NMN supplement-based elderly female immature oocyte in-vitro maturation culture system and method

Abstract

The invention relates to the technical field of assisted reproduction, and discloses an in-vitro maturation culture system and method for immature oocytes of elderly women based on NMN supplement. The culture system is an in vitro maturing (IVM) culture solution containing 10 [mu] M of NMN (Nitrogen Morphocyte Nitride), and the culture system is an in vitro maturing (IVM) culture solution containing 10 [mu] M of NMN; the culture method comprises the following steps: preparing an IVM culture solution; culturing in vitro; carrying out in-vitro fertilization; and early embryo development observation. According to the application, NAD < + > is improved through targeted supplement of NMN, the mitochondrial function is further repaired, a brand new path for cooperatively regulating meiosis is achieved, and the problem that in the prior art, only oxidative stress is relieved, but the energy supply defect cannot be solved is solved. Experimental data show that the IVM system added with the NMN can remarkably improve the blastocyst formation rate of oocytes of elderly women, the mitochondrial function is recovered, and the NMN-containing IVM system has a remarkable clinical transformation value.

Description

Technical Field

[0001] The present application relates to the field of assisted reproductive technology, and more specifically, it relates to an in vitro maturation culture system and method for immature oocytes of advanced females based on NMN supplementation. Background Art

[0002] During controlled ovarian hyperstimulation (COH) cycles, the number of oocytes obtained from older women is limited, and often contains a significant number of immature oocytes. The low quality and developmental potential of in vitro matured oocytes are bottlenecks facing current in vitro maturation (IVM) culture technology. Mitochondrial dysfunction in oocytes from older women (decreased mtDNA copy number, increased abnormal mitochondrial structure, and decreased NAD+ levels) leads to insufficient energy supply for meiosis. Conventional IVM culture media has poor maturation effects on older oocytes and does not improve blastocyst formation rates. Existing technologies, such as the addition of coenzyme Q10 and resveratrol, only alleviate oxidative stress but do not target the NAD+ metabolic axis and are unable to reverse mitochondrial dysfunction. Nicotinamide mononucleotide (NMN) has been shown to increase NAD+ levels in oocytes in mouse models, but has not been applied to the IVM system for older human oocytes.

[0003] Therefore, exploring the role of NMN in the IVM system of human aged oocytes is expected to break through the bottleneck of IVM of aged oocytes and provide new strategies for assisted reproductive technology. Summary of the Invention

[0004] The purpose of the present invention is to improve the maturation rate of immature oocytes in elderly women and the potential for subsequent embryonic development. To this end, the present invention optimizes the culture system and method for in vitro maturation of immature oocytes in elderly women (≥35 years old) by adding NMN to solve the above technical problems.

[0005] NAD+ is a crucial intracellular co-metabolic factor involved in regulating important biological processes such as cellular metabolism, energy production, and oxidative stress. Maintaining appropriate NAD+ levels during oocyte development is crucial for cellular metabolism and function. Reduced NAD+ synthesis in aged oocytes leads to a significant decrease in the NAD+ / NADH ratio, mitochondrial dysfunction, and impaired oocyte meiosis, resulting in in vitro maturation of immature oocytes and poor developmental potential.

[0006] NMN is a precursor molecule for NAD+ and can be converted to NAD+ through the NAD+ synthesis pathway. Adding NMN to in vitro maturation culture media can provide additional NAD+ synthesis and increase intracellular NAD+ content. By increasing NAD+ levels in oocytes, cellular metabolic processes can be optimized, increasing cellular resistance to oxidative stress, and promoting DNA repair and cell signaling. This will help enhance the developmental potential of in vitro matured oocytes and is expected to provide new possibilities for the improvement and development of IVM technology.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an in vitro maturation culture system for immature oocytes of advanced females based on NMN supplementation, wherein the culture system is an IVM culture medium containing NMN.

[0009] Furthermore, the NMN is 10 μM.

[0010] Furthermore, the culture system also includes 75mIU / mL FSH and 75mIU / mL LH.

