The aim of this study was to investigate if urinary glutamyl aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), Klotho and hydroxyproline can be considered as potential biomarkers of renal injury and fibrosis in an experimental model of obesity. Male Zucker lean (ZL) and obese (ZO) rats were studied from 2 to 8 months old. Kidneys from ZO rats at the end of the study (8 months old) developed mild focal and segmental glomerulosclerosis as well as moderate tubulointerstitial injury. Urinary excretion of Klotho was higher in ZO rats at 2, 5, and 8 months of study, plasma Klotho levels were reduced and protein abundance of Klotho in renal tissue was similar in ZL and ZO rats. GluAp and AlaAp urinary activities were also increased in ZO rats throughout the time-course study. ZO rats showed an augmentation of hydroxyproline content in renal tissue and a significant increase of tubulointerstitial fibrosis. Correlation studies demonstrated that GluAp, AlaAp, and Klotho are early diagnostic markers of renal lesions in Zucker obese rats. Proteinuria and hydroxyproline can be considered delayed diagnostic markers because their contribution to diagnosis starts later. Another relevant result is that GluAp, AlaAp, and Klotho are related not only with diagnosis but also with prognosis of renal lesions in Zucker obese rats. Moreover, strong predictive correlations of aminopeptidasic activities with the percentage of renal fibrosis or with renal hydroxyproline content at the end of the experiment were observed, indicating that an early increased excretion of these markers is related with a higher later extent of fibrosis in Zucker obese rats. In conclusion, GluAp, AlaAp, and Klotho are early diagnostic markers that are also related with the extent of renal fibrosis in Zucker obese rats. Therefore, they have a potential use not only in diagnosis, but also in prognosis of obesity-associated renal lesions. TRenal injury is characterized by an increased time related proteinuria, focal segmental glomerulosclerosis and tubulointerstitial damage that culminates ultimately in renal failure and death .Renal clinical practice requires highly sensitive and specific diagnostic and prognostic biomarkers for acute and chronic kidney disease (CKD). The early identification of renal disease onset and risk stratification in CKD progression are essential for early treatment of patients to ameliorate their comorbidity burden, particularly cardiovascular disease (CVD), and prevent the development of end stage renal disease (ESRD).Enzymes released from damaged tubular cells and excreted into urine are the most promising biomarkers for an early detection of kidney injury. They have an obvious diagnostic benefit because their measurements may provide detailed information about the nature, size and site of the damage to tubular cells and their possible necrosis or dysfunction . Alanyl In previous works, we determined the activity of these aminopeptidases as an index of renal damage in hypertensive hyperthyroid rats under normal, high and low saline intake  and in cKlotho is a transmembrane protein that is expressed mainly in the kidney. Parathyroid gland and choroid plexus are also sites of abundant Klotho expression . The solIn the kidney, Klotho is expressed in the distal convoluted tubules, in the proximal convoluted tubule  and the Under physiological conditions, the kidney has dual effects over soluble αKlotho homeostasis, producing and cleaving αKlotho from the membrane of the renal tubular epithelial cells into the circulation and removing αKlotho from the blood into the tubular urinary lumen through transcytosis to cross renal tubules .Soluble αKlotho is the main functional form in the circulation , cerebroIn this work we characterized the histopathological manifestations of obesity –induced renal injury in obese Zucker (ZO) rats; and investigated the urinary enzymatic activities of AlaAp and GluAp, plasma, urinary and renal Klotho levels and urinary and renal hydroxyproline as potential biomarkers of the renal injury observed in ZO rats.All experimental procedures were carried out in accordance with the principles of the Basel Declaration and recommendations of European Union Guidelines to the Care and Use of Laboratory Animals. The protocol was approved by the Ethical Committee of the University of Jaén and authorized by Junta de Andalucía with the approval identification number 450-5297 (02/21/2014).n = 10 each group) at 2 months old were purchased from Charles River Laboratories  and studied up to 8 months old. These rats were kept in a room maintained at 24 ± 1°C and humidity of 55 ± 10%, with a 12 h light/dark cycle and had free access to rat chow and tap water.Male Zucker lean and obese rats  once a month during 24 h with free access to food and drinking. Food and fluid intakes were measured and 24 h urine samples were collected. Urine samples were centrifuged for 15 min at 1000 g and stored at -80°C. Finally, rats were killed by pentobarbital overdose , and kidneys and heart were quickly removed and weighed.After completion of the experimental period (at 8 months old), rats were anesthetized with pentobarbital, 50 mg/kg, i.p. Blood samples were then drawn by abdominal aortic puncture to determine plasma variables. Blood samples were centrifuged for 15 min at 1000 Plasma variables were: glucose, urea, creatinine and Klotho. Tibial length was measured to normalize heart and kidney weight, since body weight cannot be used for this purpose in the present experimental setting. One kidney was fixed in 10% neutral-buffered formaldehyde solution for 48 h and subsequently placed in 70% ethanol for histological studies.Proteinuria and urine creatinine were determined in urine samples by an autoanalyzer Spin120. Plasma creatinine, urea and glucose were also measured in this instrument. Reagents for proteinuria, urea, and creatinine-Jaffé method were provided by Spinreact . Plasma and urinary Klotho levels were determined with an enzyme immunoassay kit, Bioassay Technology Laboratory . Renal and urinary hydroxyproline was measured with a Hydroxyproline Assay Kit . Protein content in renal tissue was analyzed with DC Bio-Rad protein assay .g for 2 min. 50 μl of each supernatant were transferred to a 69-well plate and dried in a 60°C oven until dryness. 100 μl of chloramine T/oxidation buffer mixture, 50 μl of 4-(dimethylamino) benzaldehyde concentrate and 50 μl of perchloric acid/isopropanol solution were added to each well and plates were heated for 90 min at 60°C. Absorbance was measured at 560 nm. The amount of hydroxyproline in each well was calculated from a standard curve of hydroxyproline containing 0–1 μg/well.Hundred microliter of urine sample or 100 μl of renal tissue (100 mg/ml in water) were hydrolyzed with the same volume of 37% HCl at 120°C overnight. 5 mg of activated charcoal were added to samples, mixed and centrifuged at 13,000 Kidneys were homogenized using RIPA lysis cell buffer containing protease inhibitors cocktail . 30 μg of protein were subjected to SDS-PAGE electrophoresis using 50 μL/well ready gels purchased from Bio-Rad . Proteins were transferred to a nitrocellulose membrane which was probed with 1 μg/mL rabbit anti-Klotho-antibody , and 20 ng/mL goat anti-rabbit IgG antibody HRP-conjugated . Bands were visualized with an enhanced chemiluminescence system  in a CCD camera image system and quantified with Image J software (v 1.48) .L-glutamic acid γ-2-naphthylamide or 10 mM alanyl-2-naphthylamide in pH 8.7 50 mM HCl-Tris). This buffer was also used to homogenate kidneys. Substrates had been previously dissolved in 1 ml of dimethyl sulfoxide and stored at -20°C. The amount of 2-naphthylamine released as a result of the aminopeptidase activities was measured fluorimetrically at an emission wavelength of 412 nm with an excitation wavelength of 345 nm, and quantified using a standard curve of 2-naphthylamine (0–200 nmol/ml). Fluorimetric data from samples and standard curve were taken each minute. Specific aminopeptidase activities were calculated from the slope of the linear portion of enzymatic assay, and expressed as nanomol of substrate hydrolyzed per minute per mg of urine creatinine (mU/mg Cr) per ml of plasma or per mg of total protein content in renal tissue.GluAp and AlaAp fluorimetric activities were determined in a kinetic fluorimetric assay using L-glutamic acid γ-2-naphthylamide or alanyl-2-naphthylamide  as substrates, respectively. 20 μl of urine, plasma or kidney homogenate were incubated during 30 min at 37°C with 80 μl of substrate solution  and re-hydrated in ethanol of decreasing gradation . Tissue sections were stained with hematoxylin-eosin, Masson’s trichrome and periodic acid-Schiff (PAS). The presence of glomerular lesions  was assessed in at least 200 glomeruli. Tubulointerstitial damage  was also studied. Injury was graded according to Shih, Hines, and Neilson on a semiquantitative scale of 0–4  . The morSamples were fixed in buffered 10% formalin, embedded in paraffin and serially sectioned at 5 μm thickness. Afterwards, they were stained with 1% picro Sirius red F3BA  for image analysis quantification. To improve staining, tissue sections were kept after deparaffination for 3–5 days in 70% alcohol as mordent. Picro Sirius red stains connective fibers deep red and cell nuclei and cytoplasmatic structures light red/bright yellow . To semiTo study the time course of biological variables and urinary markers, we used a factorial ANOVA for repeated measures, taking each rat as the subject and the group (ZL or ZO) as the between-subjects factor. Interactions between factors were analyzed using Bonferroni method. We used StatGraphics Centurion XVII software.t-test for the analysis of variables with normal distribution and equal variances. Welch modification of t-test was used for data with normal distribution and unequal variances and Mann–Whitney W (Wilcoxon) test was used to analyze the differences when data did not correspond to a normal distribution. Shapiro–Wilk test was used to analyze the normality of distributions. Differences were considered statistically significant at p < 0.05 level. We used StatGraphics Centurion XVII software.Morphological and biological variables measured at the end of the experiment were compared using a p < 0.05).Simple linear regressions were analyzed with Statgraphics Centurion XVII software. Multiple linear regressions were also analyzed with this software to establish the optimal correlation between urinary markers as independent variables and each renal lesion as dependent variable. The model of regression with best information criteria (Akaike information criteria) was selected. This model eliminates the markers that do not significantly contribute to regression, obtaining an optimal fitted model that includes all markers with a significant contribution to regression  was used for the analyses. Results are presented as mean ± standard error in the case of fibrosis, or median and interquartilic range for categorical data. A comparison of the biological variables between ZO and ZL rats was performed (data not shown). The variables analyzed were: body weight, food intake, water intake, diuresis and water balance. Body weight was obviously greater in ZO with respect to ZL rats. Food intake was increased in ZO rats, whereas water intake was reduced along the study. Unexpectedly, diuresis was higher in ZO rats; and consequently, water