Label-free, three-dimensional multiphoton microscopy of the connective tissue in the anterior vaginal wall.

INTRODUCTION AND HYPOTHESIS: Multiphoton microscopy (MPM) is a nonlinear, high-resolution laser scanning technique and a powerful approach for analyzing the spatial architecture within tissues. To demonstrate the potential of this technique for studying the extracellular matrix of the pelvic organs, we aimed to establish protocols for the detection of collagen and elastin in the vagina and to compare the MPM density of these fibers to fibers detected using standard histological methods. METHODS: Samples of the anterior vaginal wall were obtained from nine patients undergoing a hysterectomy or cystocele repair. Samples were shock frozen, fixed with formaldehyde or Thiel's solution, or left untreated. Samples were imaged with MPM to quantify the amount of collagen and elastin via second harmonic generation and autofluorescence, respectively. In six patients, sample sections were also histologically stained and imaged with brightfield microscopy. The density of the fibers was quantified using the StereoInvestigator and Cavalieri software. RESULTS: With MPM, collagen and elastin could be visualized to a depth of 100 µm, and no overlap of signals was detected. The different tissue processing protocols used did not result in significantly different fiber counts after MPM. MPM-based fiber quantifications are comparable to those based on conventional histological stains. However, MPM provided superior resolution, particularly of collagen fibers. CONCLUSIONS: MPM is a robust, rapid, and label-free method that can be used to quantify the collagen and elastin content in thick specimens of the vagina. It is an excellent tool for future three-dimensional studies of the extracellular matrix in patients with pelvic organ prolapse.

Current Treatment of Stress Urinary Incontinence by Bulking Agents and Laser Therapy-An Update.

Stress urinary incontinence (SUI) affects around 20% of women. In addition to the established suburethral sling insertion, two less invasive approaches are of interest today: urethral bulking agents and vaginal laser therapy. This review discusses articles through December 2023 identified by a PubMed literature search using the keywords "incontinence" and "bulking" or "laser". Although the two approaches are less effective than sling insertions, there are specific conditions in which one or the other technique is more advantageous. Injecting bulking agents into the urethra only takes some minutes and works without general anesthesia. The method is particularly suited for elderly, frail, or obese patients with multiple comorbidities, but is also applicable for all patients and in combination with other therapies. Generally, the safety profile is good but differs between bulking materials. Two laser types-the Erbium:YAG laser with SMOOTH-mode and the fractional ablative CO2 laser-deliver heat into the tissue to induce tissue tightening and regeneration. Intravaginal laser therapy improves mild to moderate SUI, while studies describe how intraurethral laser therapy is also beneficial for severe SUI. Young women between childbirths, as well as postmenopausal women, may benefit from laser therapy. The method is safe, can be performed on an outpatient basis, and does not require any artificial material.

Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.