Expression of the alternative oxidase in mtDNA mutator mice improves their blood phenotype but enhances inflammatory and stress responses in skeletal muscle. Ciona intestinalis alternative oxidase (AOX), which attenuates ROS production, affects murine stem cell pools. We found that AOX does not affect neural stem cells but delays the progression of mutator-driven anemia. Furthermore, when combined with the mutator, AOX potentiates mitochondrial stress and inflammatory responses in skeletal muscle. These differential cell type-specific findings demonstrate that AOX expression is not a global panacea for curing mitochondrial dysfunction. ROS attenuation must be carefully studied regarding specific underlying defects before AOX can be safely used in therapy.Energetic insufficiency, excess production of reactive oxygen species (ROS), and aberrant signaling partially account for the diverse pathology of mitochondrial diseases. Whether interventions affecting ROS, a regulator of stem cell pools, could modify somatic stem cell homeostasis remains unknown. Previous data from mitochondrial DNA mutator mice showed that increased ROS leads to oxidative damage in erythroid progenitors, causing lifespan-limiting anemia. Also unclear is how ROS-targeted interventions affect terminally differentiated tissues. Here, we set out to test in mitochondrial DNA mutator mice how ubiquitous expression of the   Genetic mutations that disrupt mitochondrial gene expression can lead to the defective assembly of respiratory chain (RC) complexes and appear to be a frequent cause of mitochondrial diseases. Myopathies, muscle diseases, are the most common mitochondrial disease phenotype in adults , 2, 3. E“Mutator” mice are a commonly used model to study mitochondrial dysfunction , 5. TheyThe excess production of ROS partially causes the mutator stem cell homeostatic defect. In adult mutators, bone marrow shows highly increased oxidative stress within mitochondria, delayed mitochondrial exclusion from red blood cell (RBC) precursors, and extended iron loading by transferrin, leading to excess ROS generation via the Fenton reaction and oxidative damage in erythrocyte membranes . In othe2 to H2O, bypassing the enzymes ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase (COX), that is, complexes III and IV of the respiratory chain, respectively, and decreasing mitochondrial membrane potential and ATP synthesis  resides in the inner mitochondrial membrane as part of the RC  and is pynthesis . This reynthesis . AOX froand mice , 16, 17. animals , 23, 24. animals , 20, and animals . DiffereIn this study, we used the combination of AOX and mutator mice to determine whether and eventually how AOX expression affects (a) somatic stem cell function in healthy mice, (b) stem cells subjected to mtDNA mutagenesis, and (c) respiratory chain function in key postmitotic tissues including skeletal muscle. We report that AOX expression has tissue-specific consequences in mutator mice. It alleviates anemia in mutators by shifting erythroid precursors toward a more mature state in bone marrow, whereas also inducing the mitochondrial integrated stress response (ISRmt) and inflammatory pathways in skeletal muscle. Our findings indicate that AOX expression may be helpful in hematopoietic stem cells experiencing mitochondrial stress but potentially harmful in postmitotic tissues via the induction or exacerbation of metabolic stress and inflammation.C. intestinalis AOX (AOX mice) and those with a knock-in mutation (p.D257A) inactivating the proof-reading exonuclease domain of DNA polymerase gamma  have been described previously in references The mouse strains used in the study, that is, those expressing tion p.D27A inactiAOX–mutator double-transgenic mice’s gross phenotype and outward appearance were indistinguishable from that of mutator mice, with signs of progeria starting at 6 mo. Neural stem cells (NSCs) and postmitotic skeletal and heart muscles were confirmed to express AOX .Mutator mice manifest anemia and increased amounts of reticulocytes , 8, 11. AOX–mutators showed higher hemoglobin (Hb) and lower circulating reticulocyte (immature RBC) counts than mutators . AOX expBecause of the effect of AOX on mutator hematopoiesis, we proceeded to look at its effect on NSCs. Previously, we demonstrated that mutator NSCs show decreased “stemness,” presenting a decreased ability to self-renew in clonal culture . AOX expThese data demonstrate differential effects of AOX expression on different somatic stem cell compartments, but only under conditions of mitochondrial dysfunction. In WT mice, AOX expression did not affect erythroid, lymphatic or neural progenitors or their differentiation pattern.After examining the effect of AOX on stem- and progenitor-cell compartments, we asked whether it could provide a therapeutic benefit and alleviate RC dysfunction previously demonstrated in the postmitotic skeletal muscle of mutators , 8. We dWe proceeded to look at RC components at the protein