Mara D. Saenz-de-Juano

Last Name

Saenz-de-Juano

First Name

Mara D.

ORCID

0000-0002-2860-2252

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03999 - Ulbrich, Susanne / Ulbrich, Susanne

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Publications1 - 10 of 23

One-week storage of refrigerated bovine milk does not affect the size, concentration, or molecular properties of extracellular vesicles
Saenz-de-Juano, Mara D.; Silvestrelli, Giulia; Ulbrich, Susanne E. (2024)
Journal of Dairy Science
Milk extracellular vesicles (EV) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EV with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2 quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EV size, concentration, or morphology. We did not detect any changes in the EV cargo regarding the amount of protein and RNA, nor in the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low SCC. Overall, we observed fewer CD81 and CD9 markers in quarters with high SCC. Moreover, we found no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to 1 wk is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EV are highly stable in the raw milk matrix.
Mastitis induces proteomic cargo changes in a subpopulation of CD81 extracellular vesicles in milk
Saenz-de-Juano, Mara D.; Silvestrelli, Giulia; Krivitsky, Vadim; et al. (2024)
Methods for isolating Extracellular Vesicles from Bovine Mammary Epithelial Cells in Culture
Silvestrelli, Giulia; Ulbrich, Susanne E.; Saenz-de-Juano, Mara D. (2021)
Reproduction in Domestic Animals
Mastitis-related Staphylococcus aureus-derived extracellular vesicles induce a pro-inflammatory response in bovine monocyte-derived macrophages
Saenz-de-Juano, Mara D.; Silvestrelli, Giulia; Buri, Samuel; et al. (2025)
Scientific Reports
Staphylococcus aureus (S. aureus) is one of the most common causative agents of mammary gland infection and mastitis, but the specific role of S. aureus-derived extracellular vesicles (SaEVs) in mastitis has been poorly studied to date. Here, we aimed to investigate the response of bovine monocyte-derived macrophages (boMdM) to SaEVs of the genotype B (GTB) mastitis-related strain M5512B. Specifically, we evaluated the effects on the actin cytoskeleton, gene expression, and the SaEV proteomic cargo. Furthermore, we assessed to what extent the cellular and molecular response of boMdM to SaEVs differed from peripheral mononuclear blood cells (PBMCs) used for in vitro derivation of the former. We observed that SaEVs induced morphological changes in boMdM, leading to a pro-inflammatory and pyroptosis-related increased gene expression. Additionally, our study revealed that boMdM and PBMCs exhibited stimulus-specific differing responses. The proteomic analysis of SaEVs identified clusters of proteins related to virulence and antibiotic resistance, supporting the theory that S. aureus might use EVs to evade host defences and colonize the mammary gland. Our results bring new insights into how SaEVs might impact the host during an S. aureus infection, which can be useful for future S. aureus vaccine development.
Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles
Saenz-de-Juano, Mara D.; Silvestrelli, Giulia; Weber, Andres; et al. (2022)
Biology
In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells.
In vitro models to study extracellular vesicles in bovine mammary gland
Silvestrelli, Giulia; Ulbrich, Susanne E.; Saenz-de-Juano, Mara D. (2019)
Proceedings of the 3rd CellFit Annual Metting: The Extracellurlar Vesicles Paradigm of Intra and Intercellular Communication
The major urinary protein gene cluster knockout mouse as a novel model for translational metabolism research
Greve, Sarah; Kuhn, Gisela A.; Saenz-de-Juano, Mara D.; et al. (2022)
Scientific Reports
Scientific evidence suggests that not only murine scent communication is regulated by major urinary proteins, but that their expression may also vary in response to metabolism via a yet unknown mechanism. Major urinary proteins are expressed mainly in the liver, showing a sexually dimorphic pattern with substantially higher expression in males. Here, we investigate the metabolic implications of a major urinary protein knockout in twelve-week-old male and female C57BL/6N mice during ad libitum feeding. Despite both sexes of major urinary protein knockout mice displayed numerically increased body weight and visceral adipose tissue proportions compared to sex-matched wildtype mice, the main genotype-specific metabolic differences were observed exclusively in males. Male major urinary protein knockout mice exhibited plasma and hepatic lipid accumulation accompanied by a hepatic transcriptome indicating an activation of lipogenesis. These findings match the higher major urinary protein expression in male compared to female wildtype mice, suggesting a more distinct reduction in energy requirements in male compared to female major urinary protein knockout mice. The observed sex-specific anabolic phenotype confirms a role of major urinary protein in metabolism and, since major urinary proteins are not expressed in humans, suggests the major urinary protein knockout mouse as a potential alternative model for translational metabolism research which needs to be further elucidated.
Study of the correlation between somatic cell count variation and EVs miRNA cargo in milk from healthy quarters
Saenz-de-Juano, Mara D.; Silvestrelli, Giulia; Ulbrich, Susanne E. (2022)
Subclinical mastitis, the inflammation of the mammary gland devoid of clinical signs, is one of the most prevalent and costly diseases in dairy farming worldwide. It usually derives from an intramammary infection (IMI) that induces a rise in the milk’s somatic cell count (SCC). For that reason, the SCC is a standard detection method with 200’000 cells/ml as the threshold. However, the SCC is highly variable, and apart from an IMI, other factors are of influence, such as lactation stage, parity, season, breed or productivity. Extracellular vesicles (EVs) and their miRNA cargo have been suggested as potential biomarkers for mammary gland health. The current study aimed to evaluate whether changes in SCC are reflected in changes in the miRNA cargo of milk EVs.
Endometriosis-associated infertility alters the microRNA signatures of cumulus cells with a particularly pronounced effect in oocytes that failed fertilization
Almiñana, Carmen; Makieva, Sofia; Bauersachs, Stefan; et al. (2025)
Biological Research
Background: Endometriosis (E) is multifactorial disease affecting around 10% of women worldwide. The association between E and infertility is clinically well recognized. For E patients to achieve a successful pregnancy, assisted reproductive technologies (ART) are considered as a treatment option. However, the impact of E on oocyte quality, its potential to be fertilized as well as pregnancy rates, is still under debate and with very few molecular clues explaining the clinical data. Alterations in protein-coding RNAs in cumulus cells (CCs), cells surrounding the oocytes and contributing to oocyte maturation, have been reported in E patients. But there is a lack of information regarding microRNAs (miRNAs), which control protein translation. Thus, we aimed: (1) to identify altered miRNA expression in CCs of E patients versus patients without the disease (control, C); and (2) to unveil if in E patients, CCs from fertilized oocytes display a different miRNA profile versus oocytes that failed fertilization. Small RNA-sequencing was performed on CCs from patients undergoing ART. Results: A total of 85 differentially expressed (DE) miRNAs were identified in E versus C patients (FDR < 0.05). In E patients, 25 DE miRNAs were found between fertilized oocytes and oocytes that failed fertilization, while 13 DE miRNAs in C patients (FDR < 0.05). Comparisons among DE miRNAs highlighted three notable miRNA sets: Set (1) 35 DE miRNAs specific to E; Set (2) 27 DE miRNAs affected by both E and the potential to be fertilized; and Set (3) 6 DE miRNAs characteristic of a competent oocyte successfully fertilized despite the disease. Target gene analysis of DE miRNAs unveiled genes involved in oocyte meiosis, progesterone-mediated oocyte maturation pathway, embryo development, mitochondria and spindle alterations, calcium signaling, and oxidative stress. Conclusion: This study identified for the first time an altered miRNA signature in CCs of E patients, pointing towards compromised oocyte competence. Besides, in E patients, a characteristic CCs miRNA footprint for oocytes that can be successfully fertilized despite the disease has been revealed. The study charts new territory for non-invasive diagnosis and personalized treatments based on miRNAs to improve oocyte competence in E patients under ART treatments.
DNA methylation and mRNA expression of imprinted genes in blastocysts derived from an improved in vitro maturation method for oocytes from small antral follicles in polycystic ovary syndrome patients
Saenz-de-Juano, Mara D.; Ivanova, Elena; Romero, Sergio; et al. (2019)
Human Reproduction
Study Question: Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients? Summary Answer: No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts. What Is Known Already: Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity. Study Design, Size, Duration: For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts. Participants/Materials, Setting, Methods: COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3–5 days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24 h containing C-type natriuretic peptide), followed by an IVM step (30 h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets. Main Results and the Role of Chance: CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups. Limitations, Reasons for Caution: The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known. Wider Implications of the Findings: A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS Study Funding/Competing Interest(s): IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.’s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest.

Publications1 - 10 of 23

Mara D. Saenz-de-Juano

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