Homo sapiens dispersal and resilience in a mosaic of environments that were affected by significant climate changes.Documenting the subsistence strategies developed by early modern humans is relevant for understanding the success of their dispersal throughout Eurasia. Today, we know that there was not a single colonization event and that the process was progressive while coping with the MIS3 abrupt climatic oscillations. Modern humans expanded into the continent by adapting to different topographic situations and by exploiting resources in diverse ecological niches. The northern part of Italy is one of the first European regions where early modern humans are documented. Here, we present the subsistence regimen adopted by the Protoaurignacian groups in two different levels in Fumane Cave based on archaeozoological data. New radiocarbon dates confirm an overlap between Uluzzian and Protoaurignacian occupations, around 42 and 41,000 cal BP, and reveal that modern humans occupied the cave from GI10 to GS9, the last level coinciding with the Heinrich Event 4. The data indicate seasonal site occupations during late spring/summer and that prey exploitation was focused mostly on ibex and chamois, killed in nearby areas. The whole faunal assemblage suggests the presence of early modern humans in a cold environment with mostly open landscapes and patchy woodlands. The estimation of net primary productivity (NPP) in Fumane, compared with other contemporaneous Italian sites, reflects how the NPP fluctuations in the Prealpine area, where Fumane is located, affected the biotic resources in contrast to known Mediterranean sites. From a pan-European perspective, the spatiotemporal fluctuation of the NPP versus the subsistence strategies adopted by Protoaurignacian groups in the continent supports rapid  Current evidence of human remains found in Grotte Mandrin in France may attest to a much earlier presence of H. sapiens in SW Europe between 56.8 and 51.7 ka cal BP4, followed by Bacho Kiro in Bulgaria6, Grotta del Cavallo, Riparo Bombrini and Grotta di Fumane in Italy8 between ~ 45 to 41 ka cal BP. This combined cultural and biological process ultimately resulted in the gradual replacement of the late Neanderthal populations of the entire continent18. During the earliest Upper Palaeolithic, the AMH populations, coming from the Levant, progressively settled in the various areas of the continent following both the Mediterranean and Centro-European routes13. This process is characterized by the appearance of new cultural traits, such as symbolic material including artworks and personal ornaments24, bone and antler technology29, found across a variety of cultural technocomplexes such as the Uluzzian, Neronian, Bachokirian, Bohunician, LJR, Early Ahmarian/Kozarnikian/Protoaurignacian, etc.33. The Aurignacian is one of these first technocomplexes attributed to the spread of AMH from east to west39, dated to around 43–42 ka cal BP47. Within the Aurignacian, the earliest phase is known as the Protoaurignacian, most frequent in the Mediterranean region, while the Early Aurignacian is spread across the continental areas49. Differences in chronology, technology and human adaptative systems between the Proto- and Early Aurignacian, played out against the background of deterioration of the environment at the onset of Heinrich Event 4 (HE4), are still a matter of debate54. While Banks et al.50 proposed that the EA started at the onset of HE4, immediately after the PA , other studies have pointed out that some PA assemblages are dominated by cold steppe animal species and, for this reason, this technocomplex was still present during the HE4 in some regions, such as central and southwestern France55.Recent studies about the dispersal of Anatomically Modern Humans (AMH) across Western Eurasia reformulate prevailing ideas, especially those based on their chronological expansion and arrival in different continental areas and how colonization was influenced or motivated by the climate and broader environmental conditions that AMH had to face56, rather than divergence in hunting strategies or dietary shifts67. However, documenting the subsistence strategies employed by early modern humans in their continental dispersal is key to understanding their success. Not only detailed information on the archaeozoology of faunal assemblages, but also high-resolution chronological, subsistence and Net Primary Productivity data are thus needed to provide accurate insights on human-environmental interactions during the early phases of modern humans on a regional scale.However, while in the last decade intensive work has been done to disentangle the main technological features of the Aurignacian dispersal and its complex synchronic and diachronic variability, few studies exist about the subsistence and the ecological settings and biodiversity exploited by the makers of the new technology. Research has mainly focused on the subsistence differences between Neanderthals and modern humans. On a broader scale, scholars agree that there was no significant change in the range of hunted taxa or differences in the abundance of the species by modern humans, remarking that economic diversifications can be ascribed to environmental changes that led to fundamental socio-economic developments68  to provide, for the first time, a detailed understanding of the subsistence and ecosystems exploited by modern humans in this region. Thirdly, the calculation of Net Primary Productivity (NPP) is undertaken to compare how this is correlated with the ungulate species exploited at Fumane throughout time and, also, to compare it with the NPP of other Italian Protoaurignacian sites. Finally, the archaeozoological and ecological data from other European early Upper Palaeolithic macrofaunal assemblages have been correlated with the NPP in each site to address the spatio-temporal ecological niches and prey consumed from the Black Sea to Atlantic Portugal.Fumane Cave, in north-eastern Italy, contains a long, amply dated and well-established stratigraphic sequence that includes Mousterian, Uluzzian and Protoaurignacian levels, thus making it a key site for deciphering the Middle to the Upper Palaeolithic transition in south-central Europe68 Fig. , Fig. S171; Uluzzian with A374; Protoaurignacian with A2–A1, late Protoaurignacian with D6 and D376, and D1d with early Gravettian77. In the Upper Palaeolithic sequence, numerous lithic and bone artefacts, dwelling structures with hearths and a toss zone were identified , while the Uluzzian seems to have ended shortly after 42 ka cal BP72. In this study, two new radiocarbon dates from A1 and D3 and two from A3, using ultrafiltration protocols on human-modified animal bone, were undertaken to reinforce the chronology of the early Upper Palaeolithic levels located at the entrance of the present-day cave.Currently available radiocarbon dates for the Upper Palaeolithic levels were obtained on ultra-filtered animal bone collagen and ABOx-treated charcoal samplesAlthough Protoaurignacian levels A2 and A1 were excavated as single units, here they are grouped because A1 is a thin anthropogenic level in the cave entrance with horizontal bedding that is indistinguishable from A2 in innermost excavated zone. A2 thus extends throughout the whole present-day cave. The larger faunal remains were recovered and plotted individually and specimens smaller than 3 cm were recovered in general bags from each 33 × 33 cm sub-square after wet-sieving the sediments with 2 mm mesh.80, specimens related to some anthropogenic structures were analysed by Broglio et al.81. Preliminary analyses of cementochronology on ungulates were performed by Facciolo et al.82, and taxonomical determinations of bone retouchers were carried out by Jéquier et al.83. Here we provide, a complete archaeozoological and taphonomical assessment for the entire sequence of these levels, considering previous analyses and providing new data about the subsistence patterns and the ecosystems exploited by the modern human groups.The archaeozoological and taphonomic analyses of units A2–A1 (Protoaurignacian) and D3 (late Protoaurignacian) are presented here. The macrofaunal material was recovered from a 16 sq m area documented in the cave entrance and near the combustion features found during the excavations between 1991 to 2006 Fig. . We haveCapra ibex, Rupicapra rupicapra, Capreolus capreolus, Canis lupus); size 4, medium/large mammals between 100 and 300 kg ; size 5, large mammals of > 300 kg . When species determination was undistinguishable, elements were reported to their family  or genus  level.The studied material was recorded using a database purposely designed for the Subsilience project. Taxonomy was determined using the osteological comparative collection based at the EvoAdapta Laboratory of the University of Cantabria. Due to the high fragmentation and the lack of any distinctive landmark, taxonomically unidentifiable specimens were grouped according to their body size into five mammals categories: size 1: smaller than rabbit; size 2, small mammals weighing < 20 kg ; size 3, medium mammals between 20 and 100 kg 88 by the MNI. The assemblage diversity was examined through the Inverse of Simpson’s Index (1/D)89. The higher the value, the wider the diet breadth90. For regional and European comparison of hunting preferences, the Inverse of Simpson’s Index with NISP and MNI was estimated, whenever possible. The ratio of high to low prey ranks to evaluate the selection of the prey hunted was also calculated. Among the determined species at Fumane, roe deer and chamois were considered low‐ranked species, while the rest of the ungulate preys were considered to be high‐ranked , in accordance with previous studies by Marín-Arroyo91, here adapted to the specific case of Fumane.The assemblage was quantified by applying the following indices: Number of Remains (NR), Number of Identified Specimens (NISP), Minimum Number of Individuals (MNI), Minimum Number of Skeletal Elements (MNE) and Minimum Animal Units (MAU) following Marín-Arroyoone Fig. . The rep92, Mariezkurrena93 and Tomé and Vigne94 were followed. For caprids, Couturier95, Habermehl96 and Pflieger97 were used. Once the age of death was determined, individuals were grouped into five age groups: F ; J (Juvenile—light worn on deciduous teeth and erupted M1); SAd (sub-Adult—moderate worn on deciduous teeth and erupted M2); Ad ; S (old Adult or Senile—advanced wear). The ratio between juvenile and adult individuals was estimated to measure the pressure on low-return younger prey and the exploitation prey type.The ungulate mortality pattern was assessed by both dental eruption and wear stage and bone fusion. For cervids, the studies by Azorit98), FUI , CFUI (Corrected Food Utility Index—Morin and Ready100) were undertaken. %MAU and Maximum Bone Density101 was applied to assess the influence of attrition in the faunal assemblage. To determine if the bone breakage patterns were the result of marrow extraction, correlations between %MAU and the Marrow Index98 were calculated. In addition, %MAU of high survival skeletal elements was correlated with the unsaturated marrow index (UMI) proposed by Morin102. Spearman’s correlation coefficient was used to test the quantitative validity of these correlations and determine the level of statistical significance (p) thereof. Furthermore, to overcome the limitations of previous indices on evaluating the skeletal profiles and attrition at the site, a Bayesian method based on a Monte Carlo Markov Chain sampling that uses the available skeletal information to constrain the possible degrees of attrition and carcass processing strategies was applied103. This method considers two parameters, alpha (α) and beta (β), that are active during the assemblage formation and history, α informs about the transport preference based on butchering efficiency in skeletal elements and it can take any value between − 1  and 1 (> appendicular contribution). The degree of attrition (β), follows the definition established by Rogers105, which related the survivorship of bone elements to their maximum bone density. This method aims specifically to overcome the problem of equifinality in skeletal profile interpretations, analysing two factors simultaneously106.To evaluate the carcass exploitation