The impact of maternal age on gene expression during the GV to MII transition in euploid human oocytes.

STUDY QUESTION: Are there age-related differences in gene expression during the germinal vesicle (GV) to metaphase II (MII) stage transition in euploid human oocytes? SUMMARY ANSWER: A decrease in mitochondrial-related transcripts from GV to MII oocytes was observed, with a much greater reduction in MII oocytes with advanced age. WHAT IS KNOWN ALREADY: Early embryonic development is dependent on maternal transcripts accumulated and stored within the oocyte during oogenesis. Transcriptional activity of the oocyte, which dictates its ultimate developmental potential, may be influenced by age and explain the reduced competence of advanced maternal age (AMA) oocytes compared with the young maternal age (YMA). Gene expression has been studied in human and animal oocytes; however, RNA sequencing could provide further insights into the transcriptome profiling of GV and in vivo matured MII euploid oocytes of YMA and AMA patients. STUDY DESIGN, SIZE, DURATION: Fifteen women treated for infertility in a single IVF unit agreed to participate in this study. Five GV and 5 MII oocytes from 6, 21-26 years old women (YMA cohort) and 5 GV and 6 MII oocytes from 6, 41-44 years old women (AMA cohort) undergoing IVF treatment were donated. The samples were collected within a time frame of 4 months. RNA was isolated and deep sequenced at the single-cell level. All donors provided either GV or MII oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cumulus dissection from donated oocytes was performed 38 h after hCG injection, denuded oocytes were inserted into lysis buffer supplemented with RNase inhibitor. The samples were stored at -80°C until further use. Isolated RNA from GV and MII oocytes underwent library preparation using an oligo deoxy-thymidine (dT) priming approach (SMART-Seq v4 Ultra Low Input RNA assay; Takara Bio, Japan) and Nextera XT DNA library preparation assay (Illumina, USA) followed by deep sequencing. Data processing, quality assessment and bioinformatics analysis were performed using source-software, mainly including FastQC, HISAT2, StringTie and edgeR, along with functional annotation analysis, while scploid R package was employed to determine the ploidy status. MAIN RESULTS AND THE ROLE OF CHANCE: Following deep sequencing of single GV and MII oocytes in both YMA and AMA cohorts, several hundred transcripts were found to be expressed at significantly different levels. When YMA and AMA MII oocyte transcriptomes were compared, the most significant of these were related to mitochondrial structure and function, including biological processes, mitochondrial respiratory chain complex I assembly and mitochondrial translational termination (false discovery rate (FDR) 6.0E-10 to 1.2E-7). These results indicate a higher energy potential of the YMA MII cohort that is reduced with ageing. Other biological processes that were significantly higher in the YMA MII cohort included transcripts involved in the translation process (FDR 1.9E-2). Lack of these transcripts could lead to inappropriate protein synthesis prior to or upon fertilisation of the AMA MII oocytes. LARGE SCALE DATA: The RNA sequencing data were deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo), under the accession number: GSE164371. LIMITATIONS, REASONS FOR CAUTION: The relatively small sample size could be a reason for caution. However, the RNA sequencing results showed homogeneous clustering with low intra-group variation and five to six biological replicates derived from at least three different women per group minimised the potential impact of the sample size. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the effects of ageing on the oocyte transcriptome could highlight the mechanisms involved in GV to MII transition and identify biomarkers that characterise good MII oocyte quality. This knowledge has the potential to guide IVF regimes for AMA patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Medical Research Council (MRC Grant number MR/K020501/1).

Influence of advanced age on the blastocyst development rate and pregnancy rate in assisted reproductive technology.

OBJECTIVE: To evaluate the percentage of blastocysts developing, the pregnancy rate, the implantation rate, and the abortion rate in women >40 years of age using a cell-free culture system for the development of viable human blastocysts. DESIGN: Retrospective clinical study. SETTING: Private IVF units. PATIENT(S): Two hundred ninety-three cycles in patients undergoing IVF treatment for infertility. Sixty-two cycles were in patients > or =40 years of age, and 231 cycles were in patients <40 years of age. INTERVENTION(S): Pronucleate oocytes obtained from IVF were cultured in vitro for 5-6 days. One to four embryos were transferred. MAIN OUTCOME MEASURE(S): Blastocyst development rate, pregnancy rate, implantation rate, and abortion rate. RESULT(S): From 293 cycles, 3,115 pronucleate oocytes were cultured, producing 1,175 blastocysts. In the women >40 years of age, the blastocyst development rate was 22.2%, and in the younger group, the rate was 40.5%. The pregnancy rate and implantation rate in the > or =40-year age group were 21.1% and 8.9%, respectively; corresponding rates in the younger group were 44.6% and 19.9%. The abortion rate was increased for the > or =40-year age group (25% versus 13.3%). CONCLUSION(S): Success rates for the development of viable human blastocysts, pregnancy, and implantation decline significantly in women > or =40 years old.

Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilization program.

OBJECTIVE: To determine if pituitary suppression is still maintained if GnRH agonist (GnRH-a) is discontinued as gonadotropin stimulation is begun in a long protocol. DESIGN: Prospective, randomized study. SETTING: An outpatient IVF-GIFT program. PATIENTS: One hundred seventy-three patients entering an IVF-GIFT program. INTERVENTIONS: Gonadotropin-releasing hormone agonist in long protocol was either discontinued or continued as gonadotropin stimulation was begun. MAIN OUTCOME MEASURES: Luteinizing hormone (LH), E2, and P levels, egg numbers, fertilization rate, number of embryos transferred, day of gonadotropin stimulation, and pregnancy rates (PRs). RESULTS: Pituitary suppression was maintained although GnRH-a was discontinued as gonadotropin stimulation was begun. No spontaneous LH surge was seen, and PRs were increased in the IVF patients. CONCLUSION: This study indicates that the advantages gained from use of GnRH-a in the long protocol are not compromised by its early discontinuation.

Oocyte donation in menopausal women aged over 40 years.

In order to investigate the pregnancy potential of menopausal women over 40 years of age by use of donor eggs, we retrospectively analysed the results of our ovum donation programme. Forty-one clinical pregnancies were established in 134 recipient cycles. The recipients were divided into three age groups (40-43, 44-47 and > or = 48 years) in order to investigate the implantation rate with respect to age. The 30.6% pregnancy rate per embryo transfer cycle and 9.7% implantation rate per embryo appeared to be constant in all age groups studied. Fourteen pregnancies ended in miscarriage, there was one ectopic pregnancy and 25 healthy babies have been delivered. The oldest woman to deliver was 54 years of age. This report highlights the question of age limit for application of the new reproductive technologies, and especially of oocyte donation.