Age-related gene expression profiles of immature human oocytes.

STUDY QUESTION: What is the difference between the gene expression profiles of single human germinal vesicle (GV) oocytes from women of different ages? SUMMARY ANSWER: There were no statistically significant differences in gene expression profiles of human GV oocytes from women of different ages (range: 25-43). WHAT IS KNOWN ALREADY: It is well established that reproductive capacity declines as women age, which is attributed to oocyte quality since this decline is counterbalanced in older women receiving young donor oocytes. Altered gene expression of human oocytes at different stages of development in relation to female age is one of the suggested mechanisms that could explain the decrease in oocyte quality. STUDY DESIGN, SIZE, DURATION: Between 2012 and 2014, 40 human GV oocytes of 40 women were obtained during follicular aspiration as part of routine ICSI treatment. Gene expression profiles of 38 GV oocytes were determined in four different age groups: 25-30, 31-35, 36-38 and 39-43 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: GV oocytes were donated for research and frozen between 3.5 and 7.5 h after follicular aspiration. Subsequently, GV oocytes were thawed and prepared for gene expression profile analysis using Agilent microarrays containing ~42 000 Human Gene Expression probe-sets. Gene expression profiles were visualized by hierarchical clustering and the top 500 most differing genes were determined by multidimensional scaling (MDS). Transcripts were analysed in a class comparison between the four age groups and for indicators of biological age: antral follicle count (AFC) and the total dosage of FSH needed for ovarian stimulation. Individual transcripts were analysed using linear regression. A false discovery rate <0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Visualization of gene expression profiles of GV oocytes with hierarchal clustering and MDS demonstrated no clear grouping of samples based on female age, AFC or FSH dosage. The gene expression profile of GV oocytes classified in four age groups revealed no significantly differentially expressed genes between the four different age groups. There were also no significantly differentially expressed genes in the linear regression analysis for individual transcripts against age. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Immature (GV) oocytes obtained from ovarian stimulation cycles were used. Findings may therefore differ for oocytes at other developmental stages and for in-vivo matured oocytes under physiological conditions. Due to our relatively large, but still limited study sample (40 GV oocytes), we cannot exclude that there might be smaller age-related gene-expression differences, i.e. due to a lack of power. WIDER IMPLICATIONS OF THE FINDINGS: We did not find an effect of female age on gene expression profiles of individual human GV oocytes. Other studies have suggested that gene-expression profiles are affected in mature oocytes, which might imply that female age affects oocyte maturation. Alternatively, other mechanisms in human oocytes might cause the age-related fertility decline. STUDY FUNDING/COMPETING INTEREST(S): This study received no external funding and there are no competing interests.

Factors affecting the gene expression of in vitro cultured human preimplantation embryos.

STUDY QUESTION: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? SUMMARY ANSWER: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture. WHAT IS KNOWN ALREADY: Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking. STUDY DESIGN, SIZE, DURATION: Donated, good quality, day 4 cryopreserved human preimplantation embryos (N = 89) were randomized to be cultured in one of two culture media (G5 medium or HTF medium) and one of two oxygen concentrations (5% or 20%), with stratification for maternal age. Next to these variables, developmental stage after culture was taken into account in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37). MAIN RESULTS AND THE ROLE OF CHANCE: Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. LIMITATIONS, REASONS FOR CAUTION: Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RT-PCR validation was not performed due to scarcity of the material. WIDER IMPLICATIONS OF THE FINDINGS: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines. STUDY FUNDING/COMPETING INTERESTS: This study received no external funding and there were no competing interests.

Embryo culture media and IVF/ICSI success rates: a systematic review.

BACKGROUND The media that are used to culture human preimplantation embryos are considered to be an important factor for the success rates of IVF/ICSI. Here, we present a systematic review of randomized controlled trials (RCTs) on the effect of culture media on IVF/ICSI success rates. METHODS RCTs published between January 1985 and July 2012 were eligible for inclusion. The primary outcome was live birth. Secondary outcomes were health of babies born, ongoing pregnancies, clinical pregnancies, miscarriages, multiple pregnancies, implantation rate, cryopreservation rate, embryo quality and fertilization rate. For those media that were evaluated in more than one comparison, an unconventional meta-analysis was performed by pooling the data of the media they were compared to. RESULTS Twenty-two RCTs were included that evaluated 31 different comparisons. Conventional meta-analysis was not possible for any of the outcomes as nearly all trials compared different culture media. Only four trials reported on live birth, and one of them reported a significant difference. Nine trials reported on ongoing and/or clinical pregnancy rates, of which four showed a significant difference. Pooling the data did not reveal a superior culture medium. CONCLUSIONS It is yet unknown what culture medium leads to the best success rates in IVF/ICSI. Given the potential importance of culture media for treatment outcome, rigorously designed RCTs are needed for currently available, as well as newly introduced culture media.

Newborn screening for hemoglobinopathies using capillary electrophoresis technology: Testing the Capillarys Neonat Fast Hb device.

OBJECTIVES: To diagnose hemoglobinopathies in newborns by separating and measuring the Hb fractions on high throughput capillary electrophoresis. To test and validate the Capillarys Neonat Fast Hb device (Sebia) on fresh and dry blood samples. DESIGN AND METHODS: The Hb fractions in 1.600 cord blood samples from the multi ethnic Dutch population were separated and measured. Further, the sensitivity, specificity and reproducibility of the device in detecting abnormalities and measuring the Hb fractions were estimated. RESULTS: The apparatus separated all significant Hb fractions that should be detected during newborn screening (NBS) with 100% sensitivity. The reproducibility of the migrations guaranteed putative specificity for the few relevant frequent variants observed (HbS, C, and E). The estimation of the HbA and F fractions proved reliable using a well-designed integration mode. DISCUSSION: Due to the limited number of samples no cases with sickle cell disease or ß-thalassemia major were found in this cohort. However, the heterozygous state for the common variants associated with these diseases was clearly recognizable. The measurements were sufficiently precise to recognize sickle cell disease, ß-thalassemia major and intermedia and to identify carriers including possible ß-thalassemia. Therefore, Capillarys Neonat Fast Hb (Sebia) can be considered as a valid instrument for NBS of the Hemoglobinopathies on fresh and dry blood samples.