Good Egg or Bad Egg: Developing markers of oocyte competence for Assisted Reproductive Interventions.

The oocyte is the foundation of life. It develops from a single fertilized cell to a multicellular organism capable of an independent existence. Competence to achieve this maximum potential is acquired following the protracted, but, highly co-ordinated process of growth and subsequently, maturation. The environment in which all or part of these processes occur, can ultimately have long-term consequences for female fertility and the health of resulting offspring.The pressure to identify and select oocytes or embryos with the highest developmental potential has intensified as the number of patients and the range of options available to them have increased. In particular, as in vitro maturation and single embryo transfer become more routine in assisted reproductive technology, selection is critical to a successful outcome. Moreover, the identification of markers of oocyte health and quality is essential to monitor the impact of these technologies on gametes and embryos.Technologies, such as transcriptomics, proteomics and metabolomics, offer more sophisticated methods for oocyte and embryo selection, with the emphasis on the predictive value of non-invasive protocols which profile follicular fluid, follicle/ granulosa cells and cumulus cells, for assessment of oocyte quality. Using the bovine as a model, we have employed a range of approaches and identified many potential markers, such as oocyte and cumulus candidate proteins and transcripts and follicular fluid fatty acids and amino acids. The models, technologies, and future strategies will be discussed in detail

Genetic merit for fertility traits in Holstein cows: VI. Oocyte developmental competence and embryo development

peer-reviewedThe hypothesis of this study was that cows with good genetic merit for fertility traits (Fert+) would produce oocytes and embryos of greater quality than cows with poor genetic merit for fertility traits (Fert−) and that mRNA expression of candidate genes would reflect the observed differences in quality. The aim of the study, therefore, was to determine the effect of genetic merit for fertility traits on morphological classification and mRNA abundance of key genes in immature oocytes and cumulus cells following ovum pick-up and in embryos following superovulation, artificial insemination (AI), and uterine flushing. In experiment 1, 17 Fert+ and 11 Fert− cows, ranging from 54 to 84 d in milk, were submitted to ovum pick-up on 4 occasions during a 2-wk period. Recovered cumulus–oocyte complexes (COC) were morphologically graded. Oocytes and cumulus cells were separated, and mRNA abundance of genes associated with oocyte developmental competence was measured. There was no effect of genotype on the distribution of COC grades or on the mRNA abundance of the candidate genes in grade 1 COC. In experiment 2, 20 Fert+ and 19 Fert− cows, ranging from 71 to 189 d in milk, were submitted to superovulation and AI. The uteri of cows that responded to the superovulation protocol (17 Fert+ and 16 Fert− cows) were nonsurgically flushed 7 d postovulation. Recovered embryos were morphologically graded, and mRNA abundance of genes associated with embryo development was measured in grade 1 blastocysts. The response to the superovulation protocol was assessed by counting the number of codominant follicles on the day of AI, which was similar for both genotypes (22.0 ± 9.7 and 19.8 ± 8.2 for Fert+ and Fert− cows, respectively). There was no effect of genotype on the proportion of transferable embryos recovered or on the mRNA abundance of the candidate genes tested in the grade 1 blastocysts. Of the total embryos classified as blastocysts, however, the Fert+ cows tended to have a greater proportion of grade 1 blastocysts compared with Fert− cows (90% vs. 64%, respectively). In conclusion, genetic merit for fertility traits had a no effect on mRNA abundance of the candidate genes that were examined in immature oocytes and cumulus cells and in embryos recovered after superovulation. The observed differences in morphological blastocyst quality following superovulation would suggest that the superior reproductive performance of Fert+ cows could arise during the later stages of embryo development from d 7 until maternal recognition of pregnancy

Biochemical alterations in the follicular fluid of bovine peri-ovulatory follicles and their association with final oocyte maturation

