Reproductive biotechnologies are important strategies to enforce the production of genetically superior animals. The fixed-time artificial insemination is widely used in Brazil and it is especially important in farms located in remote areas, contributing to a better management and increase in genetics and productivity in areas such the Lower Amazon. We evaluated two different protocols of fixed-time insemination using two different hormones in the Nellore heifer raised under tropical conditions. The protocol of the fixed-time artificial insemination using equine chorionic gonadotrophin presented a better outcome in the pregnancy rate compared to the protocol using follicle stimulating hormone. Our tested protocol may be the most suitable alternative to increase conception rates in animals that are raised in an extensive system under tropical conditions in the Amazon.Experiment I evaluated the ovarian follicular dynamics in 64 Nellore heifers submitted to different FTAI protocols (n = 32/group) using either FSH or eCG. In Experiment II, the pregnancy rate was evaluated in 195 heifers submitted to FTAI protocols and divided into two groups: FSH (n = 97) and eCG (n = 98). In Experiment I, the ultrasound examination showed that the maximum diameter of the dominant and preovulatory follicles and the ovulation time were similar between the FSH and eCG groups (p > 0.05). However, the ovulation rate was higher in the eCG group when compared to FSH (p = 0.014). In Experiment II, females that received eCG presented a higher pregnancy rate (58.1%) when compared to FSH (40.2%) (p = 0.012). The use of eCG in the FTAI protocol in Nellore heifers promoted a higher ovulation rate and increased pregnancy rate and may be the most suitable alternative to increase conception rates in animals that are raised in an extensive system under tropical conditions in the Amazon.This study aimed to evaluate follicular dynamics and pregnancy rates in Nellore heifers submitted to fixed-time artificial insemination (FTAI) protocols associated with equine chorionic gonadotrophin (eCG) or follicle stimulating hormone (FSH). Nellore heifers (n = 259) were used, divided into two studies.  The reproductive efficiency of cattle herds is influenced by several factors, such as the breeding system, the breed used, the management employed on the farm, and the sanitary and nutritional status of the animals ,2. DespiBos indicus postpubertal heifers have resulted in lower pregnancy rates than in anestrous cows [In this context, it is worth mentioning fixed-time artificial insemination (FTAI), which is a technique that eliminates difficulties faced in conventional artificial insemination, thereby removing the need for detection of estrus since it uses pharmacological protocols that allow synchronized ovulation, increases the rate of service and conception and favors the genetic improvement of the herd ,8. The uous cows , but FTAous cows ,15.Among the available hormone protocols for heifers, the use of follicle stimulating hormone (FSH) and equine chorionic gonadotropin (eCG) are widely used in Brazil . FSH is Differently from FSH, equine chorionic gonadotropin (eCG) is naturally produced in the mare’s endometrial calyces during gestation (40 to 130 days). Its use in FTAI programs in cattle can improve fertility rates, especially in heifers and in recent postpartum cows , with a In this study, we adopted the hypothesis that the adoption of the hormonal FTAI protocol using eCG would promote an increase in the ovulation and pregnancy rates in Nellore heifers raised under pasture conditions in the Amazon, compared to the FSH protocol. Given the context, the present study aimed to evaluate the follicular dynamics and pregnancy rate in Nellore heifers subjected to FTAI protocols associated with eCG) or FSH.All animals were managed on a commercial farm during routine management procedures and FTAI. Cows were treated according to best practices in veterinary care and animal welfare. The present study was approved by the Ethics Committee on Animal Use of the Institute of Biodiversity and Forestry, Santarém, Brazil (CEUA/IBEF) under protocol number 04.002/13).The experiments were conducted on a farm in the municipality of Mojuí dos Campos, Pará, Brazil (2°28′ and 4°23′ S and 54°31′ and 55°40′ W). The climate is hot and humid (Am4) with annual rainfall between 1900 to 2100 mm, an average annual air temperature of 25.6 °C, and relative humidity ranging from 84 to 86% . The raiBrachiaria brizanhta cv. Marandu) and had free access to water and mineral mixture. The females were randomized, considering the BCS to form the experimental groups. Nellore heifers (n = 259) were divided into two experiments. Experiment I evaluated follicular dynamics using either FSH  or eCG  FTAI protocols. Experiment II evaluated the pregnancy rate of heifers submitted to FTAI with a protocol containing FSH (n = 97) or eCG (n = 98). A limitation of this study was the fact that the reproductive tract score was not performed at the time of the randomization of the experimental groups; however, since it was a homogeneous group of animals from the same property, this limitation did not affect the validity of the results obtained.The females were healthy, without any reproductive alterations, cyclic, with a mean age of 21 ± 2 months, mean weight 340 ± 1.9 kg, mean body condition score (BCS) 3.1  . The heiExperiment I, the females were subjected to two different hormonal FTAI protocols (®) was introduced, then 2.0 mg of estradiol benzoate  was administered intramuscularly (IM). On day eight (D8) at 8 h, the progesterone implant was removed and administered by IM 2.5 mL of PGF2α  and 1 mg of estradiol cypionate (ECP®). At this time, heifers randomly received 300 IU of eCG (Novormon®) or 10 mg of FSH (Folltropin-V®) via IM. All hormones were from Zoetis  with exception of FSH which was bought from Vetoquinol Saúde Animal Ltd. . On day ten (D10) from 8 am, the FTAIs were performed. In Experiment II, we evaluated the pregnancy rate of Nellore heifers subjected to FTAI with the same protocols containing FSH or eCG (In rotocols . On day H or eCG .Experiment I were divided into two groups: FSH Group (n = 32) or eCG Group (n = 32). An ultrasound (US) device  equipped with a 7.5 MHz linear transducer (Nanshan®) was used to assess the presence of corpus luteum of heifers on day zero (D0), and the diameter of the dominant follicle on day eight (D8) and of the pre-ovulatory follicle on day ten (D10), and thereafter every 12 h until ovulation or until 96 h after FTAI. Ovulation time was defined when detected by US. Ovulation rate considered the heifers that ovulated and failed to ovulate within the 96 h post-FTAI interval, when the US exam was performed sequentially.For assessing follicular dynamics, females in To evaluate the pregnancy rate, 195 heifers that were subjected to hormonal protocol containing FSH (n = 97) or eCG (n = 98) were used. The pregnancy diagnosis was performed by US 35 days after the FTAI, and the presence of a viable embryo with a heartbeat indicated pregnancy.Experiment I, the data referring to the number of hours to ovulation were submitted to the Kolmogorov–Smirnov test, and since they presented a non-Gaussian distribution, they were compared by the Kruskal–Wallis test to assess whether the treatment had interference in the ovulation moment. The significance level adopted was 5% and the analyses were performed in statistical software .The analyses were performed separately, considering experiments I and II. The data were analyzed by the Chi-square test, used to determine whether there was a significant influence of the treatments (eCG or FSH) on ovulation and conception rates. Additionally, in Experiment I, the maximum diameter of the dominant and pre-ovulatory follicle and the ovulation time were similar between the FSH and eCG groups (p > 0.05). However, the ovulation rate was higher in the eCG group (87.5%) when compared to the FSH group (78.1%) (p = 0.014). Similar results were observed in a study with Nellore cows using eCG and FSH, where the ovulation rate was lower in the FSH [ the FSH . This isG group 8.5% when  the FSH . Previou the FSH .p = 0.003) and ovulation rate . [The eCG supplies the lack of LH and provides the follicle with the ability to develop until the pre-ovulatory phase, which contributes to increase the production of follicular estrogen. In this way, the gradual increase in plasma estrogen causes positive feedback in the hypothalamus, determining the pre-ovulatory peak of LH . Neverth 0.006). .p > 0.05) in the diameters of the dominant and pre-ovulatory follicles between the FSH and eCG groups, which may indicate that FSH promotes an adequate development in the final growth of the follicle in Nellore heifers. However, other studies have shown results indicating that FSH reduced follicular development compared to eCG when used in FTAI protocols in heifers [There were no differences ( heifers .Regarding pregnancy rate, it was found that females submitted to the pharmacological protocol with eCG showed better results when compared to FSH. The eCG positively influences the pregnancy rate, providing an increase in the fertility of the bovine herd ,27. SimiBoth of the treatments used efficiently induced follicular synchronization in Nellore heifers, which can probably be related to their good body condition score. The use of eCG in the FTAI protocol in Nellore heifers promoted a higher ovulation rate and increased pregnancy rate, and may be the most suitable alternative, aiming to raise the conception rates in animals bred in an extensive system under tropical conditions in the Amazon.