and transport type, bivariate correlations between %MAU and different indices such as MGUI , hammerstone percussion marks , type and angle of fracturing ; impact flakes (positive flake scars); adhering flakes, percussion pits and crushing marks118. According to diagnostic characteristics, impact flakes were defined and distinguished from those made by carnivores119: ventral face with the point of detachments and bulb; greater breadth than length; absence or reduction of the cortical surface. Thermoalterations colour degrees were recorded following Stiner et al.120. The study of bone retouchers was carried out following Mallye121. Carnivore activity was classified in pits, scores, punctures, furrowing, gnawing, crenulated edge and corrosion of gastric acid according to Blumenschine122, Domínguez-Rodrigo and Piqueras123, Domínguez-Rodrigo and Barba124. Other biological and physicochemical alterations, such as weathering125, root etching, insect/fungus activity, carbonate deposits, polishing128, or formation of mineral coatings  was examined under a LEICA S8 APO stereoscope with 10× eyepieces in search of bone surface biostratinomic and diagenetic alterations, such as cut marks  and the existence of insurmountable barriers. Besides, to define the preferred biotopes for plain and mountain species within the boundaries determined by the catchment area, a threshold value of 30% slope was fixed to differentiate areas related to one or the other group of faunal taxa. Beyond that value, grazing suitability is depleted140. In the current study, NPP was estimated over an area of 50 km2 around the Fumane cave and in the surrounding area of 26 European archaeological sites with dated Protoaurignacian, Early Aurignacian and early Upper Palaeolithic techno-complexes associated with faunal remains. NPP was estimated with the Lund-Postdam-Jena General Ecosystem Simulator (LPJ-GUESS) v.4.0. LPJ-GUESS is a coupled biogeography-biogeochemistry model which incorporates process-based representations of terrestrial vegetation dynamics at regional and global scales141. Vegetation composition in the model results from growth and competition for light, space and soil resources among plant communities according to specific photosynthetic rates, stomatal conductance, phenology, allometric calculations, and carbon and nutrient allocation according to specific Plant Functional Types (PFTs)142. PFTs are a group of species with similar bioclimatic limits, morphology, phenology, photosynthetic pathway and life history strategy143. Different PFTs can be incorporated and simulated in LPJ-GUESS according to their specific functional traits; in the current study, we used the standard global PFT set described in Smith et al.142 with the ‘cohort’ mode. The model was run in 100 replicate patches with an area of 0.1 ha, and we did not use nitrogen limitation because nitrogen deposition is unknown for the Pleistocene144. For details on the global parameters used, see Table Net primary productivity (NPP) is the biomass per unit of land surface and time of all autotroph organisms. Therefore, NPP represents the base of the food chain for all terrestrial ecosystems−2), and rainy days (days/month). These input data were obtained from the HadCM3B-M2.1 general circulation model145 after performing a delta bias correction with the CRU v.4 datasets146. We used the HadCM3B-M2.1 general circulation model because previous studies compared this palaeoclimatic model with the palynological record obtained from different European regions, providing evidence for the accuracy and reliability of these climatic simulations in the Northern Hemisphere148. The model also used, as input data, the atmospheric carbon dioxide concentration (ppm) obtained from Lüthi et al.149 and the specific soil classes obtained from Zobler150. All simulations were initialised with ‘bare ground’ conditions, and the model was spun up for 500 years until the simulated vegetation was in approximate equilibrium. This spin-up phase used palaeoclimatic conditions between 55 and 54.5 ka BP145. After that, the model was run at a monthly resolution spanning between 54.5 and 30 ka BP. The NPP of the Protoaurignacian units of Fumane were compared with the other archaeological Mousterian and Uluzzian units of Fumane with a Mann–Whitney–Wilcoxon test. To assess whether fluctuations in the estimated NPP affected the species diversity recovered in each archaeological unit of Fumane, a Spearman’s rank correlation test was used.The input climate variables for LPJ-GUESS are monthly temperature (°C), precipitation (mm/month), incoming shortwave radiation  and A2–A1 and D3 units and, with start and end Boundaries. The difference between the probability density functions of the start and end boundaries was also calculated to estimate the likely duration of the phase. All radiocarbon determinations were given a 5% prior likelihood of being an outlier within the General t-type Outlier Model151, so that the model could test their reliability. Convergence was greater than 95% and the model agreement index was 81   appear occasionally in both levels. Among the carnivores, the diversity of species is represented by a few remains of each species. Canis lupus and Vulpes vulpes are the most common species, followed by an element each of Lynx lynx, Ursus sp. and Martes foina along the sequence. Leporids appear exclusively in A2–A1 Unit. Several bird elements are represented in both units, mostly in A2–A1.Among the herbivores, 159. Although carnivore activities were documented, it was in a limited percentage in comparison with the anthropogenic activity.The taphonomic study is key for discerning the origin of the deposit and the main bone accumulators. Previous studies confirmed that mammals, and especially ungulates, recovered in the Mousterian and Uluzzian units at Fumane were mainly brought to the site by humans attested by the significant amount of butchering marks, fresh bone breakage, thermoalteration and skeletal profiles representation of the herbivores consumed160, and also on meat-bearing anatomical portions162.In both Protoaurignacian occupations, anthropogenic modifications were identified on herbivores, including butchering activities, deliberate breakage activities (percussion marks—mainly impact flake—and percussion notches) and thermoalterations Table . Fresh bAmong the complete assemblage, 37% and 24% in A2–A1 and D3 units, respectively showed thermoalteration and different burning categories from brown to black colouration were recognised , unidentified remains on long bone shafts and just a few on flat bones and epiphysis. Any other taxa do not bear carnivore bone modifications Table . Pits, sCervus elaphus and medium-size such as Capra ibex, Rupicapra rupicapra and Capreolus capreolus played an essential role in the diet of the human groups at that time. In terms of MNI, during A2–A1 unit, ibex, red deer and chamois, are the most exploited species with 38%, 21% and 17%, respectively, followed by limited roles of aurochs, roe deer and giant deer, with a total of 8%, while during D3 unit occupations, red deer decreases significantly to 7% and roe deer increases up to 20%. Both montane caprine species continue playing the most significant role in the human diet, with a slight decrease in ibex (27%) and a remarkable increase in chamois (33%). Giant deer maintains its representation with 7% of the ungulates  than in unit A2–A1 (4.3) and both values likely reflect the exploitation of a high-relief, mosaic landscape surrounding the cave. The NISP values for D3 (3.47) is higher than for A2–A1 (2.44), which could be partially influenced by the assemblage fragmentation, or other factors such as the duration of occupation at that time, seasonality or human group size, among other factors.82.During both units, there is a predominance of prime-age individuals 54 and 53%), followed by juveniles (46 and 47%) in A2–A1 and D3 units, respectively  was applied to disentangle the prey’s transport and the attrition that occurred at each unit. This new method was applied only with ibex from unit A2–A1, as it was the only species with an MNE higher than 50 corresponding to nine different individuals  following Marín-Arroyo and Ocio98. Also, Spearman’s correlation coefficient between Grease Index and %MAU shows a positive and significant correlation with ibex. Bone density was also determined for this species (Table There were abundant signs of marrow extraction for ibex at A2–A1 unit. The degree of fragmentation (measured as the quotient between NISP and MNE) correlates positively and significantly with Marrow Indexes Table , which cTable As can be seen in Fig. The mountainside cave location also might explain a low amount of ibex carcass processing at the kill sites, which is suggested by the mainly complete transport of these prey during the Protoaurignacian. The hunting preference, calculated as the ratio between mountain and fluvial plain habitat species, does not reveal drastic changes between units Table . Only fo68 also support the picture these locally limited human mobility patterns.Despite the fact that within a 1.2 h walk from the site, the catchment area above a 30% slope makes up only 15%, this area would have provided access to chamois and ibex , confirming the use of the nearby environment by the human groups. Within 2.15 h there is a shift of 5% of the mountain area in favour of the plain areas, but this small shift does not affect the hunting preferences undertaken by the different human groups occupying the cave Table . In summ2/year on average (SD 0.08). However, during the stadial phases, the mean NPP dropped to 0.189 kg/m2/year on average (SD. 0.09). During the Protoaurignacian occupation recorded in unit D3, the mean NPP was 0.181 kg/m2/year (SD 0.02) and, in A2–A1 unit, the NPP was 0.218 kg/m2/year (SD 0.06). Accordingly, the NPP of the Protoaurignacian units (A2–A1 and D3) of Fumane are significantly lower than that of the previous late Mousterian (A4) and Uluzzian (A3) units , Uluzzian (A3) and Protoaurignacian units that have revealed human occupations, the results indicate that in Fumane, during the interstadial conditions of MIS3, the Net Primary Productivity (NPP) was 0.327 kg/mNPP fluctuations did not affect significantly herbivore diversity in Fumane , but it did the carnivore richness  and the whole mammalian diversity  recovered in each archaeological unit Fig. . The NPPNew insight into the subsistence strategies adopted by early modern human groups that occupied Fumane has been developed through the taphonomic and archaeozoological study of the complete macrofaunal assemblage of units A2–A1 and D3 located outside the cave. The data provide details about the palaeoeconomic behaviour of the different human groups that made the Protoaurignacian and late Protoaurignacian technocomplexes identified in those units. Altogether these new data expand our understanding of the activities carried out at the site, human mobility patterns and the types of environments that surrounded Fumane when AMH occupied the cave Table .89. The earlier published subsistence patterns indicate that humans targeted red deer and ibex, as well as giant deer, roe deer, bison and chamois of all age classes158. Compared to the Mousterian, the Uluzzian assemblage records a cooling of climatic conditions correlated with modifications in the game that was hunted comparable to those of the Protoaurignacian. Wolf, fox and brown bear were exploited as well158.The chronometric dates for the site Table  cover thHowever, occupation of unit A2–A1 was more intensive than during D3, likely caused by the less favourable conditions during H4. After humans left the cave, carnivores hardly scavenged the human leftovers.164. Humans exploited the montane areas surrounding the site, where both ibex and chamois dwelled, as well as forest species like cervids, and likely large bovines that could have occupied wooded areas. Interestingly, the exploitation of large bovines seems was relatively limited, although the protein yield provided by these large animals might have been significant for the human diet.Protoaurignacian groups mainly exploited prime-age ibex and chamois, followed by red deer. Although a similar diet breath exists between both units, there is a reduction in red deer individuals