balance was found reduced in these animals. The time course of glucosuria was not significantly different in ZO and ZL rats when expressed in concentration, but it was significantly increased from 6 to 8 months old when data were normalized by creatinine excretion.Final body weight was significantly increased in ZO rats by 1.35-fold when compared to ZL rats. Kidney weight and heart weight in absolute values or relative to tibial length were increased in ZO rats when compared with the ZL group  in ZO rats when compared with ZL group. The protein abundance of Klotho in renal tissue was similar in ZO and ZL rats. These data are shown in Figure The urinary excretion of Klotho was higher in ZO rats at 2, 5, and 8 months old when expressed as total daily excretion (ng/rat/day), urinary concentration (ng/ml) or relative to creatinine excretion (ng/mg Cr). The first two ways of expression are not presented. Plasma Klotho concentration was reduced to the half  were pooled in a common regression line, reaching the strongest correlation with urinary GluAp Figure .p < 0.001). These data are displayed in Figure Renal hydroxyproline content was increased in renal tissue of obese rats and morphometrical quantification with Sirius red stain and polarized light of renal cortex showed a significant increase of tubulointerstitial fibrosis in Zucker obese rats  study demonstrate that albuminuria augmented during the development of diabetic kidney disease and that, in addition, is a marker of the progression of diabetes . In our Proteinuria from the classic point of view is secondary to alterations of the glomerular barrier membrane, in fact podocyte injury underlies the progression of focal segmental glomerulosclerosis in the fa/fa Zucker rat , but nowThis study shows increased urinary levels of Klotho, a reduction in plasma and normal values of protein abundance in the renal tissue. These last data agree with the results of The possible explanation for the absence of uniformity of our Klotho data is that tubular injury determines an increased urinary excretion of Klotho as happens with aminopeptidases that are released to urine. This wasting of Klotho through the urine, might contributes to the reduced plasma level, since urinary αKlotho, at least in part, may arise from the plasma. Thus, exogenously injected labeled αKlotho was detected in the urine of rats . In suppAs reported in the Introduction section, plasma soluble Klotho may operate as an endocrine agent on distant organs , 2015, wUrinary hydroxyproline excretion has been related in previous works with the development of different renal lesions including fibrosis in a murine model of nephrotoxicity evoked with melamine and cyanuric acid . In our The correlation studies shown in Tables Another relevant result is that GluAp, AlaAp and Klotho are related not only with diagnosis but also with prognosis of renal lesions in Zucker obese rats. Results reported in Figures Urinary aminopeptidases and Klotho are early diagnostic biomarkers of renal injury, and urinary levels of these biomarkers are also related with the extent of renal fibrosis in Zucker obese rats. Measurements of GluAp, AlaAp, and Klotho may represent a novel, specific and non-invasive diagnostic approach to assess kidney fibrosis and may have a potential use not only in diagnosis but also in prognosis of obesity-associated renal lesions.AO, FV, and RW: conception and design of the experiments. AL-C, AQ, FO’V, NM-M, SM-M, and RW: collection, analysis and interpretation of the data. FO’V, AO, FV, and RW: drafting the article or revising it critically for important intellectual content. All authors reviewed and approved the final version of the manuscript.RW, FV, and AO are co-authors of the patent “Glutamyl aminopeptidase as a marker of renal damage” with publication number 2382960. There is not any other relevant declaration related to employment, consultancy, patents, products in development or modified products. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To compare the capsule edges ultrastructure obtained by two femtosecond laser-assisted cataract surgery (FLACS) platforms and manual continuous curvilinear capsulorhexis (CCC) using scanning electron microscopy (SEM).  Eye Clinic, University of Trieste, Italy.  Experimental comparative study.  150 anterior capsules were collected and divided into three groups as follows: Group 1 (50 capsules) obtained with manual CCC, Groups 2 and 3 (each with 50 capsules) obtained with the Catalys Laser and the LenSx Laser, respectively. All samples were imaged by means of SEM and regularity of the cut surface, and thickness of the capsule edge were evaluated and compared. p < 0.05). Group 1 had a significantly lower thickness of the capsule edge than the FSL groups (p < 0.05). No statistically significant difference in the capsule edge thickness between the FSL groups was found (p=0.244).  All femtosecond laser (FSL) capsules were perfectly circular, whereas some alteration of the circular shape was observed in the manual ones. Group 1 showed a smooth and regular capsule edge without any surface irregularity, conversely Groups 2 and 3 showed postage-stamp perforations on the capsule edge. The cut surface irregularity value in Group 2 was 1.4 ± 0.63, while it was 0.7 ± 0.49 in Group 3 ( Despite the presence of slight cut surface irregularities, both FSL capsulotomies showed a better geometry and circularity than the manual ones. Capsulotomy specimens obtained using both FSL capsulotomies showed laser-induced alterations of the capsule edge when compared with smooth and regular edges obtained using manual CCC. Cataract surgery is the most commonly performed ophthalmic surgery in the world , 2.Nowadays, phacoemulsification with intraocular lens (IOL) implantation is not only a treatment for visual rehabilitation but also a refractive procedure, with particular demands to achieve optimal functional results.Anterior capsulotomy is one of the most critical and important steps in cataract surgery that may influence the correct position and centration of the IOL –6.A well-centered, regular, and size-desired capsulotomy is particularly important when implanting advanced technology IOL, such as multifocal and toric IOLs, which requires a precise centration in order to maximize the IOL performance .Moreover, although new IOL designs have diminished the incidence of posterior capsular opacification, a precise anterior capsulotomy is crucial in preventing the migration of lens epithelial cells , 9.The most frequently used technique to obtain an anterior capsulotomy is the manual continuous curvilinear capsulorhexis (CCC) first described by Gimbel and Neuhann . This teCurrently, the introduction of femtosecond laser-assisted cataract surgery (FLACS) has changed the way of undertaking cataract surgery by automating key steps in the surgical procedure , 14, 15.Although consistent circularity and predictability in size have been demonstrated with all commercially available FSL platforms, irregularities on the capsulotomy cutting edge, such as laser induced perforations and postage-stamp perforations, have been reported , 19–21.These irregularities, not noticeable during surgery, are probably due to several factors such as fixational eye movements, pulse laser energy, and patient interface and could lead to an increased rate of anterior capsule tears , 22.The aim of this study was to compare the capsule edge ultrastructure of the anterior lens capsule specimens obtained by two commercially available FSL platforms for cataract surgery with manual CCC by means of scanning electron microscopy (SEM). Using the same energy setting for both FSL systems, we evaluated which factors might lead to achieve the capsulotomy with the best cutting edge surface.This is an experimental comparative study, following the tenets of the Declaration of Helsinki. All patients signed an informed consent after full explanation of the procedure.All procedures were performed by the same experienced surgeon (DT) under topical anaesthesia.150 capsule specimens of 150 eyes with senile cataract undergoing lens removal and IOL implantation were collected for this study.Group 1 (50 eyes) included anterior capsule specimens obtained after manual CCCGroup 2 (50 eyes) included anterior capsule specimens obtained after FSL capsulotomy using the Catalys Precision Laser System Group 3 (50 eyes) included anterior capsule specimens obtained after FSL capsulotomy using the LenSx Laser System Specimens were divided into the following 3 groups:Manual CCC was performed using capsulorhexis forceps under an ophthalmic viscosurgical device (OVD).The laser parameters included the capsule diameter, laser pulse energy, spot separation, and layer separation. Spot separation represents the distance between two adjacent spots, whereas layer separation represents the distance to the next row.µJ in both FSL groups.Both FSL platforms were planned to achieve a 4.9 mm diameter anterior capsulotomy centered on the pupil. The pulse energy was 4 µm, and the layer separation was 10 µm, whereas in Group 3, the spot separation and layer separation was 4 µm.According to the suggestion of the manufacturers of the two FSL platforms, in Group 2, the spot separation was 5 The two laser systems have different patient interfaces. Catalys Laser has a fluid interface  without cornea applanation, while LenSx Laser has a curved interface with a soft contact lens (SoftFit) that applanated the cornea.Once completed laser treatment, the suction was released, the patient interface was removed, and the patient was slowly undocked from the laser. Immediately, the patient was transferred under the surgical microscope  to complete the surgical procedure. The paracentesis and primary incision were opened, and an OVD was injected into the anterior chamber above the anterior capsule. Subsequently, the anterior capsule was gently removed using capsulorhexis forceps.After anterior capsule removal, standard phacoemulsification with IOL implantation was performed in all cases.Once removed, capsule specimens were immediately placed in a sterile container filled with fixative fluid and prepared for SEM analysis.The samples were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer and then dehydrated in an ascending series of alcohol rinses.Specimens were mounted on aluminium stubs covered with conductive double-sided carbon adhesive tape. Next, the samples were sputtered with gold  and immediately analysed by means of SEM  operated in secondary electron detection mode. The working distance was adjusted in order to obtain the suitable magnification, and the accelerating voltage was set to 30 kV. The thickness was assessed with ten measurements taken along the capsulorhexis. The roughness of the cut surface, which included microgrooves, surface pitting, and notches, and overall irregularity of the cut surface, was graded on a scale of 0 to 3 .p was considered statistically significant when <0.05.The Shapiro–Wilk test was used to analyse the distribution of the data. The Kruskal–Wallis and Mann–Whitney tests were used to statistically compare the differences among the anterior capsule edges characteristics. Statistical analysis was performed with SPSS software . For all tests, Low-magnification SEM images of all capsule specimens in FSL groups showed a perfectly circular geometry , whereasµm in size near the capsule edge (µm in Group 2 (µm in Group 3 (p < 0.05).Regularity and thickness of the capsulotomy cutting edge was assessed at high-magnification . Group 1ule edge . These p Group 2  and betw Group 3 . The cutp < 0.05). No statistically significant difference regarding the capsule edge thickness between the FSL groups was found (p=0.244).Group 1 had a significantly lower thickness of the capsule edge than the FSL groups (All surgeries were uneventful. No anterior capsule tears or incomplete procedures were observed.Nowadays, FSL technology has changed the way of undertaking cataract surgery.This technology offers the possibility of automating crucial steps of the surgery allowing to achieve repeatable and better results than the manual technique , 15, 23.One of the greatest benefits in using femtosecond laser in cataract surgery is the possibility to obtain a predictable capsulotomy –18.Achieving a round, well-centered, and size-desired anterior capsulorhexis is mandatory in order to obtain a perfect IOL positioning, especially with advanced technology IOL .Capsulorhexis obtained with FSL had a higher circularity index than the manual technique, resulting more similar to a perfect circle . Nagy etSimilarly, low-magnification SEM images of our FSL capsule specimens showed a perfectly circular geometry, whereas some deformations such as folding and tears were observed in the manual CCC group.Several studies reported irregularities of the capsule cutting edge obtained by means of FSL , 19, 21.Several factors might influence capsule irregularities such as laser pulse energy, laser focus, and patient interface –21. MastSeveral studies reported that a soft contact lens interface resulted in a smoother capsule cutting edge than a rigid curved contact lens , 25–27. Differently to other studies, we performed an interplatform comparison using the same energy setting for both FSL systems and retrospectively analysed which FSL features might allow to obtain the ideal edge shape. Regarding the patient interface, we observed that at the same energy setting, LenSx Laser with soft contact lens interface performed significantly smoothest capsule cutting edges than that obtained with a fluid interface of the Catalys platform. Moreover, we did not find differences in the cut surface irregularities between LenSx Laser capsulotomy and manual CCC. Our results are in concordance with results reported by Bala et al.  who showOther laser parameters, such as spot separation and layer separation, should be considered to better understand the microstructural differences of capsulotomy after FSL.μm and using a dense spot separation result in an improvement of the cut quality and a reduction of the number of tags on the capsulotomy cutting edge [Studies on the Catalys Laser showed that increasing the layer separation from 10 to 15 ing edge , 29.μm compared with Catalys Laser with spot separation of 5 μm was found, suggesting a better cut surface morphology using dense spot spacing.In our interplatform comparison, a smoothest capsulotomy cutting edge in the LenSx Laser with spot separation of 4 In conclusion, the Catalys and LenSx femtosecond laser platforms allow the surgeon to perform a better capsulorhexis in geometry and circularity. As previously described, we confirm the presence of cut surface irregularities in the capsulotomies performed by means of FSL. Moreover, our study showed the smoothest capsulotomy cutting edge in the LenSx Laser if compared with Catalys Laser at the same laser energy setting, suggesting a better cut surface morphology using a soft liquid-free curved patient interface and dense spot separation. Further studies with different laser settings should be performed to find, for each commercially available FSL platform, the ideal laser parameters to achieve better surface regularity of the capsulotomy cutting edge after FLACS.


Despite being the fourth largest island in the Mediterranean basin, the genetic variation of Corsica has not been explored as exhaustively as Sardinia, which is situated only 11 km South. However, it is likely that the populations of the two islands shared, at least in part, similar demographic histories. Moreover, the relative small size of the Corsica may have caused genetic isolation, which, in turn, might be relevant under medical and translational perspectives. Here we analysed genome wide data of 16 Corsicans, and integrated with newly  and previously generated samples from West Eurasia and North Africa. Allele frequency, haplotype-based, and ancient genome analyses suggest that although Sardinia and Corsica may have witnessed similar isolation and migration events, the latter is genetically closer to populations from continental Europe, such as Northern and Central Italians. Nowadays, approximately ~339,000 people inhabit the island1.Corsica, located south of the shore of Côte d’Azur (France), and west of Tuscany , is separated from Sardinia to its south by the Strait of Bonifacio. It is the fourth largest Mediterranean island 4. Archaeological and genetic data suggest episodic and discontinuous settlements during Mesolithic and transitional phases between Mesolithic and Neolithic periods6. The permanent human presence in Corsica is attested in the Neolithic period since the 6th millennium BC and possible interactions with mainland and other islands are suggested by the wider appearance of non-local lithic resources and similar ceramic traditions over a larger Western Mediterranean region7.The understanding of the peopling of Corsica has remained incomprehensive. From a geological perspective, during the last glaciation, Corsica and Sardinia formed a single landmass and its distance to Italy was reduced, possibly increasing connections with mainlandth century, followed by Roman Papacy from the 8th century onwards. From the end of the 11th century Corsica was under the government of Pisa. Then, with the intermittent period dominated by the kings of Aragon, Corsica was under the rule of Genoa from the 13th to the 18th century, when it was incorporated into France in 17688.In the last three millennia, the Corsican population witnessed several dramatic demographic changes due to conquests, epidemics outbreaks and economic crises. The Greeks established the city of Alalia (today Aleria) in 565 BC, which is also the first mention of Corsica in historical records. Subsequently, it witnessed numerous intrusions and conquests by different populations. Carthaginians and Etruscans dominated the island until the Roman occupation in the third century BC. Successive invasions by the Vandals, Ostrogoths and Saracens, took place until the beginning of the Byzantine influence in the 612, such as a marked North-South differentiation, confirmed by surname and linguistic studies13, and mirroring the geographic features of the island.Previous genetic surveys on Corsican population have shown regional differentiation16, while conversely, some have suggested genetic distinction18. Furthermore, the extent of Corsica’s genetic relationships with mainland populations is controversial. Close genetic connections have been observed between Corsica and continental Mediterranean populations20, whereas other studies have reported limited genetic influences from mainland12. Furthermore, it has been previously suggested that different regions in Corsica have received genetic influences from distinct sources, showing diverse affinities with surrounding populations12.Despite their geographic proximity, different investigations based on unilinear and autosomal markers have produced contradicting results in characterizing the relationship between Sardinia and Corsica. Some studies have found genetic affinity between the two islands22. Sardinia is a well-known example (see for instance23 and references therein). Preliminary studies have suggested Corsican potential in this context: an analysis on chromosome X microsatellite markers demonstrated an overall LD decrease in the innermost part of Corsica24 and some medical and association studies have been conducted on Corsican population27.Regardless of its genetic affinity, it is unclear to what extent the Corsican population shows a signature of demographic bottleneck or population decrease, as expected in a long term isolation scenario. Previous studies have demonstrated that isolated populations are valuable resources in genome-wide association studies. In geographically and/or culturally isolated conditions evolutionary forces and population dynamics can lead to the high level of homozygosity, reduced genetic diversity and increase in peculiar allele frequencies, which makes easier to trace genetic variants affecting medical or phenotypic traitsDespite its anthropological and epidemiological relevance, a genome-wide characterisation of Corsican population is not available so far. In order to evaluate the within-population genetic variation, and the affinity with other modern and ancient European populations, here we genotyped 16 Corsican samples collected from different locations of the island , togethe29. Corsicans show an excess of RoHs indicated by the high median values of total number and total length of RoHs, next to Sardinians and French Basques . RoH are uninterrupted segments of homozygous genotypes present in individuals. The number and extension of RoH stretches are correlated to the level of identity by descent and are largely affected by population’s demographic historyues Fig. . This ho31 implemented in the program ADMIXTURE32. At the level of lowest cross-validation index (K = 6), Corsicans and geographically related populations are composed mainly by three ancestry components - “Sardinian”, “Northern and Eastern Europe”, “Caucasus and Middle East”  than the latter. However, the large standard errors associated to the f3 statistics suggest that the differences in affinity of Corsica with Sardinian/Basque or mainland populations are negligible or non-significant. The tests of admixture based on the f3 statistics revealed an overall complexity in the admixture history of Corsica. Out of 2,652 tests performed, 18 were statistically significant (|Z| > 3), although only 8 remained significant using a more conservative threshold (|Z| > 4)  . Regardlj as a mosaic of genomic fragments inherited by n donor samples34, both in terms of total number (chunkcounts) or length of fragments . The resulting j x n coancestry chunkcount matrix was then employed to identify and characterise homogenous groups of individuals, in the form of a dendrogram. fineSTRUCTURE clustering results together with the ChromoPainter coancestry chunkcount matrix were subsequently used to identify, date and describe admixture events with GLOBETROTTER35.We inferred the fine-scale population structure harnessing the haplotype-sharing patterns among individuals. In detail, using ChromoPainter we reconstructed each analysed individual The complete fineSTRUCTURE dendrogram is represented in At a finer inspection, out of the 16 Corsican samples analysed, 12 formed a population specific cluster (Corsicans2) in the Southern European clade, related to North and Central Italian group. Three samples fell into three different clusters composed by North and Central Italians , French (French) and Iberians (Iberia2), respectively Fig. , possiblIn order to explore the relationships between populations, we performed a Principal Component Analysis (PCA) based on the chunkcount coancestry matrix. The first two PCs explain a large