NOTCH3 gene is an important cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) disease modifier of age at first stroke and white matter hyperintensity (WMH) volume. No study has yet assessed the effect of other known CADASIL modifiers, that is, cardiovascular risk factors and sex, in the context of NOTCH3 EGFr group. In this study, we determined the relative disease-modifying effects of NOTCH3 EGFr group, sex and cardiovascular risk factor on disease severity in the first genotype-driven, large prospective CADASIL cohort study, using a comprehensive battery of CADASIL clinical outcomes and neuroimaging markers.A retrospective study has shown that EGFr  group in the Patients with CADASIL participated in a single-center, prospective cohort study  between 2017 and 2020. The study protocol included a clinical assessment, neuropsychological test battery and brain magnetic resonance imaging on a single research day. Multivariable linear, logistic and Cox regression models were used to cross-sectionally assess the effect of CADASIL modifiers on clinical severity  and neuroimaging markers .NOTCH3 EGFr 1–6 variant and 97 an EGFr 7–34 variant. NOTCH3 EGFr 1–6 group was the most important modifier of age at first stroke , lacune volume , WMH volume , and peak width of skeletonized mean diffusivity . EGFr 1–6 patients had a significantly higher WMH volume in the anterior temporal lobes and superior frontal gyri and a higher burden of enlarged perivascular spaces. After NOTCH3 EGFr group, male sex and hypertension were the next most important modifiers of clinical outcomes and neuroimaging markers.Two hundred patients with CADASIL participated, of which 103 harbored a NOTCH3 EGFr group is the most important CADASIL disease modifier not only for age at first stroke and WMH volume but also strikingly so for a whole battery of clinically relevant disease measures such as lacune volume and peak width of skeletonized mean diffusivity. NOTCH3 EGFr group is followed in importance by sex, hypertension, diabetes, and smoking. NOTCH3 gene (NOTCH3cys).2 SVD severity in CADASIL is highly variable, ranging from first stroke in the third decade to symptom-free survival up to the eighth decade.4 The variability in CADASIL disease severity is poorly understood, but NOTCH3cys variant location in one of the 34 EGFr  domains of the NOTCH3 ectodomain has been shown to be the most important modifier in CADASIL in retrospective studies: NOTCH3cys variants located in one of the first 6 EGFr domains (EGFr 1–6) have been found to be associated with a higher risk of stroke7 and higher white matter hyperintensity (WMH) volume5 than NOTCH3cys variants located in one of EGFr domains 7 to 34 (EGFr 7–34). However, much is still to be learned about the differences in SVD phenotype between EGFr 1–6 and EGFr 7–34 patients with CADASIL, as no study has yet compared the burden of all relevant SVD imaging markers, clinical symptoms and cognitive function between the NOTCH3cys EGFr groups. Moreover, all previous studies investigating cysNOTCH3 EGFr group were retrospective in nature, which could have led to substantial bias.Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary small vessel disease (SVD) caused by pathogenic cysteine altering variants located in the NOTCH3cys EGFr group, cardiovascular risk factors (CVRF)17 and sex15 have been shown to modulate CADASIL disease severity. However, the relative impact of the various modifiers is unknown, as EGFr group has never been included in studies addressing CADASIL disease modifiers.Next to Here, we performed a prospective, genotype-driven, single-center cohort study of 200 uniformly characterized patients with CADASIL called DiViNAS , with an equal representation of patients with EGFr 1–6 and EGFr 7–34 variants. We investigated for the first time differences in disease severity between these EGFr groups using extensive clinical and neuroimaging phenotyping, including clinical data of the affected parent of each participant. We also included less well studied CADASIL imaging markers, such as enlarged perivascular spaces and diffusion tensor imaging (DTI) metrics, and determined the relative disease-modifying effects of EGFr group, CVRF, and sex on clinical and neuroimaging SVD outcomes.The data that support the findings of this study are available from the corresponding authors upon reasonable request.NOTCH3cys variant were contacted and asked to participate in the DiViNAS prospective, genotype-driven cohort study. All individuals in the DiViNAS cohort were 20 years or older and were included between November 2017 and December 2020. Each participant completed the full study protocol at the research site , with clinical assessment, neuropsychological test battery and brain magnetic resonance imaging performed on a single day at the same study site by the same researchers for all participants.All patients and presymptomatic family members in the Dutch CADASIL registry with a genetically confirmed 18The study was approved by the medical ethics committee of the Leiden University Medical Center (P18.164 and P17.170). All participants gave written informed consent. This manuscript follows the STROBE  reporting guideline.Supplemental Methods.19The following clinical outcomes were compared between EGFr 1–6 and EGFr 7–34 patients: age at onset and presence of stroke, transient ischemic attack, dementia, depression, apathy, migraine with/without aura, encephalopathy, walking disabilities, seizures, cognitive test scores, and disability assessed with modified Rankin Scale (mRS). Details about the definition of the clinical symptoms and CVRF can be found in the Also, the risk of stroke and death was assessed in the affected parent of each participant by determining the age at first stroke and age at death on clinical record, family history, and the Personal Records Database of the Dutch government, respectively. The affected parent of each participant was determined based on genetic testing (n=81) or on clinical and family history (n=46). The affected parent could not be determined in 13 participants.Laboratory evaluation included hemoglobin A1C and a nonfasting lipid panel .20 Details about the calculation of cognitive test scores can be found in the Supplemental Methods.22The cognitive examination battery included Montreal Cognitive Assessment, Trail Making Test A and B, Stroop Test, Rey Auditory Verbal Learning Test, Verbal Fluency animal and professions, Wechsler Adult Intelligence Test IV Block design, Coding, and Digit Span. An established composite score of processing speed was calculated by averaging t scores of Trail Making Test A and B.23 The brain magnetic resonance imaging protocol included the following sequences: 3-dimensional T1-weighted images, T2-weighted images, fluid-attenuated inversion recovery, susceptibility-weighted images, and diffusion-weighted imaging sequences. Acquisition parameters are presented in the Supplemental Methods.Brain magnetic resonance imagings were all performed on the same 3 Tesla MR system  and evaluated according to consensus criteria.23: normalized WMH volume (nWMHv), normalized lacune volume (nLV), cerebral microbleed (CMB) count, brain parenchymal fraction (BPF), and burden of enlarged perivascular spaces (ePVS). BPF was defined as the ratio of brain parenchymal volume to the intracranial volume expressed as percentage. WMH and lacune volume were normalized to the intracranial volume . ePVS were evaluated in 4 specific regions: the global white matter, basal ganglia, subinsular region and anterior temporal lobes (ATL), according to a 4-grade semiquantitative scale.24 As WMH in the ATL and superior frontal gyri (SFG) have been shown to be frequently present in CADASIL and lead to a higher brain volume due to increased water content,26 nWMHv was also assessed in these regions separately and used as a covariate in the analyses with BPF as outcome.The following conventional SVD markers were evaluated according to consensus criteria27 is the difference between the 95th and 5th percentiles of the voxel-based mean diffusivity values within the skeleton.The following DTI metrics were quantified on preprocessed diffusion-weighted images: peak width of the skeletonized mean diffusivity (PSMD), fractional anisotropy, mean, axial, and radial diffusivity. PSMD, which is a proven robust imaging marker for SVD,Supplemental Methods.28Details about the quantification of the conventional SVD imaging markers and DTI metrics can be found in the t tests. Non-normally distributed continuous variables were summarized using median with interquartile range. Binary categorical variables were compared between 2 groups using Fisher exact Tests.Normally distributed continuous variables were summarized using mean±SD and compared between 2 groups using unpaired 10[x+1]), fractional anisotropy (squared), other diffusion metrics , Trail Making Test t scores (squared), Montreal Cognitive Assessment score (reflect and cube root). CMB count , and nLV were divided in quartiles.In all analyses, continuous variables were standardized by dividing their value by their SD. The following variables were transformed to obtain a normal distribution or a linear relationship between dependent and independent variables in multiple linear regression: total nWMHv (square root), nWMHv in ATL and SFG (ln transformed), nLV (cube root), CMB count (logPage). In time-to-event analysis, log-rank test was used to compare age at onset of clinical symptoms between the 2 groups (PLR).Multiple linear and logistic regression models were used to compare clinical symptoms, cognitive scores and SVD neuroimaging markers between EGFr 1–6 and EGFr 7–34 patients, corrected for age .Multiple linear, logistic, and Cox regression models were