in the D3 unit, likely caused by forest reduction which is broadly consistent with evidence from the environmental proxies at the site165.The proximity of the ibex kills might well explain the complete transport of this prey to the site. Various butchering activities related to the consumption and processing of the carcasses were carried out at the cave, as revealed by the different kinds of anthropic cut marks and evidence of green bone breakage. In addition, traces of wolf and fox fur preparation were attested, thus confirming human interest in these and other carnivores such as lynxThe mortality profiles reveal a profitable prey selection of prime-age individuals with significant, but non-majority representation of juveniles. Some of them were infantile ibex individuals that reveal late spring/summer hunting episodes, likely because humans were massacring female herds with young.166, which is driven by the flow of energy through trophic levels. Food chain length increases along gradients of NPP167, so a higher NPP is not only associated with a greater abundance of herbivores, but also with the frequency of secondary consumers166. In this study, the results have revealed that the NPP is more correlated with the diversity of secondary than of primary consumers, which is probably because herbivore remains were mainly accumulated by humans and, therefore, are biased by hunting choices/preferences. In any case, the results show that the productivity-diversity relationships explain the mammalian richness recorded through the different Fumane units. Therefore, climatic conditions during the Protoaurignacian occupation reduced the base of the food chain  and, as a consequence, mammalian diversity and abundance were likely constrained. Under these ecological conditions before and during Heinrich Event 4 (H4), the resource availability for secondary consumer species decreased and, consequently, the diversity of secondary consumers also came down. Such a scenario is documented for the Protoaurignacian of Fumane, which suggests that early modern humans successfully coped with severe environmental conditions that had negatively affected the productivity and diversity of the ecosystem. This is also seen in the micromammalian assemblages of these units that indicate a slight decrease in woodland formations163. Except for the Mousterian A5–A6 unit, in the Protoaurignacian units, the NPP was significantly lower than in the other archaeological units of Fumane  and its significant correlation with the herbivore species diversity recorded in the Protoaurignacian Italian sites.If we want to evaluate the NPP in the Italian Peninsula at the time of arrival of the Protoaurignacian modern humans, in terms of a similar temporal span, technological culture and ecological setting, only Riparo Bombrini174. Thus, it has been proposed that the stadial-interstadial cycles of the MIS3 generated a Neanderthal demographic crisis or even vacuum in some regions, which favoured the rapid spread of modern humans during the interstadial phases176. However, recent studies suggest that modern humans also spread through Europe during cold stadial conditions2. This idea is reinforced by the Protoaurignacian occupations of Fumane, where the arrival of modern humans was coeval with harsh environmental circumstances even including occupation of the site during the Heinrich Event 4. However, this association between the arrival of modern human groups and severe environmental conditions cannot be extended to all European sites or regions (Fig. 2.It has been argued that peopling of Europe by modern humans could be contingent on the environmental conditionsons Fig. . Therefoons Fig. , the Proons Fig. . These rons Fig. . In thisons Fig.  similar In summary, the data obtained in this research have provided a wider knowledge about the earliest modern humans’ presence in Italy. Fumane has been proven to be a significant site to study the organisation of the Protoaurignacian human groups in the Prealpine region. The archaeozoological data of both units reveal evidence of subsistence and landscape changes in the vicinity of Fumane. These changes could have been caused by the severe condition during H4 when the D3 unit was accumulated. Human occupations were probably more sporadic and, due to the vegetation cover reduction, focused on the montane-adapted animals located in the vicinity of the cave. Evaluating the subsistence strategies adopted by those groups by comparing them with the Net Primary Productivity at that time, has helped to understand how those groups adapted their strategies and also reveal the resilience of our ancestors to cope with different ecosystems and stadial/interstadial phases in their wave of migration from the Near East to the western part of the continent. The low productivity of particular ecosystems did not stop the rapid migration of the Proto- and Early Aurignacian technocomplexes. In effect, the settlement of those groups was, mainly, in areas where high NPP is proven which might explain their successful advance and colonization of the continent.Supplementary Information.Supplementary Tables.


Medial patellofemoral ligament (MPFL) reconstruction is an important surgical therapy for recurrent patellar dislocation. However, few studies have focused on exercise therapy after MPFL reconstruction. Therefore, the first purpose was to compare the active and traditional postoperative exercise therapies on the recovery of knee joint function and reduction of muscle atrophy after MPFL reconstruction, and the second purpose was to compare the active and traditional postoperative exercise therapies on the patellar stability after MPFL reconstruction.The cases of 31 patients with recurrent patellar dislocation treated with patella double semi-tunnel anatomical MPFL reconstruction from February 2016 and February 2019 were retrospectively reviewed. The clinical outcomes, including the patellar tilt angle (PTA), lateral patellofemoral angle (LPFA), thigh circumference reduction, Kujala score, and Lysholm score, were compared between two groups  preoperatively, 3 months postoperatively, 6 months postoperatively, 12 months postoperatively, and 24 months postoperatively.P < 0.01), 6 months postoperatively , 12 months postoperatively , and 24 months postoperatively . Similarly, there was a higher Lysholm score in the active exercise group compared with traditional exercise group 3 months postoperatively , 6 months postoperatively , 12 months postoperatively , and 24 months postoperatively . Significantly lower thigh circumference reduction was reported in the active exercise group compared with that in the traditional exercise group 3 months postoperatively  and 6 months postoperatively . No statistical difference was observed between the two groups in terms of PTA (P > 0.05) or LPFA postoperatively (P > 0.05).The Kujala score was significantly higher in the active exercise group than traditional exercise group 3 months postoperatively (80.06 vs. 74.80, Our results suggested that active exercise therapy might benefit the early recovery of knee joint function and reduction of muscle atrophy in patients with recurrent patellar dislocation after MPFL reconstruction. Patellar dislocation is a common knee injury, accounting for 2% to 3% of all knee injuries worldwide , 2. RecuAn appropriate exercise therapy after MPFL reconstruction is as important as the surgical process in the treatment of recurrent patellar dislocation given its vital effect on the recovery of the knee joint function; however, there is no consensus on the postoperative exercise therapy recommend . SeveralTherefore, this study was retrospectively performed on patients with recurrent patellar dislocation treated with MPFL reconstruction. The first purpose of this study was to compare the active and traditional postoperative exercise therapies on the recovery of knee joint function and reduction of muscle atrophy after MPFL reconstruction, and the second purpose was to compare the active and traditional postoperative exercise therapies on the patellar stability after MPFL reconstruction.This study was approved by the ethics committee of our hospital, and informed consent was obtained from each patient included in the research. The cases of patients with recurrent patellar dislocation who had undergone patella double semi-tunnel anatomical MPFL reconstruction between February 2016 and February 2019 were retrospectively reviewed.Q angle >20° (The inclusion criteria were as follows: (1) patellar dislocation occurred at least two times; (2) patellar instability following the initial dislocation persisted for more than three months; (3) magnetic resonance imaging indicated the injury, and even rupture, of the MPFL; (4) grade 1 or 2 focal articular cartilage defects according to the Outerbridge standard ; (5) patgle >20° ; (6) patgle >20° , and (8)gle >20° .The details of the surgical technique were described in our previous studies , 23. TheAll the patients were divided into the active exercise group and the traditional exercise group on the basis of the training schedule of the knee flexion. In the active exercise group, patients were required to start the knee flexion exercise 5 days postoperatively with the assistance of an adjustable brace  . The degWith respect to weight training, all patients began to gradually bear the body weight with the assistance of a brace 2 weeks postoperatively, and they would be permitted to bear full body weight at 4 weeks postoperatively. For strength training, ankle pump exercises were used to promote blood circulation and prevent thrombosis after surgery. The straight leg raise exercises started at the third day postoperatively, and the half-squat exercises started at 6 weeks postoperatively, and both of them stopped at 12 weeks postoperatively. Moreover, all the patients started the patella-shifting exercise by shifting the patella medially at 3 days postoperatively until the knee joint reached its full range of motion (ROM) at 8 weeks postoperatively to reduce the tension of the reconstructed MPFL and promote the healing of soft tissues.All patients could walk normally without the support of a brace at 8 weeks postoperatively. Patients could perform short-distance accelerated running and long-distance slow running at 12 weeks postoperatively. They could do normal physical exercise, such as running and rotational motion of the knee, at 26 weeks postoperatively.The ROM of the knee was measured using a universal goniometer. Thigh circumference was measured 5 cm above the patella superior border by using a medical ruler . Thigh ct-test or Mann-Whitney U test was used for the continuous variables, and the Fisher exact test was used for the categorical variables to determine the difference between the two groups. The paired sample t-test or Wilcoxon signed rank test was used to detect changes during the treatment for continuous variables for the same one patient. Normally distributed data were represented by mean ± standard deviation (SD), and non-normally distributed data were represented by a median with a range. A statistical difference was indicated by P < 0.05. All the analyses were performed using SPSS 20.0 .The independent sample n = 2), lost to the follow-up (n = 2), and insufficient data (n = 1). Finally, 31 patients were included in this study.A total of 36 patients underwent MPFL reconstruction for recurrent patellar dislocation in our hospital from February 2016 and February 2019. Among them, 5 patients were excluded from this study because of the following reasons: bilateral patellar dislocation (2. The median follow-up time was 30 months (range: 25–48 months). The active exercise group comprised 16 patients, while the conventional exercise group consisted of 15 patients. No significant difference was observed between two groups in terms of gender (P = 1.00), age (P = 0.90), surgical side (P = 0.72), BMI (P = 0.58), interval between the first dislocation and the surgery (P = 0.35), or follow-up time (P = 0.07). All patients completed the exercise plan, and there was no complication relevant to the exercise therapy during the regular follow-up, including recurrent dislocation and patellar instability.The demographic data of all the patients are provided in P > 0.05). Kujala score, Lysholm