Detection of AZF microdeletions and analysis of reproductive hormonal profiles in Hainan men undergoing assisted reproductive technology.

BACKGROUND: Male infertility has become a global health problem, and genetic factors are one of the essential causes. Y chromosome microdeletion is the leading genetic factor cause of male infertility. The objective of this study is to investigate the correlation between male infertility and Y chromosome microdeletions in Hainan, the sole tropical island province of China. METHODS: We analyzed the semen of 897 infertile men from Hainan in this study. Semen analysis was measured according to WHO criteria by professionals at the Department of Reproductive Medicine, the First Affiliated Hospital of Hainan Medical University, where samples were collected. Y chromosome AZF microdeletions were confirmed by detecting six STS markers using multiple polymerase chain reactions on peripheral blood DNA. The levels of reproductive hormones, including FSH, LH, PRL, T, and E2, were quantified using the enzyme-linked immunosorbent assay (ELISA). RESULTS: The incidence of Y chromosome microdeletion in Hainan infertile men was 7.13%. The occurrence rate of Y chromosome microdeletion was 6.69% (34/508) in the oligozoospermia group and 7.71% (30/389) in the azoospermia group. The deletion of various types in the AZF subregion was observed in the group with azoospermia, whereas no AZFb deletion was detected in the oligozoospermia group. Among all patients with microdeletions, the deletion rate of the AZFc region was the higher at 68.75% (44 out of 64), followed by a deletion rate of 6.25% (4 out of 64) for the AZFa region and a deletion rate of 4.69% (3 out of 64) for the AZFb region. The deletion rate of the AZFa region was significantly higher in patients with azoospermia than in patients with oligozoospermia (0.51% vs. 0.39%, p < 0.001). In comparison, the deletion rate of the AZFc region was significantly higher in patients with oligozoospermia (3.08% vs. 6.30%, p < 0.001). Additionally, the AZFb + c subregion association deletion was observed in the highest proportion among all patients (0.89%, 8/897), followed by AZFa + b + c deletion (0.56%, 5/897), and exclusively occurred in patients with azoospermia. Hormone analysis revealed FSH (21.63 ± 2.01 U/L vs. 10.15 ± 0.96 U/L, p = 0.001), LH (8.96 ± 0.90 U/L vs. 4.58 ± 0.42 U/L, p < 0.001) and PRL (263.45 ± 21.84 mIU/L vs. 170.76 ± 17.10 mIU/L, p = 0.002) were significantly increased in azoospermia patients with microdeletions. Still, P and E2 levels were not significantly different between the two groups. CONCLUSIONS: The incidence of AZF microdeletion can reach 7.13% in infertile men in Hainan province, and the deletion of the AZFc subregion is the highest. Although the Y chromosome microdeletion rate is distinct in different regions or populations, the regions mentioned above of the Y chromosome may serve an indispensable role in regulating spermatogenesis. The analysis of Y chromosome microdeletion plays a crucial role in the clinical assessment and diagnosis of male infertility.

Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency.

BACKGROUND: OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usually needs 14-18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency. METHODS: In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them. RESULTS: OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group. CONCLUSION: The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.

Can CT-Based Arterial and Venous Morphological Markers of Chronic Obstructive Pulmonary Disease Explain Pulmonary Vascular Remodeling?

RATIONALE AND OBJECTIVES: We analyzed changes in quantitative pulmonary artery and vein parameters to investigate pulmonary vascular remodeling characteristics in chronic obstructive pulmonary disease (COPD) patients. MATERIALS AND METHODS: This retrospective study recruited healthy volunteers and COPD patients. Participants undergoing standard-of-care pulmonary function testing (PFT) and computed tomography (CT) evaluations were classified into five groups: normal and Global Initiative for Chronic Obstructive Lung Disease (GOLD) grades 1-4. Artery and vein analyses (volumes, numbers, densities, and fractions) were performed using artificial intelligence. RESULTS: Among 139 subjects (136 men; mean age, 64years±8 [SD]) with GOLD grade 1 (n = 13), grade 2 (n = 49), grade 3 (n = 42), grade 4 (n = 17) and control subjects (n = 18) enrolled, differences in arterial volumes (BV5-10, BV10+, pulmonary arterial volume) and venous densities (BV5 density, BV10+ density, pulmonary venous density, pulmonary venous branch density) among control and GOLD grades 1-4 were statistically significant (P < .05). Higher pulmonary arterial volumes and lower number were observed with more advanced COPD. The number and volumes of pulmonary veins were lower in GOLD grades 2 and 3 than in GOLD grade 1 but higher in GOLD grade 4 than in GOLD grade 3. The numbers and volumes of pulmonary arteries and veins showed varying positive correlations (γ = 0.18-0.96, P < .05). Pulmonary vascular densities were mildly to moderately correlated with PFT results (γ = 0.236-0.495, P < .05) and were moderately negatively correlated with the emphysema percentage (γ = -0.591 to -0.315, P < .05). CONCLUSION: Patients with COPD exhibited pulmonary vascular remodeling, which occurred in the arteries at the early grade of COPD and in the veins at the late grade. CT-based quantitative analysis of pulmonary vasculature may become an imaging marker for early diagnosis and assessment of COPD severity.

The Effect of the cAMP Signaling Pathway on HTR8/SV-Neo Cell Line Proliferation, Invasion, and Migration After Treatment with Forskolin.