used to assess the effect of EGFr group, sex, and CVRF on 8 different SVD outcomes: age at first stroke, processing speed, mRS score, nWMHv, nLV, BPF, CMB count, and PSMD. To assess the association of hemoglobin A1C or nonfasting lipid levels  with disease severity, diabetes and hypercholesterolemia were excluded as covariates, respectively. Bonferroni correction was used to correct for multiple testing of 8 different SVD outcomes . Two-sided NOTCH3cys variant located in one of EGFr domains 1–6 and 103 patients in one of EGFr domains 7–34 (Table S1).A total of 200 patients with CADASIL from the Dutch CADASIL registry were recruited in the DiViNAS cohort, of which 97 patients harbored a P=8.6×10-5; Table P=0.012). Although there were no significant differences in CVRF, EGFr 7–34 patients more often had diabetes type 1 or 2 (P=0.08), hypertension (P=0.15), and hypercholesterolemia (P=0.14).EGFr 1–6 patients were significantly younger than EGFr 7–34 patients: mean age 48.9±12.3 years  versus mean age 55.6±11.3 years  and transient ischemic attack  and a lower life expectancy  or cognitive test scores  between EGFr 1– 6 and EGFr 7–34 patients (Table S2). There was also no significant difference in presence and age at onset of any of the following: migraine with aura, dementia, depression, seizures, walking disability, and CADASIL encephalopathy.EGFr 1–6 patients had a significantly earlier onset of stroke than EGFr 7–34 patients , PSMD (P=2.2×10-8) and nLV . Additionally, EGFr 1–6 patients had a significantly higher nWMHv in the ATL and SFG (P=2.1×10-15), which was still significant after adjustment for total nWMHv  and subinsular regions (P=0.006), but not in the basal ganglia or the white matter , but this was no longer significant after adjustment for nWMHv in the ATL and SFG (Table S4).After adjustment for age, EGFr 1–6 patients had a significantly higher nWMHv , nLV (P=0.004) and unspecific nWMHv . There was no independent association between mRS score and nWMHv in the ATL and SFG (P=0.46) or ePVS score in any region.In multivariable analysis, the following SVD imaging markers were significantly associated with mRS score: BPF . For disability, the most important modifier was hypertension , followed by male sex , diabetes  and EGFr 1–6 group .In multivariate analysis including EGFr group, sex, and CVRF, only EGFr 1–6 group was significantly associated with an earlier onset of stroke  and PSMD , followed by male sex  and hypertension , followed by diabetes , EGFr 1–6 group  and packyears , followed by male sex . Nonfasting levels of LDL-C, HDL-C, triglycerides, hemoglobin A1C were not significantly associated with any of the clinical outcomes or neuroimaging markers in multivariable models.In multivariate analysis of SVD imaging markers, EGFr 1–6 group was the only disease modifier significantly associated with a higher nWMHv , and in these population cohorts are associated with a very broad spectrum of cerebral SVD and even nonpenetrance.5 Therefore, EGFr 7–34 patients in CADASIL cohorts, although milder than EGFr 1–6 patients, still constitute the severe end of the EGFr 7–34 SVD spectrum.In this prospective, genotype-driven, single-center CADASIL cohort study, addressing for the first time the relative disease-modifying effects of NOTCH3cys EGFr 1–6 variants are not only associated with a higher WMH volume and an earlier onset of stroke7 but also with other important CADASIL neuroimaging markers which are associated with disease severity, namely lacune volume and microstructural white matter damage as measured by DTI,27 as well as enlarged perivascular spaces. These findings can improve CADASIL patient stratification and inclusion for clinical studies, as the most severe patients are clearly those with EGFr 1–6 variants, and their disease severity is reflected by the clinically most relevant neuroimaging markers.27A novel finding of this study is that 26 Indeed, we found that nWMHv in CADASIL-specific regions was significantly associated with a higher BPF. Possibly, NOTCH3cys-EGFr 1–6 variants lead to a more pronounced blood-brain barrier dysfunction in these regions.30EGFr 1–6 patients had a higher burden of WMH and ePVS in CADASIL-specific regions, that is, ATL, SFG and subinsular region, previously shown to be associated with increased water content and brain swelling.31 In addition, older EGFr 1–6 patients were underrepresented because they were more likely to be too disabled to participate or to be deceased, or were diagnosed with CADASIL at a younger age.Despite the substantially higher burden of SVD neuroimaging markers, EGFr 1–6 patients did not have higher disability scores than EGFr 7–34 patients, and they performed equally well on cognitive tests. Potential differences in cognitive function and disability may have been masked by the fact that EGFr 1–6 patients had a higher educational level than the EGFr 7–34 participants, which has been shown to protect against the detrimental effects of brain damage in patients with CADASIL.7 A recent study points towards a potential pathomechanistic explanation for the differences between EGFr groups, as EGFr 1–6 variants were shown to be associated with significantly higher NOTCH3 aggregation load in CADASIL vessels than EGFr 7–34 variants.32 However, there are some variants in EGFr 7–34 which are frequent in numerous CADASIL cohorts, suggesting that some EGFr 7–34 variants may be more severe and the current 1–6 versus 7–34 model may be too simplistic.EGFr 1–6 variants are extremely rare in the population, which has been the rationale for the cutoff between EGFr group 1–6 and EGFr group 7–34 in this and other studies.13. The fact that we were able to discern this effect is likely due to our study design, which includes not only EGFr group but also all CVRF. Possibly, the effect of sex is modulated by the enhanced vasodilatory and neuroprotective effect of premenopausal female hormones.35After EGFr group, we found sex to be the most important disease modifier; male sex was associated with increased disability, risk of stroke, lacune volume, CMB count, and lower BPF. This important modifying effect of sex has not been consistently observed in prior studies16. Here, we found that hypertension was significantly associated with nLV, CMB, and disability but not with nWMHv9 and stroke.12 Also, packyears of smoking were associated with a lower brain volume and lower processing speed in DiViNAS participants but not with an increased risk of stroke as 2 prior studies.15 Differences in study design, cohort composition and definition of CVFR likely account for the differences that are found between CVFR and disease severity measures in the large CADASIL cohort studies performed to date.16 Taken together, however, hypertension is consistently found to be an important and actionable disease modifier in CADASIL, which suggests blood pressure monitoring and antihypertensive treatment may be beneficial for patients with CADASIL. However, therapeutic trials are warranted as cerebrovascular reactivity and autoregulation are impaired and lowering blood pressure could cause cerebral hypoperfusion.36 In contrast, in line with the European Academy of Neurology consensus statement,37 standard preventive prescription of statins seems not to be indicated in patients with CADASIL with normal lipid levels, as hypercholesterolemia has never been found to be associated with any of the CADASIL outcome measures, a finding which we corroborate in this study.34 However, statin therapy is not contraindicated in patients with CADASIL, and statins can be prescribed for other indications such as coronary artery disease.Hypertension has been described to be associated with a number of neuroimaging markers and stroke in patients with CADASIL.Only 13 individuals in the DiViNAS cohort had diabetes type I or II, but despite the small sample size we still found a significant association between diabetes and a lower BPF. There was also a trend towards higher disability in DiViNAS participants with diabetes, suggesting a potential strong effect of diabetes on CADASIL disease expression. Larger cohorts are needed to accurately estimate the effect of diabetes on CADASIL disease severity.Although EGFr group and CVRF have now unequivocally been shown to be important CADASIL disease modifiers, a significant proportion of disease variability is still unaccounted for. Identifying these (genetic) modifiers will contribute to improved personalized disease prediction and may lead to the identification of novel biological treatment targets for CADASIL.A limitation of this study is the selection bias towards relatively mildly affected patients, and the consequent relatively low frequency of clinical stroke in this cohort. Strengths are the genotype-driven study design, the prospectively and uniformly collected data including high-resolution neuroimaging, and the fact that both EGFr groups are equally represented.NOTCH3cys EGFr group is the most important modifier of CADASIL known to date, followed by male sex, hypertension, diabetes, and smoking. These findings are important for the improvement of disease prediction, for guiding patient inclusion in clinical trials and for developing guidelines for secondary disease prevention.In conclusion, this prospective, genotype-driven cohort study has validated and extended the substantial differences in disease severity between EGFr 1–6 and 7–34 CADASIL patient. Our results show that The authors wish to acknowledge all the patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) who participated in our studies. Dr Hack designed and conceptualized the study and contributed to acquisition of data, analyzed and interpreted the data, and drafted the manuscript. Dr Cerfontaine and S. Tap contributed to acquisition of data and critical revision of the manuscript for important intellectual content. Drs Gravesteijn, Hafkemeijer, van der Grond, Witjes-Ané, and Baas contributed to critical revision of the manuscript for important intellectual content. Dr Rutten and Lesnik Oberstein designed and conceptualized the study and contributed to acquisition of data, interpreted the data, critical revision of the manuscript for important intellectual content, and supervision.This study was funded by Netherlands Organisation for Health Research and Development (ZonMW 91717325) and The Netherlands Brain Foundation (HA2016-02-03).Dr Hack and Cerfontaine are funded by the Netherlands Organisation for Health Research and Development (ZonMW 91717325). Dr Rutten has financial research support from the Netherlands Organisation for Health Research and Development (ZonMW 91717325) and the Netherlands Brain Foundation (HA2016-02-03). Dr Lesnik Oberstein has financial research support from the Netherlands Organisation for Health Research and Development (ZonMW 91717325) and the Netherlands Brain Foundation (HA2016-02-03), and receives grants from Leiden University Medical Center. The other authors report no conflicts.Supplemental MethodsTable S1–S5Figure S1STROBE ChecklistReference 27