level by immunofluorescent staining for the COX1 subunit, which was also decreased in mutators and not rescued by AOX  and S4A.We also asked whether the alteration in COX activity and protein amount in mutators could be related to a shift in muscle-fiber type. We explored myosin heavy-chain isoforms preferentially associated with either oxidative or glycolytic metabolism in skeletal muscle. Based on immunohistochemistry for myosin heavy chain expression, which enables different muscle-fiber types to be distinguished , 29, mutIn deletor mice, a well-characterized model of mitochondrial myopathy, RC deficiency manifests in a mosaic pattern in skeletal muscle, alongside mosaicism for the promotion or prevention of mitophagy . Our finIn the brain, aging mutator mice show diminished COX activity in multiple brain regions  with a smaller decrease in the cerebral cortex , 32. In Supplemental Data 1).In plants, AOX modifies mitochondrial signaling and affects nuclear transcription programs . To gainSupplemental Data 1.Significantly altered transcripts in RNA sequencing of skeletal muscle.Chrna9, Prg4, Zbp1, and Zdbf2) were among those with the greatest fold change . Overall, inflammatory and immune-related pathways, such as STAT3 and interferon signaling and phagocytosis, were down-regulated in the skeletal muscle of AOX mice .The histone methyltransferase Setd5 was the most significantly altered transcript in AOX versus WT , whereasd change . Moreoved change   cells and IL-8 signaling and phagocytosis, were predicted to be up-regulated in mutator skeletal muscle . FurtherMost activated and inhibited upstream regulators in ingenuity pathway analysis of RNA sequencing of skeletal muscle.Table S2. Supplemental Data 1). Furthermore, the unfolded protein response and folate signaling—both components of ISRmt—were activated , including IRF3, a critical player in the cGAS–STING pathway , which icGAS and STING, and the cGAS–STING–TBK1–IRF3 signaling cascade, were up-regulated by the expression of AOX and/or the mutator phenotype , and thiTfam+/−) mice (Aberrant activation of the type-I IFN response may aggravate the mutator phenotype . This ha/−) mice  and in m/−) mice . ActivatDespite much research, no specific therapy is available for pathological oxidative phosphorylation defects. AOX expression has been proposed as a tool not only to study the mechanisms of respiratory chain dysfunction but also to alleviate its pathological consequences , 18. TheAOX mice were generated and characterized by Szibor et al . MutatorNSC extraction from WT (n = 4), AOX (n = 2), mutator (n = 4), and AOX–mutator (n = 3) mice embryos was performed from the lateral ventricular wall of E11.5–E15.5 mouse brains as previously described . NeurospWhole-cell protein extraction from NSCs was performed as in reference Blood was collected from euthanized mice into EDTA tubes (Greiner Bio-One) by heart puncture. A blood count was performed from whole-blood samples on the same day using an Advia 2120i analyzer (Siemens).6 cells; BD Pharmingen) was used to inhibit possible nonspecific binding of the antibodies. Fluorescence-conjugated antibodies against CD71 (BD Pharmingen), CD11b (BD Pharmingen), Ter119 (eBioscience), and B220 (BD Pharmingen) were used (each 1 μg/1 × 106 cells) for staining. Propidium iodide was used as a dead cell marker. Unstained and fluorescence-minus-one controls were used to determine the autofluorescence of the cells and gates for the cell populations of interest, respectively. One hundred thousand cells per sample were analyzed.Euthanized mice’s femoral and tibial bones were cut out and cleaned of soft tissue. Bone marrow was flushed out using a syringe and 1 ml of PBS + 5% FBS and filtered through a 40-μm strainer (Dako). Cells were counted and analyzed immediately. Hematopoietic lineages of adult bone marrows were analyzed using a BD Influx Cell Sorter (Beckton Dickinson). CD16/CD32 blocker (1 μg/1 × 10m. quadriceps femoris) using TRIzol reagent (Invitrogen) and purified using RNeasy Mini Kit (QIAGEN). The samples were homogenized using TRIzol reagent (Invitrogen), and chloroform was added to allow homogenate separation. The lysate–ethanol mix was then purified using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Extracted RNA was treated with RNase-free DNAse . 