proportion of the total variance , similarly to Italian, Spanish and Balkan populations  and Iranian Neolithic (Iran_N) were considered in the same analysis . Compared to Corsicans, French and Spanish samples were characterised by a smaller proportion of Iranian Neolithic , and a slightly higher contribution from Western Hunter Gatherers (14% and 17%).In order to better characterize the “ancestral” composition of Corsican and European populations, we inferred their genetic relationship with a set of ancient individuals using qpAdmons Fig. . In addi41, which is situated only 11 km South. However, it is likely that the two populations have shared at least part of their demographic history, given their geographic proximity and similarities in the archaeological record. In addition, the relatively small size of Corsica may have contributed to create isolation conditions affecting the genetic variability of the autochthonous population24.Despite being the fourth largest island in the Mediterranean basin, the genetic variation of Corsica has not been explored as exhaustively as SardiniaOur analysis revealed that Corsican population shares several genomic features with Sardinia and North-Central Italy, creating a unique blend of genomic ancestries Fig. –3.30, relatively high homozygosity found in the current study confirms Corsican population as a potentially valuable resource for association studies, as also suggested by some previous surveys of autosomal markers27 and chromosome X microsatellite LD24 variation in Corsican population.The Corsican population shows a relatively high homozygosity characterised by high variance, suggesting that it witnessed both an isolation and migration phase. A similar pattern has been observed for Sardinians Fig. . As isolAllele frequency-based assignment algorithm and genetic distance methods showed that Corsica is more closely related to mainland populations from France, Italy, Spain and Greece rather than Sardinia Fig. . In contTo better characterise the genetic relationship of Corsica with other Mediterranean populations we have harnessed the information embedded in haplotype configurations. The analyses of the haplotype sharing patterns performed using ChromoPainter and fineSTRUCTURE provided substantial evidence of higher affinity between Corsica and Central or Northern Italian populations rather than Sardinian, French and Iberian populations Fig. . In fact36.When we investigated admixture evidence of Corsican population through f3 analysis, Corsicans could be described as a combination of allele frequencies from Sardinia, Northern Europe, Caucasus, North Africa and Arabian Peninsula . A simil42. In fact, we have found a similar proportion of Steppe Bronze Age (~19%) and Iranian Neolithic (~14%) in Corsicans.Lastly, we have evaluated the genetic relationship of Corsica and other Mediterranean populations with ancient Eurasian individuals through PCA and qpAdm Fig. . CorsicaIn conclusion, the genetic characterisation of Corsica is consistent with a closer genetic affinity with Northern and Central Italian populations rather than Sardinians, although sharing with the latter a noteworthy proportion of ancestry and similar demographic and isolation processes. The analysis of larger sample sizes from different regions of the island and genetic material from ancient specimen may help to further evaluate the genetic structure in the island and its demographic history.12 in order to maximize geographic coverage  were controlled with software KING v1.457 and two samples (one Yemen and one North Kannadi) were randomly removed from detected relative pairs. For some analyses SNPs in strong linkage disequilibrium (pairwise genotypic correlation R2 > 0.4) in a window of 1,000 SNPs, sliding the window by step of 25 SNPs, were excluded. Exact numbers of individuals, populations and markers used in each analysis are specified in DNA was extracted from blood/saliva samples and genotyping was carried out on Illumina 660 K platform (Human660W-Quad BeadChip). New samples were combined with data from previous studiesanalysed . The mer56, with sliding window of 50 SNPs , allowing for one heterozygous and five missing calls per window. RoH were defined as regions of at least 50 consecutive homozygous SNPs spanning at least 1,500 kb, with a gap of less than 1,000 kb between adjacent regions. The required minimum density was set at 50 kb/SNP58.Runs of homozygosity (RoH) were inferred using PLINK v1.932 was used to infer putative ancestral components in the Corsican population in a worldwide context. Clustering was performed 100 times at K = 2 to K = 15  individuals were randomly selected from each population. The inferred parameters were finally used in ChromoPainter specifying all the available samples as donors and recipients. This resulted in two different matrices, the “chunkcount” and the “chunklength”; the former summarises, for each recipient, the number of fragments inherited from each donor, while in the latter the same statistics is expressed in total genomic (in centimorgan) length. The coancestry matrices based on the length (chunklength) and number (chunkcount) of fragments were then obtained by combining the different chromosomal outputs with ChromoCombine. The obtained chunkcount coancestry matrix was harnessed to identify homogeneous groups of individuals using fineSTRUCTURE. In detail, two different runs of 4,000,000 iterations were performed, discarding the first 1 million as burn-in and using a thin interval of 10,000. For each of the clustering approaches, a hierarchical tree was inferred by taking advantage of the “Tree” method and the “maximum concordance state” approach, performing 1 million iterations. In order to assess the robustness of the clustering process, the pairwise coincidence statistics among individuals was evaluated  was carried out using the coancestry matrix of coping vectors created with ChromoPainter. On the whole, 624 samples were used, including all the samples from Europe, the Caucasus and Anatolia and the sub-set of Near Easterners, that on fineSTRUCTURE tree , formed 35. In detail, the painting profile obtained by ChromoPainter was harnessed by testing for any evidence of admixture using the options null.ind = 1 prop.ind = 0, and performing 100 bootstrap iterations. For each of the inferred admixture events, only those characterised by bootstrap values for time of admixture between 1 and 400 were considered. Subsequently, the time of admixture was estimated by repeating the same steps with options null.ind = 0 and prop.ind = 1. For the “non-local” analysis the same procedure has been repeated excluding clusters from the Southern European group as possible sources of the target. We considered a generation time of 30 years38.The time of admixture and mixture profile were estimated for all the previously inferred clusters (targets) using GLOBETROTTERet al. 201763 and Olalde et al. 201864 were used, from which non relevant individuals and all the samples with more than 70% of missingness were removed. After the final filtering 222 samples were retained  the tested sources were significantly different and b) the target may be reconstructed using a specific combination of sources. A p-value threshold of 0.01 was used, as well as the following set of sources (The admixture profile of all the analysed populations was reconstructed with qpADM sources :Anatolia_BA, Anatolia_N, Steppe_EMBA, WHG, EHG, Iran_N, Minoan_Lasithi, Mycenaean, Yorubas, Levant_N, CHG, Europe_EN, Europe_LNBA, Europe_MNChLand the following set of Outgroups:AfontovaGora3, EHG, ElMiron, GoyetQ116-1, Iran_N, Kostenki14, Levant_N, MA1, Mota, Natufian, Ust_Ishim, Vestonice16, CHG.All the tests for which the fitted model was supported were shown in Supplementary Figures and TablesSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5


In the rabbit model, this bone scaffold provided less ability to enhance tissue healing compared with a commercial bone scaffold. Moreover, persistence of scaffold material and absence of integration with connective tissue around the scaffolds were observed. These results raised some concerns about the osteochondral use of this chitosan composite scaffold, especially for the bone layer. Further studies are needed to explore the best formulation of chitosan-reinforced composites for osteochondral treatment.Several biomaterials have recently been developed to address the challenge of osteochondral regeneration. Among these, chitosan holds promises both for cartilage and bone healing. The aim of this in vivo study was to evaluate the regeneration potential of a novel hybrid magnesium-doped hydroxyapatite (MgHA), collagen, chitosan-based scaffold, which was tested in a sheep model to ascertain its osteochondral regenerative potential, and in a rabbit model to further evaluate its ability to regenerate bone tissue. Macroscopic, microtomography, histology, histomorphometry, and immunohistochemical analysis were performed. In the sheep model, all analyses did not show significant differences compared to untreated defects ( Chondral and osteochondral defects are challenging problems, since the native structure of cartilage and subchondral bone cannot be regenerated with any of the available treatments ,2. The mco-glycolic acid) (PLGA)–calcium–sulfate construct showed poor quality of repair tissue [Several biomaterials have been investigated as potential substrates for osteochondral repair ,5, and sr tissue ,7, a morr tissue . Howeverr tissue  or unicor tissue , were cor tissue ,12. In fr tissue ,14. EvenChitosan is attracting the interest of biologists and material scientists, due to its good biocompatibility and reabsorption property; moreover, it can be combined with collagen by intermolecular interactions, to create scaffolds with enhanced mechanical strength ,16,17. TTherefore, in the present study a hybrid osteochondral cell-free scaffold, obtained by a blending process, to achieve a collagen and chitosan (col+chit)-reinforced blend, and by a bio-inspired mineralization process, to grow MgHA nanoparticles on col+chit blends (MgHA/(col+chit)), was developed and tested in vivo. A pilot study was performed on a sheep model to ascertain the potential for the treatment of critical size osteochondral defects. Furthermore, due to the key role of subchondral bone for the success of osteochondral treatment, a rabbit model was used to evaluate bone isolated from the articular environment, and its contribution toward the overall osteochondral regeneration induced by this chitosan-reinforced scaffold.Anesthesia procedures and postoperative healing period were uneventful for all animals. When femoral condyles were retrieved, a macroscopic analysis revealed some fibrotic peri-articular tissues in one case for the control group and in five cases for the experimental group. Three of the 12 samples, both in the control and in the experimental group, were not included in the evaluation because the scaffolds were not fully surrounded by trabecular bone, not allowing a fair assessment and comparison with fully trabecular bone-surrounded implants.1) in the experimental group (See p < 0.05). Finally, no intergroup differences were observed on peri-implant bone 3D morphometric parameters.Limited growth of new bone inside the defect and a thin trabecular new bone structure were detected in the control group , whereasroup See , with loResiduals of scaffold material were described in three cases in the control group. They were surrounded by connective tissue a with anConversely, the scaffold material MgHA/(col+chit) was always recognizable in the bone defect area in the experimental group. No direct contact between bone tissue and scaffold was observed in seven cases d, due top = 0.138 and p = 0.176, respectively.Finally, statistical analysis revealed no significant differences between experimental scaffold and control material regarding dynamic histomorphometric parameters   .1)  . 