Pre-eclampsia (PE) is thought to be related to placental dysfunction, particularly poor extravillous trophoblast (EVT) invasion and migration abilities. However, the pathogenic mechanism is not fully understood. This article describes the impact of the cyclic adenosine monophosphate(cAMP) signaling pathway on EVT behavior, focusing on EVT proliferation, invasion, and migration. Here, we used the HTR8/SV-neo cell line to study human EVT function in vitro. HTR8/SV-neo cells were treated with different concentrations of forskolin (cAMP pathway-specific agonist) to alter intracellular cAMP levels, and dimethyl sulfoxide (DMSO) was used as the control. First, a cAMP assay was performed to measure the cAMP concentration in HTR8/SV-neo cells treated with different forskolin concentrations, and cell proliferation was assessed by constructing cell growth curves and assessing colony formation. Cell invasion and migration were observed by Transwell experiments, and intracellular epithelial-mesenchymal transition (EMT) marker expression was evaluated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB). According to our research, the intracellular cAMP levels in HTR8/SV-neo cells were increased in a dose-dependent manner, and HTR8/SV-neo cell proliferation, invasion and migration were significantly enhanced. The expression of EMT and angiogenesis markers was upregulated. Additionally, with the increase in intracellular cAMP levels, the phosphorylation of intracellular mitogen-activated protein kinase (MAPK) signaling pathway components was significantly increased. These results suggested that the cAMP signaling pathway promoted the phosphorylation of MAPK signaling components, thus enhancing EVT functions, including proliferation, invasion, and migration, and to a certain extent, providing a novel direction for the treatment of PE patients.

Ecological regulation of water level should be combined with seed supplementation for lakeshore Carex community restoration in Yangtze-disconnected lakes.

Carex communities in most Yangtze-disconnected lakes have been degraded severely due to alterations in water level fluctuations. To explore the feasibility of restoring the lakeshore Carex communities through ecological regulation of water level, the present study selected the Yangtze-connected Qili Lake (the lakeshore was dominated by Carex) and the Yangtze-disconnected Wuchang Lake (the lakeshore was dominated by Zizania latifolia) as model systems, and analyzed the lakeshore seed bank characteristics and seed-related quantitative, morphological, and germination traits of three representative Carex species. According to the results, although Carex seed density in the Qili Lake seed bank was obviously higher than that in Wuchang Lake, their contribution to the total seed density in both lakes was extremely low, with no significant difference between the two lakes. The results indicate that restoration of the degraded Carex communities using existing seed bank in Yangtze-disconnected lakes exclusively through water level regulation is not feasible. In addition, the seed densities of aboveground parts of Carex cinerascens, Carex dimorpholepis, and Carex argyi in Qili Lake were 6.9 × 104, 45.1 × 104, and 3.6 × 104 seeds/m2, respectively, which can provide high numbers of seeds continuously for lakeshore Carex restoration. The results of seed germination experiments showed that light, burial depth, and their interaction had significant effects on seed germination of the three species, whereas water condition had a significant effect only on C. dimorpholepis seed germination. The average germination rates of the three Carex species were 16.63 %, 19.06 %, and 7.78 %, respectively. However, considering the high seed densities in the aboveground parts of the three species, there are considerable numbers of seeds that can be used for Carex restoration. Therefore, the restoration of Carex communities in lakeshore zones of Yangtze-disconnected lakes is still possible if water level regulation can be combined with natural or artificial seed supplementation.

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

Cardiac specification during gastrulation - The Yellow Brick Road leading to Tinman.

The question of how the heart develops, and the genetic networks governing this process have become intense areas of research over the past several decades. This research is propelled by classical developmental studies and potential clinical applications to understand and treat congenital conditions in which cardiac development is disrupted. Discovery of the tinman gene in Drosophila, and examination of its vertebrate homolog Nkx2.5, along with other core cardiac transcription factors has revealed how cardiac progenitor differentiation and maturation drives heart development. Careful observation of cardiac morphogenesis along with lineage tracing approaches indicated that cardiac progenitors can be divided into two broad classes of cells, namely the first and second heart fields, that contribute to the heart in two distinct waves of differentiation. Ample evidence suggests that the fate of individual cardiac progenitors is restricted to distinct cardiac structures quite early in development, well before the expression of canonical cardiac progenitor markers like Nkx2.5. Here we review the initial specification of cardiac progenitors, discuss evidence for the early patterning of cardiac progenitors during gastrulation, and consider how early gene expression programs and epigenetic patterns can direct their development. A complete understanding of when and how the developmental potential of cardiac progenitors is determined, and their potential plasticity, is of great interest developmentally and also has important implications for both the study of congenital heart disease and therapeutic approaches based on cardiac stem cell programming.

Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development.

During the phylotypic period, embryos from different genera show similar gene expression patterns, implying common regulatory mechanisms. Here we set out to identify enhancers involved in the initial events of cardiogenesis, which occurs during the phylotypic period. We isolate early cardiac progenitor cells from zebrafish embryos and characterize 3838 open chromatin regions specific to this cell population. Of these regions, 162 overlap with conserved non-coding elements (CNEs) that also map to open chromatin regions in human. Most of the zebrafish conserved open chromatin elements tested drive gene expression in the developing heart. Despite modest sequence identity, human orthologous open chromatin regions recapitulate the spatial temporal expression patterns of the zebrafish sequence, potentially providing a basis for phylotypic gene expression patterns. Genome-wide, we discover 5598 zebrafish-human conserved open chromatin regions, suggesting that a diverse repertoire of ancient enhancers is established prior to organogenesis and the phylotypic period.