Trophectoderm cells of human mosaic embryos display increased apoptotic levels and impaired differentiation capacity: a molecular clue regarding their reproductive fate?

STUDY QUESTION: Are there cell lineage-related differences in the apoptotic rates and differentiation capacity of human blastocysts diagnosed as euploid, mosaic, and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A) based on concurrent copy number and genotyping analysis? SUMMARY ANSWER: Trophectoderm (TE) cells of mosaic and aneuploid blastocysts exhibit significantly higher levels of apoptosis and significantly reduced differentiation capacity compared to those of euploid blastocysts. WHAT IS KNOWN ALREADY: Embryos diagnosed as mosaic after PGT-A can develop into healthy infants, yet understanding the reasons behind their reproductive potential requires further research. One hypothesis suggests that mosaicism can be normalized through selective apoptosis and reduced proliferation of aneuploid cells, but direct evidence of these mechanisms in human embryos is lacking. Additionally, data interpretation from studies involving mosaic embryos has been hampered by retrospective analysis methods and the high incidence of false-positive mosaic diagnoses stemming from the use of poorly specific PGT-A platforms. STUDY DESIGN, SIZE, DURATION: Prospective cohort study performing colocalization of cell-lineage and apoptotic markers by immunofluorescence (IF). We included a total of 64 human blastocysts donated to research on Day 5 or 6 post-fertilization (dpf) by 43 couples who underwent in vitro fertilization treatment with PGT-A at IVI-RMA Valencia between September 2019 and October 2022. A total of 27 mosaic blastocysts were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study consisted of two phases: Phase I (caspase-3, n = 53 blastocysts): n = 13 euploid, n = 22 mosaic, n = 18 aneuploid. Phase II (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), n = 11 blastocysts): n = 2 euploid, n = 5 mosaic, n = 4 aneuploid. Following donation for research, vitrified blastocysts were warmed, cultured until re-expansion, fixed, processed for IF, and imaged using confocal microscopy. For each blastocyst, the following cell counts were conducted: total cells (DAPI+), TE cells (GATA3+), inner cell mass (ICM) cells (GATA3-/NANOG+), and apoptotic cells (caspase-3+ or TUNEL+). The incidence of apoptosis was calculated for each blastocyst by dividing the number of caspase-3+ cells (Phase I) or TUNEL+ cells (Phase II) by the number of TE or ICM cells. Statistical analysis was performed according to data type and distribution (P < 0.05 was considered statistically significant). MAIN RESULTS AND THE ROLE OF CHANCE: Phase I: Mosaic blastocysts displayed a similar number of total cells (49.6 ± 15 cells at 5 dpf; 58.8 ± 16.9 cells at 6 dpf), TE cells (38.8 ± 13.7 cells at 5 dpf; 49.2 ± 16.2 cells at 6 dpf), and ICM cells (10.9 ± 4.2 cells at 5 dpf; 9.7 ± 7.1 cells at 6 dpf) compared to euploid and aneuploid blastocysts (P > 0.05). The proportion of TE cells retaining NANOG expression increased gradually from euploid blastocysts (9.7% = 63/651 cells at 5 dpf; 0% = 0/157 cells at 6 dpf) to mosaic blastocysts (13.1% = 104/794 cells at 5 dpf; 3.4% = 12/353 cells at 6 dpf) and aneuploid blastocysts (27.9% = 149/534 cells at 5 dpf; 4.6% = 19/417 cells at 6 dpf) (P < 0.05). At the TE level, caspase-3+ cells were frequently observed (39% = 901/2310 cells). The proportion of caspase-3+ TE cells was significantly higher in mosaic blastocysts (44.1% ± 19.6 at 5 dpf; 43% ± 16.8 at 6 dpf) and aneuploid blastocysts (45.9% ± 16.1 at 5 dpf; 49% ± 15.1 at 6 dpf) compared to euploid blastocysts (26.6% ± 16.6 at 5 dpf; 17.5% ± 14.8 at 6 dpf) (P < 0.05). In contrast, at the ICM level, caspase-3+ cells were rarely observed (1.9% = 11/596 cells), and only detected in mosaic blastocysts (2.6% = 6/232 cells) and aneuploid blastocysts (2.5% = 5/197 cells) (P > 0.05). Phase II: Consistently, TUNEL+ cells were only observed in TE cells (32.4% = 124/383 cells). An increasing trend was identified toward a higher proportion of TUNEL+ cells in the TE of mosaic blastocysts (37.2% ± 21.9) and aneuploid blastocysts (39% ± 41.7), compared to euploid blastocysts (23% ± 32.5), although these differences did not reach statistical significance (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: The observed effects on apoptosis and differentiation may not be exclusive to aneuploid cells. Additionally, variations in aneuploidies and unexplored factors related to blastocyst development and karyotype concordance may introduce potential biases and uncertainties in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate a cell lineage-specific effect of aneuploidy on the apoptotic levels and differentiation capacity of human blastocysts. This contributes to unravelling the biological characteristics of mosaic blastocysts and supports the concept of clonal depletion of aneuploid cells in explaining their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) and Generalitat Valenciana (APOTIP/2019/009). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

