Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma.

BACKGROUNDS: Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. AIMS: First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. MATERIALS & METHODS: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. RESULTS: In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. DISCUSSION: A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. CONCLUSION: Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

Influence of FGF23 and Klotho on male reproduction: Systemic vs direct effects.

Currently, no treatment exists to improve semen quality in most infertile men. Here, we demonstrate systemic and direct effects of Fibroblast growth factor 23 (FGF23) and Klotho, which normally regulate vitamin D and mineral homeostasis, on testicular function. Direct effects are plausible because KLOTHO is expressed in both germ cells and spermatozoa and forms with FGFR1 a specific receptor for the bone-derived hormone FGF23. Treatment with FGF23 increased testicular weight in wild-type mice, while mice with global loss of either FGF23 or Klotho had low testicular weight, reduced sperm count, and sperm motility. Mice with germ cell-specific Klotho (gcKL) deficiency neither had a change in sperm count nor sperm motility. However, a tendency toward fewer pregnancies was detected, and significantly fewer Klotho heterozygous pups originated from gcKL knockdown mice than would be expected by mendelian inheritance. Moreover, gcKL mice had a molecular phenotype with higher testicular expression of Slc34a2 and Trpv5 than wild-type littermates, which suggests a regulatory role for testicular phosphate and calcium homeostasis. KLOTHO and FGFR1 were also expressed in human germ cells and spermatozoa, and FGF23 treatment augmented the calcium response to progesterone in human spermatozoa. Moreover, cross-sectional data revealed that infertile men with the highest serum Klotho levels had significantly higher serum Inhibin B and total sperm count than men with the lowest serum Klotho concentrations. In conclusion, this translational study suggests that FGF23 and Klotho influence gonadal function and testicular mineral ion homeostasis both directly and indirectly through systemic changes in vitamin D and mineral homeostasis.

Presence of the vitamin D inactivating enzyme CYP24A1 in human sperm and prediction of the success of intrauterine insemination: A prospective study.

Male fertility is routinely evaluated by semen analysis, although semen quality variables such as; sperm count, motility and morphology have low predictive value for spontaneous pregnancies and fertility treatment outcomes. Vitamin D has been suggested to be beneficial for male reproduction. The vitamin D receptor and the vitamin D inactivating enzyme CYP24A1 are co-expressed in high quality sperm. Presence of CYP24A1 at the annulus of human sperm can distinguish between sperm from healthy and infertile men with high specificity and is positively correlated with semen quality. The high expression level in the testis of FAM57B2, which is activated by 24,25OH2D3, indicates an uncharacterized biological role for CYP24A1 in male reproduction. Moreover, activated vitamin D has been shown to induce sperm motility and promote fertilization in vitro. Here, we prospectively investigated whether the fraction of CYP24A1 positive sperm was a better predictor of clinical pregnancy than semen analysis by including 240 fertility treatments (169 couples) from a single fertility centre in Denmark. ROC-curve based analysis showed that the percentage of sperm expressing CYP24A1 was a better predictor of successful pregnancy outcome after intrauterine inseminations (IUI) than both sperm concentration and motility (p < 0.05). Interestingly, samples with CYP24A1 staining >67% of the sperm increased the likelihood of achieving pregnancy 4-fold after IUI compared with samples having fewer sperm with detectable CYP24A1 (p < 0.05). Neither CYP24A1 nor any of the other assessed semen quality variables were predictive for the treatment outcome of the more invasive assisted reproductive techniques (IVF and ICSI). In conclusion, our results provide proof of principle for a CYP24A1-based sperm test to improve fertility outcome for infertile patients referred for IUI and supports a role for vitamin D metabolites during fertilization.

A Liposome-Based Adjuvant Containing Two Delivery Systems with the Ability to Induce Mucosal Immunoglobulin A Following a Parenteral Immunization.