1,000 ng of total RNA was used to generate cDNA using a MAXIMA cDNA synthesis kit .Total RNA was extracted from skeletal muscle  (RNA from WT (n = 5), AOX (n = 5), mutator (n = 5), and AOX–mutator (n = 3) was submitted for transcriptomic analysis. RNA quality-control analysis was done using Tapestation 4200 (Agilent). RNA-seq was performed using a “Bulkseq” 3′UTR-counting gene expression profiling method based broadly on BRBseq/Dropseq with high-output (1 × 75 bp) read lengths. The Biomedicum Functional Genomics Unit provided the service at the Helsinki Institute of Life Science and Biocenter Finland at the University of Helsinki. Primary data analysis was done using the DeSeq2 package from Bioconductor release 3.9  in R Stuen-ipa/) .m. quadriceps femoris, QF), heart muscle, and brain were collected from WT, AOX, mutator, and AOX–mutator mice aged 40 wk. The tissues were harvested immediately after sacrificing the mice, embedded in OCT Compound embedding medium (Tissue-Tek), and snap-frozen in a 2-methylbutane bath in liquid nitrogen. In situ histochemical COX and SDH activities were analyzed from frozen tissue sections (12 μm) using standard protocols , COX-I (complex IV), nuclei, and laminin in the muscle of WT (n = 3), AOX (n = 3), mutator (n = 4), and AOX–mutator (n = 2) mice was done by adapting a previously published protocol . PrimaryAdditional immunofluorescent staining of COX-I and TOM20 was done on muscle of WT (n = 3), AOX (n = 3), Mutator (n = 4), and AOX-Mutator (n = 1) mice. COX-I was stained as above. For TOM20, the primary antibody used was anti-TOMM20 rabbit  and the secondary antibody was chicken anti-rabbit IgG (H+L) Alexa Fluor 594 . Slides were mounted with Fluoromount . Imaging was done by light microscopy  at 20X magnification. Co-localization of COX-I and TOM20 was quantified using CellProfiler .m. quadriceps femoris) of WT (n = 5), AOX (n = 7), mutator (n = 8), and AOX–mutator (n = 5) mice using a protocol adapted from previous reports  of WT (n = 3), AOX (n = 3), mutator (n = 4), and AOX–mutator (n = 1) mice. Fresh frozen 10-μm muscle sections were used. The primary AOX antibody , * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), and **** (P ≤ 0.0001). Graphs show the mean with SD with individual measurements shown.One-way ANOVA for groups displaying normal distribution followed by Tukey’s multiple comparisons test was performed on independent observations using GraphPad Prism version 9.0.2 for Mac, GraphPad Software, PRJNA1006295.All original data are available from the authors upon request. The RNA-sequencing data from this publication have been deposited to the NCBI Sequence Read Archive (SRA) database and assigned the BioProject ID


Tubal patency assessment is considered a crucial component of initial infertility evaluation with several evaluation tests available, such as hysterosalpingography (HSG), hysterosalpingo-contrast sonography (HyCoSy), and hysterosalpingo-foam sonography (HyFoSy), the latest tubal patency assessment, utilizing ultrasonography and a foam-based contrast agent. An additional side-benefit of these assessment tests is a fertility-enhancing effect, best studied with the application of HSG. In this report, we present a case of a 28-year-old woman with unexplained infertility who spontaneously conceived in the same menstrual cycle that the HyFoSy exam with ExEm Infertility is defined as the inability to conceive within 12 months of regular intercourse without contraception, with up to 12% of couples worldwide being affected by it [Traditionally, laparoscopy with chromopertubation was used as the main tubal patency assessment and remains the gold standard today . HoweverDespite the availability of HyFoSy and the disadvantages of more conventional methods, many fertility specialists opt for the latter, as, apart from their imaging capabilities, they have also demonstrated a positive effect on fertility, whereas the same has not been adequately demonstrated for ultrasonographic methods in the available literature . In the MTHFR double heterozygosity for C677T and A1298C mutations.A 28-year-old female, patient, gravida 0, para 0, presented to our fertility clinic with primary infertility for the past 14 months. Her BMI was within normal range, she had regular menstrual cycles and reported no dysmenorrhea or menorrhagia. She reported regular intercourse with her husband during those 14 months, without any sexual dysfunctions. From her past medical history, of note was her history of cervical lesions  I, CIN II) and genital condylomas, while she had received no prior fertility treatment or undergone surgery. She was a smoker, smoking approximately 20 cigarettes per day. Her hormonal profile was within normal range, except for anti-Müllerian hormone (AMH) levels, which were low for her age. On transvaginal ultrasound (TVUS) examination, no congenital or acquired structural abnormalities were noted and she had good ovarian reserves . Genetic testing for thrombophilia revealed Her partner was a 32-year-old healthy man with no reported loss of libido or other intercourse dysfunctions and no urinary complaints. He reportedly smoked and consumed alcohol socially and mentioned taking protein and creatine supplements for three months in the past. Basic semen analysis revealed a borderline sperm count.Prior to any fertility treatment, we performed a HyFoSy with ExEm foam and subsequently a three-dimensional 3D) TVUS scan. The HyFoSy was conducted on the sixth day of the menstrual cycle, with low to no resistance. Both fallopian tubes were visualized and their patency was verified Figure . The uteD TVUS scIn this report, we presented the case of a young woman experiencing