Broothaers, K., O. B. Pascottini, M. Hedia, D. Angel-Velez, T. De Coster, S. Peere, E. Polfliet, E. Van den Branden, J. Govaere, A. Van Soom, et al., "Oocyte holding and in vitro maturation duration between 28 and 34 hours do not affect equine OPU-ICSI outcomes.", Theriogenology, vol. 233, pp. 64-69, 2025 Feb. Abstract

Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in three different time windows using quartiles, with 465 oocytes (22.0 %) between 28 and 30 h (first quartile), 1078 oocytes (51.0 %) >30 and 31.7 h (second and third quartiles), and 571 oocytes (27.0 %) >31.7 and 34 h (fourth quartile). Using logistic regression models, the effect of duration of IVM with and without holding was tested on nuclear maturation, cleavage, blastocyst, and pregnancy rates. The three IVM intervals did not show differences in nuclear maturation (respectively 64.5 ± 0.48 %, 65.7 ± 0.47 %, and 67.3 ± 0.47 %), cleavage (respectively 59.7 ± 0.49 %, 58.5 ± 0.49 %, and 64.8 ± 0.48 %), blastocyst (respectively 17.5 ± 0.38 %, 19.0 ± 0.39 %, and 20.8 ± 0.41 %) nor pregnancy rates (respectively 65.4 ± 0.49 %, 70.3 ± 0.46 %, and 74.2 % ± 0.44) (P ≥ 0.38). Oocyte holding prior to IVM did not affect the results either (P ≥ 0.15). In conclusion, oocyte holding and IVM duration between 28 and 34h do not significantly affect outcomes, allowing flexibility in the planning of clinical OPU-ICSI in horses.

Fernández-Montoro, A., E. Araftpoor, T. De Coster, D. Angel-Velez, M. Bühler, M. Hedia, K. Gevaert, A. Van Soom, K. C. Pavani, and K. Smits, "Decoding bull fertility in vitro: a proteomics exploration from sperm to blastocyst.", Reproduction (Cambridge, England), vol. 169, issue 4, 2025 Apr 01. Abstract

IN BRIEF: Bulls are selected for field fertility and semen quality, but traits such as polyspermy are not considered and can increase aneuploidy during in vitro embryo production. This study links bull-specific proteomic signatures to polyspermy and embryo quality, further refining bull selection criteria.

ABSTRACT: Male fertility plays a pivotal role in the success rates of in vitro embryo production. While livestock breeding programs rigorously select bulls according to their predicted field fertility, specific traits such as polyspermy rates are not routinely evaluated. Despite the known negative impact of polyspermy on embryo survival, the paternal factors involved remain unclear. In this study, we aimed to address this gap by evaluating the in vitro outcomes of four bulls, focusing on sperm motility, fertilization rates, polyspermy incidence, embryo development and quality. In addition, we analyzed the proteome profiles of sperm, 2-4 cell stage embryos and blastocysts derived from those bulls to identify potential molecular factors associated with male fertility. Bulls with comparable sperm motility parameters displayed varying in vitro fertilization outcomes. Notably, the bull with the highest polyspermy rate achieved blastocyst rates similar to those of bulls with lower polyspermy rates. The number of apoptotic cells in the blastocysts was bull-dependent. Proteomic analysis revealed bull-specific signatures in sperm and blastocysts, with no differences at the 2-4 cell stage. Differences in the sperm proteome suggested that bull-dependent penetration and polyspermy rates might be associated with the ability of the sperm to undergo capacitation and acrosomal reaction. At the blastocyst level, the bull with the highest polyspermy rates produced lower quality blastocysts due to imbalances in key proteins and pathways for embryo development. In conclusion, bulls with similar blastocyst rates may differ in polyspermy rates and resulting embryo quality underscoring the importance of careful bull selection for in vitro embryo production.

Fakhar-I-Adil, M., D. Angel-Velez, E. Araftpoor, Q. A. Amin, M. Hedia, M. Bühler, K. Gevaert, B. Menten, A. Van Soom, S. M. Chuva de Sousa Lopes, et al., "Biphasic CAPA-IVM Improves Equine Oocyte Quality and Subsequent Embryo Development Without Inducing Genetic Aberrations.", International journal of molecular sciences, vol. 26, issue 12, 2025 Jun 08. Abstract