Worldwide, enteric infections rank third among all causes of disease burdens, and vaccines able to induce a strong and long-lasting intestinal immune responses are needed. Parenteral immunization generally do not generate intestinal IgA. Recently, however, injections of retinoic acid (RA) dissolved in oil, administered multiple times before vaccination to precondition the vaccine-draining lymph nodes, enabled a parenteral vaccine strategy to induce intestinal IgA. As multiple injections of RA before vaccination is not an attractive strategy for clinical practice, we aimed to develop a "one injection" vaccine formulation that upon parenteral administration induced intestinal IgA. Our vaccine formulation contained two liposomal delivery systems. One delivery system, based on 1,2-distearoyl- sn-glycero-3-phosphocholine stabilized with PEG, was designed to exhibit fast drainage of RA to local lymph nodes to precondition these for a mucosal immune response before being subjected to the vaccine antigen. The other delivery system, based on the cationic liposomal adjuvant CAF01 stabilized with cholesterol, was optimized for prolonged delivery of the antigen by migratory antigen-presenting cells to the preconditioned lymph node. Combined we call the adjuvant CAF23. We show that CAF23, administered by the subcutaneous route induces an antigen specific intestinal IgA response, making it a promising candidate adjuvant for vaccines against enteric diseases.

Selection of High-Quality Spermatozoa May Be Promoted by Activated Vitamin D in the Woman.

Context: The vitamin D receptor (VDR) and enzymes involved in activation (CYP2R1, CYP27B1) and inactivation (CYP24A1) of vitamin D are expressed in ovary, testes, and spermatozoa. Objective: Determine responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in spermatozoa from normal and infertile men, and identify the site of exposure and how 1,25(OH)2D3 influences sperm function. Design: Spermatozoa expressing VDR, CYP2R1, CYP27B1, and CYP24A1 were analyzed in normal and infertile men. 25-Hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D3], and 1,25(OH)2D3 were measured in serum, seminal fluid, cervical secretions, and ovarian follicular fluid. 1,25(OH)2D3 was tested on human spermatozoa. Setting: Tertiary center for fertility. Participants: Protein expression in spermatozoa and semen quality were assessed in 230 infertile and 114 healthy men. Vitamin D metabolites were measured in fluids from 245 men and 13 women, while 74 oocytes and 17 semen donors were used for sperm-function tests. Main Outcome Measures: VDR and CYP24A1 expressions in spermatozoa, fluid concentrations of 25-OHD, 24,25(OH)2D3, and 1,25(OH)2D3, and 1,25(OH)2D3-induced effects on intracellular calcium concentration ([Ca2+]i) and sperm-oocyte binding in vitro. Results: VDR and CYP24A1 were expressed in a >2-fold higher fraction of spermatozoa from normal than infertile men (P < 0.01). Concentrations of 25-OHD, 24,25(OH)2D, and 1,25(OH)2D3 were undetectable in seminal fluid but high in ovarian follicular fluid. Follicular concentrations of 1,25(OH)2D3 induced a modest increase in [Ca2+]i and sperm-oocyte binding in vitro (P < 0.05). Conclusion: Presence of VDR and CYP24A1 mainly in spermatozoa of higher quality supports that 1,25(OH)2D3 available in the female reproductive tract may promote selection of the best gametes for fertilization.

Possible influence of vitamin D on male reproduction.

Vitamin D is a versatile signaling molecule with an established role in the regulation of calcium homeostasis and bone health. In recent years the spectrum of vitamin D target organs has expanded and a reproductive role is supported by the presence of the vitamin D receptor (VDR) and the vitamin D metabolizing enzymes in the gonads, reproductive tract, and human spermatozoa. Interestingly, expression levels of VDR and the vitamin D inactivating enzyme CYP24A1 in human spermatozoa serve as positive predictive markers of semen quality and are higher expressed in spermatozoa from normal than infertile men. VDR mediates a non-genomic increase in intracellular calcium concentration, sperm motility, and induces the acrosome reaction. Furthermore, functional animal model studies have shown that vitamin D is important for sex steroid production, estrogen signaling, and semen quality. Cross-sectional clinical studies have supported the notion of a positive association between serum 25-hydroxyvitamin D (25-OHD) level and semen quality in both fertile and infertile men. However, it remains to be determined whether this association reflects a causal effect. The VDR is ubiquitously expressed and activated vitamin D is a regulator of insulin, aromatase, and osteocalcin. Hence, it is plausible that the influence of vitamin D on gonadal function may be mediated indirectly through other vitamin D regulated endocrine factors. Recent studies have indicated that vitamin D supplementation may be beneficial for couples in need of assisted reproductive techniques as high serum vitamin D levels were found to be associated with a higher chance of achieving pregnancy. Randomized clinical trials are needed to determine whether systemic changes in vitamin D metabolites can influence semen quality, fertility, and sex steroid production in infertile men. In this review known and possible future implications of vitamin D in human male reproduction function will be discussed.