follows the principle of symptomatic treatment, lacking specific treatment methods, and requires multiple treatment methods to be carried out in parallel, such as mental health management, fertility treatment, hormone therapy, non-hormone therapy, etc.

[0115] Hormone therapy. Currently, the "European Society of Human Reproduction and Embryology Clinical Practice Guidelines" and the domestic "Clinical Practice Expert Consensus" indicate that unless there are absolute contraindications, all POI patients should receive estrogen therapy to reduce the risk of osteoporosis and cardiovascular diseases. However, current research on the optimal regimen for hormone replacement therapy is limited, and there are still significant differences in clinical practice in terms of the administration route, dosage, and types of estrogen and progesterone preparations. The hormone replacement therapy regimen recommends continuous medication, which should be started at the time of diagnosis and continued until the average natural menopause age to induce sexual development in patients, maintain secondary sexual characteristics and bone mass, improve the quality of life of patients, and reduce the incidence and mortality of long-term complications.

[0116] Treatment and health management of other complications. For patients with related systemic complications, personalized treatment and management should be provided, including bone health, cardiovascular and metabolic health, neurological health, urogenital health, and sexual health. Since POI is the main cause of the above complications, the treatment principle still focuses on the first-line treatment for POI, and bone mineral density should be monitored regularly.

[0117] Non-hormone therapy. For POI patients with contraindications to hormone replacement therapy or those who are temporarily unwilling or unsuitable to receive hormone replacement therapy, non-hormone preparations can be selected to relieve low estrogen symptoms. However, there is less research data on the benefits of non-hormone therapy (such as phytoestrogens, traditional Chinese medicine, etc.) for the bone health and cardiovascular and metabolic health of POI patients, and there is no consistent conclusion.

[0118] Treatment with mesenchymal stem cells (MSCs). In previous studies, the use of MSCs from various sources such as bone marrow, placenta, and umbilical cord has been shown to be effective in animal models of POI. The mechanism of action is mainly the paracrine effect of stem cells, including the release of various cytokines, promoting neovascularization, anti-inflammation, anti-apoptosis, anti-fibrosis, and immunomodulation. In the context of POI, it has been observed that conditioned medium from stem cells can protect the ovary from age-related damage, highlighting the key role of the paracrine mechanism in improving ovarian function. Further studies have shown that MSCs can upregulate the release of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, IL-10, IL-11, and IL-15, while reducing the secretion of tumor necrosis factor-alpha (TNFα) and IL-6. However, the impact of stem cell differentiation on POI patients has not been determined. It is still unclear whether MSCs can differentiate into oocytes, although previous animal studies have shown that MSCs can differentiate into granulosa cells, indicating their potential to restore ovarian endocrine function and folliculogenesis in rodents. Notably, the possibility of stem cell malignancy cannot be ignored.

[0119] Platelet-rich plasma PRP PRP is a platelet (PLT)-rich protein concentrate extracted from fresh whole blood, also known as autologous conditioned plasma. It exerts anti-inflammatory and regenerative functions by centrifuging to remove red blood cells. Over the past two decades, PRP has gradually attracted great attention from medical professionals due to its potential in enhancing the regeneration process. Multiple clinical studies have found that PRP can be used for tissue regeneration and repair in various fields, such as dermatology, osteoarthritis, intervertebral disc degeneration, and infertility.

[0120] Intrauterine injection of PRP is beneficial to the maturation of follicles in all aspects and has a positive impact on the pregnancy outcomes of patients with unexplained repeated implantation failure. In addition, PRP treatment also has a positive significance in improving ovarian physiological structure and reserve function.

[0121] Regarding the mechanism by which PRP improves ovarian function, on the one hand, it is believed that it can enhance the viability of primary follicles and preantral follicles and promote the development of isolated human primordial follicles and primary follicles to the preantral stage. On the other hand, the mechanism of action is that there are abundant PLTs and α-granules in PRP. Activated PLTs can release various cytokines such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), VEGF, IGF-1, thrombospondin 1 (THBS1), EGF, and HGF, etc. By reducing oxidative stress and promoting tissue angiogenesis, it thus promotes the recovery of the ovarian microenvironment. The third mechanism holds that the restorative effect of PRP on the ovary is achieved through sphingosine-1-phosphate (S1P). And the α-granules of PLTs contain abundant S1P, which is released during activation. It is measured that the S1P content in each 1×10 7 PLTs exceeds 300 nM. If there is a linear relationship between the S1P concentration and the PLT count, it is estimated that in the study of infusing activated PRP into the ovary, the amount of S1P delivered is approximately 9 μM. Currently, small-scale non-first-line experimental treatments of PRP for POI have been carried out clinically. Some evidence-based medicine shows that PRP injection (intravenous / intraovarian) can improve ovarian reserve and related functions.

[0122] However, although a large number of animal models and clinical evidence have proven the effectiveness of PRP in the treatment of POI, currently the preparation and injection protocols of PRP vary among different centers. Due to the heterogeneity of PRP sources and the differences in administration methods, the degree of improvement in ovarian function by intraovarian injection of PRP remains controversial, and there is no clinical consensus on the optimal protocol. Therefore, it is urgent to develop PRP alternative drugs with clear components for standardized clinical application.

[0123] In summary, although PRP has achieved certain effects in clinical trials, due to factors such as its heterogeneity, accessibility, and differences in preparation methods, the clinical treatment effects are unstable. Since PRP is isolated from the patient's own peripheral blood, on the one hand, if the patient is older (e.g., ≥35 years old), their own PRP also has the risk of aging, which will affect the treatment effect on POI; on the other hand, if the patient has other underlying diseases and is not suitable for collecting the peripheral blood required for PRP preparation, the accessibility of PRP is restricted; thirdly, the current preparation methods of PRP have not been unified, resulting in unclear dosing (such as the number of PLTs per milliliter of PRP), leading to unstable treatment effects.

[0124] The platelets of the present invention The platelets of the present invention are the in vitro regenerated platelets provided by the present invention. The starting cells for the in vitro regeneration of the platelets of the present invention are hematopoietic stem / progenitor cells (HSPCs) isolated from the mobilized peripheral blood of healthy people meeting the inclusion and exclusion criteria or human ESC / iPSC cells.

[0125] The present invention applies the in vitro regenerated PLTs to the treatment of POI patients, aiming to solve the problems that PRP has not been able to solve. For the in vitro regenerated PLTs involved in the present invention, for humans, ovarian injection is performed under ultrasound guidance at a dose of (1 - 4)×10⁹ per ovary, and single or regular injections are determined according to the treatment purpose and effect.

[0126] The main advantages of the present invention include: (a)The present invention provides an application of in vitro regenerated platelets in the treatment of premature ovarian insufficiency, providing a potential therapeutic drug with high efficiency, stability, high safety, and no obvious side effects for premature ovarian insufficiency.

[0127] (b)The in vitro regenerated platelets provided by the present invention are not restricted by the differences in autologous platelet-rich plasma of patients, have a clear dosing, are simple to prepare, have uniform properties, stable performance, are safe, and have strong accessibility, and can make up for the disadvantages of autologous platelet-rich plasma in clinical applications.

[0128] (c)After activation, the in vitro regenerated PLTs can release abundant cytokines, including but not limited to PDGF, TGF-β, bFGF, VEGF, IGF-1, THBS1, EGF, and HGF, etc. The above cytokines are important factors for regulating the repair of the ovarian tissue microenvironment. The cytokine release of the regenerated PLTs produced by a specific process is clearly controllable, and the dose is easy to control in clinical practice applications.

[0129] (d)Based on the in vitro regenerated platelets provided by the present invention, the present invention provides a treatment method for premature ovarian insufficiency that is safe, efficient, stable, controllable, easy to operate, and easy to implement.

[0130] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Qiao Zilin et al., "Experimental Guidance for Animal Cell Culture Engineering" (2024), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0131] Example 1: Experimental method.

[0132] First, a NOD-SCID mouse POI model was constructed by a single intraperitoneal injection of 1.2 mg / kg busulfan and 12 mg / kg cyclophosphamide for 7 days to obtain the model group. Subsequently, grouping and drug administration were carried out according to the protocol shown in Table 1.

[0133] Table 1 Experimental protocol

[0134] Among them, the positive control (Positive) product is PRP isolated from donor-derived peripheral blood that meets the same inclusion and exclusion criteria as the test product. The isolation method refers to previous studies, and the PLT count after absolute counting is the same as that of the test product. Among them, the donor is a young and healthy subject. However, in the actual autologous platelet-rich plasma, due to factors such as the patient's older age and underlying diseases, the PLT quantity and quality in PRP are mostly significantly inferior to those of the PRP used in the examples of the present invention.

