Bi-allelic variants in DNAH3 cause male infertility with asthenoteratozoospermia in humans and mice.

STUDY QUESTION: Are there other pathogenic genes for asthenoteratozoospermia (AT)? SUMMARY ANSWER: DNAH3 is a novel candidate gene for AT in humans and mice. WHAT IS KNOWN ALREADY: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. STUDY DESIGN SIZE DURATION: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. MAIN RESULTS AND THE ROLE OF CHANCE: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.

The novel lactoferrin and DHA-codelivered liposomes with different membrane structures: Fabrication, in vitro infant digestion, and suckling pig intestinal organoid absorption.

Inspired by membrane structure of breast milk and infant formula fat globules, four liposomes with different particle size (large and small) and compositions (Single phospholipids contained phosphatidylcholine, complex phospholipids contained phosphatidylcholine, phosphatidylethanolamine and sphingomyelin) were fabricated to deliver lactoferrin and DHA. In vitro infant semi-dynamic digestive behavior and absorption in intestinal organoids of liposomes were investigated. Liposomal structures were negligible changed during semi-dynamic gastric digestion while damaged in intestine. Liposomal degradation rate was primarily influenced by particle size, and complex phospholipids accelerated DHA hydrolysis. The release rate of DHA (91.7 ± 1.3 %) in small-sized liposomes (0.181 ± 0.001 µm) was higher than free DHA (unencapsulated, 64.6 ± 3.4 %). Complex phospholipids liposomal digesta exhibited higher transport efficiency (3.4-fold for fatty acids and 2.0-fold for amino acids) and better organoid growth than digesta of bare nutrients. This study provided new insights into membrane structure-functionality relationship of liposomes and may aid in the development of novel infant nutrient carriers.

Specific protection mechanism of oligosaccharides on liposomes during freeze-drying.

Liposomes have been received much attention during the past decades as bioactive compounds carriers in food field. However, the application of liposomes is extremely limited by the structural instability during processing such as freeze-drying. In addition, the protection mechanism of lyoprotectant for liposomes during freeze-drying remains controversial. In this study, lactose, fructooligosaccharide, inulin and sucrose were used as lyoprotectants for liposomes and the physicochemical properties, structural stability and freeze-drying protection mechanism were explored. The addition of oligosaccharides could significantly suppress the changes in size and zeta potential, and the amorphous state of liposomes was negligible changed from XRD. The Tg of the four oligosaccharides, especially for sucrose (69.50 °C) and lactose (95.67 °C), revealed the freeze-dried liposomes had formed vitrification matrix, which could prevent liposomes from fusion via increasing the viscosity and reducing membrane mobility. The decrease in Tm of sucrose (147.67 °C) and lactose (181.67 °C), and the changes in functional group of phospholipid and hygroscopic capacity of lyophilized liposomes indicated oligosaccharides replaced water molecules to interact with phospholipids by hydrogen bonds. It can be concluded that the protection mechanism of sucrose and lactose as lyoprotectant was attributed to the combination of vitrification theory and water replacement hypothesis, while the water replacement hypothesis was dominated by fructooligosaccharide and inulin.

Effect of oligosaccharides as lyoprotectants on the stability of curcumin-loaded nanoliposomes during lyophilization.

Nanoliposome is a promising delivery system, whereas its commercial application is limited by the structural instability, cargo leakage and particles aggregation during the processing such as freeze-drying. In this study, the effect of four oligosaccharides, fructo-oligosaccharides, lactose, inulin and sucrose (control), on the physicochemical properties, structural stability, and in vitro semi-dynamic digestion behavior of curcumin-loaded nanoliposomes were investigated before and after lyophilization. The results showed that the addition of the oligosaccharides inhibited the changes in particle size and reduced curcumin leakage from lyophilized nanoliposomes. Oligosaccharides significantly improved the physical stability of lyophilized nanoliposomes and delayed curcumin release during in vitro digestion. In addition, oligosaccharides could decrease the hydrophobicity of liposomal membrane and the tightness of phospholipid molecule arrangement, with the increase in micropolarity and fluidity of the bilayer membranes. These results suggested that fructo-oligosaccharides, lactose and inulin could be effective lyoprotectants for lyophilized nanoliposomes.

Structural degradation and uptake of resveratrol-encapsulated liposomes using an in vitro digestion combined with Caco-2 cell absorption model.

Resveratrol (RES), a polyphenol with strong antioxidant capacity but poor bioavailability and light instability, urgently needs an effective delivery technique to overcome its drawbacks. As it is a highly biocompatible delivery system, liposomes were used to carry RES to form resveratrol-encapsulated liposomes (RES-LPS). Results showed that the diameter of RES-LPS was 333 ± 50 nm and the encapsulation efficiency was 84.69 ± 0.02 %, with a spherical shape and double-layered structure. Morphology showed that RES-LPS, could maintain an intact membrane structure during stomach digestion, as well as while under hydrolysis, mimicking intestinal conditions, before releasing RES. Moreover, Caco-2 cells uptake study also demonstrated that the digesta of RES-LPS resulted in a better cell absorption efficiency and a stronger ability to reduce reactive oxygen species when compared with free RES. Thus, these results indicate that liposomes play a key role in improving the bioavailability of RES, demonstrating the promising role of liposomes as a delivery system for food supplements.

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform.

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

Establishment of human embryonic stem cell line with a complex abnormal karyotype and the stable XIST expression.

The embryonic stem cell line chHES-3XISTa was derived from heterogeneous chHES-3 cells. chHES-3XISTa showed a new abnormal karyotype of 46,XX with 8 derivation chromosomes and expressed X inactive specific transcript (XIST) in continual culture. Tri-methylation of H3 Lys-27 (H3K27me3) punctate enrichment located in RNA Polymerase II hole was also found in all chHES-3XISTa cells. Pluripotent markers and differentiate capability in vitro were confirmed by immunochemistry staining.

Tumor progression of culture-adapted human embryonic stem cells during long-term culture.

Human embryonic stem cells (hESCs) during long-term culture acquire chromosomal changes similar to those occurring in tumorigenesis. This was raised concerns about the progression from hESCs to malignant cells. This study aimed to investigate the changes in chromosomes, cell phenotype, and genes in culture-adapted hESCs to ascertain whether tumorigenic transformation occurred. By cytogenetic analysis we found progressive karyotypic changes from simple to complex in chHES-3, one of the hESC lines established in our laboratory, during a long-term suboptimal culture. We further compared chHES-3 cells at different karyotypic stages in cell surface markers, in vivo differentiation, cell cycle, apoptosis, and gene expression profiles. We found that the karyotypically aberrant chHES-3 had higher S-phase fraction in cell cycle distributions and antiapoptosis ability. In vivo differentiation of karyotypically normal chHES-3 resulted in relatively mature teratoma, whereas karyotypically aberrant chHES-3 formed immature teratoma (grade III), in which more primary neural epithelium was revealed by pathological analysis. The microarray analysis and real-time PCR results showed that some oncogenes were upregulated in karyotypically aberrant chHES-3 cells, whereas the genes related to differentiation were downregulated, and that Wnt signal pathway was activated. In conclusion, chHES-3 cells underwent deregulation of self-renewal and dysfunction of related genes in long-term culture adaptation, leading to malignant transformation.