The establishment of a molecular diagnostic platform for mitochondrial diseases: from conventional to next-generation sequencing.

BACKGROUND: The aim of this study was to create a molecular diagnostic platform and establish a diagnostic pipeline for patients highly suspected of mitochondrial disorders. The effectiveness of three methods, namely, traditional restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), Sanger sequencing for hotspot detection and whole mitochondrial DNA (mtDNA), and third-generation (Nanopore) whole mtDNA sequencing, will be compared in diagnosing patients with suspected primary mitochondrial diseases (PMDs). The strengths and limitations of different methods are also discussed. MATERIAL AND METHODS: A single-center prospective cohort study was conducted to validate the diagnostic pipeline for suspected mitochondrial diseases. In the first stage, a PCR-based method with five sets of primers was used to screen for eight hotspots (m.3243A>G, m.3460G>A, m.8344A>G, m.8993T>G, m.9185T>C, m.11778G>A, m.13513G>A, and m.4977deletion) using either RFLP or direct Sanger sequencing. Sanger sequencing was also used to confirm the RFLP-positive samples. In the second stage, for samples with negative screening results for the eight hotspots, mitochondrial whole-genome sequencing was performed using Sanger sequencing or third-generation nanopore sequencing. RESULTS: Between June 2020 and May 2023, 30 patients from ages 0 to 63 with clinically suspected mitochondrial disease were enrolled. The positive yield for the diagnosis of PMDs was 8/30=26.7%, and the sensitivity of the heteroplasmy level for the RFLP-based method was approximately 5%. The remaining 22 patients who tested negative at the first stage were tested using Sanger sequencing or the third-generation sequencing Nanopore, and all tested negative for pathological mtDNA mutations. Compared to the Sanger sequencing method, the results of RFLP-PCR were compromised by the limitations of incomplete RFLP enzyme digestion. For whole-genome sequencing of mtDNA, Sanger sequencing, instead of nanopore sequencing, is preferred at our institution because of its cost-effectiveness. CONCLUSIONS: In our highly selective cohort, most tested positive in the first stage of the 8 hot spots screen. Sanger sequencing is a conventional and accurate method for mitochondrial disease screening, at least for the most common hot spots in the region. The results revealed that Sanger sequencing is an accurate method with the benefit of being more cost-effective. This integral platform of molecular diagnosis bears the advantages of being relatively low cost and having a shorter reporting time, facilitating crucial identification of patients with clinical evidence of such disorders. This diagnostic flowchart has also been translated into routine clinical use in the tertiary hospital.

A Preliminary Investigation of the Roles of Endometrial Cells in Endometriosis Development via In Vitro and In Vivo Analyses.

Endometriosis is a complex gynecological disease that affects more than 10% of women in their reproductive years. While surgery can provide temporary relief from women's pain, symptoms often return in as many as 75% of cases within two years. Previous literature has contributed to theories about the development of endometriosis; however, the exact pathogenesis and etiology remain elusive. We conducted a preliminary investigation into the influence of primary endometrial cells (ECs) on the development and progression of endometriosis. In vitro studies, they were involved in inducing Lipopolysaccharide (LPS) in rat-isolated primary endometrial cells, which resulted in increased nuclear factor-kappa B (NF-κB) and vascular endothelial growth factor (VEGF) mRNA gene expression (quantitative polymerase chain reaction analysis, qPCR) and protein expression (western blot analysis). Additionally, in vivo studies utilized autogenic and allogeneic transplantations (rat to rat) to investigate endometriosis-like lesion cyst size, body weight, protein levels (immunohistochemistry), and mRNA gene expression. These studies demonstrated that estrogen upregulates the gene and protein regulation of cytoskeletal (CK)-18, transforming growth factor-ß (TGF-ß), VEGF, and tumor necrosis factor (TNF)-α, particularly in the peritoneum. These findings may influence cell proliferation, angiogenesis, fibrosis, and inflammation markers. Consequently, this could exacerbate the occurrence and progression of endometriosis.

The effect of various air pollution and participants' age on semen quality in southern Taiwan.

OBJECTIVE: This study aimed to investigate the association between semen quality and air pollution in southern Taiwan. MATERIALS AND METHODS: In this retrospective study, 4338 males aged 21-70 years were recruited between 2001 and 2018 from a reproductive medical center. Semen quality was assessed according to standardized methods outlined in the World Health Organization (WHO) Laboratory Manual 1999 and 2010, including total sperm count, progressive sperm motility (%), rapid progressive sperm motility (%), and sperm with normal morphology (%). All designated national air quality automatic continuous monitoring stations measured the levels of air pollution [particulate matter (PM10 and PM2.5), sulfur dioxide (SO2), nitrogen dioxide (NO2), carbon monoxide (CO), ozone (O3)], and was documented by Environmental Protection Administration in Taiwan. We collected data on the levels of air pollution based on the participants' residential addresses. RESULTS: In our study, we found that progressive and rapid progressive sperm motility significantly decreased annually (p < 0.05). In addition, increasing age influenced total sperm count, progressive sperm motility, rapid progressive sperm motility, and sperm with normal morphology (p < 0.05). Among different air pollution, we observed SO2 was associated with lower rapid progressive sperm motility and lower sperm with normal morphology (ß = -0.103, p = 0.043; ß = 0.118, p = 0.001, respectively). However, NO2 was associated with higher rapid progressive sperm motility and a high number of sperm with normal morphology (ß = 0.129, p = 0.002; ß = 0.127, p < 0.001, respectively). CONCLUSIONS: The semen quality in southern Taiwan appears to have declined in recent years. The participant's age for semen analysis was most strongly associated with semen parameters, Moreover, a significant association between SO2 and NO2 levels and semen motility was observed, even after adjusting for multiple comparisons. Further study is required to analyze the dose-dependent effect of SO2 and NO2 on semen parameters.