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).

Evaluating the value of day 0 of an ICSI cycle on indicating laboratory outcome.

A number of oocyte characteristics have been associated with fertilization, implantation and live-birth rates, albeit without reaching a consensus. This study aims to delineate possible associations between oocyte characteristics, oocyte behavior during intracytoplasmic sperm injection (ICSI), fertilization potential, and laboratory outcomes. Four-hundred and seventy-seven patients, yielding 3452 oocytes, were enrolled in this prospective observational study from 2015 to 2018. Οoplasm granularity was associated with poor embryo quality and higher probabilities of post-ICSI oocytes and embryos discarded in any developmental stage and never selected for embryo transfer or cryopreservation (p < 0.001). Both sudden or difficult ooplasm aspiration, and high or lack of resistance during ICSI were associated with either a poor Zygote-Score or fertilization failure (p < 0.001). Sudden or difficult ooplasm aspiration and high resistance during ICSI penetration were positively associated with resulting to a post-ICSI oocyte or embryo that would be selected for discard. Evaluation of oocyte characteristics and oocyte behavior during ICSI may provide early information regarding laboratory and cycle outcomes. Particularly, ooplasm granularity, and fragmentation of polar body, along with sudden or difficult ooplasm aspiration and high or lack of resistance during ICSI penetration may hinder the outcome of an ICSI cycle. The associations presented herein may contribute towards development of a grading system or a prediction model. Taking into account information on oocytes and ICSI behavior may effectively assist in enhancing IVF outcome rates.

Should the flexibility enabled by performing a day-4 embryo transfer remain as a valid option in the IVF laboratory? A systematic review and network meta-analysis.

PURPOSE: The present systematic review and network meta-analysis aims to uniquely bring to literature data supporting the true place of the alternative practice of day-4 embryo transfer (D4 ET) in an IVF laboratory, beyond the one-dimensional option of facilitating a highly demanding program. METHODS: A systematic search was conducted in the databases of PubMed/Medline, Embase, and Cochrane Central Library, resulting to six prospective along with nine retrospective cohort studies meeting eligibility criteria for inclusion. A comparison of D4 ET with day-2 (D2), day-3 (D3), and day-5 (D5) ET, respectively, was performed employing R statistics. RESULTS: The sourced results indicate no statistically significant difference regarding clinical pregnancy rates, and ongoing pregnancy/live birth rates stemming from the comparison of D4 with D2, D4 with D3, and D4 with D5 ET, respectively. Additionally, no statistically significant difference could be established in respect to cancelation, and miscarriage rates, following the comparison of D4 with D3 and D4 with D5 ET. Interestingly, we report statistically significant lower preterm birth rates associated with D4 ET, in contrast with D5 ET (RR, 0.19; 95% CI, 0.05-0.67; p value = 0.01). CONCLUSIONS: The aforementioned results may serve as advocates buttressing the option of D4 ET as a valid candidate in the ET decision-making process. Possible limitations of the current study are the publication bias stemming from the retrospective nature of certain included studies, along with various deviations among studies' design, referring to number and quality of transferred embryos, or different culture conditions referring to studies of previous decades.

Risks in Surrogacy Considering the Embryo: From the Preimplantation to the Gestational and Neonatal Period.

Surrogacy is an assisted reproduction-based approach in which the intended parents assign the gestation and birth to another woman called the surrogate mother. The drivers of surrogacy refer largely to infertility, medical conditions, same-sex couples' parenting, and cases of diversity regarding sexual identity and orientation. Surrogacy consists of a valid option for a variety of conditions or circumstances ranging from medical to social reasons. However, surrogacy may be associated with risks during the preimplantation, prenatal, and neonatal period. It became obvious during the exhaustive literature research that data on surrogacy and its association with factors specific to the IVF practice and the options available were not fully represented. Could it be that surrogacy management adds another level of complexity to the process from the ovarian stimulation, the subsequent IVF cycle, and the techniques employed within the IVF and the Genetic Laboratory to the fetal, perinatal, and neonatal period? This work emphasizes the risks associated with surrogacy with respect to the preimplantation embryo, the fetus, and the infant. Moreover, it further calls for larger studies reporting on surrogacy and comparing the surrogate management to that of the routine IVF patient in order to avoid suboptimal management of a surrogate cycle. This is of particular importance in light of the fact that the surrogate cycle may include not only the surrogate but also the egg donor, sperm donor, and the commissioning couple or single person.

The effect of platelet-rich plasma (PRP) combined with a bone allograft on human periodontal ligament (PDL) cells.

The prominent purpose of the study was the evaluation of the in vitro mitogenic effect of three different homologous platelet-rich plasma (PRP) preparations (PRPa, PRPb, PRPc) on three different lines of periodontal ligament (PDL) cells (PDL(1,2,3)), cultured alone or in combination with a demineralized freeze-dried allograft (DFBA). PDL cell cultures were derived from the mid root of three maxillary caries-free premolars extracted for orthodontic reasons. Cells were grown and reached confluence. To evaluate the mitogenic effect of all exogenous factors (PRPa, PRPb, PRPc and DFBA) on PDL cells, specific number of cells (10.000/well) was cultured in the presence or absence of the above factors. Each PRP preparation (5% v/v) was added in all cell lines, in the absence or presence of 10 mg/ml of DFBA. The cells were also treated with 25 ng/ml bFGF (positive control). The mitogenic effect was evaluated 24 h after incubation, using the Trypan blue exclusion assay. The results revealed that all PRP preparations act as potent mitogens as they significantly induced cell proliferation on PDL(1,2,3) lines. All PRP preparations when added alone in the PDL cell cultures, exhibited a significant advantage over the positive control (bFGF). The addition of DFBA to PRP did not influence significantly cell proliferation in all cell lines, comparatively to PRP alone, at the time -period studied. The findings of this study demonstrate the beneficial role of PRP alone or combined with the bone graft on periodontal ligament cells in vitro, suggesting that it may be considered as a potential biological approach in periodontal regeneration.