primary infertility for more than a year. The patient, without having received any fertility treatment, managed to achieve spontaneous conception following HyFoSy examination, within the same menstrual cycle.Fallopian tube patency assessment is known to possess fertility-enhancing effects for certain patients, in addition to fallopian tube visualization. As early as 1966, it was suggested by Wahby et al. that HSG may enhance fertility and promote post-procedure spontaneous pregnancies . They obThe application of HyCoSy has also been shown to have a positive impact on spontaneous pregnancy rates, like HSG, especially within the month following the examination. Bisogni et al. noted that one-third of patients with bilateral or unilateral patency had a spontaneous pregnancy 50 days following HyCoSy . A prospAt present, HyFoSy represents a growing trend in tubal patency evaluation. It involves no exposure to radiation and minimal adverse reactions, while being less costly than HSG and performed by the fertility specialist within the clinic. Additionally, HyFoSy is reportedly less painful and better tolerated than HSG and possesses remarkable imaging capabilities ,13. RegaTo our knowledge, this is the first case of spontaneous pregnancy within a month after HyFoSy ever reported from Greece. Our observations are in concordance with the observed trends in the literature, namely higher spontaneous conception rates within the first months, higher rates for patients under 35 years old, and infertility duration of less than 18 months -19. In oHyFoSy is a very promising diagnostic method for tubal patency assessment with many advantages over HyCoSy and HSG. The tubal flushing effect has already been proposed to have a positive impact on fertility patients in the past, with certain studies demonstrating a similar effect after HyFoSy as well, an observation also confirmed in the current case. Research should focus on solidifying the existence and extent of this fertility-enhancing effect, via prospective, comparative, or controlled studies, as well as investigate the risk of possible pregnancy or maternal complications after HyFoSy, in order to ensure patient safety and optimal outcomes.


Pakistan has an inadequate vital event registration system, leading to fewer than half of all births being registered, and this issue is further exacerbated by systematic recall errors and omission of births. This study aims to evaluate direct and indirect methods of fertility estimation to analyze the trends and patterns of fertility rates in Pakistan from 1990 to 2018.Indirect methods are utilized in this study to evaluate the direction and extent of changes in total and age-specific fertility rates, and these findings are compared to direct estimates. The study draws data on livebirths from four waves of the Pakistan Demographic and Health Survey that took place between 1990 and 2018. To ensure the quality of data, graphical methods and Whipple and Myers indices are employed. Additionally, the Brass Relational Gompertz model was used to analyze the data.The Relational Gompertz model revealed that total fertility rates (TFRs) were higher than direct estimates by 0.4 children and age-specific fertility rates (ASFR) were higher for all age groups except the oldest. The difference was more significant among younger women aged 15–24, and less so for age groups 29 and above. The gap in estimated fertility between direct and indirect methods decreased with age.The indirect method is an invaluable tool in situations where direct measurement of fertility rates is challenging or impossible. By utilizing this method, policymakers can gain important insights into the fertility patterns and trends of a population, which is crucial for making informed decisions on fertility planning.The online version contains supplementary material available at 10.1186/s12905-023-02435-8. Pakistan lacks a complete registration system of vital events, resulting in over half of under the age of five (58%) being unregistered  .The Pakistan Demographic and Health Survey (PDHS) serves as the primary source for examining current and lifetime fertility. It collects birth history data in temporal sequence from all women of child-bearing age 15–49 years) –49 years classifiThe direct estimation of fertility can be done in three possible ways depending on the availability of data. In the first approach to direct estimation of fertility, the vital registration system is used. The second method uses full birth history data collected in the survey, and the third approach employs summary fertility measures collected routinely in censuses for the estimation of recent fertility. Direct estimates of fertility are helpful to assess the changes in the pattern of fertility .In the indirect estimation of fertility, information on the lifetime fertility of younger women collected through the census or survey is used in conjunction with direct estimates of fertility to adjust them. However, response errors