E.P.A. Jorssen

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocytes developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocytes requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.

Single in vitro bovine embryo production: Coculture with autologous cumulus cells, developmental competence, embryo quality and gene expression profiles

Studies concerning oocyte quality markers, oocyte/embryo metabolism or commercial OPU settings treating donors with low oocyte yields, indicate a need for optimization of IVP protocols to culture single oocytes to the blastocyst stage. However, culture conditions for single oocyte usually impair development, although previous research showed that single oocyte culture on a monolayer of cumulus cells can lead to similar developmental competence than group oocyte culture. Aiming to develop a fully single IVP procedure, Experiment 1 and 2 revealed that individual maturation, fertilization and culture in 20 μL droplets, using a monolayer of heterologous (SSSm, Exp 1) or autologous cumulus cells in coculture (SSSa, Exp 2), resulted in 23.9% and 15.1% of blastocysts 8 days p.i., respectively, which is significantly less compared to regular group IVP (GGGc, 33.5% (Exp 1) and 26.2% (Exp 2), respectively). In a third Experiment, day 7 p.i. blastocyst quality was analyzed in four treatment groups: regular group IVP (GGGc), group IVP with coculture (GGGm), in group produced zygotes, singly cultured on a heterologous cumulus cell monolayer (GGSm) and individually matured and fertilized zygotes, singly cultured on a monolayer (SSSm). Mean cell number and apoptotic cell index, were similar for all treatment groups. Moreover, mRNA abundance relative to H2AFZ was equal for 9 qualitatively linked genes (TP53, BAX, SHC1 SHC, IGF2R, PTGS2, AKR1B1, PLAC8, SLC2A1, and MNSOD). Only GPX1, involved in detoxification and mtDNA protection to oxidative stress, was significantly downregulated (ANOVA, P < 0.05) in singly produced blastocysts (SSSm), compared to the other treatments. In conclusion, a valuable individual IVP system was established and autologous cumulus cells in coculture showed to partly neutralize hampered individual culture conditions. Additionally, to our knowledge this is the first report in which blastocyst quality, in terms of cell number, apoptosis and gene expression, of singly produced embryos was investigated and shown to be similar to in group produced embryos, implicating that the single IVP system can be applied as a tool in oocyte and embryo quality studies.

Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations

Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (=control); (2) elevated (75 µM) stearic acid (SA) concentrations (=HIGH SA); and (3) elevated (425 µM) NEFA concentrations (=HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.

Individual bovine in vitro embryo production and cumulus cell transcriptomic analysis to distinguish cumulus-oocyte complexes with high or low developmental potential

Studying cumulus cell (CC) transcriptome is of great interest as it could provide a noninvasive method to assess oocyte quality. In cattle, the search for quality markers has not been done with cumulus-oocyte complexes (COCs) cultured individually from maturation to blastocyst stage. Here, differences between high- and low-potential COCs were examined by transcriptomic analysis of CC biopsies obtained from COCs of 2 to 6 mm follicles (n = 249; eight replicates) before individual in vitro maturation, fertilization, and culture until Day 8 after fertilization. Each COC was individually tracked and categorized based on his fate: embryo at blastocyst stage (CC-Blast) or embryo arrested at 2- to 8-cell stage (CC-2-8-cells). Average blastocyst rates were 27.7% for individual culture and 31.2% for group control (not significantly different). For transcriptomic analysis, five cumulus biopsies per replicate were pooled for each fate. Three CC replicates underwent transcriptomic analysis using RNA microarray assay. Some clear differences in gene expression between the CC-Blast and the CC-2-8-cell groups were identified. Considering a 1.5-fold change (P < 0.05), 68 genes were differentially expressed between the CC-Blast and CC-2-8-cells. Quantitative reverse transcription-polymerase chain reaction validations were performed for 12 selected genes: six upregulated genes for each COC fate. Higher expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) (lipid metabolism), Chloride intracellular channel 3 (CLIC3), Keratin 8 (KRT8), and Lumican (LUM) (molecular transport) was observed in CC-2-8-cells (P < 0.05). The CC-Blast fate analysis revealed a significantly higher expression of Glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM) (posttranslational modification, amino acid metabolism, and free radical scavenging). This newly identified set of genes could provide new markers to distinguish COCs associated with good quality embryos from COCs with limited developmental potential.