3D morphometric analysis on bone inside the defect and around the implant did not show significant differences between groups with a large overall variability. Moreover, the 2D distribution of bone inside and surrounding the defect was analyzed by sections and did not show any significant difference between groups.Histology assessment of the control group confirmed the macroscopic results, showing amorphous fibrous tissue filling the defect in one sample, and an incomplete defect filling with irregular bone–cartilage surface in the other cases. Further analysis revealed an altered chondrocyte distribution, grouped in clusters, in comparison with native cartilage tissue.In six samples of the experimental group, fibrocartilage tissue was filling the defect site, with intense proteoglycan red staining with Safranin-O . In the Both the untreated and MgHA–collagen–chitosan groups revealed type I and II collagen staining either negative or positive in scattered areas . A compaThe main finding of this study is that the hybrid MgHA–collagen–chitosan scaffolds failed to provide both bone and cartilage regeneration in vivo.The regenerative potential of this scaffold was tested in two animal models: A pilot study on a sheep model was performed to investigate the osteochondral regenerative potential, and a rabbit model was used to investigate the specific contribute of the bone layer to the overall osteochondral regeneration provided by this scaffold. The two models contributed to demonstrate the lack of regenerative potential at both the bone and cartilage level of this MgHA–collagen–chitosan scaffold. The rabbit model had previously been shown to be suitable for studying the effectiveness of different biomaterials on bone repair and regrowth , with an®, Smith & Nephew, Andover, MA, USA) has been successfully applied in the clinical practice to treat chondral defects, after preclinical findings in an ovine model showing increased volume and hyaline-like repair tissue, with increased glycosaminoglycans (GAG) and collagen content, compared to microfracture controls [® group compared with microfracture treatment alone at 12 months [The pilot evaluation in sheep clearly provided evidence for a lack of regenerative potential, thus advising against the translation of this hybrid MgHA–collagen–chitosan osteochondral scaffold into clinical use. These negative results are in contrast with other reported applications of chitosan in humans. In fact, a chitosan-based gel  [Therefore, heterogeneous results may be obtained with different chitosan formulations, and further complexity is due to the need of blending different biomaterials with chitosan in order to improve its properties, as it lacks proper mechanical strength for bone tissue engineering and its interface does not promote easy cell adhesion . The sca, CO32−) ,43. Bio-, CO32−) . It also, CO32−) . Moreove, CO32−) . Based oEven though fibrotic peri-articular tissue and some inflammatory cells infiltrates were found, no foreign body reaction or adverse immunological rejection was documented. No significant differences were shown on peri-implant bone 3D morphometric parameters between the experimental group and control. However, direct contact between implant and bone tissue was absent in most of the samples, which could explain the less abundant formation of new bone inside the defect in the experimental group. These microtomography findings were also confirmed by histology and histomorphometry analyses. While in the control group the scaffold was reabsorbed and bone tissue progressively grew from the host bone to the area originally occupied by the scaffold, in the experimental group the scaffold material was always recognizable together with the bone defect area, with scarce or absent contact at the interface. Thus, the lack of bone integration potential of this new biomaterial may have impaired the overall regenerative properties of the osteochondral scaffold as well. To this regard, it has been underlined that the molecular weight of chitosan is inversely proportional to the hydration capacity of the chitosan itself, being a greater molecular weight correlated to lower swelling . For thiMoreover, the crosslinking treatment performed on the material with the aim of improving the scaffold stability in in vivo conditions, may have induced a too high cross-linking degree of the matrix, with a strong impact on its biodegradability properties. This aspect is coherent with in vivo studies reported by Abarrategi et al. , showingTherefore, our results raise some concerns on the osteochondral application of chitosan, particularly regarding the subchondral bone layer, which is essential in supporting the overlying chondral regeneration. While chitosan seems to be a promising option for osteochondral regeneration thanks to its safety, low cost, and overall positive literature findings, the choice of the starting material, as well as trimming material properties and interaction with other materials, deserve careful consideration. In fact, beside the biological rational and some promising findings, this study further underlines the importance for any biomaterial formulation to be tested in preclinical animal models for each specific tissue application, in order to provide empirical data and explore its potential as well as its limitations in vivo, before osteochondral regeneration can be targeted in the clinical practice.A 70:30 wt.% ratio collagen–chitosan blend was prepared and subjected to bio-inspired mineralization to obtain a hybrid magnesium-doped HA/(collagen and chitosan) composite (MgHA/(col+chit)), in the shape of a 3D scaffold .3PO4  solution (0.040 M) were added to the collagen and chitosan (70/30 wt.%) blend. This acidic gel was then dropped into a basic suspension obtained dispersing 1.20 g of Ca(OH)2  in 200 mL of distilled water supplemented with Mg2+ ions  to yield a composite material MgHA/(col+chit) with a theoretical ratio of 70:30 wt.% and with the aspect of a cream-colored gel. This process was carried out to induce the heterogeneous nucleation of apatite nano-nuclei presenting magnesium substitution.Two hundred and forty-four mL of HThe MgHA/(col+chit) gel was centrifuged until a dense gel was obtained, in order to achieve a 3D low-porous and hard construct, which was then poured into multi-well plates  and freeze-dried  at −40°C for complete water elimination and pore formation, in order to achieve a highly porous structure to allow high cell penetration and migration from the subchondral bone-like layer to the cartilaginous one. Only the bone layer was used for the medium-size animal study.Low molecular weight chitosan  was dissolved in a 0.5 M acetic acid solution to obtain a 1 wt.% chitosan concentration; 21.4 g of this solution was slowly mixed with 50 g of 1 wt.% type I collagen , suspended in acetic buffer . This suspension was then precipitated through a cofibration process by adding NaOH 0.5 M to increase the pH from 3.5 to 5.5. The formation of small fibrous aggregates of 70:30 wt.% col+chit blend was observed. The homogeneous precipitate was then washed three times with 300 mL of water, centrifuged, and freeze-dried.Both materials were treated with the cross-linking agent 1,4-butanediol diglycidyl ether ) through immersion for 48 h (24 h at room temperature followed by 24 h at 5 °C) in a BDDGE aqueous solution (2.5 mM), thus setting up a BDDGE:collagen ratio equal to 1 wt.%; they were then washed three times to remove any residual that did not react.® sheet  producing thin uniform layers that were interpenetrated to obtain monolithic bi-layer constructs and then freeze-dried (MgHA–collagen–chitosan). Finally, the monolithic scaffolds were gamma-sterilized at 25 Kgray before in vivo implantation.Both gels, mineralized and not mineralized, were spread on a MylarIn vivo studies were performed complying with the European and Italian Law on animal experiments, after the approval (N.0044454 date 27/12/2012 and N.0044451 date27/12/2012) of the research protocols by the Ethical Committee of Rizzoli Orthopedic Institute and by the responsible public authorities in agreement with EU regulations . All animals were purchased from authorized farms and submitted to a quarantine period before their utilization.Twelve adult male New Zealand rabbits , were housed in standard and controlled conditions  and fed with standard diet  and water ad libitum.® , under anesthesia. Femoral epiphyses were then processed for histological, histomorphometric and microtomographic evaluations.Animals underwent surgery under general anesthesia. The lateral distal femoral epiphysis was exposed through a 2 cm longitudinal skin incision and cylindrical critical size defects  were created bilaterally by using a drill. Scaffolds were implanted according to the group allocation: Group 1 received the RegenOss scaffold  and group 2 the MgHA/(col+chit) scaffold. Soft tissues were then sutured and routine antibiotics and analgesics were administered postoperatively. After 8 weeks, animals were euthanized with i.v. administration of Tanaxn = 2) and group 2 treated with the osteochondral MgHA–collagen–chitosan scaffold (n = 4). Surgery was conducted under general anesthesia with the animals in dorsal recumbence. The right medial and lateral condyles were exposed through a median skin incision and parapatellar joint approaches. A specifically designed drill was used to create a cylindrical osteochondral defect  in the weight-bearing area of both condyles. In group 2, grafts were implanted with a press-fit technique, while in the control group defects were left empty. Routine antibiotics and analgesics were administered postoperatively. All animals were euthanized 6 months after surgery by Tanax®  injection under general anesthesia. Stifle joints were carefully opened and macroscopically evaluated for signs of inflammation, such as tissue reddening, hypertrophy of the villous part of the synovial membrane, tissue adhesions, appearance of a fat pad, and clearness and color of the synovial fluid. The macroscopic appearance of the implants was blindly assessed using the modified Fortier [Six crossbred adult sheep, 65 ± 5 kg b.w. , were housed in single boxes in standard and controlled conditions  and fed with standard pellet diet  and water ad libitum. Animals were divided into 2 groups: Group 1 untreated  at a voltage of 50 kV, current of 500 μA, and with an aluminum filter of 0.5 mm.Each sample was rotated by 180° with a rotation step of 0.4° according to the following set up: Rabbit, average frame of 2, nominal resolution 9 μm; sheep, average frame of 3, nominal resolution 17.5 μm. The acquired images were reconstructed by the NRecon software (1.6.8.0) with corrections for alignment beam hardening and ring artefact reduction, and 3D models were created to allow visualizations of the samples in space.1: cylindrical VOI with diameter of 6 mm and height of 8 mm or