The Reliability and Accuracy of a Fall Risk Assessment Procedure Using Mobile Smartphone Sensors Compared with a Physiological Profile Assessment.

Falls in older people are a major health concern as the leading cause of disability and the second most common cause of accidental death. We developed a rapid fall risk assessment based on a combination of physical performance measurements made with an inertial sensor embedded in a smartphone. This study aimed to evaluate and validate the reliability and accuracy of an easy-to-use smartphone fall risk assessment by comparing it with the Physiological Profile Assessment (PPA) results. Sixty-five participants older than 55 performed a variation of the Timed Up and Go test using smartphone sensors. Balance and gait parameters were calculated, and their reliability was assessed by the (ICC) and compared with the PPAs. Since the PPA allows classification into six levels of fall risk, the data obtained from the smartphone assessment were categorised into six equivalent levels using different parametric and nonparametric classifier models with neural networks. The F1 score and geometric mean of each model were also calculated. All selected parameters showed ICCs around 0.9. The best classifier, in terms of accuracy, was the nonparametric mixed input data model with a 100% success rate in the classification category. In conclusion, fall risk can be reliably assessed using a simple, fast smartphone protocol that allows accurate fall risk classification among older people and can be a useful screening tool in clinical settings.

Dinophysis Species and Diarrhetic Shellfish Toxins: 20 Years of Monitoring Program in Andalusia, South of Spain.

In Andalusia, the official monitoring program for toxic phytoplankton and marine biotoxins was launched in 1994 to comply with European legislation. Since then, there have been numerous episodes of DST (Diarrhetic shellfish toxins) associated with the proliferation of Dinophysis species. This article reviews two decades of time series data and assesses the effectiveness of the program established. The testing of lipophilic toxins and toxic phytoplankton is based on official methods harmonized and accredited since 2007 according to the standard UNE-EN-ISO 17025. The major species of Dinophysis identified were D. acuminata complex, D. caudata, D. acuta and D. fortii, with the main growth season being from early spring until the end of autumn. Both D. acuminata complex and D. acuta have been clearly associated with toxicity in molluscs. Despite the complexity of data obtained through monitoring programs, it is possible to provide early warning of potential health risks for most situations. This is the first report of Dinophysis species and their relation to DST events in a time series from Andalusia.

Amphidoma languida (Amphidomatacea, Dinophyceae) with a novel azaspiracid toxin profile identified as the cause of molluscan contamination at the Atlantic coast of southern Spain.

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC-MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160µg AZA-1 eq. kg-1 (reaching maximum levels of >500µg AZA-1 eq. kg-1 in Chamelea gallina and >250µg AZA-1 eq. kg-1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins.

A comparison of lumbopelvic motion patterns and erector spinae behavior between asymptomatic subjects and patients with recurrent low back pain during pain-free periods.

OBJECTIVES: The purpose of this study was to determine the patterns of lumbopelvic motion and erector spinae (ES) activity during trunk flexion-extension movements and to compare these patterns between patients with recurrent low back pain (LBP) in their pain-free periods and matched asymptomatic subjects. METHODS: Thirty subjects participated (15 patients with disc herniation and recurrent LBP in their pain-free periods and 15 asymptomatic control subjects). A 3-dimensional videophotogrammetric system and surface electromyography (EMG) were used to record the angular displacements of the lumbar spine and hip in the sagittal plane and the EMG activity of the ES during standardized trunk flexion-extension cycles. Variables were maximum ranges of spine and hip flexion; percentages of maximum lumbar and hip flexion at the start and end of ES relaxation; average percentages of EMG activity during flexion, relaxation, and extension; and flexion-extension ratio of myoelectrical activity. RESULTS: Recurrent LBP patients during their pain-free period showed significantly greater ES activation both in flexion and extension, with a higher flexion-extension ratio than controls. Maximum ranges of lumbar and hip flexion showed no differences between controls and patients, although patients spent less time with their lumbar spine maximally flexed. CONCLUSIONS: This study showed that reduced maximum ranges of motion and absence of ES flexion-relaxation phenomenon were not useful to identify LBP patients in the absence of acute pain. However, these patients showed subtle alterations of their lumbopelvic motion and ES activity patterns, which may have important clinical implications.

Reliability and validity of a new objective tool for low back pain functional assessment.

STUDY DESIGN: Classification and functional assessment model for nonspecific low back pain (LBP) patients and controls on the basis of kinematic analysis parameters. OBJECTIVE: Develop a logistic regression model using kinematic analysis variables to (1) discriminate between LBP patients and controls and (2) obtain objective parameters for LBP functional assessment. SUMMARY OF BACKGROUND DATA: Functional assessment of spinal disorders has been carried out traditionally by means of subjective scales. Objective functional techniques have been developed, which usually involve the application of external loads or the analysis of highly standardized trunk flexion-extension maneuvers. Few studies have used everyday activities such as sit-to-stand or lifting an object from the ground. They have shown that the motion patterns of LBP patients differ from those of healthy subjects. Nevertheless, very few studies have tried to correlate objective findings to the results of subjective scales, and no previous study has developed a LBP classification and functional assessment model on the basis of kinematic analysis of everyday activities. METHODS: Sixteen controls and 39 LBP patients performed a sit-to-stand task, and lifted three different weights from a standing position. The vertical forces exerted and the relative positions of the lower limb and the cervical, thoracic, lumbar, and sacroiliac regions were recorded. Reliability was determined from repetitions of the tests performed by the control group. Binary logistic regression analyses were computed. The results of the selected regression equation were correlated to the Oswestry Disability Index scale results, to check the validity of the procedure for the measurement of functional disability. RESULTS: Reliability of the parameters was good. The selected regression model used two variables, and correctly classified 97.3% of the patients. High correlations were found between the results of this regression equation and the Oswestry Disability Index scale. CONCLUSION: It is possible to distinguish LBP patients from healthy subjects by means of the biomechanical analysis of everyday tasks. This kind of analysis can produce objective and reliable indexes about the patients' degree of functional impairment.

Neck motion patterns in whiplash-associated disorders: quantifying variability and spontaneity of movement.

BACKGROUND: whiplash-associated disorders have usually been explored by analyzing changes in the cervical motor system function by means of static variables such as the range of motion, whereas other behavioural features such as speed, variability or smoothness of movement have aroused less interest. METHODS: whiplash patients (n=30), control subjects (n=29) and a group of people faking the symptoms of whiplash-associated-disorders (Simulators, n=30) performed a cyclical flexion-extension movement. This movement was recorded by means of video-photogrammetry. The computed variables were: range of motion, maxima angular velocity and acceleration, and two additional variables that quantify the repeatability of a motion and its spontaneity. Two comparisons were made: Control vs. Patients and Patients vs. Simulators. At each comparison we used ANOVA to detect differences between groups and discriminant analysis to evaluate the ability of these variables to classify individuals. FINDINGS: comparison between Controls and Patients showed significant reductions in the range of motion, and both the maximum of angular velocity and acceleration in the Patients. The most efficient discriminant model only included the range of motion and maximum angular velocity. Comparison between Patients and Simulators showed a significant reduction in all measured variables in the Simulators. The best classification model was obtained with maximum angular velocity, spontaneity and repeatability of motion. INTERPRETATION: our results suggest that the pathological patterns differ from those of Controls in amplitude and speed of motion, but not in repeatability or spontaneity of movement. These variables are especially useful for detecting abnormal movement patterns.

Production of functionally active palytoxin-like compounds by Mediterranean Ostreopsis cf. siamensis.

BACKGROUND AND PURPOSE: Dinoflagellates from the genus Ostreopsis have been related to the production of palytoxin and analogues. Based on that, this paper describes functional studies of crude extracts from Ostreopsis cf. siamensis collected in the Mediterranean Sea in order to biochemically characterize their toxic compounds. METHODS: We compared the effects of 5 crude dinoflagellates extracts with a commercially available palytoxin and a purified Ostreopsis ovata extract on metabolic activity, membrane potential, and cytosolic calcium levels by using fluorescent dyes. RESULTS: All the extracts resulted to be neurotoxic. In addition, all of them induced a membrane depolarization and a calcium increment that were abolished when preincubating with ouabain, an inhibitor of the Na(+)/K(+) pump. CONCLUSION: The effects observed were quite close to those induced by palytoxin and the Ostreopsis ovata extract as well, suggesting that Ostreopsis cf. siamensis is actually producing palytoxin-like compounds that are highly toxic and functionally active.

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells.

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.