概要: Study Question: What effects do maternal age and oocyte maturation stage have on the human oocyte transcriptome that may be associated with oocyte developmental potential? Summary Answer: Although polyadenylated transcript abundance changes during human oocyte maturation irrespective of age, young (YNG) and advanced maternal age (AMA) metaphase II (MII) oocytes exhibit divergent transcriptomes. What is known already: Maternal age and maturation stage affect oocyte polyadenylated transcript abundance in human oocytes. Although RNA-Seq analysis of single human MII oocytes has been conducted, comparison of the germinal vesicle (GV) and MII oocyte transcriptomes has not been investigated using RNA-Seq, a technique that could provide novel insight into oocyte maturation and age-associated aberrations in gene expression. Participants / materials, settings, methods: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided egg retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 hour incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads in HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, Weighted Gene Correlation Network Analysis (WGCNA), 3’UTR motif analysis, Ingenuity Pathway Analysis) were performed. Main results and the role of chance: Within the 12,770 expressed genes in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least 3 of 5 replicates for a minimum of one sample type), 458 and 3,506 genes significantly (adjusted p < 0.05 and log2 fold change > 1) increased and decreased in polyadenylated transcript abundance during oocyte maturation, respectively. The differentially expressed genes were enriched (FDR < 0.05) for biological functions and canonical pathways related to cell cycle and mitochondrial function. The majority (76%) and minority (25%) of up- and down-regulated transcripts with a complete 3’UTR were predicted to be targets of cytoplasmic polyadenylation (910 total genes), respectively. Differential gene expression analysis between young and advanced maternal age oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted p < 0.1 and log2 fold change > 1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5, and PRDX1, which have been reported to affect oocyte developmental potential and be markers of oocyte quality. Despite similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle, and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were determined to be correlated (p < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. Limitations, reasons for caution: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by in vitro maturation of patient oocytes, which has the benefit of identifying intrinsic differences between samples, but may not be completely representative of in vivo matured oocytes. Thus, these factors should be considered when interpreting the results. Wider Implications of the findings: Transcriptome profiles of young and advanced maternal age oocytes, particularly at the MII stage, suggest aberrant transcript abundance contributes to the age-associated decline in fertility.
项目整体设计: Study design, size, and duration: The effect of oocyte maturation (cross-sectional analysis) and age (longitudinal analysis) on polyadenylated transcript abundance were analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (<30 years; average age 26.8; range 20-29) and five advanced maternal age (≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 young (5 GV and 5 MII) and 10 advanced maternal age (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis.
A single verified GV and a single in vitro matured MII oocyte were collected for each of the five YNG and five AMA patients, which were subsequently processed as individual oocytes for library preparation (total single oocyte libraries = 20). GV oocytes were either processed immediately as individual GV samples, or placed into maturation medium for 24 hours (6% CO2, 5% O2, 37℃). Maturation medium was supplemented with human recombinant BDNF (3 ng/mL; Sigma Aldrich, USA), human recombinant FSH (0.1 IU/mL, Gonal F; EMD Serono, USA), estradiol (1 μg/mL; Sigma Aldrich, USA), human recombinant EGF (10 ng/mL; Sigma Aldrich, USA), human recombinant BMP15 (100 ng/mL; R&D Systems, USA), and human serum albumin (5 mg/mL; Cooper Surgical, USA). After incubation, oocytes were examined for the presence of a polar body to confirm maturation and then processed individually as MII samples.
处理方案:
-
提取方案:
Individual GV and MII human oocytes were exposed to hyaluronidase (80 units/ml; Sigma Aldrich, USA) and manipulated with a Stripper (Origio, USA) tip to remove all remaining cumulus cells, and then washed three times in Dulbecco^ phosphate buffered saline with 0.01% PVP (Sigma Aldrich, USA). Samples were stored in PicoPure RNA extraction buffer (10 μL), heated to 42℃ for 30 minutes, and subsequently stored at -80℃ until total RNA purification as per manufacturer^ protocol (Thermo Fisher Scientific, USA), which included RNAse-free DNAse treatment (Qiagen, USA).
建库方案:
RNA eluate (9 μL/sample) was used as input for cDNA synthesis and amplification with the SMARTer Ultra Low Input RNA HV kit (Clontech, USA) following manufacturer recommendations with 13 cycles of PCR amplification. Following cDNA fragmentation with a Covaris S2 sonicator (Duty%: 10, Intensity: 5, Burst cycle: 200, Time: 5 min, Mode: frequency sweeping; Covaris, USA), the cDNA was isolated and eluted into 10 μL with the DNA Clean and Concentrator - 5 kit (Zymo Research, USA). All material isolated (3.9 ± 0.5 ng/sample) was used as input for the Thruplex DNA-seq kit (Rubicon Genomics, USA) and processed per manufacturer^ recommendations, which included 9 library amplification cycles. Illumina paired-end 100 bp sequencing was performed on a HiSeq2500 apparatus (Illumina, USA).
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2500
链特异性:
Unspecific
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文章
Differing molecular response of young and advanced maternal age human oocytes to IVM.
Human reproduction (Oxford, England) . 2017-11-01 [PMID:
29025019]