score, PTA, and LPFA improved considerably postoperatively compared with preoperatively (P < 0.05). Patients in the active exercise group had significantly higher Kujala score compared with those in the traditional exercise group 3 months postoperatively , 6 months postoperatively , 12 months postoperatively , and 24 months postoperatively . Similarly, patients in the active exercise group had significantly higher Lysholm score compared with those in the traditional exercise group 3 months postoperatively , 6 months postoperatively , 12 months postoperatively , and 24 months postoperatively . Moreover, the thigh circumference reduction in the active exercise group was significantly lower than that in the traditional exercise group 3 months postoperatively  and 6 months postoperatively , but the significant difference disappeared 12 months postoperatively (P = 0.32) and 24 months postoperatively (P = 0.20). However, no evident difference was observed between two groups in terms of PTA (P > 0.05) or LPFA (P > 0.05) postoperatively.The details of clinical outcomes are provided in Although different rehabilitation plans for improving the knee function after MPFL reconstruction have been proposed , 30, fewIn the current study, we used the traditional tunnel bioabsorbable interference screw to fix the femoral end of the ligament . SimilarPostoperative rehabilitation exercise plays a crucial role in the recovery of the knee joint function after MPFL reconstruction, however, no consensus exists regarding the exercise schedule , 17. On In recent years, an increasing number of studies have shown that early postoperative exercise helped patients return to their activity level before the injury by alleviating pain, reducing fluid accumulation, strengthening the quadriceps muscle and neuromuscular control, promoting healing, and preventing capsule contracture . Ronga eSeveral limitations should be considered when interpreting our results. First, the retrospective design might lead to selection bias, which reduced the accuracy of our results. Second, the sample size in the current study was relatively small, probably decreasing the stringency of the results. Third, follow-up time was relatively short for the assessment of PTA and LPFA, and a longer follow-up period is necessary to confirm our results. Forth, the factor of leg dominance was not considered when calculating the thigh circumference. Notably, we are conducting a prospective study that focuses on this topic with a larger sample size and a longer follow-up period. We believe our future results can eliminate the aforementioned limitations.Compared with traditional exercise, active exercise after MPFL reconstruction was beneficial for the early recovery of knee joint function and reduction of muscle atrophy in patients with recurrent patellar dislocation without harmful effects on the patellar stability.


The biological variability of semen and sperm DNA fragmentation (SDF) parameters in a longitudinal intrauterine insemination (IUI) trial over multiple IUI cycles was investigated. A TUNEL assay was used for SDF testing, both before and after density gradient centrifugation. A significant age effect was observed: while semen parameters deteriorated with advancing age, on average, higher SDF values were observed for older males. There was quite some variability observed for both semen and SDF variables. Using fertile threshold values, three patient categories were distinguished: those with a high SDF in all samples, those with low SDF in all samples and those who fluctuated between high and low during the whole IUI trial. Density gradient centrifugation increases SDF. However, the three patient categories react differently after semen processing. A large percentage of those with high SDF retain their high SDF even after gradient centrifugation. The SDF fluctuaters react with a high SDF after gradient centrifugation. The low SDF category, on the contrary, distributes itself evenly between the three categories after gradient centrifugation. SDF testing after semen processing might be indispensable for therapeutic purposes, probably influencing medical decision-making. In order to isolate fluctuaters, a second SDF testing might be advocated in certain cases. SDF after semen processing is indispensable for therapeutic management.  Although semen analysis remains the cornerstone for male factor infertility diagnosis, technical variations and biological variability  makes the correct interpretation of laboratory results difficult ,2. In thSDF is higher in subfertile men ,12 as coIntrauterine insemination (IUI) and assisted reproductive treatments (ART) such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) involve participation in one or more cycles preceded by a diagnostic semen analyses. Investigations undertaken to verify the stability of semen parameters and SDF in a longitudinal ART protocol are lacking.The objective of this study was to evaluate the degree of SDF variations in an IUI-trial over a period of 3 years.clinicaltrials.gov NCT03319654). During the period of October 2017 until October 2020, couples who were about to initiate a natural cycle IUI treatment protocol after an infertility duration of 12 months, at minimum, were invited to join the study. Women with ovulation disorders, endocrine disorders , prior ovarian surgery, prior IUI cycles, moderate to severe endometriosis and double-sided tubal disease were excluded. Men with a severe male factor  were also excluded from the study.This was a monocentric, prospective cohort study conducted at the Centre for Reproductive Medicine of the Antwerp University Hospital, Belgium  above 5 million according to the WHO criteria . The couThe Ethical Committee of the Antwerp University hospital and the University of Antwerp approved the study protocol . Informed consent to use the sperm rest fractions for DNA fragmentation testing was obtained from all the participants. I patients also gave their consent to use their clinical data for research purposes. Patients were instructed to maintain 2–7 days of sexual abstinence. All semen samples were collected at the laboratory, and any ejaculate fraction missing was reported. Samples were weighed and an analysis was initiated within 60 min after ejaculation conforming to the international standards of ISO 15189 . Standard semen parameters including sperm concentration and motility were determined using WHO  recommenAll staff members were trained in basic semen analysis  ,25 and p® . Briefly, 40% and 80% Puresperm®  density gradients were prepared using 1.5 mL of each suspension. Semen (1.0–1.5 mL) was layered on the top of each gradient and centrifuged for 20 min at 300× g. After centrifugation of the top seminal plasma layer, the 40% upper layer and the 40–80% interface were discarded and the remaining spermatozoa in the 80% pellet were collected from the bottom of the tube and washed once for 10 min with human tubal fluid  supplemented with albumin . A part of the same semen sample was treated with a two-step discontinuous density gradient  using Pu® Fixable Dead Cell Stain (far red) , after which the cells were washed 2× with phosphate buffered saline  before being incubated with 2 mM dithiothreitol  for 45 min, following which the samples were washed 2× in PBS and fixed in 3.7% formaldehyde  for 20 min at 4 °C. As we have previously shown that storage of the sample at 4 °C affects reproducibility [2O) and incubated for 5 min at 4 °C. The cells were washed 2× with PBS. The assay was performed using the fluorescein In Situ Cell Death Detection Kit  using Accuri C6 flow cytometer . For each sample, 5000–10,000 events were recorded at a flow rate of 35 µL/min. The positive control samples were treated with 5 µL of DNase I  1500 Kunitz Units for 30 min at room temperature. For a negative control, all the components of the labelling solution except the enzyme terminal deoxynucleotidyl transferase was included. Assessment of SDF was performed using the TUNEL assay described by Mitchell et al. . Brieflycibility , the assThe test was conducted on both the neat semen (ejaculate-E) and after density gradient centrifugation (gradient-G), and the results presented as follows:SDF total: percentage of the entire sperm population that was positive for DNA fragmentation.SDF vital: percentage of the sperm population that were alive and positive for DNA fragmentation.® Intrauterine Insemination Catheters, Cooper Surgical, The Hague, The Netherlands) rinsed with HTF and albumin. The inseminating volume was held constant between 0.3 and 0.5 mL [On the day of insemination, the semen sample obtained was analyzed and processed by density gradient centrifugation. In case the semen sample was deemed sufficient for IUI with an inseminating progressive motile count (IPMC) of ≥2 million , the sped 0.5 mL .A clinical pregnancy with fetal heartbeat was diagnosed by ultrasonography. Live births were registered in case of a birth occurring after 22 completed weeks of gestational age with evidence of life. Descriptive statistics , median, interquartile range (IQR) and range) are reported for the patient characteristics, semen parameters and SDF in the diagnosis and the IUI samples. Spearman correlation was calculated between SDF total-E  and patient characteristics and semen parameters in the diagnosis sample. To estimate the intraclass correlation (ICC) and the intra- and inter-variability of the SDF variables and semen parameters, a linear mixed model was fitted with these variables as the outcome, male age as a fixed effect and patient as a random effect to correct for samples coming from the same patient. In this analysis, all samples are used (diagnosis and IUI). To have assumptions of normality and homoscedasticity better satisfied, all outcomes except progressive motility were log transformed. A Bland–Altman plot comparing TPMC  between diagnosis and first IUI cycle split up according to the time interval between the measurements (> or ≤90 days) is shown. The following threshold values obtained from fertile men and sperm donors with proven fertility were used : SDF total in ejaculate : ≤13%; SDF vital in ejaculate : ≤2%; SDF total in gradient : ≤8%; SDF vital in gradient : ≤1%. The experimental results obtained led to three distinct categories: ‘Low’ SDF in all available samples, ‘High’ SDF in all available samples and ‘Fluctuaters’ who swing between the high and the low conditions. The distribution of these categories for the different SDF variables are given. The number (%) of livebirths in these categories are reported and compared with a Chi-square test or Fisher’s exact test as appropriate. Finally, a linear mixed model is fitted with the log of TPMC as outcome and a categorical variable as fixed effect indicating the SDF total-E category that the man belonged to Low/Fluctuater/High and patient as random effect. In this model, male age was also taken as a fixed effect. The same model was also fitted with the SDF total-G categories. In the case of a significant SDF category effect, categories were compared two by two using a Tukey correction for multiple testing.Analyses were conducted using R 4.1.0.SDF was assessed in 313 semen samples from 112 men  and the number of samples analyzed per patient varying between one and five. In 18 men (16.1%) only one semen sample; in 30 (26.8%), two semen samples; in 23 (20.5%), three semen samples; in 39 (34.8%), four samples; and in 2 (1.8%), five samples were analyzed. All semen samples were treated with density gradient, except for one. In 104 (92.9%) men, a diagnostic semen analyses was performed, together with SDF both before and after density gradient.Mean female age included was 30.4 years , and male age was 33  years. Of the 112 couples included, 26 (23.2%) clinical pregnancies were obtained with 21 (18.8%) live births. Basic descriptive statistics for patient characteristics, semen parameters and SDF in the diagnosis and all IUI samples are presented in p = 0.014) and progressive motility  were weakly correlated with SDF total-E ; thus, on average, higher SDF values were found for older males and lower values were found for younger males. For the semen parameters, we observed a negative coefficient, denoting that semen parameters deteriorate with advancing age. The ICC values in the table are