Artificial intelligence-powered assisted ranking of sibling embryos to increase first cycle pregnancy rate.

RESEARCH QUESTION: Could EMBRYOLY, an artificial intelligence embryo evaluation tool, assist embryologists to increase first cycle pregnancy rate and reduce cycles to pregnancy for patients? DESIGN: Data from 11,988 embryos were collected via EMBRYOLY from 2666 egg retrievals (2019-2022) across 11 centres in France, Spain and Morocco using three time-lapse systems (TLS). Data from two independent clinics were also examined. EMBRYOLY's transformer-based model was applied to transferred embryos to evaluate ranking performances against pregnancy and birth outcomes. It was applied to cohorts to rank sibling embryos (including non-transferred) according to their likelihood of clinical pregnancy and to compute the agreement with the embryologist's highest ranked embryo. Its effect on time to pregnancy and first cycle pregnancy rate was evaluated on cohorts with multiple single blastocyst transfers, assuming the embryologist would have considered EMBRYOLY's ranking on the embryos favoured for transfer. RESULTS: EMBRYOLY's score correlated significantly with clinical pregnancies and live births for cleavage and blastocyst transfers. This held true for clinical pregnancies from blastocyst transfers in two independent clinics. In cases of multiple single embryo transfers, embryologists achieved a 19.8% first cycle pregnancy rate, which could have been improved to 44.1% with the adjunctive use of EMBRYOLY (McNemar's test: P < 0.001). This could have reduced cycles to clinical pregnancy from 2.01 to 1.66 (Wilcoxon test: P < 0.001). CONCLUSIONS: EMBRYOLY's potential to enhance first cycle pregnancy rates when combined with embryologists' expertise is highlighted. It reduces the number of unsuccessful cycles for patients across TLS and IVF centres.

The driving role of the Cdk5/Tln1/FAKS732 axis in cancer cell extravasation dissected by human vascularized microfluidic models.

BACKGROUND: Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans-endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis. METHODS: Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis. RESULTS: Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression. CONCLUSIONS: These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Aqueous Humor Outflow Requires Active Cellular Metabolism in Mice.

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<<0.001), but reducing temperature did not affect ATP. Conclusions: Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Integrated Analysis of Intracellular Dynamics of MenaINV Cancer Cells in a 3D Matrix.

The intracellular environment is composed of a filamentous network that exhibits dynamic turnover of cytoskeletal components and internal force generation from molecular motors. Particle tracking microrheology enables a means to probe the internal mechanics and dynamics. Here, we develop an analytical model to capture the basic features of the active intracellular mechanical environment, including both thermal and motor-driven effects, and show consistency with a diverse range of experimental microrheology data. We further perform microrheology experiments, integrated with Brownian dynamics simulations of the active cytoskeleton, on metastatic breast cancer cells embedded in a three-dimensional collagen matrix with and without the presence of epidermal growth factor to probe the intracellular mechanical response in a physiologically mimicking scenario. Our results demonstrate that EGF stimulation can alter intracellular stiffness and power output from molecular motor-driven fluctuations in cells overexpressing an invasive isoform of the actin-associated protein Mena.

Macrophage-Secreted TNFα and TGFß1 Influence Migration Speed and Persistence of Cancer Cells in 3D Tissue Culture via Independent Pathways.

The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFß1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB-dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFß1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFß1 dual blockade as an antimetastatic strategy in solid tumors. Cancer Res; 77(2); 279-90. ©2016 AACR.

Microfluidics: A new tool for modeling cancer-immune interactions.

In recognition of the enormous potential of immunotherapies against cancer, research into the interactions between tumor and immune cells has accelerated, leading to the recent FDA approval of several drugs that reduce cancer progression. Numerous cellular and molecular interactions have been identified by which immune cells can intervene in the metastatic cascade, leading to the development of several in vivo and in vitro model systems that can recapitulate these processes. Among these, microfluidic technologies hold many advantages in terms of their unique ability to capture the essential features of multiple cell type interactions in three-dimensions while allowing tight control of the microenvironment and real-time monitoring. Here, we review current assays and discuss the development of new microfluidic technologies for immunotherapy.

Physical Factors Affecting Outflow Facility Measurements in Mice.