In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes by allowing the cytoplasm additional time to acquire essential components critical for optimal competency. This study aims to evaluate the effect of CAPA-IVM on equine oocyte quality and developmental competence. Immature cumulus-oocyte complexes (COCs) were retrieved from slaughterhouse ovaries and matured in vitro either in CAPA-IVM (short 6 h, long 24 h pre-maturation) or standard IVM. Mature oocytes from each group were analyzed for calcium-releasing potential ( = 52) and single-oocyte proteomics ( = 44), and embryo development ( = 229) was assessed after fertilization with piezo-drilled intracytoplasmic sperm injection (ICSI). Genetic analysis of developed blastocysts ( = 41) was performed to detect chromosomal aberrations. Our findings demonstrate that CAPA-IVM of equine COCs yields significantly higher maturation rates than controls. Moreover, short CAPA-IVM with six hours pre-maturation culture showed substantially higher embryo development potential than the control group (20/69 vs. 9/63, respectively). Genetic analysis revealed a high euploidy rate in equine blastocysts regardless of the maturation conditions. Live calcium imaging of the fertilized oocytes demonstrated that the majority of oocytes displayed non-continuous calcium oscillation patterns, irrespective of maturation conditions. Single-oocyte proteomics reveals a comparable proteomic landscape between mature oocytes subjected to short CAPA-IVM and standard IVM. However, we identified four enriched gene sets with positive enrichment scores after short CAPA-IVM, related to cytoskeleton regulation, ribosomal function, and cytosolic components. Our findings indicate that CAPA-IVM holds the potential to improve oocyte quality and competence in horses. However, further fine-tuning of culture conditions would benefit the effective use of these IVM systems. Moreover, given that the mare serves as an excellent model for human reproduction, the molecular trends identified in this study could provide valuable insights for advancing human artificial reproductive technologies.

Hedia, M., J. L. M. R. Leroy, S. Loomans, C. Benedetti, D. Angel-Velez, K. Chiers, J. Govaere, A. Van Soom, and K. Smits, "Lipopolysaccharide reduces progesterone and cytokines in equine follicular fluid without affecting oocyte development in vitro.", Theriogenology, vol. 249, pp. 117673, 2026 Jan 01. Abstract

Lipopolysaccharide (LPS) in follicular fluid impairs steroid production and oocyte developmental competence in cows and mice. This study assessed LPS concentrations in equine follicular fluid and their association with steroid and some cytokine levels. Additionally, we evaluated whether LPS exposure during in vitro maturation (IVM) affects equine oocyte developmental competence. In experiment 1, follicular fluid from large follicles (>30 mm in diameter) was collected from 16 slaughterhouse mares, and concentrations of LPS, estradiol, progesterone, TNF-α, and IL-6 were measured. In experiment 2, cumulus-oocyte complexes (COCs) were held overnight, then matured in vitro with (1 ng LPS/1 mL; LPS group) or without LPS (control group), and mature oocytes (n = 47 and 45, respectively) were fertilized using ICSI. Follicular fluid concentrations of LPS ranged between 5.21 and 12.08 endotoxin unit (EU)/mL (10 EU = 1 ng) and were negatively correlated with progesterone, IL-6, and TNF-α levels. Compared to controls, LPS exposure during IVM did not significantly affect maturation (56 % vs. 61 %; P = 0.432), cleavage (71 % vs. 65 %; P = 0.873), or blastocyst rates (21 % vs. 19 %; P = 0.227). In conclusion, this is the first report detecting LPS in the follicular fluid of clinically healthy mares and showing its negative association with progesterone, IL-6, and TNF-α. Exposing equine COCs to 1 ng/mL LPS during in vitro maturation had no significant effect on blastocyst rates. However, further research is needed to determine whether blastocysts derived from oocytes matured under LPS exposure can establish pregnancy after transfer.

Broothaers, K., D. Angel-Velez, F. L. G. Molto, M. Hedia, T. De Coster, J. Govaere, A. Van Soom, B. Menten, and K. Smits, "Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and Improves Intracytoplasmic Sperm Injection-Results in Horses.", Andrology, 2025 Oct 29. Abstract

BACKGROUND: In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications.

OBJECTIVES: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates.

MATERIALS AND METHODS: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI with washed frozen-thawed semen. Cleavage and blastocyst rates were assessed and compared across the three groups using one-way ANOVA. Shallow whole genome sequencing was performed on embryos obtained from IVF and ICSI cap.

RESULTS: We established a repeatable protocol for IVF with frozen-thawed semen resulting in higher blastocyst rates per collected oocyte (22.4%) when compared to control ICSI (16.4%) (p = 0.048). Furthermore, the use of semen incubated under capacitating conditions for ICSI resulted in higher blastocyst rates than washed sperm, with 69.0% versus 50.0% blastocysts per cleaved embryo (p = 0.03) and 27.8% versus 16.4% blastocysts per collected oocyte (p = 0.04), respectively. It also yielded higher blastocyst rates per cleaved embryo than IVF, with 69.0% versus 45.9% (p = 0.04). The average day of blastocyst formation was not different between the three groups (p = 0.73). Shallow whole genome sequencing revealed no differences in aneuploidy rates between IVF (1/17) and ICSI cap (0/18) (p = 0.49).