[0135] Among them, the PLT preservation solution is a phosphate buffer solution that meets the drug excipient standards.

[0136] Superovulation and co-housing of male and female mice: On the 21st day after modeling, 10 IU PMSG and 10 IU hCG were injected at 48-hour intervals for superovulation, and 3 female mice in each group were caged with 1 male mouse.

[0137] Humane endpoint of experimental animals: During the experiment, if the mice showed abnormal behavior, paralysis, or abnormal breathing, they were euthanized.

[0138] Body weight detection: Body weight was detected once before modeling and twice a week during modeling and the treatment period.

[0139] Clinical observation: Clinical observation was carried out once a day during the adaptation feeding period and the experiment. The observation content included but was not limited to the mental state and diet of the animals.

[0140] Estrous Cycle Monitoring: Vaginal smears and Giemsa staining were used to monitor the estrous cycle of mice for 21 days after dosing. During the proestrus stage, vaginal smears were dominated by nucleated epithelial cells with a small number of leukocytes. During the estrus stage, vaginal smears were dominated by anucleated keratinized squamous cells, which were large, flat, and had irregular margins. During the metestrus stage, the keratinized epithelium decreased, and numerous nucleated epithelial cells and leukocytes appeared. During the diestrus stage, leukocytes predominated.

[0141] Serum hormone level detection: At the end of the first estrus and 21 days after administration, venous blood was collected from mice to separate serum, which was frozen and stored. Reproductive hormone (FSH, LH, E2, AMH) levels were then detected according to the kit instructions.

[0142] Endpoint sampling and processing: 48 hours after cohabitation, mice were killed and their ovaries were removed, and blastomeres in the oviduct were collected for counting and statistics. The remaining mice underwent oviduct retrieval on the day after hCG injection for morphological observation and statistics, and eggs, granulosa cells, and ovaries were collected. The ovaries were fixed in 4% paraformaldehyde for paraffin section preparation and HE staining.

[0143] Statistics of MII stage oocytes and two-cell embryos: The number of first polar bodies released and two-cell embryos in the collected oocytes was counted.

[0144] Ovarian tissue pathology: Paraffin sections were prepared from fixed ovarian tissues. Each section was 3 μm thick. After HE staining and scanning, the ovarian morphology and histological changes were observed. The number of follicles and corpora lutea at each level was counted.

[0145] Statistical Analysis: Results are expressed as mean ± SEM. P < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0146] Example 2: Effects of the PLT of the present invention on the body weight and ovary weight of mice.

[0147] There were no abnormalities in the activity and eating status of the experimental animals during the experiment, and all mice reached the end point of the experiment.

[0148] The mouse body weight results were as follows Figure 1 As shown in Figure A, compared with the control group, the model group had a significant decrease in body weight, and the body weight increased after treatment with the PLT of the present invention. The weight change indicates that the health of the mice has improved after treatment.

[0149] Ovarian weight results Figure 1As shown in Figure B, by weighing the ovaries of the terminal mice, it was found that the ovarian weight of the POI model mice decreased significantly. After treatment with the PLT of the present invention and the positive control, the ovarian weight increased significantly, suggesting the promoting effect of the drug on ovarian tissue regeneration.

[0150] Example 3: Effect of the PLT of the present invention on the estrous cycle of mice.

[0151] The results are as Figure 2 shown. The results indicate that, compared with the control group, the estrus stage of the model mice was significantly shortened, while the diestrus stage was significantly prolonged.

[0152] After treatment with the PLT of the present invention, the number of days in the estrus stage increased significantly to more than twice that of the model group, while the diestrus stage was significantly shortened to 2 / 3 of the model group. The improvement of the estrous cycle suggests that the ovarian function was significantly improved after treatment.

[0153] The effect of the positive control on the estrous cycle of mice was basically equivalent to that of the PLT of the present invention, indicating that the same amount of the PLT of the present invention and the PLT derived from PRP from healthy young donors had equivalent effects on ovarian function.

[0154] Example 4: Effect of the PLT of the present invention on the hormone levels of mice.

[0155] At the end of the first estrous cycle after administration, the reproductive hormone levels of each group of mice were detected. The hormone levels of each group of mice are as Figure 3 A- Figure 3 shown in D.