are common in birth history data . The errApart from that, systematic recall errors are also a big issue . For exaThe impact of these intrinsic errors can be reduced by using indirect methods of fertility estimations . The RelThe Relational Gompertz Model is commonly used as an indirect method to determine the shape and level of fertility in a population while accounting for irregularities such as age misreporting of children and births in the data .In Pakistan, the total fertility rate has shown a declining trend over the years. From 1970 to 1980, the rate was between six to seven children per woman. , and in The total fertility rate in Pakistan was reported to be more than six children in 1990–91 . However, a further fall was observed in the following years, with a TFR of 4.1 in 2006–07 and 3.8 in 2012–13. Between 2012–13 and 20,171–18, there was a negligible decrease of 5% in TFR  [Previous studies have projected that Pakistan would achieve the replacement-level of fertility by 2020 . To suppTherefore, it can be assumed that fertility decline trends reported by previous studies based on direct methods were not precise. Limited literature exists on the implications of using the direct versus indirect methods for estimating total and age-specific fertility rates in Pakistan , 18, 19.This study fills the research gap by examining the pattern and trends of the total and age-specific fertility rates (from 1990 to 2018) with the help of indirect methods. It uses the Brass P/F ratio and Relational Gompertz model, which accounts for errors and omissions in births. Further, it compares fertility estimates obtained by direct methods with those derived from indirect methods. The study uses four waves of Pakistan Demographic and Health Survey covering the period of 28 years. The research findings help to explain the fertility puzzle in Pakistan and adds to the empirical evidence on fertility transitions in a developing country, which is crucial for devising population control strategies and programs.This study used four waves of Pakistan Demographic and Health Surveys: . Sample size comprised of 6611, 10,023, 13,557 and 15,068 women respectively for each survey wave. We have used the PDHS for the computation of TFR and considered the women of reproductive age group 15–49 year. Our analysis is restricted to the mean number of children born to a biological mother. Therefore, the children co habiting with the family at the time of survey were not the part of the analysis. DHS collects data on birth histories from all eligible married women of 15–49 years of households randomly selected for the interview.The surveys were implemented by the National Institute of Population Studies in close coordination with the Ministry of National Health Services, Regulations, and Coordination Surveillance. Surveys followed a two-stage stratified random design. Firstly, regions were stratified into urban and rural. Secondly, the primary sampling units (PSUs) were selected using the information on the enumeration blocks and following a probability rule, probability proportional to size or PPS, from the sampling frame developed by the Pakistan Bureau of Statistics.Finally, secondary sampling units (SSU) or households were selected through systematic random sampling from the selected Enumeration Blocks (EBs) of urban areas. In rural areas, households were chosen from mouzas/dehs/villages. This study mainly used data from birth histories in each survey. Total fertility rates used were from the 36 months prior to each survey. Similarly, age-specific fertility rates were computed for births occurring within the previous three years of each survey.This study is divided into two parts: data evaluation and fertility estimation using indirect methods of estimation. Data evaluation is necessary to strengthen our confidence in the reliability of estimates obtained. To avoid documenting extensive details of the data evaluation procedures and results in the paper, a supplementary file has been prepared and attached to the manuscript.The study used the refined form of the Brass P/F technique, Known as the Relational Gompertz model (RGM), to estimate the level and trend of fertility –22. BrasThe RGM serves as the best-suited diagnostic tool to identify data errors. If Brass P/F departs from a hypothesized value of “1”, it implies some errors or omissions in the fertility data. Two types of errors occur in the data if the value of Brass P/F deviates from “1”. First, if women underreport recent births, irrespective of their age, it inflates the P/F value. Second, if older women underreport their lifetime fertility, it decreases the numerator, and consequently, P/F ratio deflates , 12.The RGM estimates ASFRs and TFRs by defining the fertility schedule form with the help of the data on birth histories and shaping the pattern from reported average parities of younger women . The ferThe cumulative function of the Gompertz distribution has a sigmoid shape (S-shaped), representing the