diameter of 7 mm and height 5 mm for rabbits and sheep, respectively. These were then used to evaluate the material and the new bone formation into the defect. TV2: VOI of the trabecular bone of the condyle in the area around the defect with the same height of TV1 .Two specific volumes of interest (VOIs) were defined in each sample. TV1 (%), ratio between the volume of newly formed bone within the bone defect and the total volume of the bone defect;Defect BV/TV1;Defect Trabecular Thickness Tb.Th (mm), calculated in a model-independent way  over the−1), the number of intersections through a trabecular structure per unit length of a random linear path through TV1;Defect Trabecular Number Tb.N (mmDefect Trabecular Separation Tb.Sp (mm), calculated as the Tb.Th;2 (%), ratio between the volume of trabecular bone around the bone defect and the total volume of interest TV2;Peri-implant BV/TV2)  see .Rabbits: Femoral distal epiphyses were processed without prior decalcification. After fixation and dehydration, samples were embedded in poly-methylmetacrilate , then sectioned perpendicularly to the bone surface into sections (100 ± 10 μm thick) that were then grounded to a thickness of 15 ± 5 µm . Three sections for each sample were left unstained for oxytetracycline incorporation assay, evaluating mineral apposition rate , and bone formation rate (BFR/B.Pm μm2/μm/day). Three more sections were stained with Stevenel’s Blue and van Gieson Pichrofucsin for histological investigations [igations .Sheep: Each medial and lateral sample was cut in half along the central axis of the implant. One half was not decalcified and was processed for resin embedding; the other half was decalcified and embedded in paraffin. The latter samples were fixed in 10% formalin and decalcified in a nitric-formic acid solution. After decalcification, samples were dehydrated in a graded series of alcohols and then processed for paraffin embedding. Five-micrometer-thick sections were obtained with a Microm HM340E  and stained with hematoxylin and eosin.The non-decalcified specimens were first fixed in 4% paraformaldehyde, dehydrated in graded series of alcohols, and then embedded in polymethyl methacrylate . Blocks were sectioned along a plane parallel to the long axis of the osteochondral transplant . Sections were then grounded up to a thickness of 40 ± 10 µm , stained with Safranin-O/Fast Green, and finally evaluated histologically .The Pineda score was used for semiquantitative evaluation of the specimens .Sheep: Two sections of decalcified and paraffin-embedded samples were immunostained for col I, col II, and VEGF. After fixation, sections were extensively rinsed in PBS and permeabilized by incubation in 0.3% hydrogen peroxide in PBS solution. Sections to be immunostained were pre-treated for antigen unmasking with 0.2% Pronase  solution in PBS. Subsequently, 10% normal serum was added to block nonspecific antibody binding, and primary antibodies  were applied. After rinsing in PBS, slides were incubated with appropriate biotinylated secondary antibody and with horseradish peroxidase-streptavidin complex . Sample reaction was developed with 3,3-diaminobenzidine substrate and permanently mounted.p < 0.05. After having verified normal distribution and homogeneity of variance, the non-parametric Kruskal Wallis test, followed by U Mann–Whitney, was used to analyze the data.Statistical analysis was performed using IBM SPSS Statistics v.21. Data are reported as mean ± SD at a significance level of The hybrid MgHA–collagen–chitosan biomaterial used in this study clearly failed to promote bone and osteochondral regeneration in two different animal models. This underlines not only the challenges of regenerating the osteochondral unit, but also the necessity of in-depth in vivo testing before introducing newly developed, although promising, biomaterials into clinical use. Further studies are needed to explore the best formulation of chitosan-reinforced composites for bone and cartilage treatment.


Women with moderate and severe pain reported using pain relievers , paracetamol etc., p < 0.0005). Finally, activities affected by severe pain were class attendance (p = 0.01), personal studying (p < 0.0005), exercising (p < 0.0005), and socializing (p < 0.0005). Exam attendance (p = 0.27) and clinical placement attendance (p = 0.48) were not affected by severe dysmenorrhea. Dysmenorrhea has a high prevalence among nursing students and seems to affect important aspects of wellbeing and academic performance when the pain intensity is severe. The present findings lay the foundation for further investigation of dysmenorrhea both in the Greek population and cross-culturally.Dysmenorrhea (pain during menstruation) is one of the most common medical conditions among women of reproductive age. Dysmenorrhea has been studied around the world but not yet in Greece. The aim of the present study was to investigate the prevalence, characteristics, and impact of dysmenorrhea on the wellbeing  among nursing students in Greece. A cross-sectional study of 637 nursing students was conducted by administering a questionnaire at a university in Athens. The prevalence of dysmenorrhea was 89.2% and the rate of severe intensity was 52.5%. Factors that were associated with severe dysmenorrhea were family history ( Dysmenorrhea is one of the most common medical conditions and complaints voiced by women during their reproductive life. It is also a leading cause of absenteeism from school or college and work ,2. The GDysmenorrhea can occur as primary dysmenorrhea or secondary dysmenorrhea. Primary dysmenorrhea is characterized by pain during menstruation without organic lesion in pelvis, caused by increased endometrial prostaglandin production ,8. SeconPrevalence of primary dysmenorrhea varies from 45 to 95 percent globally . AlthougSome studies have evaluated health behavior factors, such as eating habits, smoking, physical activity, or lifestyle, which have been shown to be predictors of the painful menses e.g., ,16,17.It has been shown that dysmenorrhea, either primary or secondary, affects women’s daily living, influencing their productivity  ,19,20,21Although dysmenorrhea is a condition which is globally examined, there are no recent studies that have investigated this common female medical condition in Greece. Therefore, the aim of the present study was to assess the prevalence of dysmenorrhea among university nursing students in Greece, to describe symptoms and pain characteristics and to evaluate impact of dysmenorrhea on students’ wellbeing concerning academic performance, physical exercise, and social functioning.We conducted a descriptive cross-sectional study at the Nursing School Undergraduate Program of a university in Athens between May and October 2017. Female participants were recruited through convenience sampling among undergraduate nursing students during the academic semester. The inclusion criteria were a good knowledge of the Greek language and consent to participate in the study. Students that were on menopause or reported having a menstrual cycle duration of over 50 days were excluded from the analysis as oligomenorrhea might be indicative of transition to menopause   or attriWe designed and used a questionnaire of 40 items, to assess and determine the characteristics of dysmenorrhea. The questionnaire consisted of 3 parts. The first part included questions regarding demographic characteristics , health behaviors  and menstruation characteristics . The second part focused on the characteristics of menstrual pain including questions on its appearance, onset, intensity, areas of pain, other symptoms, mood during menstruation  and use of pain relievers. Pain intensity was assessed using a Visual Analogue Scale (VAS) which ranged from 0 cm  to 10 (worst possible pain). Scores were categorized as such: 1–3.9 mild; 4–7.9 moderate; 8–10 severe. Finally, the third part included questions on the impact of dysmenorrhea on nursing students’ academic life. The questionnaire was prepared by the authors of the study and was reviewed by an expert in gynecology who was not part of the research team.The present study was approved by the general committee of the university nursing department. Prior to participating, participants were informed on the aims of the study, assured of the confidentiality of their data, and given verbal consent according to the principles provided by the Declaration of Helsinki.2: underweight 18.5–24.9 kg/m2: normal weight, 25.0–29.9 kg/m2: overweight and ≥30.0 kg/m2: obese. To simplify analysis and meet the chi-squared test assumption of equal or more than 5 expected counts, we recoded the variables on the functioning impact as such: “never” and “a few times” were categorized as “less affected”, and “quite often” and “always” were categorized as “more affected”. Descriptive statistics  were used to assess prevalence of dysmenorrhea, demographic and clinical characteristics, menstrual pain, and menstruation characteristics, mean age and mean age of menarche. Categorical variables were presented as n(%) and quantitative variables as mean ± standard deviation. Associations between dysmenorrhea status and health and clinical characteristics and dysmenorrhea intensity and impact on academic life  were assessed using Pearson’s χ2 when the assumption of the chi-squared test (≥20% of the cells with less than 5 expected counts) was met and Fisher’s exact test when this assumption was violated. To further analyze the significant effects, we used the z test with a Bonferroni correction. The effect of dysmenorrhea intensity on the number of days being absent from school and on the number of pain relievers used were analyzed using Kruskal–Wallis tests. A value of p < 0.05 was considered statistically significant. To enter and analyze the data, we used the IBM SPSS Statistics for Windows Version 25.0 .The participants’ body mass indexes (BMIs) were calculated using the reported weight and height values and categorized as such: <18.5 kg/mOut of the 859 female students studying nursing, a total of 637 women (74.16%) agreed to participate in the study. However, 6 of them reported having a menstrual cycle duration of over 50 days so they were excluded from the analysis yielding several 631 participants. 2 . 