[0156] The results show that the levels of AMH and estrogen (E2) in the model group of mice decreased significantly, while the levels of FSH and LH increased significantly. After treatment with the PLT of the present invention and the positive control, the hormone levels of the mice were all improved. Specifically, after treatment, the levels of AMH ( Figure 3 A) and E2 ( Figure 3 B) increased significantly, while the levels of FSH ( Figure 3 C) and LH ( Figure 3 D) decreased significantly.

[0157] On the 21st day after administration, the sex hormone levels of each group of mice were detected again. The results are as Figure 3 E- Figure 3 shown in H.

[0158] It was found that AMH ( Figure 3 E), E2 ( Figure 3 F), FSH ( Figure 3 G) and LH ( Figure 3 H) all continued to develop in the direction of improvement, indicating that the endocrine function of the ovaries was improved.

[0159] Among them, the improvement effect of the sex hormone level of the mice in the positive control group is relatively more significant than that of the PLT of the present invention. On the one hand, the effect of the actually autologous PRP is limited by the patient's own health reasons, and the improvement effect on sex hormones should be inferior to that of the positive control group of the present invention. Therefore, the improvement effect of the PLT of the present invention on the sex hormone level may be comparable to that of the autologous PRP on the sex hormone level. On the other hand, in addition to PLT, PRP also contains various factors and other beneficial components. Therefore, after further optimizing and adding other factors to the PLT of the present invention, the improvement of the sex hormone level can be better.

[0160] Example 5: Effect of the PLT of the present invention on the follicular development of mice.

[0161] The follicular development of mice in each group is as Figure 4 shown.

[0162] As Figure 4 shown in A, the ovaries of the mice in the model group were significantly fibrotic, solid, and atrophied. After treatment with the PLT of the present invention, the texture became significantly looser.

[0163] Furthermore, as Figure 4 shown in B, after treatment with the PLT of the present invention, the total number of follicles increased significantly, and was more than that of the positive control group.

[0164] Among them, the results of growing follicles are as Figure 4 shown in C, indicating that the number of growing follicles increased significantly, and was also more than that of the positive control group.

[0165] In summary, the results of this example confirmed that after treatment with the PLT of the present invention, the organizational structure of the ovary was restored, indicating that the ovarian reserve function was improved.

[0166] In addition, although there were certain individual differences in the number of follicles in mice, the overall improvement effects of the PLT of the present invention on the organizational structure of the ovary and ovarian reserve function were better than those of the positive control group.

[0167] Example 6: Effect of the PLT of the present invention on the reproductive outcome of mice.

[0168] The reproductive outcomes of mice in each group are as Figure 5 shown. By superovulating the mice, the ovulation situation of the mice was observed.

[0169] Compared with the control group mice, the number of ovulations of the mice in the model group was significantly reduced ( Figure 5 B), the number of abnormal eggs was the majority ( Figure 5 A), the number of MII-phase oocytes ( Figure 5 C) and two-cell embryos ( Figure 5 D) was significantly reduced.

[0170] After PLT treatment of the present invention, the number of ovulated eggs in mice increased to about 2 times, the number of deformed eggs decreased, the number of MII eggs increased by about 4 times, and the number of two-cell embryos increased by about 3 times.

[0171] The positive control had a similar effect on mouse ovulation as the PLT of the present invention, indicating that the PLT of the present invention and the PLT derived from PRP of healthy young people with the same quantity had a comparable impact on reproductive outcomes.

[0172] The results of this example show that the PLT treatment of the present invention effectively improved the reproductive outcomes of mice.

[0173] Example 7: Anti-apoptotic effect of the present invention in an in vitro cell model.

[0174] To verify the functional effect of the product of the present invention in vitro, a POI cell model was constructed using the human ovarian granulosa cell line KGN.

[0175] The results are as Figure 6 shown. The results show that significant apoptosis occurred in KNG treated with cyclophosphamide (CTX). Subsequently, the model cells were treated with the supernatant of PLT culture medium containing a large amount of cytokines, and it was found that the apoptotic state of the cells was significantly restored, indicating that the product of the present invention counteracts the apoptosis of POI ovarian cells by releasing cytokines.

