Spain lacks detailed data on hip fracture trends despite being the country with the greatest increase in the pensioner-to-provider ratio in Europe. We reproduced a study on hip fracture incidence in a region of northern Spain (Cantabria) carried out 14 years ago to determine whether a secular trend to change is taking place. If such a trend could be found, our objective was to determine whether the effect is solely due to ageing or whether additional variables are involved. We assessed the incidence of hip fracture in patients aged > or =50 years through clinical records from Emergency Units and Orthopedic Surgical Units of all hospitals in the region of Cantabria in 1988 and 2002. A total of 318 new cases of hip fracture were recorded in 1988 and 490 in 2002 (54% increase; p<0.001). No significant changes were noticed following an adjustment for age. Women accounted for the increase in crude hip fracture incidence [246 women and 72 men suffered a hip fracture in 1988 compared to 404 women and 86 men in 2002 (64% increase in women and 19% increase in men; p<0.005 and not significant, respectively)]. The female:male ratio was 3.4 in 1988 versus 4.7 in 2002; following age-adjustment, no significant changes were found (1.8 in 1988 and 1.9 in 2002). The increase in crude hip fracture incidence was greater at cervical (versus trochanteric) sites. Patient residence, time of the year, site of fracture, kind of injury, previous contralateral hip fracture, length of stay, and peri-operative mortality did not differ significantly. In conclusion, a crude hip fracture incidence increase of about 50% in the northern Spanish region of Cantabria has taken place over the last 14 years. This effect does not persist after adjustments have been made for age. The crude rate increase occurred mainly at the expense of women, with a more noticeable rise in cervical fractures as opposed to trochanteric lesions.

Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation.

Wnt 22 β-Catenin 23 DNA methylation 24 Fractures 25 Bone diseases 26We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in 27 osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic 28 and epigenetic mechanisms involved. 29 β-Catenin gene expression and nuclear levels were analyzed by real time PCR and confocal immunofluorescence. 30 Increased nuclear β-catenin was found in osteoblasts isolated from patients with osteoarthritis (99 ± 4 31 units vs. 76 ± 12, p = 0.01, n = 10), without differences in gene transcription, which is consistent with 32 a post-translational down-regulation of β-catenin and decreased Wnt pathway activity.33 Twenty four single nucleotide polymorphisms (SNPs) of genes showing differential expression between fractures 34 and osteoarthritis (WNT4, WNT10A, WNT16 and SFRP1) were analyzed in DNA isolated from blood of 853 pa-35 tients. The genotypic frequencies were similar in both groups of patients, with no significant differences. 36 Methylation of Wnt pathway genes was analyzed in bone tissue samples (15 with fractures and 15 with osteo-37 arthritis) by interrogating a CpG-based methylation array. Six genes showed significant methylation differences 38 between both groups of patients: FZD10, TBL1X, CSNK1E, WNT8A, CSNK1A1L and SFRP4. The DNA demethylating 39 agent 5-deoxycytidine up-regulated 8 genes, including FZD10, in an osteoblast-like cell line, whereas it down-40 regulated other 16 genes. 41 In conclusion, Wnt activity is reduced in patients with hip fractures, in comparison with those with osteoarthritis. 42 It does not appear to be related to differences in the allele frequencies of the Wnt genes studied. On the other 43 hand, methylation differences between both groups could contribute to explain the differences in Wnt activity.44

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.

Klotho gene codes for a protein with glucuronidase activity and is thought to influence bone and vascular homeostasis. We studied the relationship of a common T/G polymorphism, resulting in a phenylalanine (F) to valine (V) substitution at aminoacid position 352, with bone mineral density (BMD) and osteoporotic fractures. The study group comprised 914 Spanish women, including 438 control subjects, 190 patients with osteoporosis, 198 with hip fractures, and 88 patients with severe osteoarthritis. BMD was measured by DEXA in 540 women from the control and osteoporosis groups. Allele frequencies were 86% and 14%, for the F and V alleles, respectively. In comparison with the most common FF genotype, postmenopausal women with FV/VV genotypes had higher hip BMD (femoral neck: 0.673 +/- 0.011 vs. 0.644 +/- 0.006 g/cm(2); P = 0.02; total hip: 0.807 +/- 0.014 vs. 0.774 +/- 0.008 g/cm(2); P = 0.03). Klotho alleles explained about 1.5% of BMD variance, but were not associated to the risk of osteoporotic spine or hip fractures. The Klotho genotype was not associated to BMD in premenopausal women. In conclusion, the F352V Klotho polymorphism is associated with BMD in postmenopausal women, suggesting that Klotho gene variants influence skeletal aging.

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Objective. Estrogen deprivation is a central mechanism in the development of osteoporosis with aging.Results from recent studies also suggest the involvement of estrogens in the pathophysiology of osteoarthritis (OA). Aromatization of androgenic precursors in peripheral tissue is the main source of estrogens in postmenopausal women and in men. However, the importance of aromatase expression in bone is a subject of controversy. This study was undertaken to determine aromatase expression in bone samples from patients with hip fracture and patients with OA.Methods. We studied 104 patients with hip fracture (n ‫؍‬ 60) or primary hip OA (n ‫؍‬ 44). Aromatase expression was determined in trabecular bone samples from the femoral neck and in osteoblast cultures grown by the primary explant technique (n ‫؍‬ 62), using real-time reverse transcriptase-polymerase chain reaction.Results. Aromatase RNA was detected in bone samples at levels similar to those found in adipose tissue. Transcript levels were significantly lower in bone tissue samples obtained from patients with OA than in those obtained from patients with fracture (P ‫؍‬ 0.00001). Likewise, primary cultures of osteoblast cells from OA patients revealed lower aromatase expression than those of cells from fracture patients (P ‫؍‬ 0.012). Results were independent of age or sex differences. Conclusion. Our findings indicate that the aro-

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