Follicular fluid (FF), a product of vascular transudate and granulosa and thecal cell secretions, is the milieu that has evolved to support oocyte growth and maturation which plays a central role in oocyte quality determination. Therefore, a suboptimal FF composition may be reflected in compromised oocyte progression through maturation, fertilization, or embryo development. To date, the composition of bovine FF remains understudied. To address this, we comprehensively characterized the metabolomic constituency of bovine FF in the period during which the oocyte undergoes meiotic maturation. More specifically, FF from pre (−24 h) and peri (−2 h)-ovulatory follicles was profiled by high-throughput untargeted ultra-HPLC tandem mass spectroscopy. A total of 634 metabolites were identified, comprising lipids (37.1%), amino acids (30.0%), xenobiotics (11.5%), nucleotides (6.8%), carbohydrates (4.4%), cofactors and vitamins (4.4%), peptides (3.6%), and energy substrates (2.1%). The concentrations of 67 metabolites were significantly affected by the stage of follicle development, 33.3% (n = 21) were reduced (P ≤ 0.05) by a mean of 9.0-fold, whereas 46 were elevated (P ≤ 0.05) by a mean of 1.7-fold in peri- vs pre-ovulatory FF. The most pronounced individual metabolite concentration decreases were observed in hypoxanthine (98.9- fold), xanthine (65.7-fold), 17β-oestradiol (12.4-fold), and inosine (4.6-fold). In contrast, the greatest increases were in retinal (4.9-fold), 1-methyl-5-imidazoleacetate (2.7-fold), and isovalerylcarnitine (2.7-fold). This global metabolomic analysis of bovine FF temporal dynamics provides new information for understanding the environment supporting oocyte maturation and facilitating ovulation that has the potential for improving oocyte quality both in vivo and in vitro

Differentially Expressed Genes in Endometrium and Corpus Luteum of Holstein Cows Selected for High and Low Fertility Are Enriched for Sequence Variants Associated with Fertility

peer-reviewedDespite the importance of fertility in humans and livestock, there has been little success dissecting the genetic basis of fertility. Our hypothesis was that genes differentially expressed in the endometrium and corpus luteum on Day 13 of the estrous cycle between cows with either good or poor genetic merit for fertility would be enriched for genetic variants associated with fertility. We combined a unique genetic model of fertility (cattle that have been selected for high and low fertility and show substantial difference in fertility) with gene expression data from these cattle and genome-wide association study (GWAS) results in ∼20 000 cattle to identify quantitative trait loci (QTL) regions and sequence variants associated with genetic variation in fertility. Two hundred and forty-five QTL regions and 17 sequence variants associated primarily with prostaglandin F2alpha, steroidogenesis, mRNA processing, energy status, and immune-related processes were identified. Ninety-three of the QTL regions were validated by two independent GWAS, with signals for fertility detected primarily on chromosomes 18, 5, 7, 8, and 29. Plausible causative mutations were identified, including one missense variant significantly associated with fertility and predicted to affect the protein function of EIF4EBP3. The results of this study enhance our understanding of 1) the contribution of the endometrium and corpus luteum transcriptome to phenotypic fertility differences and 2) the genetic architecture of fertility in dairy cattle. Including these variants in predictions of genomic breeding values may improve the rate of genetic gain for this critical trait

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies

Abstract Background Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Results Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. Conclusion By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Abstract Background Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results 8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology

The contribution of the maternal immune system to the establishment of pregnancy in cattle

Immune cells play an integral role in affecting successful reproductive function. Indeed, disturbed or aberrant immune function has been identified as primary mechanisms behind infertility. In contrast to the extensive body of literature that exists for human and mouse, studies detailing the immunological interaction between the embryo and the maternal endometrium are quite few in cattle. Nevertheless, by reviewing the existing studies and extrapolating from sheep, pig, mouse, and human data, we can draw a reasonably comprehensive picture. Key contributions of immune cell populations include granulocyte involvement in follicle differentiation and gamete transfer, monocyte invasion of the peri-ovulatory follicle and their subsequent role in corpus luteum formation and the pivotal roles of maternal macrophage and dendritic cells in key steps of the establishment of pregnancy, particularly, the maternal immune response to the embryo. These contributions are reviewed in detail below and key findings are discussed

Investigating the acquisition of DNA methylation at maternally imprinted genes during bovine oocyte growth

43rd Annual Meeting of Society for the Study of Reproduction, “The Intersection Between Genetics, Genomics, and Reproductive Biology”, 30 July–3 August 2010, Milwaukee, WisconsinScience Foundation IrelandConference detailshttp://www.ssr.org/10Meeting.shtmlCould not find it in cited journal: Title corresponds to abstract 307 in http://www.ssr.org/Documents/2010-07-16Abstracts.pdf; other abstract 97 was published in said journal. Conference URL: http://www.ssr.org/10Meeting.shtml. Programme does not mention abstract 307: http://www.ssr.org/10Schedule.shtml. Will treat it as unpublished - AV 27/01/201