[0176] In summary, a POI mouse model was constructed by chemotherapy injury. By administering in vitro regenerated PLT derived from human HSPCs for treatment, the development and function of POI ovaries were significantly improved in terms of ovarian volume, texture, follicular development, hormone levels, and reproductive outcomes. At the same time, by constructing an in vitro cell model and treating it with the cytokines released by PLT, a significant anti-apoptotic effect of this product was observed. And it can be concluded that the PLT of the present invention has a better effect on improving premature ovarian insufficiency than the autologous-derived platelet-rich plasma for improving premature ovarian insufficiency, or is basically comparable thereto. By detecting the content of various cytokines in the platelets of the present invention, including the levels of TGF-β1, EGF, BDNF, FGF, VEGF, PDGF-AB, PDGF-BB, and IGF, it was found that the contents of FGF and VEGF in the platelets of the present invention were significantly higher than those of PLT isolated from human peripheral blood. Specifically, in the induced hPL of the present invention, the content of bFGF increased by 2 times compared with the hPL from peripheral blood, and the content of VEGF increased by 2 times. In the induced hPL of the present invention, the levels of other cytokines did not show significant changes compared with the corresponding cytokine levels from peripheral blood.

[0177] The mechanisms by which the product of the present invention exerts its functions include: 1) promoting the regeneration and development of ovarian tissue through the cytokines released after the activation of PLT; 2) regulating the ovarian microenvironment through the "cross-talk" between PLT and ovarian granulosa cells; 3) promoting the maturation of oocytes and the development of granulosa cells through other molecular mechanisms.

[0178] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A pharmaceutical composition for treating premature ovarian insufficiency in a subject, characterized in that, The pharmaceutical composition comprises in vitro regenerated platelets and / or the culture supernatant of the in vitro regenerated platelets, and the in vitro regenerated platelets have the following characteristics: The level of FGF is significantly increased and / or the level of VEGF is significantly increased.

2. A kit for the companion diagnosis of premature ovarian insufficiency in a subject, characterized in that, The kit contains: (i) A reagent for detecting premature ovarian insufficiency; and (ii) The pharmaceutical composition according to claim 1.

3. Use of in vitro regenerated platelets, characterized in that, For preparing a pharmaceutical composition for use in one or more of the following: (Z1) Treating premature ovarian insufficiency in a subject; (Z2) Promoting ovarian regeneration in a subject; (Z3) Improving the estrous cycle of a subject; (Z4) Improving the sex hormone levels of a subject; (Z5) Improving the ovarian reserve function of a subject; (Z6) Improving the fertility outcome of a subject.

4. The use according to claim 3, characterized in that, The improvement of the estrous cycle of the subject means that after administering the pharmaceutical composition, the estrus period of the subject is significantly increased and / or the diestrus period of the subject is significantly reduced.

5. The use according to claim 3, characterized in that, The improvement of the ovarian reserve function of the subject means that the total number of follicles of the subject is significantly increased and / or the number of growing follicles is significantly increased.

6. The use according to claim 3, characterized in that, The improvement of the fertility outcome of the subject includes: the number of ovulations of the subject is significantly increased, the number of abnormal eggs is significantly reduced, the number of MII-phase oocytes is significantly increased, and / or the number of two-cell embryos is significantly increased.

7. The use according to claim 3, characterized in that, The culture system of the in vitro regenerated platelets includes a first-stage medium, a second-stage medium, a third-stage medium, and / or a fourth-stage medium, wherein the first-stage medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021; The second-stage medium includes StemSpan™-ACF Erythroid expansion medium, BMP4, VGEF, bFGF; The third-stage medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA; The fourth-stage medium includes a basal medium, TPO, SCF, PFHM-II.

8. The use according to claim 7, wherein, The method for preparing the in vitro regenerated platelets includes the following steps: (S1) Culturing and expanding ESC / iPSC cells with a stem cell medium; (S2) Performing mesoderm differentiation culture on the cells obtained in step (S1) with the first-stage medium; (S3) Performing hematopoietic endothelial and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S2) with the second-stage medium; (S4) Continuing to culture the cells obtained in step (S3) with the third-stage medium to obtain megakaryocytes; (S5) Continuing to culture the cells obtained in step (S4) with the fourth-stage medium to obtain platelets.

9. The use according to claim 3, wherein The in vitro regenerated platelets have the following characteristics: The level of FGF is significantly increased and / or the level of VEGF is significantly increased.

10. An in vitro regenerated platelet for treating premature ovarian insufficiency in a subject, characterized in that, The in vitro regenerated platelets have the following characteristics: The level of FGF is significantly increased and / or the level of VEGF is significantly increased.

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