65% of the students had a normal weight whereas 14.8% and 6.6% were overweight and obese, respectively.Regarding health, 32.2% of the participants reported smoking, 54% of them reported exercising regularly and 59.2% reported following a healthy diet. The mean BMI was 22.66 ± 4.33 kg/mFinally, regarding menstruation characteristics, the mean menarche age was 12.74 ± 1.57 years, the mean duration of menstruation was 5.85 ± 1.23 and the average menstrual cycle duration was 28.76 ± 3.4 days.The prevalence of menstrual pain experience in the present sample was 89.2% (563 out of 631 women). p > 0.1). There was a marginal effect of duration of menstruation on dysmenorrhea status (p = 0.051). Z tests with Bonferroni correction revealed no significant differences among the groups. However, it revealed a significant difference between the < 7 days and 7 ≥ days of menstruation on the severe pain group (p < 0.05). The count of the 7 ≥days subgroup was higher than the expected count  whereas the count of the < 7 days subgroup was lower than the expected count  suggesting a possible association of menstruation duration over 7 days and pain severity.The prevalence of dysmenorrhea severity across the clinical and menstrual characteristics was also assessed . There wp < 0.05). Z tests with Bonferroni correction on healthy nutrition showed no significant differences among groups on nutrition. Post-hoc Z tests on family history revealed that the severe group tended to report family history of dysmenorrhea more frequently  than the no pain group  (p < 0.05) but no other differences among groups were noticed. Post-hoc comparisons on menarche age revealed that in the subgroup that reported experiencing their first menstruation at 13–15 years, there was a statistically significant difference between the severe pain and no pain groups (p < 0.05) in which the count of the severe group was lower than the expected count  whereas the count of the no pain group was higher than the expected one . No other differences across dysmenorrhea groups were noticed. However, regarding the differences across the menarche age subgroups, in the severe pain group, there was a statistically significant difference between the ≤12 age and the 13–15 age subgroups, where the count of the first subgroup was higher than the expected count  and the count of the second subgroup was lower than expected  showing that more participants of the severe group reported experiencing their first menstruation at age younger than 13 years old than experiencing at older ages. Finally, multiple comparisons on the age effect revealed that in the over 40 years subgroup, the severe group differed significantly with a lower frequency than the expected one  from the no pain group  (p < 0.05). Moreover, in the no pain group, the over 40 years subgroup had a higher count than the expected  relatively to what was observed in both the 18–20 years  and the 21–29 age subgroups  (p <.05). This analysis suggests that in the present sample women over 40 years old experience less frequently severe menstrual pain than women younger than 30 years old. No other differences were yielded regarding participants’ age.There were significant effects of healthy nutrition, family history of dysmenorrhea, menarche age, and participants’ age (p = 0.43). However, there were significant differences between the groups on pain duration, the use of pain relievers and the number of pain relievers received (p < 0.05). Post-hoc comparisons in the pain duration revealed that for the less than 1 day pain duration subgroup, the mild pain group had a significantly higher percentage  than both the moderate pain  and the severe pain groups  (p < 0.05). For the 1–3 days pain duration, the mild pain group showed a significantly smaller percentage  than both the moderate pain  and the severe pain groups  (p < 0.05). Moreover, in the mild pain group, significantly more participants reported a pain duration of less than 1 day than a duration of 1–3 days or more than 4 days (p < 0.05). In the severe pain group, women reported significantly more frequently than expected a pain duration of 1–3 days and over 4 days  relatively to reporting a less than 1 day of pain duration (p < 0.05). This showed that women with moderate and severe pain tend to report a longer pain duration than women with mild menstrual pain.Characteristics of pain across dysmenorrhea status groups are shown in z = 8.1) than in the mild pain  and the moderate pain groups . Similarly, the mild pain group reported a significantly less frequent pain relievers use than the moderate pain group (p < 0.05). Post-hoc p correcting testing on the number of different pain relievers used during menstruation also revealed than the severe pain group reported using more different pain relievers in combination than the mild pain  and moderate pain groups . The moderate pain group also reported receiving more such drugs than the mild pain group .Multiple comparisons on pain relievers use, showed that a significantly higher percentage of participants in the severe pain group reported using pain relievers . Post-hoc testing revealed that significantly more women with severe pain reported never experiencing a time period with no pain  than women with either mild  or moderate menstrual pain . More women with moderate pain reported experiencing a pain free time period for a few times  than women with mild  and severe pain . However, significantly more women with mild pain reported having pain free time periods quite frequently  than women with either moderate  or severe pain .Finally, there was a significant association between time periods during menstruation with no pain and dysmenorrhea intensity (p > 0.1). However, there was a marginal effect of dysmenorrhea on failing a module due to menstrual pain (p = 0.08). Z tests with Bonferroni correction revealed no significant differences among the groups. However, it revealed a significant difference between the less affected and more affected subgroups on the severe pain group where the count of the more affected subgroup was higher than the expected count  whereas the count of the less affected subgroup was lower than the expected count  suggesting a possible impact of severe pain on exam performance.p < 0.05). Multiple comparisons on absenteeism showed that menstrual pain affected attending school more in the severe pain group  than in the moderate pain group . Post-hoc testing on the duration of absenteeism showed that severe pain group reported being absent from school more days than both the moderate pain group  and the mild pain group . Multiple comparisons on personal studying variable revealed that more women with severe pain reported stopping studying due to menstrual pain  than women with either mild  or moderate pain . Similarly, regarding exercising, the severe pain group reported significantly more frequently postponing physical exercising  than both the mild pain  and the moderate pain groups . Finally, the severe pain group also reported impact of dysmenorrhea on socializing significantly more frequently  than the mild pain  and the moderate pain groups .However, there were significant effects of dysmenorrhea intensity on absenteeism from school, Days being absent from school, postponing personal studying, postponing exercising, and refraining from socializing  in the present study was high 89.2%) as most students reported experiencing it. To the best of our knowledge, this is the first study measuring the prevalence of dysmenorrhea in women residing in Greece. A wide range of dysmenorrhea prevalence across different countries has been reported previously ranging between 16–91% % as most,26. ThisThe lower abdomen was reported as the most common pain region in our sample (61.2%) accompanied by other menstrual symptoms such as swollen breast (24.1%), swollen abdomen (21.6%) and headache (13.3%). Regarding mood during menstruation, almost half of the participants with dysmenorrhea reported experiencing anger during menstruation (43.8%) whereas 24.8% reported feeling depressed. These symptoms were similar to what was reported previously e.g., ,27,28. GThe effect of various clinical and menstrual characteristics that have been linked with dysmenorrhea previously was also investigated in the present study. We did not find differences in dysmenorrhea status (pain intensity and no pain) depended on smoking, exercising, BMI and menstrual cycle frequency. Previous research have shown a higher risk for dysmenorrhea in women with lower BMI, smoking behavior and a longer cycle but a lower risk in women that exercise regularly . HoweverNevertheless, we found associations between family history of dysmenorrhea, menarche age, duration of menstruation, participants’ age, and dysmenorrhea status. An association between family history and dysmenorrhea has also been shown previously e.g., ,19,29,30We also showed that there were more participants with severe pain that they menstruate for over 7 days than expected suggesting a possible association between longer menstruation and severe pain. We did not find differences across the other pain severity groups. Previous findings in this association are contradictory. Previous studies showed that longer duration of menstrual flow (over 6–7 days) was risk factor for dysmenorrhea ,34 and tThe use of pain relievers was also assessed in the present study. Most of the students with dysmenorrhea reported receiving pain relievers to manage their menstrual pain (71.4%). More participants with severe pain (86.3%) reported using pain relievers than participants with moderate (63.4%) and mild pain (36.6%). Participants with moderate pain also reported using pain relievers at higher rate than those with mild pain. High rates of pharmacologic pain relief has also been reported previously in Turkey e.g., ,29, BrazRegarding the impact of dysmenorrhea on functioning and wellbeing of the students during menstruation, we showed that severe pain negatively affected attendance of classes, personal studying, exercising, and socializing during menstruation. In all these variables, participants with severe pain significantly reported more negative impact than those with moderate or mild pain. Similar findings have also been observed in previous studies e.g., ,26,29,34Avoidance of exercise due to menstrual pain is counterproductive. Exercise has been shown to increase circulating endorphin levels  and haveRefraining from socializing due to severe menstrual pain alongside psychological symptoms may contribute to the decreased quality of life during menstruation. Given that perceived social support has been shown to moderate the association between psychological symptoms and menstrual pain , decreasAs it was reported above, severe dysmenorrhea was associated with postponing personal studying. We also find a marginal effect of dysmenorrhea on exam performance in which more than expected students with severe dysmenorrhea reported having failed an exam due to the menstrual pain. Given that previously women with dysmenorrhea have reported experiencing decreased concentration e.g., while attending class due to their menstrual pain ,43,44, iThe present study was not free of limitations. Firstly, as it aimed to investigate the prevalence of dysmenorrhea generally in a sample of nursing students in Greece, there was no distinction between primary and secondary dysmenorrhea. Future studies in Greece could investigate the prevalence of these types of dysmenorrhea separately. Moreover, as the sample was limited to nursing students in an Athenian University, generalizability of the present results is probably limited. Given that students of health sciences might be more health conscious than other students, the present results might not generalize in women with no such training. Another limitation had to do with the cross-sectional nature of the present study. Given that pain intensity and impact in everyday activities might not be stable overtime and symptoms may improve across time , a futurThe present study showed a high prevalence of dysmenorrhea in a sample of women residing in Greece. It also showed some factors being associated with mostly severe dysmenorrhea, menstrual pain characteristics varying across the dysmenorrhea intensities, and impact of this condition in wellbeing . The present results highlight the need for implementing and testing the efficacy of pain management strategies in Greece and conducting further studies to better understand the prevalence, characteristics, correlates, and impact of dysmenorrhea in Greek women.


