Does a matrix-free culture system supplemented with neurotrophic factor 4 (NT4) improve human in vitro follicular development and meiotic maturation, ultimately resulting in fertilizable oocytes? NT4 supplementation of in vitro culture significantly enhances the growth, steroid hormone production, and maturity potential of human secondary follicles derived from fresh ovarian medulla (from post- and pre-pubertal patients), thereby yielding fertilizable oocytes. Reconstituting folliculogenesis in vitro is of paramount importance in the realms of fertility preservation, reproductive biology research, and reproductive toxicity assessments. However, the efficiency of in vitro culture systems remains suboptimal, as the attainment of fertilizable oocytes from in vitro growth (IVG) of human follicles remains unachieved, with the data being particularly scant regarding follicles from prepubertal girls. We have previously found that mouse oocytes from secondary follicles derived from IVG are deficient in neuroendocrine regulation. NT4 and its corresponding receptor have been identified in human follicles. Significantly, the addition of NT4 during the IVG process markedly enhances both follicle growth and oocyte maturation rates in mice. Fresh medulla tissue obtained during tissue preparation for ovarian tissue cryopreservation (OTC) were collected from 10 patients aged from 6 to 21 years old, all of whom had undergone unilateral oophorectomy as a means of fertility preservation. Isolated secondary follicles were individually cultured in vitro with or without NT4 in a matrix-free system. Secondary follicles, extracted via enzymatic digestion and mechanical disruption from each patient, were randomly allocated to either a control group or an NT4-supplemented group (100 ng/ml), followed by individual culture on an ultra-low attachment plate. Follicle growth and viability were assessed by microscopy. Levels of anti-Müllerian hormone (AMH), estradiol, and progesterone in the medium were quantified. An oocyte-specific marker was identified using confocal fluorescence microscopy following DEAD box polypeptide 4 (DDX4) staining. The competence of individual oocytes for maturation and fertilization were assessed after IVM and ICSI with donated sperm samples. Overall, isolated follicles from both groups survived up to 6 weeks with increasing diameters over the duration (P < 0.05), reaching terminal diameters of almost 1 mm with confirmed steroidogenesis and expression of oocyte marker (DDX4), and producing morphologically normal MII oocytes. When compared with the control group, the NT4 group had a similar initial follicular diameter (206 ± 61.3 vs 184 ± 93.4 μm) but exhibited a significant increase in follicular diameter from the ninth day of culture onwards (P < 0.05). From Week 3, estradiol and progesterone production were significantly increased in the NT4 group, while no significant difference was observed in AMH production between groups. The proportion of ‘fast-growth’ follicles in the NT4 group was significantly higher than that in the control group (13/23 vs 6/24, P < 0.05). An increased efficiency of MII oocyte maturation per live follicle in the NT4 group was also observed (control group vs NT4 group, 4/24 vs 10/23, P < 0.05). It is noteworthy that an MII oocyte obtained from the control group exhibited abnormal fertilization after ICSI. In contrast, an MII oocyte acquired from the NT4 group progressed to the blastocyst stage and showed potential for transfer. N/A. The cohort examined in this study was all patients diagnosed with beta-thalassemia major. Whether this culture system is effective for patients with other diseases remains unknown. Since the chosen dose of NT4 was established based on dose finding in mice, the optimal dose for use in a human IVG system needs further confirmation. The oocytes and embryos procured from this study have not been quantified for ploidy status or epigenetic signatures. Fresh medulla tissue obtained during tissue preparation for OTC may serve as a precious source of fertilizable oocytes for female fertility preservation, even for pre-pubertal girls, without the threat of tumour reintroduction. After further characterization and optimization of the system, this culture system holds the potential to provide a powerful future research tool, for the comprehensive exploration of human follicular development mechanisms and for conducting reproductive toxicity evaluations. This work was supported by the National Key R&D Program of China (grant number 2022YFC2703000) and National Natural Science Foundation of China (grant numbers 82271651 and 81871214). The medium used in human follicle in vitro culture in this study has been applied for a national invention patent in China (No. 202211330660.7). The inventors of the patent, in order, are: Y.G., C.F., and X.L.