quite low in most cases, meaning that there is quite some variation within a person . Intra-iThe different IUI treatments were extended far beyond 3 months of diagnosis, a period typically representing one spermatogenic cycle. Mean time interval between two samples was 80 days . Bland and Altman plots for SDF reveal no difference as far as time interval is concerned , but TMPUsing fertile threshold values, we distinguished three patient categories : those wWe observed that 66.7% of those with high SDF total-E retain their high SDF total even after the gradient. The SDF total-E fluctuaters (56.3%) react with a high SDF total after the gradient . The lowWhen considering the potential effect of the different SDF categories on IUI outcome, we observed no significant difference in live births between these three categories .p = 0.775).In a linear mixed model, with log TPMC as outcome and with male age and different SDF total-E categories as fixed effects, there is no significant effect of the SDF total-E categories (p = 0.001). Comparing the categories (with Tukey correction), we observed a significance between the high SDF and the fluctuaters (p = 0.003) and between the high SDF and the low SDF groups (p = 0.003). The geometric mean in the fluctuaters  is twice as high as in the high SDF group and the same is observed in the low SDF group as compared to the high SDF . This trend is also observed in the live births (p = 0.105). On the contrary, there was no significance between the low SDF and fluctuaters (p = 0.959).Applying the same model but now using the categories after density gradient centrifugation, there is a significant effect of the SDF total-G categories  were <20% (one outlier not included), whereas for longer times, the SDF CVs (n = 45) were ≥20% in 16 out of 45 patients and lower in 29 patients [According to the group of Muratori , the lonpatients . We coulThe reliability of SDF testing has possible clinical implications for therapeutic management. In addition to intra-individual variability, it is crucial to know how often repeated test results cross the threshold levels that discriminate normal and pathological categories . Alvarez➢SDF is ‘high’ in the ejaculate or➢SDF is ‘low’ in the ejaculate but, ‘high’ after the gradientIn order to isolate ‘fluctuaters’, a second SDF testing might be advocated. Since the TUNEL assay is tedious and highly expensive, based on our observations, we could propose to stratify patients at high risk of variability by repeating analyses when:However, our results need to be substantiated first. Sergerie et al.  calculatSperm preparation via density gradient centrifugation not only result in the isolation of spermatozoa with improved morphology and motility ,43 but aThe strengths of the study lies in the methodology. However, the limited study population is a limitation. Although IUI reimbursement in Belgium takes into account female age (until 43 years of age), the patient age was limited to ≤40 years. Including women above 37 years may introduce bias in the results since the outcome measure is live births. This should be taken into consideration when substantiating our results.Finally, this article highlights the prevalence of variability of SDF in a prospective, longitudinal IUI trial. One out of three men fluctuate between the high and the low SDF groups. Repeat analysis in certain cases could help identify this group. The significance of detecting DNA damage both before and after density gradient centrifugation might be indispensable for therapeutic purposes as the high, low and the fluctuating SDF groups react differently, which alerts us to avoid such damage in the name of ‘best practice’. However, the results need to be substantiated and opportunities created to explore populations with an extreme male factor and the clinical implications in different ART.


Exosomes have a role in tumorigenesis and metastatic dissemination, their material content and size being associated with poor prognosis of colorectal cancer (CRC). Our work aims to investigate their secretion patterns in CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and their mouse xenografts, to unveil possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. Our results show that MTSs’ exosome secretion pattern depends on their structural complexity: few-layer spheroids show a lesser exosome secretion, limited to the apical domain of cancer cells; secretion increases in multilayered spheroids and is visible from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in spheroids. The influence of the surrounding environment of non-tumor cells may account for the difference in exosome secretion patterns between spheroids and xenografts.Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes’ role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them “in situ” in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs’ exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells. Epidemiological data on colorectal cancer (CRC) incidence and mortality  show that CRC is the third most commonly diagnosed cancer in males and the second in females around the world, even if marked differences in rates exist among countries . Stand-aOne strategy to reduce mortality in CRC is, on the one hand, to succeed in finding biomarkers for early detection and, on the other, the development of personalized therapies to treat patients in the more advanced stages of the disease. The oncology community is moving towards patient-tailored cancer therapy, taking into account the unique molecular profile of each patient’s cancer. This innovative approach is improving responses to therapy ,8, and tAt the same time, the role of exosomes as specific biomarkers in CRC prediction and screening is emerging . ExosomePatientA 63 aged year male underwent CRC surgery for cancer removal under the standards of the ethics committee on human experimentation of the National Institute of Health (Istituto Superiore di Sanità) authorization no.CE5ISS 09/282, as reported in .Cancer biopsies management (immediately after recovery)https://www.thermofisher.com, accessed on 27 May 2022), mixed with 3% penicillin-streptomycin-amphotericin B solution .Samples were washed 2–3 times in cold saline and transferred in Dulbecco’s modified Eagle’s medium  for 1 h at 37 °C, under shaking.Samples were washed 3–4 times in phosphate-buffered saline (PBS), sectioned into small fragments (0.5 × 0.5 mm), and incubated in DMEM (Thermo Fisher Scientific) with 1.5 mg/mL collagenase type II (Thermo Fisher Scientific) and 20 g/mL DNAse , 20 ng/mL human EGF and 10 ng/mL human basic fibroblast growth factor . For further detail, see [Resuspensions of pellets containing cells, cell clusters, and tissue fragments were cultured in CSC medium supplemented with 10 mm nicotinamide, 1 µm y-27632  and upon approval of the experimental protocol by the Italian Ministry of Health’s Animal Experimentation Committee.Animalshttps://www.jax.org, accessed on 27 May 2022). For CSC validation, 5 × 105 cells were injected subcutaneously in the flank of 3 replicate mice in 100 µL 1:1 PBS/Matrigel . In all the validated CSCs, xenografts were detectable within 3–5 weeks in at least 2/3 mice.Four- to 6-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice MTSs were fixed in a solution of 2.5% glutaraldehyde in Phosphate buffer solution 0.1 M, pH 7.4 at 4 °C for 48 h.Washing: samples were rinsed overnight in Phosphate buffer solution 0.1 M, pH 7.4 at 4 °C.Post-fixation4) at 1.33% in H2O  was used to submerge tissue fragments for 2 h.A solution of osmium tetroxide (OsO4) residuals [Washing: phosphate buffer solution 0.1 M, pH 7.4 for 20 min (10 + 10 min) was used to remove  solutions were used.Ascending alcohol series .Samples were mounted on aluminum stubs using carbon tape.Sputter coating procedure.With platinum .ObservationHitachi SU 4000 Field emission scanning electron microscope under high vacuum at 20 kV. Digital image acquisition system: DISS5 Digital Image Scanning System , Germany).Primary fixation (immediately upon recovery)MTSs and xenograft biopsies were fixed in a solution of 2.5% glutaraldehyde in phosphate buffer 0.1 M, pH 7.4 at 4 C for 48 h.Washing: samples were rinsed overnight in Phosphate buffer solution 0.1 M, pH 7.4 at 4 °C.Post-fixation4 1.33% in H2O  for 2 h.Samples were then post-fixed in a solution of OsO4) residuals.Washing: phosphate buffer solution 0.1 M, pH 7.4 for 20 min (10 min + 10 min) was used to remove (OsODehydration procedurev/v) solutions were used.Ascending alcohol series , 2 steps of 20 min each.Embedding procedureIn a mixture of 50:50 propylene oxide and epoxy resin Agar 100  overnight at 25 °C . Finally, samples were embedded in fresh epoxy resin Agar 100  and put on a stove at 60 °C for 48 h ,29,30.Semithin sections (1 m thick) were collected on glass slides, stained blue by methylene blue, to perform light microscopy observations by a Zeiss Axioskop-40  equipped with Axiovision image acquisition software.Ultrathin sections for TEM observations were cut using an ultramicrotome . Ultrathin sections were collected on 100-mesh copper grids . Staining was performed using Uranyless© solution and lead citrate 3% solution .Sectioning procedure:Observation under a transmission electron microscope  set with an accelerating voltage of 60 kV.Digital image acquisition system: CCD digital camera .Imaging procedure:t-tests, and ANOVAs with Bonferroni correction were performed, and data were plotted in histograms. All procedures were performed using Med Calc Statistical software .Exosomes and MVBs diameters  were measured on transmission electron microscopy digital images using open source Fiji software  and HitaOur morphological investigation started with the analysis of the MTSs’ three-dimensional morphology by scanning electron microscopy; we then proceeded to their ultrastructural characterization; by transmission electron microscopy, highlighting aspects related to the secretion of exosomes. Finally, an ultrastructural analysis of the xenograft was performed to compare the different experimental models and highlight similarities and differences in exosome secretion. Data on the size of exosomes and MVBs were then finally statistically analyzed.Observation of the outer morphology of MTSs by scanning electron microscopy showed that their outer surface had a variable appearance. Some MTSs appeared as compact entities with smooth surfaces or sparse, shallow furrows A. No exoObserving the internal morphology of the MTSs, at first by light microscopy on semi-thin sections, then by transmission electron microscopy on ultrathin sections, we identified three different types of MTSs, hereafter defined as A, B, and C. MTSs of group A were characterized as bi-layered or three-layered structures, containing pseudocyst-like structures  is a tissue, a more complex entity with different characteristics than MTSs, which are a 3D cultured cell mass. Simply consider the presence of necrotic and hypoxic areas, blood vessels, nerves, and fibroblasts A; altogeTo study the secretion patterns of exosomes in xenografts, images obtained by transmission electron microscopy observation of the samples were analyzed. The TEM images shown in Phenomena of exosome secretion were also observed from the basolateral domain of xenograft cells facing the tissue interstitium . In partThe different patterns of secretion in MTSs and xenografts at both the apical and basolateral membranes, secretion in MTSs and xenograft is shown in t-test results are illustrated in To provide a quantification of different secretion amounts in MTSs and xenografts, we counted the number of MVBs in epithelial cells lining pseudocystic structures (200 cells from Type C MTSs and 200 cells from xenografts). Summary statistics and MBV secretion from the apical domain of spheroid cells is five times higher than basolateral secretion. MBV secretion from the apical domain of xenograft cells is 10 times higher than basolateral secretion. MBV secretion from the apical domain of xenograft cells is massive compared to secretion from the apical domain of spheroid cells, in a ratio of 13:1. MBVs secretion from the apical domain of xenograft cells is higher than secretion from the apical domain of spheroid cells, in a ratio of 6:1.Following the observation of MTSs’ and xenografts’ different secretory modalities, we focused our analysis on the morphology and size of exosomes and MVBs.Looking at the appearance of exosomes secreted from the apical domain (in both MTSs and xenograft), we noted that they presented long filaments projecting radially from the outer aspect of the membrane. These molecules partly intertwine at their initial part, proximal to the membrane, and form a “crown” around the outer surface of the exosome. These filaments consist of glycoproteins and glycolipids of exosome membrane, highlighted by the use of tannic acid in the sample preparation process for electron microscopy A,C. ExosExosomes secreted in basal and inflammatory conditions from human epithelial cells had a size range of 30–90 nm ,34. We mAs can be seen from To verify if some difference exists in exosome diameter values in different samples (MTS vs. xenograft) and different secretion sites , an ANOVA test with Bonferroni correction was performed .The size of MVBs secreted from the apical domain of spheroid and xenograft was measured and compared. MVBs secreted by the spheroid basolateral domain were scarce (being present only in type C MTSs) concerning that of xenotransplant, and no comparison between these two groups was made. Results of statistical data analysis are reported above in t-test was performed and results are presented in To verify if some difference exists between MVB diameter value in MTSs vs. xenografts, a Cancer stem cells are a tumor subpopulation capable of self-renewal and are crucial for survival, proliferation, drug resistance, metastasis, and tumor recurrence . We receWe have previously investigated the ultrastructural features of CRC MTSs, highlighting an increased presence of stem-like cells in MTSs as compared to tumor xenografts . In thisAs reported in , MTSs frOur ultrastructural results (from a patient-derived CRC cell line) correlate for the first time the molecular data from the literature with the ultrastructural imaging of the different loose, tight, and compact MTS organizations, showing its relation with different exosome secretion patterns and amounts from both the apical and basolateral surfaces. This means that exosome production can be targeted in different pathways  to slow In xenograft models (from a patient-derived MTSs xenograft), the secretion of exosomes occurs from all domains of the tumor cells and is quantitatively greater than that observed in spheroids. The massive presence of MVBs that release their contents into the lumen of the pseudocysts or in the tissue interstitium was observed.Our ultrastructural results show for the first time the different arrangements in rows and columns (different patterns) and the difference in the amount of MBV secretion between spheroids and xenografts, xenografts being the source of a massive exosome and MVB production.Our findings suggest that targeting the pathways of MVB formation and release could be another way to slow down cancer progression and translate our findings into clinical applications.This difference in exosome secretion pattern and amount between MTSs and xenografts may be possibly due to the influence of surrounding non-tumor cells, as it has been shown that exosomes are key mediators of the communication between tumor cells and the tumor microenvironment .Statistical analysis conducted on measurements of exosome diameter shows that exosomes secreted from the different MTSs domains have the same size (about 70 nm), which is then also the same in those produced by xenograft cells. According to , the sizOur morphological data show that structural complexity influences exosome secretion of MTSs, in both intensity and pattern, if compared with xenograft models. Our observations add new knowledge to the ultrastructural features of CRC MTSs and xenografts. Future studies may define the mechanistic basis of different exosome secretion patterns in the two model systems.


TERC, the enzyme telomerase synthesizes the elongation of telomeric DNA using the 3’ end of the chromosome as a primer. TERC harbors a highly conserved template region consisting of 11 nucleotides spanning hg19 coordinates chr3:169482793–169482803. In human cell lines, when TERC was mutated to alter its template region, telomerase generated the predicted mutant telomeric repeats. However, it is unknown if this can occur in human clinical samples. Here, we report on the rare occurrence of two tumor samples where TERC template mutations were reflected in telomeric repeats.Telomerase-mediated lengthening is a mechanism by which some cancer cells avoid senescence-mediated cell cycle arrest due to shortened telomeres. By reverse transcribing an RNA template, encoded by  TERT, and was observed in 85–90% of tumors [TERC, which encodes an RNA template, the telomerase enzyme can synthesize the elongation of telomeric DNA using the 3’ end of the chromosome as a primer [Telomeres are protective DNA-protein complexes present at the ends of linear chromosomes, consisting of repetitive hexamers (TTAGGG) that shorten in length with every cell division by an average of 50–150 base pairs . If telof tumors , 5. By ra primer , 7.TERC is a lncRNA harboring a highly conserved template region consisting of 11 nucleotides spanning hg19 coordinates chr3:169482793–169482803 encoding GTTAGGGTTAG [TERC was mutated to alter its template region, telomerase generated the predicted mutant telomeric repeats [TERC template mutations were reflected in telomeric repeats.AGGGTTAG , 11. In  repeats –14. HoweTERC’s template sequence . We observed an over-representation of mutations at chr3:169482801 (position 9), seen in 68% (82) of samples with a mutation. Overall, 88% of samples harbored only one mutation in TERC’s template region, but in 12% of samples, multiple mutations in the template sequence were observed, where one mutation always impacted chr3:169482801 (position 9) and the other mutation was either at chr3:169482800 (position 8), or chr3:169482803 (position 11), or both. Interestingly, 86% (118) of the mutations identified were a transition from the reference base to an A. Of note, the observed allele frequency of these TERC template mutations was low, with a median of 0.02 , indicating that only a small proportion of tumor cells across these samples carry a TERC template mutation.Across 309,384 unique tumor samples from the Foundation Medicine dataset, we identified 120 samples carrying 137 mutations in TERC template sequence and found two samples that fit this criterion . The allele frequency of the TERC mutation in both samples was in the top 90th percentile (0.23 and 0.16). Sample 1 had a G > T mutation at chr3:169482798 (position 6) on the positive strand, which results in a C > U mutation in the middle of TERC’s template sequence. This mutation results in the priming of the synthesis of telomeric ends with TTAGTG repeats, instead of the canonical TTAGGG repeats . Sample 1 is an ovary epithelial carcinoma and had an amplification in RAD21, which we have previously shown to be associated with increased telomeric content [th percentile across all 2623 ovary epithelial carcinoma samples in our cohort and at the 62nd percentile of RAD21-altered ovary epithelial carcinomas. Sample 2 harbored an A > G mutation at chr3:169482802 (position 10) in TERC’s template sequence, which results in the synthesis of TTGGGG repeats instead of TTAGGG . Sample 2 is a skin melanoma sample and harbored a TERT promoter mutation (-124 C > T), associated with upregulation of telomerase expression. The telomeric content of sample 2 was 1281.9 TRPM, at the 64th percentile of all 5204 skin melanoma samples in our cohort and at the 67th percentile of TERTp-mutated melanomas. As shown in We then checked for the presence of mutated telomeric reads that matched the mutation of each sample’s  content . The telTERC template sequence must basepair with the sequence transcribed from positions 7–11 to enable further elongation. Sample 1 had a mutation at position 6, which avoids this guide/template constraint . Sample 2 had an alteration at position 10, which would necessitate a G:U pairing between the mutated telomeric repeat and the unmutated position 4 of TERC’s template sequence . We hypothesize that in this case, wobble-base pairing allows for binding and priming of telomeric elongation [pTERT and RAD21), played a role in incorporating mutated guide sequences into telomeric repeats.Mechanistically, positions 1–5 of the ongation , 17. We TERC template mutations was quite low (0.04%). This is consistent with the observation that the expression of mutant template RNA in cancer cell lines negatively impacts cellular survival and proliferation [Saccharomyces cerevisiae and Tetrahymena thermophila, mutations of the template sequence resulted in mutated telomeres, but negatively impacted cell survival [TERC template sequence exist in a small fraction of clinical tumor samples and rarely, this mutated template sequence can prime the synthesis of mutated telomeric repeats in clinical tumor samples.In our dataset, the prevalence of feration , 18, 19.feration . Similarsurvival –23. Our Approval for this study, including a waiver of informed consent and a HIPAA waiver of authorization, was obtained from the Western Institutional Review Board (Protocol No. 20152817). Consented data that can be released is included in the paper. Institutional review board approval of the study protocol was obtained prior to study conduct and included a waiver of informed consent.The Foundation Medicine dataset consisted of specimens sequenced as a part of routine clinical care using a targeted next-generation sequencing assay as previously described (FoundationOne CDx) , 25. TheTERC’s template were reflected in the repeat sequence.For each sample, we generated a list of possible read sequences where the read exclusively consisted of TTAGGG repeats, except that the nucleotides mutated in


Among postmenopausal women, those with two or more children showed a greater risk of idiopathic MH than those who had not been pregnant, with a hazard ratio of 1.80. In conclusion, idiopathic MH occurred earlier and greater in women. Childbirth were associated with an increased risk of MH development, and obesity was associated with a lower risk of MH.This nationwide population-based cohort study searched for demographic, comorbid, behavioral, and reproductive risk factors for idiopathic macular hole (MH) development using data provided by the Korean National Health Insurance Service. A total of 4,496,867 individuals aged 50–79 years who participated in the Korean National Health Screening Program in 2013 or 2014 were included. Participants were followed up until December 2018, and incident cases of idiopathic MH were identified. Prospective associations between incident idiopathic MH and various covariates were investigated using multivariable-adjusted Cox proportional hazard models. During an average follow-up period of 4.91 years, 3054 patients were newly diagnosed with idiopathic MHs. Women showed greater risk (hazard ratio of 1.71) and earlier presentation of idiopathic MH than men. Compared to the normal body mass index group, the obese group (≥ 25 kg/m The prevalence of idiopathic MH ranges from 0.02 to 0.33%3, and the incidence rate of idiopathic MH in the general population was reported to be 7.80 per 100,000 person-years in the Olmsted County4 and 3.14 per 100,000 person-years in South Korea5. Approximately 10–16% of patients have MH in both eyes7, which implies that certain individuals have a greater tendency to develop MH. Nevertheless, the reason why some individuals have stronger anteroposterior vitreomacular traction and eventually progress to idiopathic MH is poorly understood.The macular hole (MH) is a full-thickness defect of the retinal tissue occurring at the center of the macula. MH can occur secondary to diabetic retinopathy, pathologic myopia, and other ocular condition, but most MH cases develop without obvious secondary cause and this condition is referred to as idiopathic MH. Idiopathic MH commonly affects people over 60 years of age and is caused by anteroposterior vitreomacular traction resulting from anomalous posterior vitreous detachment5. However, risk factors for developing idiopathic MH other than age and sex are not yet known. Specifically, much remains