[0011] In the second aspect, the present invention provides a method for preparing an in vitro maturation culture system for immature oocytes of advanced females based on NMN supplementation as described in the first aspect, wherein NMN powder is dissolved in sterile deionized water to prepare an NMN storage solution; NMN storage solution is added to a commercial in vitro maturation culture medium to prepare an IVM working solution with a final concentration of 10 μM NMN, and 75 mIU / mL LSH and 75 mIU / mL LH are dissolved in it; the IVM working solution is used to make 25 μL-30 μL droplets in an embryo culture dish, covered with oil, and placed in a 37-degree incubator for equilibrium for at least 4 hours.

[0012] In a third aspect, the present invention provides a method for in vitro maturation and culture of immature oocytes from advanced-age women based on NMN supplementation, comprising the following steps:

[0013] Step 1, in vitro maturation culture medium preparation: Dissolve NMN powder in sterile deionized water to prepare NMN storage solution; add NMN storage solution to commercial in vitro maturation culture medium to prepare IVM working solution with a final concentration of 10μM NMN, and dissolve 75mIU / mL FSH and 75mIU / mL LH; Use IVM working solution to make 25μL-30μL droplets in embryo culture dishes, cover with oil, and equilibrate in a 37°C incubator for at least 4 hours.

[0014] Step 2: The immature oocytes were transferred into IVM culture medium, 2-3 immature oocytes / microdroplet, and cultured in a three-gas incubator for 24-28 hours. The polar body extrusion was observed under an inverted microscope, and the maturation rate was calculated.

[0015] Step 3: Culture the mature oocytes for intracytoplasmic sperm injection (ICSI); then transfer them to full embryo culture medium and culture them in a three-gas incubator. Observe fertilization and embryo development, and calculate the fertilization rate, cleavage rate, high-quality embryo rate, blastocyst formation rate, and high-quality blastocyst rate. Blastocysts are then vitrified.

[0016] In summary, this application has the following beneficial effects:

[0017] The present invention breaks through the limitations of existing antioxidant solutions, combines the metabolic characteristics of mitochondrial dysfunction and NAD+ metabolic defects in oocytes of advanced age women (≥35 years old), and coordinates the regulation of energy supply and mitochondrial function by targeting the NAD+ metabolic axis. It establishes and optimizes the in vitro maturation culture system of immature oocytes (GV phase / MI phase) in clinical controlled ovarian stimulation cycles, forming a more comprehensive IVM support program for advanced age oocytes. Significantly improve the embryonic development potential of in vitro matured oocytes, especially the subsequent blastocyst formation rate. And based on the safety and clinical research basis of NMN (such as the anti-aging field), develop an efficient and low-cost IVM technology that can be directly applied to human assisted reproduction, improve the utilization rate of oocytes in elderly women in assisted reproductive technology, and promote clinical translational applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Representative images of immature oocytes in the NMN-added group during in vitro maturation (from GV stage to MII stage). (A) GV stage oocyte (germinal vesicle visible); (B) MII stage oocyte (first polar body not extruded); (C) MII stage mature oocyte (first polar body extruded); (D) Observation of spindle morphology and position in MII stage oocytes.

[0019] Figure 2 Representative images of high-quality blastocysts formed from GV-stage oocytes cultured in vitro in the NMN-supplemented group. (A) 38-year-old patient, high-quality blastocyst 4BB formed on Day 5; (B) 40-year-old patient, high-quality blastocyst 4BB formed on Day 6; (C) 35-year-old patient, high-quality blastocyst 4AA formed on Day 5; (D) 39-year-old patient, high-quality blastocyst 4BA formed on Day 5.

[0020] Figure 3Mitochondrial distribution and mitochondrial membrane potential in oocytes from different origins. Mitotraker, a mitochondrial distribution probe, and TMRE, a mitochondrial membrane potential probe, were stained in oocytes from different origins. Green: MitoTracker, red: TMRE, blue: Hoechst 33342; (B) Quantitative analysis of MitoTracker fluorescence intensity in oocytes from different origins. (C) Quantitative analysis of TMRE fluorescence intensity in oocytes from different origins. Data are expressed as mean ± SD; *: P < 0.05; **: P < 0.01; ns: P ≥ 0.05. DETAILED DESCRIPTION

[0021] The technical solutions and effects of the present application are further described in detail below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0022] Example 1: Liquid Configuration

[0023] NMN powder (Sigma-Aldrich, USA) (purity ≥98%) was dissolved in sterile deionized water to prepare a 10mM stock solution. NMN stock solution (10mM) and Hemeiqi (high-purity urogonadotropin for injection) (Huiling Pharmaceuticals) solution were added to commercial oocyte in vitro maturation culture medium (Ayers (Zhejiang) Medical Technology Co., Ltd.) to prepare an IVM working solution with a final concentration of 10μM (NMN), 75mIU / mL follicle-stimulating hormone (FSH), and 75mIU / mL luteinizing hormone (LH). Use the IVM working solution to make 25μL-30μL microdrops in a 30mm embryo culture dish (BD Company, USA), cover with oil, and equilibrate in a 37-degree incubator for at least 4 hours.