Background: In-vitro-grow (IVG) of preantral follicles is essential for female fertility preservation, while practical approach for improvement is far from being explored. Studies have indicated that neurotrophin-4 (NT-4) is preferentially expressed in human preantral follicles and may be crucial to preantral follicle growth.Methods: We observed the location and expression of Tropomyosin-related kinase B (TRKB) in human and mouse ovaries with immunofluorescence and Western blot, and the relation between oocyte maturation and NT-4 level in follicular fluid (FF). Mice model was applied to investigate the effect of NT-4 on preantral follicle IVG. Single-cell RNA sequencing of oocyte combined with cell-specific network analysis was conducted to uncover the underlying mechanism of effect.Results: We reported the dynamic location of TRKB in human and mouse ovaries, and a positive relationship between human oocyte maturation and NT-4 level in FF. Improving effect of NT-4 was observed on mice preantral follicle IVG, including follicle development and oocyte maturation. Transcriptome analysis showed that the reparative effect of NT-4 on oocyte maturation might be mediated by regulation of PI3K-Akt signaling and subsequent organization of F-actin. Suppression of advanced stimulated complement system in granulosa cells might contribute to the improvement. Cell-specific network analysis revealed NT-4 may recover the inflammation damage induced by abnormal lipid metabolism in IVG.Conclusions: Our data suggest that NT-4 is involved in ovarian physiology and may improve the efficiency of preantral follicle IVG for fertility preservation.

Genetic diseases encompass a spectrum of disorders resulting from DNA variations. Preimplantation genetic testing (PGT) is a critical strategy for preventi

Background and Clinical Significance: FLNC encodes filamin C, a muscle-scaffolding protein crucial for cardiac integrity. Pathogenic FLNC variants cause diverse cardiomyopathies (hypertrophic, dilated, restrictive, and arrhythmogenic) and myofibrillar myopathies, but their role in congenital cardiac malformations is unclear. Notably, FLNC has not been implicated in structural defects such as Tetralogy of Fallot (TOF) to date. Case Presentation: Two fetuses from the same family were prenatally diagnosed with TOF via ultrasound. The trio whole-exome sequencing of the second fetus and her parents identified a novel heterozygous truncating FLNC variant (NM_001458.5:c.1453C>T, p.Q485*). Sanger sequencing confirmed the same variant in the earlier TOF fetus. The mother carried the variant but was asymptomatic. In vitro mutagenesis in rat cardiomyocytes showed that the mutant FLNC construct produced markedly reduced FLNC proteins compared to the wild type and did not form abnormal cytoplasmic aggregates. Conclusions: We report on a novel FLNC truncating variant associated with fetal TOF, extending the spectrum of FLNC-related cardiac anomalies. The variable outcomes among variant carriers—from fetal TOF to adult cardiomyopathy or no clinical manifestations—underscore the complex genotype–phenotype correlations of filaminopathy. This case highlights the importance of comprehensive genetic evaluation in families with diverse cardiac phenotypes and suggests that additional genetic factors likely influence phenotypic expression.

Genetic diseases encompass a spectrum of disorders resulting from DNA variations. Preimplantation genetic testing (PGT) is a critical strategy for preventing recurrent miscarriage, foetal malformations, and the birth of children affected by chromosomal abnormalities and monogenic disorders. Traditional PGT techniques necessitate comprehensive pedigree genetic data for haplotype linkage analysis, whereas PGT employing third-generation sequencing (TGS) has distinct advantages, particularly in cases of incomplete pedigree information, de novo mutations, and complex pathogenic variants. Nevertheless, the widespread application of TGS-based PGT in clinical practice encounters hurdles owing to its high costs. Targeted sequencing technologies present a promising solution by selectively enriching regions of interest while disregarding nontargeted areas, offering a more cost-effective and flexible alternative. In this study, we employed next-generation sequencing (NGS) and adaptive sampling-based targeted nanopore sequencing (ASTN-seq) to analyse samples from five couples who carried balanced translocations and HBB gene pathogenic mutations, as well as three additional couples with monogenic diseases caused by mutations in PKD1 , ASNS , or ALPL . ASTN-seq successfully identified various mutations and facilitated haplotype linkage analysis, confirming its accuracy and reliability. Successful embryo transfer and subsequent prenatal diagnosis in certain families underscore the potential of ASTN-seq in assisted reproduction. Compared with traditional NGS-based PGT techniques, our work highlights that ASTN-seq is a promising tool for PGT, offering cost-effective solutions for PGT, especially for incomplete pedigrees and de novo mutations.