PURPOSE: Mice are commonly used to study conventional outflow physiology. This study examined how physical factors (hydration, temperature, and anterior chamber [AC] deepening) influence ocular perfusion measurements in mice. METHODS: Outflow facility (C) and pressure-independent outflow (Fu) were assessed by multilevel constant pressure perfusion of enucleated eyes from C57BL/6 mice. To examine the effect of hydration, seven eyes were perfused at room temperature, either immersed to the limbus in saline and covered with wet tissue paper or exposed to room air. Temperature effects were examined in 12 eyes immersed in saline at 20 °C or 35 °C. Anterior chamber deepening was examined in 10 eyes with the cannula tip placed in the anterior versus posterior chamber (PC). Posterior bowing of the iris (AC deepening) was visualized by three-dimensional histology in perfusion-fixed C57BL/6 eyes and by spectral-domain optical coherence tomography in living CD1 mice. RESULTS: Exposure to room air did not significantly affect C, but led to a nonzero Fu that was significantly reduced upon immersion in saline. Increasing temperature from 20 °C to 35 °C increased C by 2.5-fold, more than could be explained by viscosity changes alone (1.4-fold). Perfusion via the AC, but not the PC, led to posterior iris bowing and increased outflow. CONCLUSIONS: Insufficient hydration contributes to the appearance of pressure-independent outflow in enucleated mouse eyes. Despite the large lens, AC deepening may artifactually increase outflow in mice. Temperature-dependent metabolic processes appear to influence conventional outflow regulation. Physical factors should be carefully controlled in any outflow studies involving mice.

Ultrastructural changes associated with dexamethasone-induced ocular hypertension in mice.

PURPOSE: To determine whether dexamethasone (DEX)-induced ocular hypertension (OHT) in mice mimics the hallmarks of steroid-induced glaucoma (SIG) in humans, including reduced conventional outflow facility (C), increased extracellular matrix (ECM), and myofibroblasts within the outflow pathway. METHODS: Osmotic mini-pumps were implanted subcutaneously into C57BL/6J mice for systemic delivery of DEX (3-4 mg/kg/d, n = 31 mice) or vehicle (n = 28). IOP was measured weekly by rebound tonometry. After 3 to 4 weeks, mice were euthanized and eyes enucleated for ex vivo perfusion to measure C, for electron microscopy to examine the trabecular meshwork (TM) and Schlemm's canal (SC), or for immunohistochemistry to examine type IV collagen and α-smooth muscle actin. The length of basement membrane material (BMM) was measured along the anterior-posterior extent of SC by electron microscopy. Ultrastructural changes in BMM of DEX-treated mice were compared against archived human SIG specimens. RESULTS: Dexamethasone increased IOP by 2.6 ± 1.6 mm Hg (mean ± SD) over 3 to 4 weeks and decreased C by 52% ± 17% versus controls. Intraocular pressure elevation correlated with decreased C. Dexamethasone treatment led to increased fibrillar material in the TM, plaque-like sheath material surrounding elastic fibers, and myofibroblasts along SC outer wall. The length of BMM underlying SC was significantly increased in mice with DEX and in humans with SIG, and in mice decreased C correlated with increased BMM. CONCLUSIONS: Dexamethasone-induced OHT in mice mimics hallmarks of human SIG within 4 weeks of DEX treatment. The correlation between reduced C and newly formed ECM motivates further study using DEX-treated mice to investigate the pathogenesis of conventional outflow obstruction in glaucoma.

The influence of genetic background on conventional outflow facility in mice.

PURPOSE: Intraocular pressure (IOP) varies between genetically distinct strains of mice. The purpose was to test the hypothesis that strain-dependent differences in IOP are attributable to differences in conventional outflow facility (C). METHODS: The IOP was measured by rebound tonometry in conscious or anesthetized BALB/cJ, C57BL/6J, and CBA/J mice (N = 6-10 per strain). Conventional outflow facility was measured by ex vivo perfusion of enucleated eyes (N = 9-10 per strain). RESULTS: Conscious IOP varied between strains, being highest in CBA/J (14.5 ± 0.9 mm Hg, mean ± SD), intermediate in C57BL/6J (12.3 ± 1.0 mm Hg), and lowest in BALB/cJ (10.6 ± 1.8 mm Hg) mice. Anesthesia reduced IOP and eliminated any detectable differences between strains. Conventional outflow facility also varied between strains, but, in contrast to IOP, C was lowest in CBA/J (0.0113 ± 0.0031 µL/min/mm Hg) and highest in BALB/cJ (0.0164 ± 0.0059 µL/min/mm Hg). Like IOP, C was intermediate in C57BL/6J (0.0147 ± 0.0029 µL/min/mm Hg). There was a strong correlation between conscious IOP and outflow resistance (1/C) from individual eyes across all three strains, revealing that 70% of the variation in IOP was attributable to variation in outflow resistance. CONCLUSIONS: Differences in IOP among three genetically distinct murine strains are attributable largely to differences in conventional outflow facility. These results motivate further studies using mice to identify the morphologic and genetic factors that underlie IOP regulation within the conventional outflow pathway.

