Cumulus expansion is an important indicator of oocyte maturation and has been suggested to be indicative of greater oocyte developmental capacity. Although multiple methods have been described to assess cumulus expansion, none of them is considered a gold standard. Additionally, these methods are subjective and time-consuming. In this manuscript, the reliability of three cumulus expansion measurement methods was assessed, and a deep learning model was created to automatically perform the measurement. Cumulus expansion of 232 cumulus-oocyte complexes was evaluated by three independent observers using three methods: (1) measurement of the cumulus area, (2) measurement of three distances between the zona pellucida and outer cumulus, and (3) scoring cumulus expansion on a 5-point Likert scale. The reliability of the methods was calculated in terms of intraclass-correlation coefficients (ICC) for both inter- and intra-observer agreements. The area method resulted in the best overall inter-observer agreement with an ICC of 0.89 versus 0.54 and 0.30 for the 3-distance and scoring methods, respectively. Therefore, the area method served as the base to create a deep learning model, AI-xpansion, which reaches a human-level performance in terms of average rank, bias and variance. To evaluate the accuracy of the methods, the results of cumulus expansion calculations were linked to embryonic development. Cumulus expansion had increased significantly in oocytes that achieved successful embryo development when measured by AI-xpansion, the area- or 3-distance method, while this was not the case for the scoring method. Measuring the area is the most reliable method to manually evaluate cumulus expansion, whilst deep learning automatically performs the calculation with human-level precision and high accuracy and could therefore be a valuable prospective tool for embryologists.

The assessment of polymorphonuclear leukocyte (PMN) proportions (%) of endometrial samples is the hallmark for subclinical endometritis (SCE) diagnosis. Yet, a non-biased, automated diagnostic method for assessing PMN% in endometrial cytology slides has not been validated so far. We aimed to validate a computer vision software based on deep machine learning to quantify the PMN% in endometrial cytology slides. Uterine cytobrush samples were collected from 116 postpartum Holstein cows. After sampling, each cytobrush was rolled onto three different slides. One slide was stained using Diff-Quick, while a second was stained using Naphthol (golden standard to stain PMN). One single observer evaluated the slides twice at different days under light microscopy. The last slide was stained with a fluorescent dye, and the PMN% were assessed twice by using a fluorescence microscope connected to a smartphone. Fluorescent images were analyzed via the Oculyze Monitoring Uterine Health (MUH) system, which uses a deep learning-based algorithm to identify PMN. Substantial intra-method repeatabilities (via Spearman correlation) were found for Diff-Quick, Naphthol, and Oculyze MUH (r = 0.67 to 0.76). The intra-method agreements (via Kappa value) at ≥1% PMN (κ = 0.44 to 0.47) were lower than at >5 (κ = 0.69 to 0.78) or >10% (κ = 0.67 to 0.85) PMN cut-offs. The inter-method repeatabilities (via Lin’s correlation) were also substantial, and values between Diff-Quick and Oculyze MUH, Naphthol and Diff-Quick, and Naphthol and Oculyze MUH were 0.68, 0.69, and 0.77, respectively. The agreements among evaluation methods at ≥1% PMN were weak (κ = 0.06 to 0.28), while it increased at >5 (κ = 0.48 to 0.81) or >10% (κ = 0.50 to 0.65) PMN cut-offs. To conclude, deep learning-based algorithms in endometrial cytology are reliable and useful for simplifying and reducing the diagnosis bias of SCE in dairy cows.

Following acute human alphaherpesvirus 1 (HSV-1) infection of oral-facial mucosal surfaces, sensory neurons in trigeminal ganglia (TG) are important sites for life-long latency. Neurons in the central nervous system, including brainstem, also harbor viral genomes during latency. Periodically, certain cellular stressors trigger reactivation from latency, which can lead to recurrent HSV-1 disease: herpes labialis, herpes stromal keratitis, and encephalitis for example. Activation of the glucocorticoid receptor (GR) by stressful stimuli enhances HSV-1 gene expression, replication, and explant-induced reactivation. GR and certain stress-induced Krüppel like factors (KLF) cooperatively transactivate cis-regulatory modules (CRM) that drive expression of viral transcriptional regulatory proteins (ICP0, ICP4, and ICP27). These CRMs lack GR response elements (GRE); however, specificity protein 1 (Sp1) binding sites are crucial for GR and KLF15 or KLF4 mediated transactivation. Hence, we tested whether Sp1 or Sp3 regulate viral replication and transactivation of the ICP0 promoter. During early stages of explant-induced reactivation from latency, the number of Sp3+ TG neurons were significantly higher relative to TG from latently infected mice. Conversely, Sp1+ TG neurons were only increased in females, but not male mice, during explant-induced reactivation. Sp1 siRNA significantly reduced HSV-1 replication in cultured mouse (Neuro-2A) and monkey (CV-1) cells. Mithramycin A, an antibiotic that has anti-tumor activity preferentially interacts with GC-rich DNA, including Sp1 binding sites, significantly reduced HSV-1 replication indicating it has antiviral activity. GR and Sp1 or Sp3 transactivated the HSV-1 ICP0 promoter in Neuro-2A and CV-1 cells confirming these transcription factors enhance viral replication and gene expression.

In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality.