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 effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Pharmacy stewardship to reduce recurrent hypoglycemia.

BACKGROUND: Inpatient hypoglycemia is associated with increased morbidity and mortality. After a hypoglycemic event, the likelihood of additional episodes increases. The Joint Commission recommends evaluating all episodes of hypoglycemia for root-cause analysis. Studies have shown that pharmacists' involvement with glycemic control protocols can prevent hypoglycemia. OBJECTIVES: This study aimed to assess whether the implementation of pharmacists' real-time assessment of hypoglycemic events using an electronic alert messaging system contributes to the reduction of the number of recurrent hypoglycemia during hospitalization. PRACTICE DESCRIPTION: A community hospital that provides a wide range of health care services. The pharmacy department provides fully decentralized clinical services and team-based specialist services. PRACTICE INNOVATION: The pharmacist-led hypoglycemia stewardship initiative included a comprehensive review of hypoglycemic alerts received via an automated message. The alerts generated in the electronic health record (EHR) every time a patient's blood glucose resulted in less than 70 mg/dL if there was a documented administration of a hypoglycemic agent 48 hours before the hypoglycemia event. Once the alert was received by the pharmacists via an EHR in-basket, a real-time review was conducted to identify the potential causes of the event and opportunities for therapy modification. EVALUATION METHODS: A single-center retrospective observational study including a pre- and post-implementation phase from January 1 to June 3, 2020, and January 1 to June 30, 2021, respectively. Continuous data were analyzed using paired and equal variance t test. Noncontinuous data were analyzed using Fisher exact and chi-square test. Descriptive statistics were used to describe distribution and frequency of data. RESULTS: There was a 5.1% absolute reduction in recurrent hypoglycemic events (P < 0.001) and a 0.6% reduction of severe hypoglycemic days (P = 0.269) in the postimplementation group. The average time to pharmacist intervention was 4 (± 3.5) hours with a 92% acceptance rate. CONCLUSION: This study demonstrated the utility of pharmacist-led hypoglycemia reviews in the reduction of recurrent hypoglycemic events in the inpatient setting.

Effects of home confinement on physical activity, nutrition, and sleep quality during the COVID-19 outbreak in amateur and elite athletes.

Introduction: Despite the progress in the management of the pandemic caused by COVID-19, it is necessary to continue exploring and explaining how this situation affected the athlete population around the world to improve their circumstances and reduce the negative impact of changes in their lifestyle conditions that were necessitated due to the pandemic. The aim of this study was to analyze the moderating influence of physical activity (PA) and dietary habits on the impact of the COVID-19 pandemic experience on sleep quality in elite and amateur athletes. Materials and methods: A total of 1,420 elite (40.1%) and amateur (59.9%) athletes (41% women; 59% men) from 14 different countries participated in a cross-sectional design study. Data were collected using a battery of questionnaires that identified sociodemographic data, sleep quality index, PA levels, dietary habits, and the athletes' perception of their experience during the COVID-19 pandemic. Means and standard deviations were calculated for each variable. The analysis of variances and the correlation between variables were carried out with non-parametric statistics. A simple moderation effect was calculated to analyze the interaction between PA or dietary habits on the perception of the COVID-19 experience effect on sleep quality in elite and amateur athletes. Results: The PA level of elite athletes was higher than amateur athletes during COVID-19 (p < 0.001). However, the PA level of both categories of athletes was lower during COVID-19 than pre-COVID-19 (p < 0.01). In addition, amateurs had a higher diet quality than elite athletes during the pandemic (p = 0.014). The perception of the COVID-19 experience as controllable was significantly higher (p = 0.020) among elite athletes. In addition, two moderating effects had significant interactions. For amateur athletes, the PA level moderated the effect of controllable COVID-19 experience on sleep quality [F (3,777) = 3.05; p = 0.028], while for elite athletes, the same effect was moderated by dietary habits [F (3,506) = 4.47, p = 0.004]. Conclusion: Elite athletes had different lifestyle behaviors compared to amateurs during the COVID-19 lockdown. Furthermore, the relevance of maintaining high levels of PA for amateurs and good quality dietary habits by elite athletes was noted by the moderating effect that both variables had on the influence of the controllable experience during the COVID-19 pandemic on sleep quality.

One-year breakthrough SARS-CoV-2 infection and correlates of protection in fully vaccinated hematological patients.

The long-term clinical efficacy of SARS-CoV-2 vaccines according to antibody response in immunosuppressed patients such as hematological patients has been little explored. A prospective multicenter registry-based cohort study conducted from December 2020 to July 2022 by the Spanish Transplant and Cell Therapy group, was used to analyze the relationship of antibody response over time after full vaccination (at 3-6 weeks, 3, 6 and 12 months) (2 doses) and of booster doses with breakthrough SARS-CoV-2 infection in 1551 patients with hematological disorders. At a median follow-up of 388 days after complete immunization, 266 out of 1551 (17%) developed breakthrough SARS-CoV-2 infection at median of 86 days (range 7-391) after full vaccination. The cumulative incidence was 18% [95% confidence interval (C.I.), 16-20%]. Multivariate analysis identified higher incidence in chronic lymphocytic leukemia patients (29%) and with the use of corticosteroids (24.5%), whereas female sex (15.5%) and more than 1 year after last therapy (14%) were associated with a lower incidence (p < 0.05 for all comparisons). Median antibody titers at different time points were significantly lower in breakthrough cases than in non-cases. A serological titer cut-off of 250 BAU/mL was predictive of breakthrough infection and its severity. SARS-CoV-2 infection-related mortality was encouragingly low (1.9%) in our series. Our study describes the incidence of and risk factors for COVID-19 breakthrough infections during the initial vaccination and booster doses in the 2021 to mid-2022 period. The level of antibody titers at any time after 2-dose vaccination is strongly linked with protection against both breakthrough infection and severe disease, even with the Omicron SARS-CoV-2 variant.

Physical activity level measured by accelerometry and physical fitness of schoolchildren / Nível de atividade física mensurada por acelerometria e aptidão física de escolares

Abstract Objective: To describe and compare physical fitness variables according to compliance with the recommendations of physical activity, measured by accelerometry. Methods: The sample gathered 120 students, 57 boys and 63 girls aged 9 to 11 years. The variables analyzed were: weight, stature, BMI, skinfolds, waist circumference, agility, flexibility, speed and strength of upper and lower limbs, and abdominal strength. Physical activity was measured objectively using an accelerometer. The students were divided into two groups: "complies with recommendations" (≥60min/day) and "does not comply with recommendations" (<60min/day). To verify the normality of the data, the Kolmogorov-Smirnov test was used. The mean values of students who do or do not comply with the physical activity recommendation were compared using Student's t and U-Mann Whitney tests. The level of significance was set at p<0.05. Results: The students who followed the recommendation showed significantly lower values compared to those who did not for adiposity (sum of 7 skinfolds); body weight; body mass index (BMI) and abdominal strength. No significant differences were found in the variables of speed and agility, and the upper limbs' strength was greater in subjects who did not comply with recommendations. Conclusions: Students who complied with physical activity recommendations had better body composition and more abdominal strength than those who did not.