[0024] Example 2: Immature oocyte collection

[0025] Thirty-six hours after hCG injection, cumulus-oocyte complexes (COCs) were retrieved under transvaginal ultrasound guidance. Blood cells and cellular debris surrounding the COCs were washed away in GMOPS PLUS (Vitrolife, Sweden) oocyte manipulation medium, and the COCs were transferred to a droplet of G-IVF PLUS (Vitrolife, Sweden) fertilization medium and cultured for 2 hours. Cumulus cells surrounding the oocytes were individually stripped using 180P and 150P (150μm) stripping needles (sunlight) in the presence of hyaluronidase (80 IU). Mature oocytes (MII stage) were used for intracytoplasmic sperm injection (ICSI). Immature oocytes were transferred to in vitro maturation medium, with 2-3 immature oocytes per droplet, and cultured in a tri-gas incubator (37°C, 6% CO2, 5% O2, saturated humidity).

[0026] Example 3: Maturity Assessment and Intracytoplasmic Sperm Injection (ICSI) Insemination

[0027] The immature oocytes collected in Example 2 were cultured for in vitro maturation for 24-28 hours, and the polar body extrusion (a marker of the MII stage) was observed under an inverted microscope.

[0028] Mature oocytes underwent ICSI and were transferred to GTL PLUS (Vitrolife, Sweden) embryo culture medium. Fertilization and embryo development were observed after 16-18 hours. Fertilization rate, cleavage rate, high-quality embryo rate, blastocyst formation rate, and high-quality blastocyst rate were calculated, and blastocysts were vitrified.

[0029] Example 4: Effect of NMN Addition on In Vitro Maturation of Immature Oocytes from Advanced-Age Women

[0030] (1) Experimental materials and methods

[0031] Immature oocyte collection: Patients underwent controlled ovarian stimulation (COH). On the day of oocyte retrieval, cumulus-oocyte complexes (COCs) were retrieved from the follicular fluid under transvaginal ultrasound guidance. Surrounding COCs were washed with GMOPS PLUS (Vitrolife, Sweden) to remove blood cells and debris. The COCs were then transferred to G-IVF PLUS (Vitrolife, Sweden) droplets and cultured for 2 hours. Cumulus cells were then removed using 180P and 150P (150μm) (sunlight) stripping needles in a hyaluronidase solution (80 IU). Oocyte maturity was observed under an inverted microscope. MII-stage mature oocytes were selected for ICSI, while immature oocytes were used for in vitro maturation.

[0032] A total of 164 immature oocytes were collected from elderly women (≥35 years old), including 108 GV stage oocytes and 56 MI stage oocytes.

[0033] (2) In vitro maturation culture system and grouping

[0034] Control group (Aged group): 72 immature oocytes (48 at GV stage and 24 at MI stage), cultured in conventional in vitro maturation medium (Zhejiang Ayers) + 75mIU / mL (LH+FSH).

[0035] Experimental group (Aged+NMN group): 92 immature oocytes (60 at the GV stage and 32 at the MI stage), 10 μM NMN was added to the conventional in vitro maturation culture medium (Zhejiang Ayers) + 75 mIU / mL (LH+FSH).

[0036] (3) Enrollment conditions

[0037] Patients with ≥2 immature oocytes remaining after ICSI on the same day were randomly assigned to an experimental or control group. Oocytes were cultured in vitro for 24–28 hours, their maturation was observed, and the oocyte maturation rate was calculated. The mature oocytes then underwent ICSI. They were then transferred to GTL PLUS, a full-scale culture medium, and placed in a tri-gas incubator for continued culture until day 6 (D6). Fertilization rate, cleavage rate, high-quality embryo rate, and blastocyst formation rate were calculated, and vitrification was performed simultaneously. To minimize the influence of confounding factors, all ICSIs were performed by a single researcher.

