245 EFFECT OF BOVINE OOCYTE MATURATION SYSTEM ON RELATIVE ABUNDANCE OF mRNA

2010 ◽  
Vol 22 (1) ◽  
pp. 280
Author(s):  
M. M. Pereira ◽  
F. Q. Costa ◽  
A. P. Oliveira ◽  
C. M. Assunção ◽  
R. V. Serapião ◽  
...  
Keyword(s):  
Relative Abundance ◽  
In Vitro Maturation ◽  
Rna Extraction ◽  
Cumulus Cell ◽  
Sao Paulo ◽  
São Paulo ◽  
Crossbred Cows ◽  
Stored Mrna

The oocyte cytoplasm contains several transcripts that are important for early pre-implantation embryo development, and alterations on the amount of these stored mRNA can disturb oocyte competence. The aim of this study was to evaluate the relative abundance of specific transcripts in oocytes matured in vivo or in vitro. For in vitro maturation, immature oocytes were obtained by ovum pickup from 4 crossbred cows (group 1: G1) or from ovaries collected at a slaughterhouse (group 2: G2) and matured in TCM-199 containing 10% estrus cow serum and 2 μg FSH for 24 h under 5% CO2 in air at 38.5°C. For in vivo maturation, the same crossbred cows used in G1 received a progesterone intravaginal implant (CIDR®, Eazi-Breed CIDRO, São Paulo, Brazil) and 2 mg of estradiol benzoate (Estrogin®, Farmavet, São Paulo, Brazil) on Day 0. On Day 4, cows were superstimulated with 180 mg FSH (Folltropin®, Bioniche, Canada) injected in 6 decreasing doses every 12 h, and on Day 6, the cows received 0.53 mg of sodium cloprostenol (Ciosin®, Cooper, São Paulo, Brazil). On Day 7, CIDR® was removed and 2.5 mg of gonadorelina (Gestran-Plus®, Tecnopec, São Paulo, Brazil) was injected. Ovum pickup was performed 18 h after gonadorelina injection. Oocytes with expanded cumulus cell were then pooled and used as in vivo-matured oocytes (group 3: G3). Oocytes from all groups were denuded and frozen in liquid nitrogen. Pools of 10 oocytes for each group were subject to RNA extraction and reverse transcription. cDNA was amplified by real-time PCR using the beta-actin gene as the endogenous reference. The transcripts analyzed are encoded by TEA domain 2 (TEAD2), high mobility group N1 (HMGN), zygotic arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor-9 (GDF9), and peroxiredoxin 1 (PRDX1) genes. Results were analyzed by REST software v.2 using the pair-wise, fixed reallocation randomization test. Data from G3 were used as calibrator. There was no difference (P > 0.05) on relative abundance of all transcripts between pools of oocytes matured in vitro or in vivo obtained from the same cows (G1 and G3, respectively). However, the relative abundance of GDF9 (0.22 ± 0.04-fold) was less (P < 0.05), whereas the relative abundance of TEAD2 transcripts (4.27 ± 2.14-fold) was greater (P < 0.05) for in vitro-matured oocytes obtained from slaugterhouse ovaries (G2) when compared with in vivo-matured oocytes (G3). No difference (P > 0.05) on relative abundance was found between G2 and G3 for the other genes. These data suggest that in vitro maturation does not alter the relative abundance of some transcripts stored into oocytes when compared with the ones stored in oocytes obtained from the same donors by means of multiple ovulation. Financial support was provided by CNPq and FAPEMIG.

scholarly journals α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes

2018 ◽  
Vol 30 (12) ◽  
pp. 1728 ◽  
Author(s):  
M. Arias-Álvarez ◽  
R. M. García-García ◽  
J. López-Tello ◽  
P. G. Rebollar ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Rabbit Model ◽  
Developmental Competence ◽  
Antioxidant Agents ◽  
Junction Protein

The developmental competence of in vitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400 µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus–oocyte complexes (COCs) after IVM with 100 μM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100 μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 μM α-tocopherol was included in the IVM medium, but remained low compared with in vivo-matured oocytes. In conclusion, the addition of 100 μM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

272 A NEW APPROACH TO IN VITRO MATURATION (IVM) AND EMBRYO IN VITRO PRODUCTION: INDUCED IVM SUBSTANTIALLY IMPROVES EMBRYO YIELD AND PREGNANCY OUTCOMES

