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Introduction: Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV‐2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio‐Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results [1]. Materials and Methods: A RT‐PCR protocol was set up using the One‐Step RT‐ddPCR Kit for Probes on the QX200 platform (Bio‐Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines [2]. Inter‐run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV‐negative plasma. The ddPCR assay was compared to a qRT‐PCR previously used in routine (LOQ 50 cop/mL [3]) using 46 clinical samples and the NIBSC international HIV‐2 RNA standard. Results: The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5′FAM‐probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter‐run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2,224 cop/mL, delta log=0.68) and 12 others were quantified by ddPCR only below 50 cop/mL (mean=16 cop/mL). Conclusions: We validated a ddPCR HIV‐2 VL assay that is more sensitive and more reproducible than the qPCR assay used as comparator, with a limit of quantification of 7 cop/mL of plasma. A careful definition of the limit of blank allows the management of false positive droplets, but the variable user‐defined positive threshold may be an issue for compliance to the quality norms., References Strain M, Lada S, Luong T, Rought SE, Gianella S, Terry VH, et al. Highly precise measurement of HIV DNA by droplet digital PCR. PLos One. 2013;8(4): e55943. Huggett [...]

Study Question: Are Sertoli cells (SCs) from adult Klinefelter men (47,XXY) capable of proliferating in vitro and maintaining their main phenotypical and functional characteristics as do SCs from adult 46,XY patients?, Summary Answer: Isolated SCs from patients with Klinefelter syndrome (KS) can be expanded in vitro while maintaining their characteristics and a stable karyotype, similar to SCs from 46,XY patients., What Is Known Already: The mechanism leading to testicular tissue degeneration in KS is still unknown. A few recent studies highlight the main role played by SCs in the physiopathology of the disease, but new study models based on co-culture or testicular organoids are needed to further understand the SC's involvement in the mechanism of testicular degeneration and fibrosis, and to find therapeutical targets. KS SC expansion could be the first step towards developing such in vitro study models. SCs have been isolated from 46,XY men and expanded in vitro while maintaining the expression of phenotypical and functional markers, but propagation of SCs from KS men has not been achieved yet., Study Design, Size, Duration: Testicular tissue was obtained during a testicular sperm extraction procedure for infertility treatment between 2019 and 2021 from three azoospermic adult KS (47,XXY) men (33±3.6 years old) and from three control patients (46,XY) (36±2 years old) presenting with obstructive azoospermia. SCs isolated from frozen-thawed tissue of KS and 46,XY patients were cultured for 60 days and compared. All patients signed an informed consent according to the ethical board approval of the study protocol., Participants/materials, Setting, Methods: Testicular biopsies obtained from KS (n = 3) and 46,XY (n = 3) adult patients were slow-frozen. After tissue thawing SCs were isolated using a double-step enzymatic digestion and differential plating, and cultured for 60 days in DMEM medium containing FBS. Analyses were performed at different culture times (passages 5 (P5) and 10 (P10)). Quantification of cells using immunofluorescence (IF) for cell type-specific markers (Sox9, GATA4, ACTA2, INSL3, MAGEA4), SCs characterization using both IF and quantitative real-time PCR for GDNF, BMP4, AR and CLDN11 and cells karyotyping were performed., Main Results and the Role of Chance: We demonstrate for the first time that a small population of human SCs isolated from frozen-thawed testis of adult KS patients can be expanded in vitro while retaining expression of characteristic markers of SCs and the 47,XXY karyotype, and exhibiting cell-specific functional proteins and gene expression (GDNF, BMP4, AR, and CLDN11) after 60 days in culture. At P10, 83.39 ± 4.2% of cultured cells from KS men and 85.34 ± 4.1% from 46,XY men expressed Sox9, and 88.8 ± 3.9% of KS cells versus 82.9 ± 3.2% of the control cells were positive for GATA4 without any differences between two groups; both Sox9 and GATA4 are typical SC markers. No differences were found between KS and 46,XY SCs in vitro in terms of cells expansion (exponential growth between P1 and P10 with an average cell count of 2.8±1.5×107 versus 3.8±1.2×107 respectively for the KS and control groups at P10). There was no significant statistical difference for functional proteins and genes expressions (GDNF, BMP4, AR, and CLDN11) neither between KS SCs and control SCs nor between P5 and P10., Limitations, Reasons for Caution: The small number of donor samples is a limitation but it is due to limited availability of tissue for research in KS populations. Although no differences were observed in SCs function in the culture of isolated SCs after 60 days, the possibility of a SCs dysfunction needs to be investigated in more complex 3-dimensional models allowing the establishment of a proper cell organization and further analyses of cell functions and interactions during longer culture periods., Wider Implications of the Findings: The demonstration of the possibility to propagate KS SCs in vitro could be useful to build new in vitro models for deciphering testicular cell interactions, determining deficient signalling pathways involved in impaired spermatogenesis, and identifying targets for infertility treatment in KS. As the cell numbers achieved in this study are higher than cell numbers used to develop testicular organoids, we may expect to be able to understand the behaviour and physiopathology of SCs in the disease during the long-term culture of these organoids. Such models could be further applied to understand other causes of deficiencies in seminiferous tubules., Study Funding/competing Interest(s): M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. The authors declare no conflicts of interest., Trial Registration Number: NCT05997706., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We have characterized the Bacillus subtilis homologs of fructoselysine 6-kinase and fructoselysine-6-phosphate deglycase, two enzymes that specifically metabolize the Amadori compound fructose-epsilon-lysine in Escherichia coli. The B. subtilis enzymes also catalyzed the phosphorylation of fructosamines to fructosamine 6-phosphates (YurL) and the conversion of the latter to glucose 6-phosphate and a free amino acid (YurP). However, their specificity was totally different from that of the E. coli enzymes, since they acted on fructoseglycine, fructosevaline (YurL) or their 6-phosphoderivatives (YurP) with more than 30-fold higher catalytic efficiencies than on fructose-alpha-lysine (6-phosphate). These enzymes are therefore involved in the metabolism of alpha-glycated amino acids.