RESUMO Objetivo: Descrever e comparar as variáveis da aptidão física de acordo com o cumprimento da recomendação de atividade física, medida por acelerometria. Métodos: A amostra foi composta de 120 escolares, sendo 57 meninos e 63 meninas, de 9 a 11 anos de idade. As variáveis analisadas foram: peso, estatura, índice de massa corpórea (IMC), dobras cutâneas, circunferências, agilidade, flexibilidade, velocidade e força de membros superiores, inferiores e abdominal. A atividade física foi mensurada de forma objetiva por meio de acelerômetro. Os escolares foram divididos em dois grupos: "cumpre a recomendação" (≥60min/dia) e "não cumpre a recomendação" (<60min/dia). Para verificar a normalidade dos dados, foi utilizado o teste de Kolmogorov-Smirnov. A comparação dos valores médios dos escolares que cumprem ou não a recomendação de atividade física foi feita pelos testes t de Student e U de Mann-Whitney. O nível de significância adotado foi de p<0,05. Resultados: Os escolares que cumpriram a recomendação, comparados com os que não cumpriram, apresentaram valores significativamente menores para adiposidade (somatória das sete dobras cutâneas), peso corporal, IMC e força abdominal. Não foram encontradas diferenças significativas nas variáveis de velocidade e agilidade; quanto à força de membros superiores (MMSS), constatou-se que ela foi maior em quem não cumpriu a recomendação. Conclusões: Crianças que cumpriram a recomendação de atividade física apresentaram melhor composição corporal e força abdominal que as crianças que não cumpriram.

Physical activity level measured by accelerometry and physical fitness of schoolchildren.

OBJECTIVE: To describe and compare physical fitness variables according to compliance with the recommendations of physical activity, measured by accelerometry. METHODS: The sample gathered 120 students, 57 boys and 63 girls aged 9 to 11 years. The variables analyzed were: weight, stature, BMI, skinfolds, waist circumference, agility, flexibility, speed and strength of upper and lower limbs, and abdominal strength. Physical activity was measured objectively using an accelerometer. The students were divided into two groups: "complies with recommendations" (≥60min/day) and "does not comply with recommendations" (<60min/day). To verify the normality of the data, the Kolmogorov-Smirnov test was used. The mean values of students who do or do not comply with the physical activity recommendation were compared using Student's t and U-Mann Whitney tests. The level of significance was set at p<0.05. RESULTS: The students who followed the recommendation showed significantly lower values compared to those who did not for adiposity (sum of 7 skinfolds); body weight; body mass index (BMI) and abdominal strength. No significant differences were found in the variables of speed and agility, and the upper limbs' strength was greater in subjects who did not comply with recommendations. CONCLUSIONS: Students who complied with physical activity recommendations had better body composition and more abdominal strength than those who did not.

Fatores do estilo de vida estão associados à adiposidade em escolares de Ilhabela? Um estudo transversal / Are lifestyle factors associated with adiposity in schoolchildren from Ilhabela? A cross-sectional study

Excesso de peso e obesidade têm aumentado no Brasil e no mundo causando grande impacto na saúde pública. O objetivo foi medir a associação do nível de atividade física, o comportamento sedentário e o tempo de sono com a composição corporal de escolares. Uma amostra por conveniência foi composta por 97 escolares participantes do Projeto Misto-Longitudinal de Crescimento, Desenvolvimento de Ilhabela, aparentemente saudáveis, 50 meninos e 47 meninas entre 9 e 11 anos (x 9,8 ± 0,7), com avaliação completa no período analisado (2015 e 2019) em estágio pré-púbere de maturação sexual. Variáveis analisadas: peso (kg); índice de massa corporal (kg/m2); adiposidade pela média de três dobras cutâneas: tríceps, subescapular e suprailíaca (mm); relação cintura quadril (cm). Nível de atividade física, comportamento sedentário e tempo de sono mensurados por acelerômetro (ActiGraph GT3X, analisado com Freedson 1998) dados em counts por minuto. Dados descritos em média (x) e desvio padrão. Normalidade dos dados obtida com o teste de Kolmogorov-Smirnov e para as associações utilizou-se o coeficiente de correlação de Spearman Rho (SPSS-20.0). Nível de significância adotado P < 0,05. Houve associações significativas entre comportamento sedentário, atividades físicas leve, moderada/vigorosa e as variáveis da composição corporal. Nas meninas, as associações entre o comportamento sedentário e as variáveis da composição corporal foram de maior número e intensidade nas correlações encontradas. Verificamos que meninos realizaram atividade física leve apresentando menores valores na composição corporal. As meninas realizaram pouca atividade física de moderada a vigorosa necessitando aumento desta variável para diminuir adiposidade. O tempo de sono não apresentou associação significativa.