Pharmacologic manipulation of conventional outflow facility in ex vivo mouse eyes.

PURPOSE: Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice. METHODS: A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 4°C. Three groups of experiments examined sphingosine 1-phosphate (S1P), S1P with antagonists to S1P(1) and S1P(2) receptors, and the prostanoid EP(4) receptor agonist 3,7-dithia PGE(1). We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E(1) (PGE(1)). RESULTS: S1P decreased facility by 39%, and was blocked almost completely by an S1P(2), but not S1P(1), receptor antagonist. The S1P(2) receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE(1) increased facility by 106% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE(1) was reduced 3-fold, yet remained statistically detectable. CONCLUSIONS: C57BL/6 mice showed opposing effects of S1P(2) and EP(4) receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S1P and EP(4) receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics.

eNOS, a pressure-dependent regulator of intraocular pressure.

PURPOSE: Pathology in the primary drainage pathway for aqueous humor in the eye is responsible for ocular hypertension, the only treatable risk factor in patients with glaucoma. Unfortunately, the mechanisms that regulate pressure-dependent drainage of aqueous humor and thus intraocular pressure (IOP) are unknown. To better understand one possible underlying molecular factor that regulates IOP, nitric oxide (NO), pressure-dependent drainage in transgenic mice overexpressing endothelial NO synthase (eNOS) was studied. METHODS: IOP was measured by rebound tonometry in mice, and pressure versus flow data were measured by ex vivo perfusion at multiple pressures between 8 and 45 mm Hg, using mock AH ±100 µM L-NAME. A subset of eyes was examined histologically using standard techniques or was assayed for fusion protein expression by Western blot analysis. RESULTS: IOP was lower (9.6 ± 2.7 vs. 11.4 ± 2.5 mm Hg; mean ± SD; P = 0.04) and pressure-dependent drainage was higher (0.0154 ± 0.006 vs. 0.0066 ± 0.0009 µL/min/mm Hg; P = 0.002) in the transgenic mice than in the wild-type animals; however, pressure-independent drainage was unaffected. The NOS inhibitor L-NAME normalized pressure-dependent drainage in transgenic animals. For IOP >35 mm Hg, the slope of the pressure-flow curve in wild-type mice increased to match that seen in transgenic mice. Shear stress in the pressure-dependent pathway at elevated pressures was calculated to be in a range known to affect eNOS expression and activity in vascular endothelia. CONCLUSIONS: Endothelial NOS overexpression lowers IOP by increasing pressure-dependent drainage in the mouse eye. Data are consistent with NO's having a mechanoregulatory role in aqueous humor dynamics, with eNOS induction at elevated IOPs leading to increased pressure-dependent outflow.

Outflow physiology of the mouse eye: pressure dependence and washout.

PURPOSE: Mice are commonly used in glaucoma research, but relatively little is known about aqueous outflow dynamics in the species. To facilitate future use of the mouse as a model of aqueous humor outflow, several fundamental physiological parameters were measured in the mouse eye. METHODS: Eyes from adult mice of either sex (C57BL/6 background) were enucleated, cannulated with a 33-gauge needle, and perfused at constant pressure while inflow was continuously measured. RESULTS: At 8 mm Hg, total outflow facility (C(total)) was 0.022 ± 0.005 µL/min/mm Hg (all values mean ± SD; n = 21). The flow-pressure relationship was linear up to 35 mm Hg. The conventional outflow facility (C(conv)) was 0.0066 ± 0.0009 µL/min/mm Hg, and the unconventional outflow (F(u)) was 0.114 ± 0.019 µL/min, both measured at room temperature. At 8 mm Hg, 66% of the outflow was via the unconventional pathway. In a more than 2-hour-long perfusion at 8 mm Hg, the rate of facility change was 2.4% ± 5.4% (n = 11) of starting facility per hour. The ocular compliance (0.086 ± 0.017 µL/mm Hg; n = 5) was comparable to the compliance of the perfusion system (0.100 ± 0.004 µL/mm Hg). CONCLUSIONS: Mouse eyes are similar to human eyes, in that they have no detectable washout rate and a linear pressure-flow relationship over a broad range of intraocular pressures. Because of the absence of washout and the apparent presence of a true Schlemm's canal, the mouse is a useful model for studying the physiology of the inner wall of Schlemm's canal and the conventional outflow tissues.