2010 ◽  
Vol 22 (1) ◽  
pp. 293
Author(s):  
R. B. Gilchrist ◽  
F. K. Albuz ◽  
J. G. Thompson
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
In Vitro Production ◽  
New Approach ◽  
Fetal Survival ◽  
Wide Range ◽  
Vitro Production

Oocyte in vitro maturation (IVM) is the rate-limiting step in the in vitro production (IVP) of embryos. Oocyte maturation in vivo is a highly orchestrated, induced process, whereby cAMP-mediated meiotic arrest is overridden by the gonadotrophin surge prior to ovulation. However, aspirated oocytes resume maturation spontaneously compromising developmental competence. Hence, we hypothesized that establishing an induced system in vitro would synchronize oocyte-somatic cell communication leading to improved oocyte quality. Abattoir-collected bovine or 129/Sv mouse oocytes were treated for the first 1 to 2 h in vitro (pre-IVM) with the adenylate cyclase activator forskolin (100 μM, 50 μM, respectively) and a nonspecific phosphodiesterase (PDE) inhibitor, IBMX (500 μM, 50 μM), which substantially increased cumulus-oocyte complex (COC) cAMP (bovine, 180 v. 2 fmol/COC, treated v. control; P < 0.001). To maintain oocyte cAMP levels and prevent precocious oocyte maturation, IVM media (VitroMat + BSA) contained an oocyte-specific (type 3) PDE inhibitor, cilostamide (20 μM, 0.1 μM), plus FSH to induce maturation. The net effect of this system (induced-IVM) was to increase oocyte-cumulus cell gap-junctional communication (bovine: 1000 ± 148 v. 340 ± 73 unit, treated v. control; P < 0.05) and to slow meiotic progression through prophase I to metaphase II, extending the normal IVM interval (bovine: 30 v. 24 h, mouse: 22 v. 18 h; treated v. control). FSH was required to complete maturation and FSH-induced maturation was prevented by an epidermal growth factor receptor inhibitor, AG1478 (2.5 μM), demonstrating induced oocyte maturation functions via secondary autocrine signaling within the cumulus cell compartment. These effects on COC functions had profound consequences for oocyte developmental potential. In completely serum-free bovine IVP, induced-IVM more than doubled blastocyst yield (69 v. 27%, treated v. control; P < 0.05) and improved blastocyst quality (186 v. 132 blastomeres). To achieve these rates, the pre-IVM phase, the modified IVM conditions, and delayed IVF were all required. Adapting the system to the mouse, induced-IVM increased blastocyst rate (86 v. 55%, treated v. control; P < 0.05), implantation rate (51 v. 25%; P < 0.01), fetal survival rate (29 v. 5%; P < 0.01) and fetal weight (0.9 v. 0.5 g; P < 0.01). All these embryonic and fetal outcomes in mice were equivalent (P > 0.05) using induced-IVM to levels obtained from in vivo-matured control oocytes (conventional IVF). Data were analyzed by ANOVA. In conclusion, induced-IVM mimics some of the characteristics of oocyte maturation in vivo and substantially improves oocyte developmental outcomes in 2 disparate mammalian species. Adaption of this new approach to clinical/field conditions should lead to new opportunities for a wide range of reproductive biotechnologies. Such a notable increase in IVM efficiency could see IVP as the preferred embryo production technology in future livestock artificial breeding programs. Funded by an Australian Research Council Linkage Grant and Cook Australia. Thanks to M. Sasseville, M. Lane, and D. T. Armstrong.

Recent insights into oocyte - follicle cell interactions provide opportunities for the development of new approaches to in vitro maturation

2011 ◽  
Vol 23 (1) ◽  
pp. 23 ◽  
Author(s):  
Robert B. Gilchrist
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Egf Receptor ◽  
Cumulus Cell ◽  
Follicle Cell ◽  
Cell Interactions ◽  
Developmental Competence ◽  
New Approaches

The last 5–10 years of research in ovarian and oocyte biology has delivered some major new advances in knowledge of the molecular and cellular processes regulating oocyte maturation and oocyte developmental competence. These new insights include, among others: (1) the knowledge that oocytes regulate granulosa and cumulus cell differentiation, ovulation rate and fertility via the secretion of soluble paracrine growth factors; (2) new perspectives on the participation of cyclic nucleotides, phosphodiesterases and gap junctions in the regulation of oocyte meiotic arrest and resumption; and (3) the new appreciation of the mechanisms of LH-induced oocyte maturation and ovulation mediated by the follicular cascade of epidermal growth factor (EGF)-like peptides, the EGF receptor and their intracellular second messengers. These recent insights into oocyte–follicle cell interactions provide opportunities for the development of new approaches to oocyte in vitro maturation (IVM). Laboratory IVM methodologies have changed little over the past 20–30 years and IVM remains notably less efficient than hormone-stimulated IVF, limiting its wider application in reproductive medicine and animal breeding. The challenge for oocyte biologists and clinicians practicing IVM is to modernise clinical IVM systems to benefit from these new insights into oocyte–follicle cell interactions in vivo.

271 NUCLEAR MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS - OOCYTE COMPLEXES

2013 ◽  
Vol 25 (1) ◽  
pp. 283
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Nuclear Maturation ◽  
Wood Bison

Methods of producing wood bison embryos in vivo and in vitro are being developed in an effort to preserve the genetic diversity of this threatened species. Previous data from our laboratory suggest that oocytes collected 24 h after LH treatment had not yet achieved nuclear maturation. The objectives of this study were (1) to determine the optimal interval of time after hCG treatment required for in vivo maturation of cumulus–oocyte complexes (COC) in wood bison, and (2) to compare the maturational characteristics of COC after in vitro v. in vivo maturation. Follicular wave emergence was synchronized among bison cows (n = 25) by follicular ablation (Day –1) from May to June. Ovarian superstimulation was induced with FSH IM diluted in 5 mg mL–1 of hyaluronan (MAP-5, Bioniche, Belleville, Ontario, Canada) given on Day 0 (300 mg) and Day 2 (100 mg). Superstimulated cows were assigned randomly to 5 groups (n = 5/group): COC collected on Day 4 with no maturation (control), or matured in vitro for 24 or 30 h, or collected 24 or 30 h after treatment with 2000 IU of hCG IM on Day 4. The COC were collected by transvaginal ultrasound-guided follicle aspiration. In vitro maturation was done in TCM-199 with 5% calf serum, 5 µg mL–1 of LH, 0.5 µg mL–1 of FSH, and 0.05 µg mL–1 of gentamicin, at 38.5°C and in 5% CO2. To assess nuclear maturation, oocytes were stained with anti-lamin AC/DAPI (4′,6-diamidino-2-phenylindole). Nuclear stages were classified as germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Comparisons among groups were made by ANOVA and Fisher’s exact test (Table 1). A mean (± SEM) of 7.6 ± 0.6 COC was collected per bison; no differences were observed among groups (P = 0.37). Cumulus cell expansion was more extensive after in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30-h group (97%; P < 0.05). No COC were expanded in the control (0 h) group, and none reached MI. Maximal nuclear maturation was achieved in vitro by 24 h; that is, there was no difference in the proportion of MII-stage COC at 24 versus 30 h. However, between 24 and 30 h of in vivo maturation, the percentage of nuclear stages GV + GVBD decreased from 54 to 24% (P < 0.05), whereas nuclear stages MI + MII increased from 39 to 74% (P < 0.05). In conclusion, nuclear maturation occurred earlier in vitro versus in vivo, but the consequences of this difference are unknown. Although more than one-third of oocytes matured in vivo for 30 h were mature enough to permit immediate IVF, whether additional in vivo maturation time would be beneficial to fertilization rates remains to be tested. Table 1.Nuclear status of wood bison oocytes after in vitro or in vivo maturation Thanks to Bioniche Canada.

179 DIFFERENT GONADOTROPIN SUPPLEMENTATIONS ALTER mRNA EXPRESSION PATTERN IN BOVINE OOCYTES DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 198
Author(s):  
C. Blaschka ◽  
B. Zimmer ◽  
C. Wrenzycki
Keyword(s):  
Mrna Expression ◽  
Expression Pattern ◽  
Relative Abundance ◽  
In Vitro Maturation ◽  
Immature Oocytes ◽  
Mrna Expression Pattern ◽  
First Polar Body ◽  
Final Maturation