We aim to assess the effect of late prematurity on exercise capacity in childhood and to compare it to early preterm (EP) (born < 300 GA), and to term healthy control (TC) (>370 week GA). Methods: Late preterm and early preterm (7–10 years) completed a cardiopulmonary exercise test (CPET) and spirometry and were compared to EP and to TC. Results: Eighty-four children  participated. Twenty-one former LP were compared to 38 EP (15 with Bronchopulmonary dysplasia (BPD) [EP+], 23 without BPD [EP−]) and to 25 TC children. Peak oxygen uptake (peakV̇O2) was statistically lower than in the TC, but within the normal range, and without difference from the EP . Lung function (FEV1) was lower than normal only in the EP+ . Respiratory and cardiac limitations were similar between all four study groups. Conclusions: This study demonstrated lower exercise capacity (peakV̇O2) in former LP children compared with healthy term children. Exercise capacity in LP was comparable to that of EP, with and without BPD. However, the exercise test parameters, specifically peakV̇O2, were within the normal range, and no significant physiological exercise limitations were found.Background: Late preterm (34 This definition of BPD, moderate or severe (or grade 2–3), predicts early childhood morbidity [The study population included three main groups: late preterm infants , and a congenital cardiac defect . Children with acute febrile or upper respiratory illness in the last 2 weeks, using systemic or inhaled steroids in the previous month or inhaled bronchodilator use in the previous 24 h were excluded.The different infants’ groups were identified using hospital’s computerized list generated according to birth gestational age or from the pediatric pulmonary institute outpatient clinic. BPD was assessed from the computerized medical records.An initial phone call was done between a neonatologist (O.H.) or a pediatric pulmonologist (R.B.-Y.) and the parents, explaining about the study and assessing the inclusion and exclusion criteria. Control group was recruited from healthy siblings and friends of the study group children and from healthy children visiting the children hospital with their families. At the day of the study, the parents received a repeated explanation and signed an informed consent.Age, sex, height, body mass, and body mass index (BMI) as well as z-scores for body mass index (BMI) based on center of disease and control (CDC) criteria were recorded . Each pa1), and maximum voluntary ventilation (MVV) were calculated using the 12-s sprint method [1 was considered exercise induced bronchoconstriction.Spirometry was performed in accordance with the European Respiratory Society and American Thoracic Society guidelines using a Quark spirometer  . Spiromet method . Pulmona2) were recorded at rest, during the test and during the recovery period. SpO2 was measured continuously using Massimo SET 2000 (Schiller) and recorded at baseline, every 120 s, peak exercise and one, two- and five-min post exercise. Criteria for terminating the test were: inability to maintain pedaling cadence (<60 rpm), in association with subjective evidence of fatigue , and one or more of the following: peakV̇O2 > 80% predicted [2 plateau (failure to increase oxygen uptake despite a continuous increase in work) [CPET was performed using a Quark CPET metabolic cart  according to ATS guidelines . All theredicted , maximalredicted , respirain work) . Breathiin work) .p < 0.05 was considered as significant. A 10% difference in peakV̇O2 was considered clinically significant and, using a significance level of 5% with 80% power, a sample of 25 children in each group was required for demonstrating a statistical significant difference in peakV̇O2.Descriptive statistics in terms of mean, standard deviation, median and percentiles were calculated to the whole parameters in the study. Normal distribution of the quantitative parameters was tested by Kolmogorov–Smirnov. As a result of this test, we used ANOVA and a Kruskal–Wallis test for differences between groups with Bonferroni or Dunn’s post-tests, respectively. SPSS  version 27 was used for all statistical analysis. Eighty-four children participated in the study: 21 LP, 38 EP , and 25 TC. All patients completed PFT and maximal CPET. No adverse events were recorded . Demogra1 (14.9 ± 75.6% predicted) and lower than all other groups (p ≤ 0.02). The FVC tended to be lower in the EP(+) group, but there was no significant difference between the groups, and all were within normal limits. None of the participants had post exercise bronchoconstriction.Pulmonary function tests are presented in 2 was within the normal range and comparable between all preterm groups but statistically lower than our healthy control group . Pre-exercise and peak SpO2 were preserved and similar between groups. Ventilatory equivalent for V̇CO2 (V̇E/V̇CO2 slope measured during ramp protocol up to the anaerobic threshold) was normal and similar between groups. Low BR was significantly more common in the LP group compared to TC  with no difference compared to low BR in the EP groups. Peak O2 pulse (V̇O2/HR% predicted) was comparable and within the normal range for all preterm groups) but statistically lower from the TC group.CPET parameters are presented in The general questionnaires and the CAPE questionnaires did not show any significant difference in daily leisure and physical activity between the three groups.2, were within the normal range and no significant physiological exercise limitations were found. Spirometry values were also within normal range in all groups; only FEV1 was significantly lower in the BPD group.This study demonstrated lower exercise capacity in former LP children aged 7–10 years compared to healthy term children. Exercise capacity in LP was comparable to that of EP, with and without BPD. However, the exercise test parameters, specifically peakV̇O2 was significantly lower than in the TC group, but similar to the EP groups, (with or without BPD). Svedenkrans et al. studied 218,820 young males (18–25 years) conscripting for military service who completed a modified maximal exercise test on a cycle ergometer and found lower exercise capacity in subjects born moderately preterm (32–36 weeks GA) vs. term; however, they did not report gas exchange parameters including peakV̇O2. Additionally, the distribution of results consisted of both LP and infants born earlier, at 32–33 weeks GA [In our LP group, peakV̇Oweeks GA .1 and V̇O2max) compared to the term-born group [Evidence from other, simpler, techniques to assess physical fitness  suggest that those born as early preterm had lower muscular fitness than controls born at term and report to engage less in leisure time physical activities ,33. Tamarn group . To summThe precise underlying cause of lower aerobic exercise capacity in children born preterm is not entirely known, but it is probably not solely due to low breathing reserve . It could be affected by a number of interrelated parameters including mechanical ventilatory constraints, impaired cardiovascular function and altered muscle bioenergetics , as well2 slope, a submaximal marker for increased ventilatory drive that is related to the amount and sensitivity of central chemoreceptors and the ventilatory dead-space) were similar between all groups and could not further explain our above findings.In our study, intriguingly, respiratory limitation  was more common in the LP group (43%) compared to TC (12%), with no statistical difference from other study groups . While this potential limitation could be explained by low pulmonary function tests in the EP+ group, we have not found an explanation in the other groups. Furthermore, the ventilatory equivalents  and significantly lower than in the TC group, lung function was significantly lower only in the BPD group. Similar to the results in our study, the diagnosis of BPD in a preterm infant was shown to be associated with impaired lung function later in life [1 [1 within the normal range (82% pred) but lower than control [Although peakV̇O in life ,37. Howe life [1 ,39. The  control ,41. On t control ,42,43.In addition to the risk of long-term deficits in lung function, prematurity (mainly EP) is also associated with cardiovascular changes including alterations in arterial distensibility , smaller2pulse parameters  were similar and within the normal range for all study groups and only peak O2pulse was statistically lower from the TC group. O2 pulse curve flattening was not observed in any of the patients. Baseline and peak exercise oxygen saturation (Sat. O2) were normal in all participants. Baseline echocardiography was not performed in this study, but no significant heart abnormalities were reported. These findings could result from possible lower habitual physical activity in the study groups compared to the control group.In our study, OAnother possible functional limitation could be the cognitive system and motor performance. In a previously mentioned study, Svedenkrans et al. showed lower cognitive function for young men born moderately preterm (32–36 weeks GA) with a positive correlation between cognition and exercise capacity . In the The strength of our study is the comparison of LP to both early preterm groups (with and without BPD) and healthy controls and using the same comprehensive tests , investigators, technicians, and equipment. The control group was chosen mostly from the same surrounding of the preterm infants . Another advantage is that it was done on children born as preterm in the “new BPD” era, while many previous studies assessed preterm infants with “old BPD”, thus less applicable to our time.Interestingly, although even the low degree of prematurity of the LP group had a significant influence on exercise capacity compared to TC, we found no significant difference between LP and EP groups. This finding is intriguing. Theoretically, all the above-mentioned explanations for lower exercise capacity  may have an effect towards a further decrease in exercise capacity as gestational age at birth decreases. However, the questionnaires in our study failed to describe any difference regarding physical activity between the LP and EP groups. Moreover, it was observed that lung function alone is not necessarily a major factor influencing exercise capacity . In the 2% predicted in our control group was 112.4 ± 16.9%. This may reflect our pediatric population characteristics; however, a selective bias of fit children cannot be excluded.Our study has several limitations. First, it is a relatively small sample size. This could result in beta error. Our relatively small number of participants was partially due to the challenge of voluntary recruitment in the COVID-19 era. The exclusion criteria of children who were unable to participate in the CPET  could create a selection bias. The participants did not have routine baseline echocardiography and validated physical activity questionnaire. The mean VO2, were within the normal range both in EP and LP, and no significant physiological exercise limitation was found. These results are encouraging. Nevertheless, a personalized respiratory, fitness, and physical activity follow-up in cases of prematurity should be encouraged, and parents of all preterm infants should actively encourage their children to participate in regular childhood physical activities and sports. Longer and larger prospective case-control studies are needed focusing on the physiological mechanisms to possible exercise limitations.In conclusion, our study demonstrated lower exercise capacity in former LP children aged 7–10 years compared to healthy term children and comparable to that of EP (with and without BPD). The PFTs in LP were within the normal range in our study and could not explain the lower exercise capacity. The etiology for lower exercise capacity in LP is unclear and may include reduced physical activity, low breathing reserve, altered muscle metabolism, and altered heart structure and function. However, the exercise test parameters, specifically peakV̇O


Tissue engineering strategies have attempted to mimic regenerating axons' environment by adding supportive types of cells other than Schwann cell to the nerve allograft. We hypothesized that allografts can be seeded with amniotic fluid-derived stem cells (AFS) to promote nerve regeneration.  ANAs with AFS cells for long-gap nerve repairs were studied using a rat model. A sciatic nerve injury was created and repaired immediately with a rat acellular nerve allograft (ANA) construct alone, an ANA construct seeded with AFS cells, or with an autograft. Walking track analysis and electrophysiology were performed to document the return of motor control at 4 months post injury. Axon morphology on the nerve segments was assessed. In vivo gait analysis showed that the ANA plus AFS cell group had significantly advanced recoveries in overlap distance, paw angle degree, paw drag, stance width, axis distance, and sciatic function index (SFI) compared with ANA alone. The ANA plus AFS cell group also demonstrated greater gastrocnemius compound muscle action potential (CMAP) ratio, sciatic axon diameter, fiber diameter, myelin thickness, G ratio /fiber diameter (FD)), and neuromuscular junction (NMJ) numbers compared to ANA. Discussion. The allograft plus AFS cell group demonstrated significantly improved functional and histological outcomes compared to allograft group alone, showing no significant difference of the nerve regeneration from the autograft group. Thus, AFS cells may be a suitable cell source to replace Schwann cells to support and accelerate peripheral nerve regeneration following large-gap nerve injury. Peripheral nerve injury remains a challenging clinical problem with residual functional deficits (motor and sensory) associated with attempted regeneration across irreparable nerve gaps. In addition to fibrosis in the nerve bed and at the site of injury, peripheral nerves have an inherent regenerative difficulty in overcoming gap defects , 2. AlthAcellular nerve allografts (ANAs) are promising alternative to autografts without the immunosuppressive concerns to the host tissue . Howeverin vitro [Amniotic fluid-derived stem (AFS) cells have multipotency to differentiate into all three embryonic germ layer cell types; they also demonstrated a lack of immunogenicity and have the potential to differentiate and take on nerve cell characteristics in the presence of biochemical cues in vitro , 15. Onein vitro , 17. Henin vitro . However6 cells were suspended in 50 μl media then injected underneath the epineurium of the allografts from both ends using a 26 G syringe. Seeded grafts were placed vertically at the bottom of a small centrifuge tube covered with DMEM containing 20% FBS overnight then transferred to a 48-well plate for an additional 48 hours before being implanted into the left sciatic site of the animal. After 60 hours of incubation, the allograft was fixed in 4% of paraformaldehyde for 1 hour and the AFS cell attachment to the allograft prior to implantation was assessed by DAPI staining  on 7 allografts  were processed by AxoGen Corp, FL. The AFS cells were a gift from NuTech, Inc, AL. The cells were cultured and expanded in modified Dulbecco's Eagle's medium  containing 20% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The 1.5 cm long decellularized sciatic nerve allograft was gently perforated of the epineurium using a microneedle array. 1.5 × 10lografts .6 AFS cells previously prepared or ANA construct without cells. For the autograft group, the sciatic nerve was transected and flipped then sewn back to repair the defect. Following nerve repair, the muscle was approximated using interrupted sutures of 5-0 coated Vicryl. The skin was closed by approximating the cut edges using stainless steel surgical wound clips and subdermal sutures 5-0 coated Vicryl. The rat was given buprenorphine for postsurgical analgesia  at the end of surgery  of Wake Forest University Health Sciences. Three groups , 12 male Lewis rats per group, underwent left sciatic nerve transection surgery. The rat was anesthetized using isoflurane (1.5-2.5 volume %), initially in an induction chamber. Anesthesia was maintained via a nosecone at 1.5 volume%. The posterior aspect of the left hind limb was shaved with clippers, cleansed with betadine scrub, and disinfected with betadine solution. Using aseptic technique, a posterolateral incision was made and the sciatic nerve was exposed by dissecting the muscle plane. A 1.5 cm nerve defect was created and repaired using 10-0 nylon and standard microsurgical technique by interposing a 1.5 cm nerve allograft seeded with 1.5 × 10 surgery .Gait recovery is an indicator of return of motor control. The DigiGait Imaging System (Mouse Specifics Inc. MA) was used to test the motor function recovery of the allograft reconstruction following sciatic nerve defect in Lewis rats . The DigCadwell EMG Sienna Wave System was used for the electrophysiology testing. 4 months after the nerve autograft, ANA, and ANA plus AFS cell implantation, rats were anesthetized with isoflurane and the regenerated and contralateral sciatic nerves were exposed. At first, tibial and peroneal branches distal to the regenerated gap were briefly stimulated to test for plantar flexion and foot eversion. After the viability of the nerve was assessed, electromyographic analysis was examined by stimulating the regenerated nerve proximally  with a monopolar cathodic electrode at 1 mA, the reference anode was placed on the rat chest. The stimulating–recording electrode distance was verified visually using a ruler. Muscle contractions were recorded by electrodes placed into the gastrocnemius muscle  and tibialis muscle of both experimental and control limbs. Compound evoked muscle action potential (CMAP) was recorded by three consecutive stimulations that were averaged for CMAP delays and amplitude measurement. The measurement of CMAP was converted to ratios of the injured side to the normal contralateral side to eliminate the influence of anesthesia .μm sections were cut 1 mm distally to the distal suture in the ANA to assess the regenerating nerve fibers penetrating to the distal nerve stump. The slides were stained with toluidine blue and examined under Zeiss light and electronic microscopes  at 200x and 3700x final magnifications. The images were analyzed using ImageJ software to measure regenerated axons. The number of axons was counted, and the outlines of myelinated axons and total axons were manually traced. The cross-section area, the number of myelinated fibers (n), myelin thickness (MT), average axonal diameter (AD), and fiber diameter (FD) were assessed using ImageJ software (Wayne Rasband). The G ratio was calculated as AD/FD, and the fiber density calculated as number of fibers per square millimeter. The axonal area (_πAD/2)2 and the fiber area (_π FD/2)2 were obtained assuming the circularity of the nerve fiber area. The myelinated area was measured as the difference between fiber area and axonal area [The animals were euthanized with intracardiac injection of saturated potassium chloride. The ANAs and contralateral nerves were harvested together with the proximal and distal nerve stumps. The nerve samples were fixed in 4% paraformaldehyde or 2% osmium tetroxide, dehydrated, and later embedded in paraffin or resin. Serial 5 μm cross sections of muscle were cut from the midline maximal area and stained with α-bungarotoxin at 1 : 2000  to visualize neuromuscular junction replenishment following nerve injury and repair. 10 continuous sections of gastrocnemius and tibialis anterior muscles were analyzed per sample.The gastrocnemius and tibialis muscles from both the experimental and contralateral side were harvested and weighed . The rat∗p < 0.05; very significant, ∗∗p < 0.01; and extremely significant, ∗∗∗p < 0.001.Results were reported as mean values and the standard error of the mean (SEM). One-way ANOVA test with Bonferroni multiple comparisons was used to determine the statistically significant differences between experimental groups. The following conventions were used: significant, 6 AFS cells were stained with DAPI and viewed under a confocal microscope . Seeded AFS cells were viable and spread evenly longitudinally through the nerve fibers 60 hours postinjection. The cells seeded into ANA at the end of 60 hours before implantation were 1.1 × 106 ± 1203 per graft (n = 7)  .Gait analysis of 24 parameters at the end of 4 months following injury indicated that there were no significant differences in stance/swing ratio, stride time, stance factor, swing stride percentage, brake stride percentage, propel stride percentage, stance stride percentage, brake stance percentage, propel stance percentage, hind limb shared stance percentage, step angle, and stride length among three groups.∗p < 0.05 and ∗∗p < 0.01 in all indices, The autograft group showed significant enhanced recovery at stance width, overlap distance, ataxia coefficient, axis distance, and SFI compared to the ANA and ANA plus AFS groups. The ANA plus AFS group exhibited improved functional recovery in stance width, overlap distance, midline distance, axis distance, paw angle, and paw drag than the ANA group alone and did not show significant differences from the autograft group in these parameters, indicating beneficial regenerating ability of AFS cells at the end of 4 months following a long nerve gap injury. In addition, although the SFI of the ANA plus AFS group did not return to the level of the autograft group, the ratio of 4 months postsurgery to the baseline was significantly higher than allograft alone, suggesting an overall superior sciatic function recovery to the ANA group . CMAP ratio of the tibialis muscle had no significant differences between the autograft and ANA plus AFS groups but was significantly higher than the ANA group alone  autograft vs. ANA vs. ANA+AFS: 10.14 ± 3.52 vs. 9.20 ± 3.33 vs. 10.32 ± 2.7; right: 34.25 ± 8.25 vs. 33.45 ± 4.2 vs. 26.37 ± 6.17; E/C ratio: autograft vs. ANA vs. ANA+AFS: 0.29 ± 0.04 vs. 0.27 ± 0.04 vs. 0.37 ± 0.05, G ratio compared to the ANA group , nerve conduction velocity, compound muscle action potential (CMAP) and histomorphometry. The conclusion was that ANAs supplemented by supporting cells had an overall comparable outcomes to autografts, some of the cell-enhanced ANAs showed superiority to other study groups . Among tRecently, gestational tissues such as placenta, placental membrane, and amniotic fluid have attracted wide attention in regenerative medicine as an abundant source of highly multipotent and immunosuppressive cells. The advantages of these gestational tissues are easy collection, which are usually discarded after birth or through routine amniocentesis and minimal ethical and legal concerns associated with the usage and the convenient application clinically. The amnion is the inner part of the amniotic sac that contains the fetus and amniotic fluid. The amnion is derived from ectoderm and mesoderm, and the amniotic fluid contains a mixture of stem cell types including amnion epithelial cells and amniotic fluid stem (AFS) cells that possess multipotent differentiation, anti-inflammation, and low immunogenicity characteristics , 14, 21.in vivo preclinical perspective. As regards motor function assessment, the treadmill computerized gait analysis system captures the locomotion of the running animal continuously and generates the digital paw prints which can be translated to dynamic gait signals. This method allows the kinematics of gait of each limb to be analyzed precisely in each animal longitudinally, and the number of animals required for each cohort group was significantly reduced. We found that the overall gait function of injured limb in all 3 groups  did not return to the extent of baseline at the end of 4 months postinjury. However, the AFS plus ANA group demonstrated improved motor functional recovery in stance width, overlap distance, midline distance, axis distance, paw angle, and paw drag parameters. Most of these indices reflect the changes of balance and postal stability affected by the lesions of sciatic nerve [It is currently unclear how human stem cell therapies contribute to peripheral nerve regeneration. A variety of different sources of stem/precursor cells are under study to determine their potential for peripheral nerve repair . Howeveric nerve . For insWe also found paw angle value was significantly decreased with AFS cell treatment. This factor is considered as the level of outward rotation of the paw and is usually used to evaluate the return of tibialis anterior muscle function. Our results were in agreement with other studies –26 that in vivo study, all 3 groups of animal had significantly impaired SFI 1 month after nerve injury, and the SFI gradually improved over time but all of the groups did not restore to the level of baseline at the end of 4 months. The ANA plus AFS group had a significantly higher average SFI value than the ANA group but still lower than that of the autograft group. This phenomenon was consistent with the observation that on the treadmill, the rats that performed better had less severe and fewer toe contractures, which is a known factor to interfere with SFI calculation.Sciatic function index (SFI) has been well accepted for reflection of the overall nerve function recovery after transection injury. In our G ratio compared with the ANA group. Along with the evidence of facilitated NMJ restoration in the end muscles, AFS cell treatment leads to beneficial outcomes in nerve regeneration, neuronal signal conduction, and muscle motor functional recovery. Thus, the results presented here have potential implications for future cell-based therapies to enhance peripheral nerve regeneration in clinical use.In addition to the functional beneficial effects, the ANA plus AFS group also showed enhanced electrophysiological and histomorphological outcomes. The AFS cells significantly facilitated CMAP in both gastrocnemius (~0.4 E/C ratio) and tibialis anterior (~0.5 E/C ratio) muscles with electrical stimulation to the distal nerve stump near suture site. We did not find any significant differences of CMAP of the injured side between the autograft, ANA, and ANA plus AFS cell groups. However, the ANA plus AFS cell group demonstrated a lower CMAP in the contralateral uninjured gastrocnemius muscle, possibly due the prolonged experimental procedure, and might result from the side effects of isoflurane exposure , 17 ThesTo date, the solution to repair large defect peripheral nerve injuries (PNI) is limited and numerous studies have focused on designing the ideal conduits for peripheral nerve regeneration. Nerve autografts are considered the gold standard to provide the most closely native neuronal microenvironment, which preserve the Schwann cells and intact architecture of endoneurial tubes. The disadvantages of autografts are limited supply and associated donor site morbidity from additional incisions, loss of sensation, and possible painful neuroma formation , 27. ANAThe supplement of supporting cells to the single lumen nerve conduit has been extensively researched over the past decades . SchwannIn this study, we have shown that the transplanted AFS cells have beneficial effects in enhancing regeneration of damaged nerve tissue, but the exact mechanisms that are responsible for these therapeutic advantages are still unknown. Several studies in rats and humans suggested that the improved nerve regeneration with AFS cell treatment are through paracrine effects , 18, 22,In conclusion, this study has shown beneficial functional, electrophysiological, and histological outcomes in AFS cell-treated animals after sciatic nerve transection and repair. Thus, these cells may be a suitable cell source to replace Schwann cells to support and accelerate peripheral nerve regeneration flowing large gap nerve injury.


The database contains over 866 georeferenced samples from 9 different ecoregions with contextual environmental information. For its integration and validation we constructed a curated database (µSudAqua[db.sp]) using samples sequenced by Illumina MiSeq platform with commonly used prokaryote universal primers. This comprised ~60% of the total georeferenced samples of the µSudAqua[db]. This compilation was carried out in the scope of the µSudAqua collaborative network and represents one of the most complete databases of continental water microbial communities from South America.The biogeography of bacterial communities is a key topic in Microbial Ecology. Regarding continental water, most studies are carried out in the northern hemisphere, leaving a gap on microorganism’s diversity patterns on a global scale. South America harbours approximately one third of the world’s total freshwater resources, and is one of these understudied regions. To fill this gap, we compiled 16S rRNA amplicon sequencing data of microbial communities across South America continental water ecosystems, presenting the first database   Due to their high abundances and activities and to their collective metabolic and phylogenetic diversity, prokaryotes support aquatic food webs and regulate the magnitude and recycling rates of major elements5. Thus, understanding the microbial diversity patterns is a fundamental topic in modern Microbial Ecology and a key step for advancing our knowledge on bacterial-mediated processes across continental water ecosystems.Microorganisms are the main drivers of biogeochemical cycles in freshwater ecosystems8. For example, a rough mapping of the worldwide distribution of amplicon sequencing studies , there are still important gaps in the study of aquatic microbial diversityies Fig. , clearly11. This water flows through five huge hydrological river basins: the Amazonas , Del Plata-Paraná/Paraguay , Orinoco , Araguaia-Tocantins , and São Francisco 13. In addition, a great number and diversity of lentic water bodies are also prominent features that tend to occur in lake districts and wetlands as a result of the main climatic and geomorphological processes acting on regional scales14.The Southern Hemisphere covers a comparatively high share of the surface and volume of the continental and marine ecosystems in the world. In particular, South America is considered the “continent of water”, harboring 6 out of the 10 largest rivers in the world in terms of water discharge, draining about 30% of the continental freshwater that reaches the ocean16. South America covers about 15% of the global land area  and spans a broad latitudinal range, extending from 12° 28′N  to 55° 59′S . According to the biogeographic regionalization by Cabrera & Willink15, South America belongs to the Neotropical region, except the southernmost area, which is assigned to the Antarctic region. Owing to the wide latitudinal coverage, a large variety of climates occur, with much of the continental mass located within the intertropical belt, large regions of Chile, Argentina, and Uruguay laying in the Southern Temperate Zone, and the southern tip of the continent extending into sub-Antarctic latitudes. Due to the high habitat heterogeneity and a complex geological history, South America is considered a hotspot of biodiversity, being the most species-rich region on Earth18. This fact, added to its location in a predominantly maritime hemisphere, offers a unique opportunity for comparing empirical patterns found originally for Northern Hemisphere aquatic ecosystems. Pioneering studies have shown a geographically biased picture of the aquatic microbiome, leading to overlook differences in structure and functioning of microbial communities19. That limits, for instance, our understanding of the expected scenarios resulting from the current pressures experienced by the aquatic ecosystems20.Furthermore, the South American continent comprises a large ecological heterogeneityµSudAqua[db] containing 866 georeferenced samples. For its integration in further works, preventing biases by the sets of primers and sequencing methodology used, we constructed a curated database (µSudAqua[db.sp]) which contains over 509 samples of the V3-V4 region of the 16S rRNA gene sequenced by the Illumina MiSeq technology with the commonly used primers proposed by Herlemann & collaborators21.In order to reduce this gap, we performed an exhaustive bibliographic search, collected and annotated data of bacterial communities from South American continental waters. We constructed the first 16S rRNA amplicon sequencing database of South America, the https://microsudaqua.netlify.app/).This work is a result of the µSudAqua collaborative network, a Latin American network in Aquatic Microbial Ecology. This network emerged as an initiative to nucleate researchers of the field to join efforts in consolidating a regional critical mass. Its main objectives are to strengthen and expand the interactions between aquatic microbial ecologists, to contribute to the development of a community feeling at the regional level, and to provide a fruitful space for long-term collaboration in research and training of human resources. More information can be found at the µSudAqua website (µSudAqua[db] database was constructed with samples from published papers and new data generated in the scope of this work (Table The rk Table . We onlyhttps://qgis.org/). Each sample was assigned to an environmental type  and an ecoregion (section Ecoregions description). Besides, the georeferenced location and procedures adopted for the sampling and sequencing were fully recovered. The complete list of metadata recovered and its description is presented in Table 22.Sample metadata were collected from the published papers or provided by the authors of the current work. The altitude was automatically extracted based on the sampling location, using the QGIS geographic information system software  Illumina MiSeq technology and; 2) the commonly used set of primers proposed by Herlemann & collaborators21.The 21. Samples of each project were indexed with Nextera XT v2 kit, and sequenced using the Illumina MiSeq technology in different sequencing facilities. The samples were obtained mostly from surface waters (0–50 cm) of continental systems with different limnological characteristics and different spatial and temporal coverage through six ecoregions  and truncLen = c. Only samples with more than 10,000 reads were analyzed.Amplicon sequences from the dada function. The chimera sequences were excluded after merging the different projects using the functions removeBimeraDenovo and mergeSequenceTables, respectively. The taxonomic classification was performed using BLAST v2.526 with the blastn algorithm  and the SILVA database (SSU Ref 132 NR 9927) as a reference. The Amplicon sequence variants (ASVs) were classified into 7 different taxonomic groups. The contribution of each group was calculated as their relative abundance to the total number of reads, and the richness was defined as the total number of ASVs. The scripts used for DADA2 and sample description are available in GitHub (https://github.com/microsudaqua/usudaquadb).To increase sensitivity to rare variants and avoid chimeras and sequencing errors, we used the “pool” option from 28 for Central, South America and the Caribbean. The characteristics of each ecoregion and subregion are briefly described below.To define the ecoregions, we adopted the level II classification proposed by Griffith & collaborators18. Central Andes18.1 Central High Andes, Chile29. The ecoregion occupies an area of 140,960 km2 and lies within the altitudinal range between 3,200 and 6,600 m15. Its climate varies from temperate to cold, with an annual average temperature between below zero and 15 °C. This region is dry, with precipitation between 250 and 500 mm per year30. It is considered as a transitional zone between the wet puna to the north and west, and the dry puna to the south. This ecoregion has several high-elevation wetlands comprising both fresh and saline lakes, salt flats, temporary endorheic basins, as well as permanent rivers and streams fed by snowmelt. They regulate water flow by retaining water during the wet season and releasing it during the dry season. The salt flats, or salares, represent remnants of extensive paleolakes29.The Central High Andes ecoregion extends from southern Peru, through Chile and Bolivia, to northern Argentina . The landscape is typically mountainous, with snow-capped peaks, plateaus and valleys19. Southern Andes19.2 Valdivian Forest Hill and Mountains2). The climate is temperate cool  with predominance of westerly winds, and annual precipitation of 1,500 mm31. The ecoregion is characterized by a profuse hydrographic system including large and deep lakes 33 and