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Background: In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation., Objectives: To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM., Materials and Methods: The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass., Results: Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts., Conclusions: These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were editorial board members of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Tutt, Guven-Ates, Kwong, Simmons, Sang, Silvestri, Canedo-Ribeiro, Handyside, Labrecque, Sirard, Emes, Griffin and Sinclair.)

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal ('coasting'). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated ('coasted') cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38 g P4] or PRID® Delta [1.55 g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F 2α ) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology125: 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P < 0.001) for stimulated than non-stimulated cycles (0.712 ± 0.0288 vs. 0.466 ± 0.0360), but the overall proportion aneuploidy was similar for both groups (0.241 ± 0.0231). Most abnormalities consisted of meiotic trisomies. Twenty in vivo derived blastocysts recovered from the same donors were all euploid, thus indicating that 24 h of maturation is primarily responsible for aneuploidy induction. Chromosomal errors in OPU-IVP blastocysts decreased (P < 0.001) proportionately as stage/grade improved (from 0.373 for expanded Grade 2 to 0.128 for hatching Grade 1 blastocysts). Importantly, there was a high degree of concordance in the incidence of aneuploidy between TE and ICM lineages. Proportionately, 0.94 were "perfectly concordant" (i.e. identical result in both); 0.01 were imperfectly concordant (differing abnormalities detected); 0.05 were discordant; of which 0.03 detected a potentially lethal TE abnormality (false positives), leaving only 0.02 false negatives. These data support the use of TE biopsies for PGT-A in embryos undergoing genomic evaluation in cattle breeding. Finally, we report chromosome-specific errors and a high degree of variability in the incidence of aneuploidy between donors, suggesting a genetic contribution that merits further investigation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)