CONCLUSION: The incubation of sperm under capacitating conditions for use in ICSI or IVF with frozen-thawed semen may represent a novel method to improve the clinical efficiency of equine IVP embryos, without affecting aneuploidy rates.

Hedia, M., D. Angel-Velez, M. Papas, S. Peere, I. Gerits, T. De Coster, E. Van den Branden, J. Govaere, A. Van Soom, J. L. M. R. Leroy, et al., "Oxidative stress in donor mares for ovum pick-up delays embryonic development.", Theriogenology, vol. 213, pp. 109-113, 2024 Jan 01. Abstract

The in vitro production of equine embryos via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has increased rapidly. There is a marked effect of the individual mare on the outcome of OPU-ICSI, but little is known about the influence of the mare's health condition. This study aimed to investigate the potential associations between the concentrations of interleukin-6 (IL-6), reactive oxygen metabolites (d-ROMs), and biological antioxidant potential (BAP) in serum of oocytes' donor mares and the subsequent embryonic development. Just before OPU, a blood sample was collected from 28 Warmblood donor mares, that were subjected to a routine OPU-ICSI program. The serum concentrations of IL-6, d-ROMs, and BAP were assayed photometrically. The maturation, cleavage and blastocyst rate as well as the kinetics of blastocyst development were recorded. The average blastocyst rate was 24.68 ± 5.16% and the average concentrations of IL-6, d-ROMs, and BAP were 519.59 ± 157.08 pg/mL, 171.30 ± 4.55 carratelli units (UCARR), and 2711.30 ± 4.55 μmol/L, respectively. Serum concentrations of IL-6, d-ROMs, and BAP were not significantly different between mares yielding at least one blastocyst (552.68 ± 235.18 pg/mL, 168.36 ± 5.56 UCARR, and 2524.80 ± 159.55 μmol/L) and mares yielding no blastocysts (468.47 ± 179.99 pg/mL, 175.85 ± 7.89 UCARR, and 2999.50 ± 300.13 μmol/L, respectively). Serum concentrations of d-ROMs were significantly lower in mares with fast growing (at day 7-8 post ICSI; 148.10 ± 8.13 UCARR) compared to those with slow growing blastocysts (≥ day 9 post ICSI; 179.41 ± 4.89 UCARR; P = 0.003). Taken together, the serum concentration of IL-6, d-ROMs, and BAP do not determine the mare's ability to produce blastocysts in vitro. Although it may be questioned whether a single sample is representative of the mare's health status, changes in serum metabolites related to oxidative stress at the time of oocyte retrieval were linked to a delayed blastocyst development in a clinical OPU-ICSI outcome.

Six, R., C. Benedetti, Y. Fan, X. Guan, Y. Gansemans, M. Hedia, O. Bogado Pascottini, K. C. Pavani, F. Van Nieuwerburgh, D. Deforce, et al., "Expression profile and gap-junctional transfer of microRNAs in the bovine cumulus-oocyte complex.", Frontiers in cell and developmental biology, vol. 12, pp. 1404675, 2024. Abstract

MicroRNAs (miRNA) are important regulators of oocyte maturation, playing a key role in modulating gene expression both in a temporal- and spatial-specific manner. These small non-coding RNAs are involved in important processes during oocyte maturation, acting as messengers between the oocyte and its surrounding cumulus cells. Despite its significance, the bidirectional communication mechanism is still unknown. To test miRNA communication between oocyte and surrounding cumulus cells through the gap junctions the gap junctions were either blocked with carbenoxolone or not. MiRNA sequencing of oocytes at 1, 6, and 22 h of maturation was then performed. Among the differentially expressed miRNAs, bta-miR-21-5p, a regulator of cumulus cell viability and oocyte maturation, was the only previously known miRNA. Furthermore, by labeling a bta-miR-21-5p mimic with FAM, crossing of this miRNA through the gap junctions within the cumulus-oocyte complex could be visualized and internalization in the oocyte was confirmed by RT-qPCR. In conclusion, this study provides, for the first time, evidence that miRNA communication within the bovine cumulus-oocyte complex is enabled through the gap junctional network.

Ibrahim, S., M. Hedia, M. Taqi, M. Derbala, K. Mahmoud, Y. Ahmed, A. Sosa, Y. Saber, M. Hasanain, M. Nawito, et al., "Extracellular vesicles in low volume uterine lavage and serum: novel and promising biomarker for endometritis in Arabian mares", BMC Veterinary Research, vol. 18, issue 1, pp. 1-12, 2022.
Tourism