small and shallow lakes35. The main rivers fed from these Andean waters, run across the plateau steppe and outflow to the Atlantic Ocean, but there are also other rivers that cross the Andes flowing towards the Pacific Ocean. Deep lakes (Zmax > 100 m) have a warm monomictic thermal behavior36. Nevertheless, small and shallow lakes (Zmax ~12 m) are dimictic or polymictic34. These lakes have very low nutrient (ranging from ultra-oligotrophic to oligotrophic status) and dissolved organic carbon concentrations, and high transparency to different wavelengths, which would imply high exposure to ultraviolet radiation40.The Valdivian Temperate Forests ecoregion is in the southern cone of South America . It covers a narrow continental strip between the western slope of the Andes and the Pacific Ocean , with dry winters and rainy summers46. Annual precipitation typically ranges from 1,200 to 1,800 mm and soil is usually acid and nutrient-poor47. The Cerrado altitude has little variation, being maximum only in the central highlands, from where important springs come out and end up contributing to form the three largest water basins in South America 48. There are very few natural lakes in this region, and most water bodies are either dammed shallow lakes or large hydroelectric reservoirs. As reservoirs are mainly found near cities, the nutrient inputs, pH and trophic state can vary50.The Cerrado is the second largest ecoregion in South America. It comprises the Brazilian central region , and covers an area over 2 million km21.4 Atlantic Forests51). This ecoregion is a wide tropical , humid biome known mainly by its long line of coastal rainforest51. The coast is humid all over the year, with an annual precipitation typically ranging from 1,800 to 3,600 mm. This ecoregion is characterized by different formations like deciduous and semi-deciduous continental forests, bogs and mangroves, and grasslands52. Landscape can be flat and lentic environments in the countryside are either human made dammed creeks used for cattle ranching and crop irrigation or large hydroelectric reservoirs. Along the Brazilian Atlantic coast, lentic ecosystems are shallow lakes dug into the mountainside, or squeezed into the narrow strip between the mountain chain and the ocean53. There are also some herbaceous/shrubby sand-dune ecosystems, called Restinga, that form perennial or temporary coastal shallow lagoons54, which encompass wide environmental gradients  that greatly influence aquatic biodiversity55.The Atlantic Forests region is mainly located in Brazil, spanning along the Atlantic coast, and extending inland to Argentina and Paraguay . Climate is humid subtropical, with annual precipitation between 900 to 1,000 mm. At this stretch, the river is characterized by a well-defined main channel and a large floodplain about 20 to 40 km wide, located by its right margin. Thousands of permanent shallow lakes and temporary environments occupy the floodplain which is flooded and drained by a well-developed and relatively stable fluvial network57. The system dynamic is subject to hydro-sedimentological pulses that occur with different magnitudes and constitute the main driving factor of the limnological features and the biota58, particularly, the microbial communities61.The Paraná River is the second largest river of South America with a mean annual discharge of ~17,000 m23. Pampas23.1 Uruguayan savanna, Uruguay2 which includes the whole country of Uruguay  and extends mostly towards the southern part of Brazil to a small section of the Argentina62. The climate of this region is temperate, without dry season, and with hot summer63. The mean annual temperature ranges between 16 and 20 °C. The mean annual rainfall lies between 1,100 and 1,400 mm and is highly variable between years. This ecoregion encompasses the outlet of the Río de la Plata basin where a dense fluvial network, along with a series of coastal lagoons and numerous artificial lakes can be found. Rivers and streams are characterized by small slopes and rapid filling and draining64. Coastal lagoons, formed due to marine regressions and transgressions in the Holocene, are located at the Atlantic coast65 and their size and age increase towards the East. They are characterized by large gradients in salinity, light penetration and nutrient concentrations, and their hydrological cycle strongly determines the composition and activity of the bacterial communities67.The ecoregion Uruguayan savanna comprises an area of 355,605 km23.2 Southern Flat Pampas32. It is an extensive plain area , except for the two, almost parallel, hill systems that cross the area in a NW–SE orientation (Sierras de Tandilia and Sierras de Ventania). The climate of this region is temperate and humid, with mean annual temperatures varying from 14 to 20 °C. The precipitation is concentrated during spring and summer months, and decreases from NE to SW 38. The ecoregion is dominated by a large number of fluvial-aeolic shallow lakes and low order rivers and streams that mostly belong to the Salado-Vallimanca basins32. Particularly, lakes are characterized by rounded contours and pan-shaped profiles. They are typically shallow, polymictic, eutrophic to hypertrophic, with highly variable water renewal time and salinity. Most of the surrounding land is devoted to agricultural practices36. This economic development directly affected shallow lakes, promoting shifts in many of them from clear regimes, characterized by the presence of submerged vegetation, to algal-dominated turbid states68.The Pampa ecoregion extends westward across central Argentina , from the Atlantic coast to the Andean foothills24. Monte-Patagonian24.2 Patagonian Tablelands32, is a complex landscape of about 600,000 km2, located in Argentina  usually remain frozen from early autumn throughout late spring, however during the ice-free period due to frequent strong winds, the water columns are continuously mixed, thus preventing the formation of stable thermoclines72.The Patagonian tablelands ecoregion  are also provided in different machine-reliable files at Zenodo22.The µSudAqua[db.sp], that comprises the samples that were sequenced with the Illumina MiSeq technology, and targeted the V3-V4 regions of the 16S rRNA gene.The technical validation was performed using the µSudAqua[db.sp]: Bacterial distribution among the ecoregions of South AmericaIn total, 509 samples and 116,687,584 reads were processed with DADA2. In order to exclude possible remaining sequencing errors or chimeras, we filtered ASVs with less than 50 reads in less than 3 samples. Thus, the final ASV table consisted of 502 samples, 25,334 ASVs and 42,188,085 reads, from: Amazon and Coastal Lowlands (96 samples), Atlantic Forests (67), Cerrados (86), Humid Chaco (59), from Southern Flat Pampas (127), Valdivian Forested Hills and Mountains (7) and, Patagonian Tablelands (67). The information of sequence processing and quality check of samples is summarized in Table µSudAqua[db.sp] database was mainly represented by Bacteria  followed by chloroplasts  and Archaea . Within Bacteria, the reads were distributed in 6 principal taxonomic groups: Actinobacteria (33.4%), Proteobacteria , Cyanobacteria (10.7%), Planctomycetes (9.5%), Bacteroidetes (8.6%) and Verrucomicrobia (6.7%). Bacteroidetes was the richest group , followed by Proteobacteria  and Cyanobacteria (776 ASVs). The relative abundance and richness of each principal taxonomic group were notably different among the studied ecoregions , taxonomy, nucleotide sequences in fasta format and ASVs table filtered with only Bacteria. Importantly, the database will grow as new samples and sequencing projects from the µSudAqua network appear. This information will be uploaded in the repository and the tables will be updated in future versions of the database. A bibliography revision and open call for new data submission will be performed once a year, and the database will be updated after data quality check, processing and integration.The links to download the raw fastq data from µSudAqua[db] and µSudAqua[db.sp] databases are the first to integrate information of microbial diversity from continental systems of South America, an important region that has been overlooked comparing to other regions and environments worldwide. These databases will open new avenues for studies on the temporal patterns and spatial distributions of microbial communities among the different ecoregions of South America. Besides, the integration of the curated data to meta-analysis of microbial communities from different ecosystems (comparison between South America and well-studied regions of the world), will be particularly important for exploring the novel microbial diversity, allowing to reveal regions with unknown organisms and functions, as well as hotspots of microbial biodiversity.The