[0038] (4) Experimental results

[0039] Table 1 Comparison of in vitro maturation rates of immature oocytes in older women

[0040] Aged Group Aged+NMN group Number of GV stage oocytes (n) 48 60 GV stage maturity rate (GV) (%, n) 81.2(39 / 48) 85.0(50 / 60) Number of oocytes at MI stage (n) 24 32 MI maturity rate (MI) (%, n) 79.2(19 / 24) 84.3(27 / 32) Total number of oocytes (n) 72 92 Overall maturity rate (%, n) 80.6(58 / 72) 83.7(77 / 92)

[0041] (5) Result analysis

[0042] Overall maturity rate: The maturity rate of the experimental group (83.7%) showed an upward trend compared with the control group (80.6%), but the difference did not reach statistical significance (P>0.05).

[0043] Subgroup analysis: GV maturity rate: the experimental group (85.0%) was 3.8 percentage points higher than that of the control group (81.2%);

[0044] The MI maturity rate in the experimental group (84.3%) was 5.1 percentage points higher than that in the control group (79.2%);

[0045] There was no statistically significant difference between the two groups (P>0.05).

[0046] like Figure 1 As shown in the figure, after the experimental group, i.e., oocytes were treated with NMN, typical morphological changes from the GV stage (A), MI stage (B) to the MII stage (C) were observed, and the spindle structure of the MII stage oocytes was intact (D), indicating that the in vitro maturation process was normal.

[0047] Conclusion: Adding NMN to the in vitro maturation medium can improve the maturation rate of immature oocytes (GV stage, MI stage) in older women, although the difference did not reach statistical significance.

[0048] Example 5: Effect of NMN on the embryonic development potential of in vitro matured oocytes in older women

[0049] (1) Experimental materials and methods

[0050] A total of 150 oocytes from elderly women (≥35 years old) that had been matured to the MII stage in vitro were selected, including 92 in the experimental group (Aged+NMN group) and 58 in the control group (Aged group).

[0051] After ICSI, all oocytes were transferred to GTL PLUS embryo culture medium to observe fertilization, cleavage, and embryonic development. Blastocysts were cultured until day 6 (D5), and the quality of blastocysts was assessed according to the Gardner criteria.

[0052] (2) Experimental results

[0053] Table 2 Comparison of embryonic development potential of in vitro matured oocytes in older women

[0054] Aged Group Aged+NMN group Number of MII oocytes (GV+MI) (n) 58 92 Oocyte degeneration rate after ICSI (%, 12.1(7 / 58) 9.8(9 / 92) Normal fertilization rate (%, n) 78.4(40 / 51) 80.7(67 / 83) Cleavage rate (%, n) 85.0(34 / 40) 89.6(60 / 67) Usable embryo rate (%, n) 29.4(10 / 34) 53.3(32 / 60)a High-quality embryo rate (%, n) 8.8(3 / 34) 31.7(19 / 60)a Blastocyst formation rate (%, n) 0(0 / 10) 31.2(10 / 32)a High-quality blastocyst rate (%, n) 0 70.0(7 / 10)a

[0055] Note: a indicates significant difference compared with the control group (P<0.05).

[0056] (3) Result analysis

[0057] There was no significant difference in the normal fertilization rate (80.7% vs 78.4%) and cleavage rate (89.6% vs 85.0%) between the experimental group (NMN-supplemented group) and the control group (both P>0.05). However, the usable embryo rate (53.3% vs 29.4%) and high-quality embryo rate (31.7% vs 8.8%) were significantly improved (both P<0.05), especially the blastocyst formation rate (31.2% vs 0%) and high-quality blastocyst rate (70.0% vs 0%) (both P<0.05), which showed a significant improvement (both P<0.05).

[0058] like Figure 2 As shown, the high-quality blastocysts (AA / AB grade) obtained in the experimental group had regular morphology, clear and dense cell clusters, and tightly arranged trophoblast cells, which met the standards for high-quality blastocysts.