During final maturation (between LH surge and ovulation) in vivo, a switch from oestradiol to progesterone dominance within the follicle is well described. The aim was to mimic the in vivo situation during in vitro maturation via the supplementation of different gonadotropins. Groups of 30 cumulus-oocyte complex (abattoir-derived ovaries) were matured in TCM 199 plus different gonadotropins (eCG/hCG; FSH/LH, each in 0.05 or 0.01 IU; only FSH 0.05 IU; without gonadotropins) using a standard protocol without oil overlay. In Experiment 1, denuded oocytes were collected at 0 h (immature) and after 24 h of in vitro maturation (IVM; exhibit first polar body). In Experiment 2, oocytes were collected at different time points [0 (immature), 4, 8, 12, 16, 20, and 24 h] after IVM in eCG/hCG-supplemented medium. They were individually stored at −80°C until analyses. Transcripts of developmental competence (BMP15, GDF9, ZAR1), glucose or steroid metabolism (G6PD, STAR), and progesterone receptors (PGR, PGRMC1/2) were examined in individual oocytes via quantitative RT-PCR (n = 5). For statistical analyses, 1-way ANOVA followed by a Tukey test was used. Relative abundance of BMP15 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group matured for 24 h with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature oocytes. Relative amount of G6PD and PGRMC2 mRNA was significantly lower (P ≤ 0.05) in mature oocytes of the group with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature ones. Relative abundance of GDF9, STAR, and ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group with eCG/hCG, FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins compared with immature oocytes. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in mature oocytes of the group with eCG/hCG than in immature oocytes, FSH/LH 0.01IU, FSH 0.05 IU, and without gonadotropins (Experiment 1). Relative amount of GDF9 transcripts was significantly lower (P ≤ 0.05) in mature oocytes collected after 24 h than in immature ones. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in oocytes collected after 20 and 24 h of IVM than in immature ones. Relative amount of ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h of IVM than in immature oocytes; likewise, they were significantly lower (P ≤ 0.05) in oocytes collected after 12, 16, 20, and 24 h than in oocytes collected after 4 h of IVM. Relative amount of STAR mRNA was significantly lower (P ≤ 0.05) in oocytes collected after 24 h than in immature ones, and significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h than in oocytes collected after 4 h of IVM (Experiment 2). The results suggest a down-regulation of most transcripts during the period of IVM with different gonadotropin supplements with exception of PGR. Furthermore, most transcripts follow a timely regulated mRNA expression pattern during the entire IVM period. We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).

P–547 Single-cell RNA sequencing identifies molecular regulations associated with poor maturation performance on rescue in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
W T Lee ◽  
K W Ng ◽  
J Liao ◽  
A C S Luk ◽  
H C Suen ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Single Cell ◽  
Rna Sequencing ◽  
Maternal Age ◽  
Polar Body ◽  
Rna Extraction ◽  
Cumulus Cell ◽  
Single Cell Rna Sequencing

Abstract Study question What is the transcriptome signature associated with rescuein vitro matured (rIVM) oocytes? Summary answer GATA–1/CREB1/WNT signaling axis was repressed in rIVM oocytes of poor quality. What is known already rIVM aims to produce mature oocytes (MII) for in vitro fertilization (IVF) through IVM of immature oocytes collected from stimulated ovaries. It is less popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress, and the absence of cumulus cell support in oocytes could account for the failure of rIVM. Study design, size, duration We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo (IVO) oocytes (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors, followed by studying the maternal age effect and ovarian responses on rIVM oocyte transcriptomes. Participants/materials, setting, methods Human oocytes were collected from donors aged 28–41 years with a body mass index of &lt; 30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analyzed. Selected genes in therIVM vs. IVO comparison were validated by quantitative real-time PCR. Main results and the role of chance The transcriptome profiles of rIVM/IVO showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least two-fold change and adjusted p &lt; 0.05) were found to be enriched in metabolic processes, biosynthesis, and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signaling in rIVM when compared with IVO oocytes. We found that estradiol level exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio). rIVM oocytes with higher MII ratio showed over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor outcomes of rIVM, we compared oocytes collected from young donors with high MII ratio versus donors of advanced maternal age and revealed CREB1was an important regulator in rIVM. Our study identified GATA–1/CREB1/WNT signaling was repressed in both rIVM condition and rIVM oocytes of low-quality. Limitations, reasons for caution In the rIVM oocytes of high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle so polar body extrusion was the only indicator. Wider implications of the findings: This study showed that GATA–1/CREB1/WNT signaling and antioxidant actions were repressed in rIVM condition and was further downregulated in rIVM oocytes of low-quality, providing us the foundation of subsequent follow-up research on human subjects. Trial registration number Not applicable

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