unknown for the reasons why the disease develops frequently in postmenopausal women. A limited number of epidemiological studies have investigated other risk factors for MH development. Among those studies, the results were often inconsistent and limited by case–control design or the small number of study subjects, less than or about a hundred cases of MH9. Recently, a large-scale cohort study in the United States investigated the risk factors for developing idiopathic MH; however, did not sufficiently evaluate covariates such as behavioral or female reproductive factors10.Previous epidemiological studies attempted to identify the risk factors of MH and revealed that older age and female sex are significant risk factors for MH formation, with a reported female-to-male ratio from 2.0 to 3.3 from population-based studiesHerein, we performed a comprehensive risk factor analysis including demographic, comorbid, behavioral, and female reproductive factors from a nationwide population-based cohort to provide epidemiological basis for MH development and advance our understanding of the pathophysiology.This was a nationwide, population-based, retrospective cohort study that used authorized data from the Korean National Health Insurance Service (NHIS) database. The study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Samsung Medical Center, Seoul, Republic of Korea (IRB File Number 2020-11-074). The board waived the requirement for informed consent based on the use of de-identified public data and the retrospective design of the study.11, a free biennial general health examination provided to all Koreans aged above 40 years. The NHIS also extends the National Cancer Screening Program (NCSP) for those receiving general health examinations through the NHSP12. The NCSP provides examinations for the stomach, liver, colorectal, breast, and cervical cancer for all registered individuals at the age indicated for cancer screening (breast cancer examination beginning at 40 years of age).The NHIS is the insurer in South Korea that provides mandatory universal medical care and holds all medical information and health claims data in the country. The NHIS runs the National Health Screening Program (NHSP)14. Detailed information regarding the database profiles has been provided elsewhere12.The NHIS database stores data on demographic information ; health claims ; and those from the NHSP and NCSP  of all registered Koreans. This database has been widely used in previous studies that identified risk factors for various diseasesThis study included two separate analyses Fig. . In AnalAmong the entire South Korean population, participants who underwent national health examinations in 2013 and 2014 were identified. We included participants aged 50–79 years at baseline as most cases of posterior vitreous detachment would have already occurred in those over 80 years, and MH occurring before 50 years would have a secondary cause for MH development. Therefore, a total of 5,965,039 participants aged 50–79 years who participated in NHSP in 2013 and 2014 were identified. Individuals who had a history of vitrectomy  from 2002 to the date of the examination were excluded from the study  as they would not have vitreous that caused vitreofoveal traction and MH development. Individuals who had been diagnosed with retinal detachment (diagnostic code: H33), retinal vascular occlusion (diagnostic code: H34), uveitis , and ocular trauma (diagnostic code: S05) from 2002 to the date of the examination were also excluded  as they could cause secondary MH development. Individuals with diabetes mellitus at baseline and previous history of degenerative myopia (diagnostic code: H44.2) were then excluded as they are related to development of diabetic and myopic MH. Finally, 4,496,867 participants were included in Analysis 1.Among those included in Analysis 1, 2,166,570 women underwent NCSP with NHSP and 1,918,134 postmenopausal women were identified. Individuals who had a history of hysterectomy  were excluded from the study as they could have undergone simultaneous oophorectomy, influencing the participant's exposure to female hormones. Finally, 1,735,189 postmenopausal women were included in Analysis 2.2 measured using blood sampling. Income level was categorized into quartiles according to the insurance premium level, which was determined by household income.Comorbid hypertension, diabetes, and dyslipidemia were identified based on the followings; (1) self-reported questionnaire responses; (2) health screening measurement results of blood pressure , fasting glucose , and total cholesterol ; (3) the presence of diagnostic codes  combined with medication prescription codes within a year before the health screening examination. The history of stroke and heart disease was identified using self-reporting based on the NHSP questionnaire. The presence of chronic kidney disease was defined as a glomerular filtration rate of < 60 mL/min/1.73 m2), and categorized as underweight (BMI < 18.5 kg/m2), normal weight (18.5 ≤ BMI < 23 kg/m2), overweight (23 ≤ BMI < 25 kg/m2), obese I (25 ≤ BMI < 30 kg/m2), and obese II (≥ 30 kg/m2), according to the Korean Society for the Study of Obesity15.Data on health-related behaviors were collected using the NHSP questionnaire. Smoking status was divided into never-smokers and past/current smokers. Drinking habits were categorized into three levels: none, mild (< 6 times a week), and heavy (≥ 6 times a week). Those who had a moderate level of physical activity for more than 30 min a day for more than 5 days per week were categorized into the regular exercise group. The body mass index (BMI) was calculated as the weight (kg) divided by height squared , and oral contraceptive pill use. Data on female reproductive factors were categorized as follows: age at menarche , age at menopause , parity , duration of HRT , and duration of oral contraceptive pill use .5. Individuals with prior history of MH would not have been included in the study population, as we excluded those who had vitrectomy for any vitreoretinal disorder before the baseline examination. The incident date was defined as the date of the earliest claim that corresponded to the criteria for each patient. Incident MH cases that occurred after the diagnosis of retinal detachment, retinal vascular occlusion, uveitis, and ocular trauma were not counted as they could be secondary MH cases. Patients were followed from the date of health check-up to the date of incident MH, death, or the end of the study period , whichever came first.Incident MH cases were defined as those with both the diagnostic code for MH (H35.33) and the surgical codes for vitrectomy (S5121 or S5122) in the same claim after the date of the baseline examinationThe incidence rates of MH were calculated by dividing the number of incident cases by the total number of person-years. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazard models. The variables adjusted for each Cox model in Analysis 1 were as follows: model 1, age and sex; model 2, demographics  and comorbidities ; model 3, demographics; comorbidities; and behavioral factors . In Analysis 2, the variables adjusted for each Cox model were as follows: model 1, age group; model 2, demographics ; comorbidities; and behavioral factors; model 3, demographics; comorbidities; behavioral factors; and reproductive factors . Additional sensitivity analyses were conducted in a broader population in which previous diabetes mellitus or degenerative myopia were not excluded. The purpose of sensitivity analyses was to examine the robustness of study results and to additionally evaluate the influence of diabetes mellitus and degenerative myopia on the development of overall MH (including diabetic MH and myopic MH). All statistical analyses were performed using SAS version 9.4 .Table 2) presented a lower risk of MH development than those with normal BMI . Individuals in the highest income quartile had a greater risk of MH than those in the lowest income quartile with HR (95% CI) of 1.17 (1.05–1.30).Table Table Table Table A total of 5,491,942 individuals were included in the additional sensitivity analyses. An average follow-up duration was 4.91 years and 3054 cases of MH developed. The age group from 65 to 69 years showed the highest risk of MH and obesity was associated with reduced risk of MH development 8. In contrast, another case–control study did not show any association between MH and HRT9. The estrogen levels in the vitreous gel of eyes with MH were not different from or higher than that of eyes with other retinal diseases in previous studies23, which implies null or even detrimental effects of estrogen on MH.Female sex was a significant risk factor for idiopathic MH development, with an HR of 1.70 compared to males. Although the female preponderance in MH has been widely reported in various epidemiological studies8. According to their study, the odds ratio (95% CI) of having MH was 1.80 (1.02–3.10) for those who had parity once or more compared to those who had never been pregnant. The biological reasons for this association are unclear. However, there has been a hypothesis expecting the linkage between parity and MH24. Mehdizadeh et al. suggested that a higher incidence of MH development might be attributable to relaxin, a hormone produced during pregnancy24. Relaxin facilitates the birth process by widening the pubic bone and softening the cervix by modulating collagen metabolism, inhibiting collagen synthesis, and enhancing its breakdown by increasing matrix metalloproteinases26. They suspected that pregnancy-induced relaxin secretion may affect collagen and hyaluronic acid metabolism in the vitreous as well, and may induce premature vitreous syneresis and early anomalous posterior vitreous detachment24. Accordingly, MH would be more prevalent and present at a younger age in women than in men. Moreover, the effect size (HR) of the female sex was similar to the effect size of parity , indicating that nulliparous females have a risk of MH development similar to that of males. Collectively, our study results suggest that parity, rather than estrogen exposure, such as the history of HRT or age at menarche and menopause, is closely associated with the risk of idiopathic MH. The precise pathophysiology underlying the linkage between parity and the increased risk of MH development should be further elucidated.In the present study, the history of postmenopausal HRT was not associated with the risk of idiopathic MH development, which does not support the estrogen theory. Other parameters related to lifetime estrogen exposure (age at menarche and age at menopause) were not associated with the risk of idiopathic MH. Instead, parity was significantly associated with the risk of developing idiopathic MH. The Eye Disease Case–Control Study Group has previously reported the same association2) had a significantly lower risk of developing idiopathic MH. The association between obesity and MH has not been previously reported. One possible explanation for this association is the association between obesity and myopia. Researchers have previously reported that people with higher BMI tend to be less myopic29. It has been suggested that obese individuals tend to have a greater amount of retrobulbar fat, which then increases retrobulbar pressure and hinders axial elongation of the globe29. Hence, obese individuals in the present study might be less myopic, which would lead to a lower risk of MH. We were unable to confirm the association between BMI and myopia and MH in the present study owing to the lack of data regarding refraction and axial length. Future epidemiological studies to confirm the association between BMI, myopia, and MH may provide insights into the underlying mechanisms of the association between obesity and MH.Additionally, those who were obese  for MH development. Further studies addressing these limitations are required.In conclusion, the present nationwide population-based cohort study revealed that age, sex, BMI, and parity in women significantly influenced the risk of future idiopathic MH development. This study would promote a better understanding of the disease entity and could serve as evidence for future research regarding the underlying biological mechanism of idiopathic MH.Supplementary Tables.