[0059] Conclusion: NMN supplementation significantly improves the embryonic development potential of in vitro matured oocytes in older women, especially in key indicators such as high-quality embryos and blastocyst formation ( Figure 2 This study provides an effective technical solution for improving the outcomes of assisted reproduction in elderly patients.

[0060] Example 6: NMN increases mitochondrial membrane potential in in vitro matured oocytes from older women

[0061] (1) Detection method:

[0062] Mitochondrial distribution was labeled with Mitotracker Green, mitochondrial membrane potential was detected with TMRE probe, and nuclear staining was performed with Hoechst 33342. Fluorescence signals were acquired using a confocal microscope, and fluorescence intensity was quantitatively analyzed using ImageJ software. Statistical analysis was performed using the t-test, with the significance threshold set at P < 0.05.

[0063] (2) Sample grouping:

[0064] Young control group: donated in vivo matured oocytes (MII stage)

[0065] Aged experimental group: Aged oocytes matured in vitro (Aged group)

[0066] Aged intervention group: in vitro matured aged oocytes supplemented with 10 μM NMN (Aged+NMN group)

[0067] (3) Experimental results:

[0068] The results of laser confocal imaging and fluorescence signal quantitative analysis are shown in the figure:

[0069] The fluorescence intensity of TMRE and Mitotracker in the Aged group was significantly lower than that in the young control group (P<0.01);

[0070] The TMRE fluorescence intensity in the Aged+NMN group was significantly enhanced compared with the Aged group (P=0.0401), and the Mitotracker fluorescence intensity was significantly increased (P=0.0466), reaching 75%-85% of the level of the young control group.

[0071] like Figure 3 : Figure 3 A. Co-staining results of mitochondrial distribution (green) and membrane potential (red), showing the differences in mitochondrial membrane potential among the three groups of oocytes; Figure 3 B Histogram of quantitative analysis of TMRE and Mitotracker fluorescence intensity, with statistical differences marked (*P<0.05, **P<0.01).

[0072] Conclusion: Adding NMN can increase mitochondrial activity and membrane potential of in vitro matured oocytes from older women and improve mitochondrial function.

[0073] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. An in vitro maturation culture system for immature oocytes of advanced-age women based on NMN supplementation, characterized in that: The culture system is an IVM culture medium containing NMN.

2. The in vitro maturation culture system for immature oocytes of advanced-age women based on NMN supplementation according to claim 1, characterized in that: The NMN was 10 μM.

3. The in vitro maturation culture system for immature oocytes of advanced-age women based on NMN supplementation according to claim 1, characterized in that: The culture system also includes 75 mIU / mL FSH and 75 mIU / mL LH.

4. A method for preparing an in vitro maturation culture system for immature oocytes of advanced-age women based on NMN supplementation, characterized in that: Dissolve NMN powder in sterile deionized water to prepare NMN storage solution; add NMN storage solution to commercial in vitro maturation culture medium to prepare IVM working solution with a final concentration of 10μM NMN, and dissolve 75mIU / mL FSH and 75mIU / mL LH; use IVM working solution to make 25μL-30μL droplets in the embryo culture dish, cover with oil, and place in a 37-degree incubator for at least 4 hours to equilibrate.

5. A method for in vitro maturation and culture of immature oocytes from elderly women based on NMN supplementation, characterized in that: The following steps are involved: Step 1, in vitro maturation culture medium preparation: Dissolve NMN powder in sterile deionized water to prepare NMN storage solution; add NMN storage solution to commercial in vitro maturation culture medium to prepare IVM working solution with a final concentration of 10μM NMN, and dissolve 75mIU / mL FSH and 75mIU / mL LH; Use IVM working solution to make 25μL-30μL droplets in embryo culture dishes, cover with oil, and equilibrate in a 37°C incubator for at least 4 hours. Step 2: The immature oocytes were transferred into IVM culture medium, 2-3 immature oocytes / microdroplet, and cultured in a three-gas incubator for 24-28 hours. The polar body extrusion was observed under an inverted microscope, and the maturation rate was calculated. Step 3: Culture mature oocytes for intracytoplasmic sperm injection; then transfer them to full embryo culture medium and culture them in a three-gas incubator to observe fertilization and embryo development. Calculate the fertilization rate, cleavage rate, high-quality embryo rate, blastocyst formation rate and high-quality blastocyst rate, and vitrify the blastocysts.

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