Unravelling the needs of singly in vitro-produced bovine embryos: from cumulus cell co-culture to semi-defined, oil-free culture conditions

Producing bovine in vitro embryos individually is a challenge as it generally leads to impaired embryo development. Earlier research optimised a single embryo in vitro production (IVP) protocol using serum, cumulus cells and oil during culture. As some of these factors are undesirable in certain circumstances, the present study investigated their necessity and possible interactions, and defined their role during single-embryo culture. Although the cumulus cell monolayer produced progesterone, it appeared not to be a key factor in supporting single-embryo development. Because in vitro culture in large medium volumes was shown to impair single-embryo development, two new oil-free culture protocols were tested. Using a 30-µL droplet of medium in 96-well plates with a small surface area resulted in comparable blastocyst rates to those obtained under oil. When serum was used, co-culture with cumulus cells seems necessary, leading to consistently high blastocyst rates. Finally, a serum-free, oil-free culture system using insulin, transferrin, selenium and BSA resulted in embryos with similar total cell numbers and apoptotic cell ratios, but blastocyst rates did not equal those obtained with serum and co-culture. This research additionally stresses the fact that specific interaction mechanisms between somatic cells and a developing in vitro embryo are far from unravelled.

Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O2) or high (20% O2) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoys 5A medium for 6 days at 37 °C, 5% CO2, and 5% or 20% O2. On Days 4 and 6, the fragments were restained by adding NR to the McCoys medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O2) and 11.78 ± 1.06 μm (20% O2) on Day 4 and 20.94 ± 1.24 μm (5% O2) and 19.12 ± 1.36 μm (20% O2) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O2 (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.

A 1 bp deletion in the dual reading frame deafness gene LRTOMT causes a frameshift from the first into the second reading frame.

A 1 bp Deletion in the Dual Reading Frame Deafness Gene LRTOMT Causes a Frameshift From the First Into the Second Reading Frame Maarten Vanwesemael, Isabelle Schrauwen, Ruben Ceuppens, Fatemeh Alasti, Ellen Jorssen, Effat Farrokhi, Morteza Hashemzadeh Chaleshtori, and Guy Van Camp* Department of Medical Genetics, University of Antwerp, 2610, Wilrijk, Belgium Cellular and Molecular Research Center, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Perfluoroalkyl acid contamination of follicular fluid and its consequence for **in vitro** oocyte developmental competence

Perfluoroalkyl acids (PFAAs) have been shown to induce negative effects in laboratory animals and in vitro experiments. Also, PFAAs have been detected in human tissues and body fluids. The ovarian follicle constitutes a fragile micro-environment where interactions between hormones, growth factors, the oocyte and surrounding somatic cells are essential to generate a fully competent oocyte. In vitro experiments suggest that PFAAs can influence this balance, but very scarce in vivo data are available to confirm this assumption. In fact, the potential PFAA-presence in the follicular micro-environment is currently unknown. Therefore, we investigated if PFAAs are present in human follicular fluid and if their presence could be a risk factor for in vivo exposed developing oocytes. Furthermore, we compared the PFAA-distribution within serum and follicular fluid. PFAAs were analyzed by LC/MS in follicular fluid (n=38) and serum (n=20) samples from women undergoing assisted reproductive technologies (ARTs). Statistical models were used to investigate PFAA-distribution in both body fluids, to compare this behavior with the distribution of lipophilic organic pollutants and to explore the relationship between patient characteristics, ART-results and follicular fluid contamination. Perfluorooctane sulfonate (PFOS) was the PFAA found in the highest concentration in follicular fluid [7.5 (0.1-30.4) ng/mL] and serum [7.6 (2.8-12.5) ng/mL]. A new variable, Principal Component 1, representing the overall PFAA-contamination of the follicular fluid samples, was associated with a higher fertilization rate (p<0.05) and a higher proportion of top embryos relative to the amount of retrieved oocytes (p<0.05), after adjusting for age, estradiol-concentration, BMI, male subfertility and the presence of other organic pollutants as explanatory variables. To conclude, overall higher PFAA-contamination in the follicular micro-environment was associated with a higher chance of an oocyte to develop into a high quality embryo. Also, PFAAs have different distribution patterns between serum and follicular fluid compared to the lipophilic organic pollutants. Further research is of course crucial to confirm these new observations.