Identify the socio-economic correlates of sugar sweetened beverage (SSB) consumption among pregnant women and analyze to what extent SSB consumption is associated with diet quality and total energy intake. Additionally, we aim to predict how diet quality scores and totally energy intakes would change if SSB consumption was artificially set to 0.Repeated Cross Sectional Study.United States.SSB consumption was estimated from 1–2 24-hour dietary recalls from 1,154 pregnant women who participated in the 1999–2006 National Health and Nutrition Examination Survey.Linear regression models were used to identify socioeconomic and demographic factors associated with SSB consumption and to assess the associations between SSB consumption and diet quality and total energy intake. Diet quality was measured with the Alternate Healthy Eating Index modified for Pregnancy (AHEI-P).The mean SSB intake was 1.3 servings per day (sd 1.5). Having a household income ≤100% of the Federal Poverty Level, being born in the United States, and not being married or living with a partner were positively associated with SSB consumption. Every 12 oz. of SSBs consumed was associated with a 2.3 lower AHEI-P score  and the consumption of 124 more calories , after adjusting for age, country of birth, race/ethnicity, educational attainment, marital status, household income, survey year and day/s of the week the recall/s were collected. Our predictive models indicated that average AHEI-P would be 6.4  higher and average total energy intakes would be 203.5 calories  lower if SSB intake was set to 0.SSB consumption is associated with poorer diet quality and higher total energy intake among pregnant women. Sugar sweetened beverages (SSBs) are the single largest contributor of added sugars to the American diet, accounting for 45% of all added sugars consumed . SSBs inAmerican pregnant women have diets that need improvement , and appThis cross-sectional study used 1999–2006 data from NHANES; an ongoing multistage survey administered by the National Center for Health Statistics that selects a nationally representative sample of the noninstitutionalized U.S. civilian population . The datThe Alternate Healthy Eating Index modified for Pregnancy (AHEI-P) was used to measure diet quality from 1–2 24 hour dietary recalls . From 19We created a categorical SSB intake variable for the number of 12 oz. servings consumed per day  to describe the distribution of SSB consumption in the sample and determine how diet quality and total energy intake varied across different levels of SSB consumption. These values were chosen because they are easy to interpret, and values were well-distributed across the categories. Both energy-adjusted and unadjusted measures of SSB intake were examined in the analysis. Energy-unadjusted SSB intakes were used when we measured an association between SSB consumption and total energy intake, to avoid adjusting for the outcome of interest. Energy-adjusted SSB intakes were used when we measured an association between SSB consumption and AHEI-P. SSB intake was adjusted for total energy intake using the residual method .st, 2nd, 3rd) were considered potential confounders. The day/s of the week the recall/s were collected  was included in all models to reduce the variability in SSB consumption, diet quality, and total energy intake. Household food security status was derived from the 18 question United States Department of Agriculture’s Standard Food Security Survey Module [Age , educational attainment , high school diploma or GED, some college or Associates Degree (AA) degree, college degree)), race/ethnicity , household income , >100% & ≤200%, >200% & ≤300%, >300%), and survey year  were considered a priori to be included as confounders in the associations between SSB consumption and diet quality and total energy intake because previous research indicated these variables were associated with SSB consumption in pregnancy and total energy intake and diet quality , 24–27. y Module .Total energy intake was measured in calories and was square-root transformed to improve normality. All measures of total energy intake reported in the manuscript were transformed back to represent calories. To ensure the sample was nationally representative, dietary sampling weights were recalculated to reflect the probability of being sampled in the 8 year period .Random effect models were implemented to identify the proportion of the total variance due to between-person variability for the SSB intakes. For the descriptive statistics, the standard deviations reflect only the between-person variability in the estimates; within-person variation due to having multiple recalls has been partitioned out.For descriptive analysis, F tests were conducted to assess the associations between SSB consumption and each variable. Wald tests were conducted on linear regression coefficients when categorical variables were assessed. To determine which of the socio-economic correlates were significantly associated with SSB consumption when all variables were considered, we conducted a linear regression that included age, country of birth, race/ethnicity, educational attainment, marital status, household income, survey year and day/s of the week the recall/s were collected. Additionally, a sensitivity analysis was conducted that included this set of variables and those not associated with SSB intake in bivariate analysis; household size, number of live births a mother had had previously, trimester, and household food security status. Similarly, linear regressions were conducted to assess the relationship between SSB consumption and AHEI-P and total energy intake and adjusted for this set of confounders.The models implemented to analyze the extent to which SSB consumption was associated with diet quality and total energy intake were also applied to generate the population average diet quality and total energy intake values that would result from removing SSBs from the population’s diet. After analyzing the associations between SSB consumption and diet quality and SSB consumption and total energy intake in separate linear regression models, these same models were applied to generate predicted diet quality scores and total energy intakes for each individual based on their covariate profiles, and assuming they did not consume SSBs. The diet quality and energy intakes that were generated by these models under the assumption that no SSBs were consumed were then compared to the observed population average intakes.STATA  was used for all statistical analysis. This study was considered exempt from review by the University of California Berkeley’s Institutional Review Board.Table 1 describes the study sample and the relationships between unadjusted SSB consumption and the covariates. On average, women were 27 ± 6 years of age. Fifty-three percent of the sample was non-Hispanic White, 17% were Mexican American, 17% were non-Hispanic Black, 5% were Other Latino and 8% were of another race or multicultural. Seventy-eight percent of the women were born in the U.S. and 72% were married or living with a partner. Pregnant women in the United States consumed an average of 1.3 ± 1.5 12 ounce unadjusted servings of SSBs per day, or 15.6 oz. SSB consumption declined from 1999–2006 from 1.4 (95% CI: 1.1–1.8) to 1.1 (0.8–1.4) servings per day  and an average total energy intake of 2,072 calories . In contrast, those who consumed 2 or more 12 oz. servings of SSBs had a lower average AHEI-P score of 39.7 (37.6–41.8), and a higher average total energy intake of 2,492 calories  (p<0.001).The average AHEI-P score was 44.5 (43.3–45.7) and the average total energy intake was 2,272 calories . Table 3). Marital status was no longer a significant predictor of SSB consumption in our sensitivity analysis, when variables not associated with SSB consumption in bivariate analysis were included in the model; household size, number of live births a mother had previously, trimester, and household food security status.Our first aim was to identify the socio-economic correlates of SSB consumption. Our linear regression analysis found having an income >300% of the federal poverty level compared to having an income ≤100% of the FPL was negatively associated with SSB consumption (p ≤ 0.05), as was being born outside of the U.S. compared to being born in the U.S. and being married or living with a partner compared to being single, widowed, divorced, or separated (Table 4). The linear regression model analyzing the association between SSB consumption and total energy intake showed that consuming an additional 12 oz. of SSBs was associated with consuming an additional 124 calories per day (85–163), adjusting for potential confounders (Table 4).Our second aim was to assess the relationship between SSB’s and AHEI-P and total energy intake. Consuming an additional 12 oz. of SSBs was associated with a 2.3 point lower AHEI-P score , adjusting for potential confounders (Table 4).Finally, our last aim was to investigate how caloric intake and diet quality would change if all pregnant women did not consume SSBs. Compared to the observed average values of diet quality and total energy intake, when we set the model to reflect no SSB consumption, the average diet quality score was 6.4 points higher (5.4–7.6) and the average total energy intake was 203.5 calories lower   per day, which is about 2.6 oz. less than observed in this sample . AlthougIncome, country of birth and marital status were found to be predictive of SSB consumption. Our finding that having a lower income predicts higher SSB consumption is consistent with past research , 25, 33,Consuming an additional 12 oz. SSB was associated with consuming an additional 124 85–163) calories per day, which is consistent with past research showing consumption of SSBs may lead to consuming extra calories 3 calorie. SimilarWe observed an average AHEI-P score of 44.5 , which is indicative of a diet that needs improvement. Consuming an additional 12 oz. serving of SSBs was associated with a 2.3 point lower AHEI-P score , which is disconcerting given past research has shown a 5 point improvement in AHEI-P is associated with lower blood glucose levels and lower odds of having preeclampsia during pregnancy . There iTo further understand the extent to which SSB consumption is associated with poor diet quality and higher total energy intake, we estimated the difference in diet quality and total energy intake by creating a prediction model using our regression coefficients for high and low levels of SSB intake. If pregnant women who currently consume SSBs no longer consumed any SSBs, the average AHEI-P score of the population would be substantially improved and the caloric intake would be lower. Although the changes in diet quality and total energy intake are significant, these models assume that the changes in SSB intake occur while no other dietary or lifestyle changes are made, which is an unverifiable assumption. Additionally, it is difficult to contextualize the lower total energy intake associated with not consuming SSBs because we do not have information on the women’s pre-pregnancy body mass indices, and therefore do not know which women are on pace to gain excessive weight. However, if we assume that women who are gaining excessive weight consume SSBs and that lower total energy intakes lead to smaller gestational weight gain, then our results suggest a decrease in SSB consumption may lead to a reduced prevalence of excessive gestational weight gain. Observing a significant negative association between SSB consumption and diet quality, and a significant positive association between SSB consumption and total energy intake supports the hypothesis that reducing SSB consumption during pregnancy may help lead to appropriate weight gain and better health outcomes.NHANES is a cross-sectional study, which prevents determining causality. Social desirability bias may have influenced women to report lower intakes of SSB consumption, especially among women with higher BMI’s . This maDuring pregnancy, income, country of birth and marital status are associated with SSB intake. A greater intake of SSBs during pregnancy was associated with poorer diet quality and greater total energy intakes. When setting the population SSB consumption to 0, we observed the average AHEI-P to be significantly higher and the average total energy intake to be significantly lower. Future research should continue to assess the correlates of SSB consumption during pregnancy so we can better identify high consumer populations to tailor public health interventions.S1 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.