We observed that TL was significantly shorter in EOCRC patients than in healthy individuals , suggesting that telomeric shortening could be associated with EOCRC susceptibility. In addition, we found a significant association between several SNPs of hTERT (rs79662648), POT1 , TERF2 (rs251796 and rs344152214), and TERF2IP (rs7205764) genes and the risk of developing EOCRC. We consider that the measurement of germline TL and the status analysis of telomere maintenance related genes polymorphisms at early ages could be non-invasive methods that could facilitate the early identification of individuals at risk of developing EOCRC.Early-onset colorectal cancer  incidence has been steadily increasing in recent decades worldwide. The need for new biomarkers for EOCRC prevention strategies is undeniable. In this study, we aimed to explore whether an aging factor, such as telomere length (TL), could be a useful tool in EOCRC screening. The absolute leukocyte TL from 87 microsatellite stable EOCRC patients and 109 healthy controls (HC) with the same range of age, was quantified by Real Time Quantitative PCR (RT-qPCR). Then, leukocyte whole-exome sequencing (WES) was performed to study the status of the genes involved in TL maintenance ( Early-onset colorectal cancer  incidence has steadily increased over the past few decades. Nowadays, EOCRC accounts for approximately 10% of the total colorectal cancer (CRC) cases diagnosed each year, and it is also showing a significant rise in associated mortality . These eTelomerase activity has been postulated as a key factor in cancer development, in terms of cellular immortalization through telomeric elongation as well as in aging and senescence through telomere shortening. In healthy cells, erosion of telomere length (TL) eventually leads to regulated cell senescence and apoptosis. However, in abnormal cells, continued cell division results in telomeric shortening that can lead to end-to-end fusion of chromosomes and chromosomal instability. Therefore, telomere shortening is a process of aging associated with genetic instability . Given tHowever, studies on germline cells focusing on CRC telomere dynamics have reported controversial results, with a recent study indicating that both long and short telomeres are associated with an increased risk of CRC . For thit-test, p < 0.001)  were found by a TL statistical analysis carried out according to the clinical features of EOCRC patients  and 109 healthy controls (HC) with the same range of age, obtained from a cohort of healthy patients controlled in Primary Care Units, was quantified by Real Time Quantitative PCR (RT-qPCR). We observed that TL was significantly shorter in the EOCRC group than in the control group  A, which  < 0.001 B. Nevertpatients .hTERT, TERC, DKC1, TERF1, TERF2, TERF2IP, TINF2, ACD, and POT1) in leukocytes from 70 sporadic EOCRC cases from the original cohort by whole-exome sequencing (WES). Although we did not find any pathogenic variant that could explain the telomeric shortening observed in our series, we found several SNPs in the hTERT (rs79662648), POT1 , TERF2 (rs251796 and rs344152214) and TERF2IP (rs7205764) genes with a differential distribution in our cohort compared to the healthy controls, who were enrolled in different databases . We also found an association between several SNPs of hTERT (rs79662648), POT1 , TERF2 (rs251796 and rs344152214) and TERF2IP (rs7205764) genes and the susceptibility to develop this disease. Therefore, our data suggest that telomeric shortening could be associated with a higher risk of developing EOCRC. In this context, it has recently been described that CpGs undergoing aging-related methylation drift were significantly altered in EOCRC, as well as that accelerated aging has been found in normal mucosa from people with EOCRC [For this reason, we analyzed the absolute leukocyte TL from 87 microsatellite-stable EOCRC patients and 109 healthy individuals in the same age range, showing that germline TL was significantly shorter in EOCRC than in healthy individuals  in leukocytes from 70 sporadic EOCRC cases from the original cohort using the WES method. We found an association between different SNPs in the hTERT, POT1, TERF2, and TERF2IP genes, as well as the susceptibility of developing EOCRC. Interestingly, 7 out of 11 SNPs were found in the POT1 gene, whose pathogenic mutations have already been related to melanoma, glioma, or CRC susceptibility [POT1 rs7794637 polymorphism, which is associated with breast cancer risk [POT1 gene SNPs, as well as their functional impact on telomere maintenance regarding EOCRC risk and/or carcinogenesis. POT1 forms part of a multiprotein complex that regulates TL and protects chromosome ends from chromosomal instability and abnormal segregation. Consequently, alterations in this protein could lead to a dysfunction of the shelterin complex, which would result in telomere shortening [In addition, we studied the status of the genes involved in the TL maintenance and the shelterin complex  . All pat−∆∆Ct) comparative method was used to calculate the relative DNA amount of each amplicon. This assay was performed in a 96-well plate and the detection was carried out in the Step-One Plus Real-Time PCR system .DNA was obtained from PB leukocytes using the phenol–chloroform method. PB sample collection from EOCRC patients was performed at diagnosis, prior to the initiation of any type of treatment. All DNA samples were stored in Eppendorf tubes at 20 °C to prevent their progressive degradation and potential contamination. The TL of the leukocytes was measured by RT-qPCR using the Absolute Human Telomere Length Quantification qPCR Assay Kit , following the manufacturer’s instructions. This technique allows the initial amount of DNA coding for telomerase (TEL) and a single copy reference gene (used as an endogenous control) to be quantified simultaneously. The difference in the amount of DNA quantified represents the relative TL of each sample. To analyze these relative changes, a reference fragment of known TL (provided by the manufacturer) was added to each assay, allowing the absolute quantification of the TL of each sample. Triplicate reactions were carried to minimize variability. The TEL and SCR fragments were amplified using 10 ng in 2 µL of DNA, 1 µL of each specific primer, and 10 µL of the FastStart SYBR Green Master Mix. The amplification program was as follows: 10 min at 95 °C followed by 40 cycles at 95 °C for 15 s, 52 °C for 30 s, and 60 °C for 1 min. Finally, the Ct (2https://evs.gs.washington.edu/EVS (accessed on 10 June 2022) [http://gnomad.broadinstitute.org (accessed on 10 June 2022) [Whole-exome sequencing was performed in FACS-sorted clonal PCs and paired T lymphocytes from 70 patients with EOCRC. Exonic sequences were enriched using the Ion Ampliseq Exome kit , sequenced with a 151 × 2 bp read length and 150× depth coverage using semiconductor technology , and were then analyzed with the Ion Reporter and Torrent Suite Software (Life Technologies). Prior to analysis, FASTQ files were assessed for read quality using the FASTQC tool (v.11.9) . Raw reane 2022)  and GNOMne 2022) , throughne 2022) , which w2) test. Comparing continuous with categorical variables, either Student’s t-test or ANOVA was used for independent samples and the Mann–Whitney U test was used for continuous variables ). SPSS version 23.0 (IBM) was used for statistical analyses. A p-value less than 0.05 was established as the threshold to consider the differences as statistically significant.Categorical variables were expressed as number of cases and percentage, and these were compared using Pearson’s Chi Square (χPOT1 gene showed that different SNPs of this gene are more represented in the EOCRC series. For that reason, we consider that the measurement of germline TL and POT1 gene polymorphisms at early ages could be non-invasive methods that could facilitate the early identification of individuals at risk of developing EOCRC. This would also facilitate the identification of those individuals who do not meet the currently established CRC screening criteria, allowing us to anticipate the starting age of screening for average-risk CRC. Thus, this could justify including TL and POT1 SNPs as a new markers in CRC prevention strategies, after consequent validation in other young population cohorts.In conclusion, our results suggest that telomeric shortening could be associated with EOCRC susceptibility. Moreover, the analysis of the