Prevalência e fatores associados à sarcopenia em homens e mulheres ativos acima de 50 anos de idade. Um estudo transversal / Prevalence and factors associated with sarcopenia in active men and women over 50 years of age. A cross-sectional study

Introdução: As alterações na composição corporal em idosos, principalmente na redução da massa muscular, provocam o declínio físico e prejuízo da qualidade de vida, aumentando o risco de quedas, institucionalizações e morte. Identificar os fatores associados à sarcopenia possibilita a realização de estratégias para prevenção e reversão deste fenômeno durante o processo de envelhecimento. Objetivo: O estudo teve como objetivo verificar a prevalência e fatores associados à sarcopenia em homens e mulheres não sedentários acima de 50 anos de idade. Tipo de estudo e local: Estudo transversal com homens e mulheres de São Caetano do Sul. Métodos: Foram avaliados 787 mulheres e 123 homens do Projeto Longitudinal de Envelhecimento e Aptidão Física de São Caetano do Sul que frequentam duas vezes na semana um programa de atividade física orientado do município. Para determinar a sarcopenia, utilizou-se o critério de redução da massa muscular (circunferência muscular do braço) e redução da força (preensão manual) ou a redução do desempenho (velocidade de andar). As variáveis independentes foram sexo, idade, índice de massa corporal (IMC), socioeconômicas, comportamentais, de saúde e nível de atividade física. Resultados: A prevalência de sarcopenia foi de 13,5%, na qual 18,4% da amostra apresentou redução da massa muscular, 34,2% diminuição da velocidade de andar e 50,9% redução da força muscular. A prevalência de sarcopenia foi maior em quem não cumpria a recomendação de atividade física (65,8%), assim como quem ultrapassava o recomendado de tempo sentado durante a semana (63,2%). Conclusão: Os fatores associados à sarcopenia são IMC, circunferência de cintura e panturrilha. PALAVRAS-CHAVE: Envelhecimento, sarcopenia, sistema musculoesquelético, força muscular, desempenho físico funcional

Valgismo associado à aptidão física, nível de atividade física e tempo sedentário em escolares. Um estudo transversal / Valgus associated with fitness physical, physical activity level and sedentary time in schoolchildren. A cross-sectional study

Introdução: Os desvios axiais dos membros inferiores estão associados ao aumento da adiposidade, desconforto na prática da atividade física, dificultando um estilo de vida fisicamente ativo. Objetivo: Associar os graus de geno valgo com a intensidade da atividade física e o tempo sedentário, assim como determinar a relação com a aptidão física em escolares de Ilhabela. Tipo de estudo e local: Estudo transversal em escolares de Ilhabela, SP. Métodos: Estudo transversal, com amostra por conveniência, de 96 escolares entre 9 e 11 anos, residentes em Ilhabela, São Paulo. O geno valgo foi avaliado por goniômetro, classificando a distância intermaleolar (DIM, cm) em leve, moderada e grave. O nível de atividade física foi avaliado por acelerômetro (ActiGraph GT3X-BT, Freedson P.S), sendo classificado em atividade física: leve, moderada, moderada a vigorosa, vigorosa e tempo sedentário. Análise estatística: Foi utilizado o Teste t de Student, análise de variância (ANOVA oneway) seguido da correlação de Pearson, por meio do software SPSS versão 20.0, adotando nível de significância de P < 0,05. Resultados: No grau leve DIM, as associações moderadas e significativas das intensidades do nivel de atividade física foram mais fortes no sexo feminino. Com o aumento do grau na classificação moderado e grave (DIM) as associações são de moderada a forte e significativas somente no sexo masculino. Conclusão: Concluímos que o aumento dos graus de geno valgo foi associado a um maior tempo sedentário e à intensidade da atividade física leve; e há um menor envolvimento com atividades de intensidades moderada e moderada/vigorosa.