average parities of women through age. Moreover, the hazard function of Gompertz distribution is right skewed, capturing the pattern of cumulated fertility of women . The GomEquation  refers tIn Eq. , \documeribution , 24. ThiThis study utilized the Microsoft Excel worksheet for data analysis, specifically the “Relational Gompertz model” (RGM) as outlined by . Direct Table Pakistan’s highest rate of decline in fertility was -1.82 during 2012 2017 , see Tables The reasons for slow fertility decline were traced by comparing changes in proximate factors Table  and seleBoth the median ages at first marriage and first birth increased by 1.8 and 1.5 years respectively between 1990–91 and 2017–18 in Pakistan. This can be considered as one of the factors contributing to the consistent decline in fertility rates in the country.Previous studies have established a consistent decline in the total fertility rate in Pakistan, particularly after 1990. However, the evidence was based on direct estimates of fertility that are prone to errors. The unusual pattern in the birth distribution was identified and reported in PDHS 2012–13. Age displacement and omission of children were the probable reasons for these discrepancies . Dead chThis study provides estimates of fertility patterns and trends in Pakistan by accounting for data errors. The Relational Gompertz model (RGM) was used instead of the Brass P/F ratio method, which makes assumptions about constant fertility that may not hold in developing countries with incomplete or distorted data . SimilarThe total fertility estimates obtained by RGM showed a decline over the years in Pakistan. According to RGM estimates, the TFR was four children per woman. The decline in total fertility rate was estimated to be 31% between 1990–91 and 2017–18. The estimates of TFR obtained by RGM were found to be consistently higher in all survey years compared to the fertility rates estimated by direct methods. Recent surveys such as the Pakistan Demographic Survey  and the Pakistan Social and Living Standards Measurement  reported TFR as 3.7 which is slightly higher than the 3.6 reported in PDHS 2017–18, supporting the argument that direct estimates marginally underestimate actual fertility rates , 31.A difference of at least 0.4 children was observed between the TFRs for direct and indirect estimations. The overall annual rate of decline in TFR for 28 years depicted by the indirect method was 1.15%  but again declined during 2017–18 Table . As α beOn the other hand, a two-month decline was observed in the median duration of breastfeeding among childbearing mothers. The median birth interval shortened by one month between 1990–91 and 2012–18, whereas it increased by one and a half months in 2017–18 [Based on the findings presented, there are several policy implications that can be drawn for fertility planning in Pakistan, as follows. 1) the findings indicate regional disparities in fertility rates, with higher rates observed in rural areas and in some provinces such as Balochistan and Khyber Pakhtunkhwa. Family planning programs should be targeted to address these regional disparities, 2) the fertility intentions of women have remained static at 4.1 children per woman, indicating the need to address underlying factors that influence fertility intentions, such as gender norms, education, and economic empowerment of women, 3) results call for investing into maternal and child health factors, such as breastfeeding duration and birth intervals, to influence fertility rates. Programs aimed at improving maternal and child health can contribute to reducing fertility rates in Pakistan, 4) accurate age-specific fertility estimation can help policymakers identify which age groups have the highest fertility rates, and subsequently design targeted interventions to reduce fertility rates in these age groups.The indirect method offers reliable estimate of fertility in countries where misreporting about childbirth and women’s age are common. It may be due to a massive illiteracy and inadequate vital event management system. This can help policymakers to better understand the fertility patterns of a population and make informed decisions about resource allocation and family planning programs.However, the accuracy of indirect methods may be limited by the assumptions underlying the model and the quality of the data used to calibrate it. Therefore, policymakers should be cautious in interpreting and using the results of indirect methods and consider them in conjunction with other sources of data and information. In addition to this, the evidence provided by this study may not be applicable to other populations or contexts. Further research would be needed to confirm these findings and explore the underlying causes and potential policy implications in similar settings.Additional file 1: Supplementary material. Data quality checks.


The cost of childbirth has been confirmed as a vital factor in families’ fertility decision-making, and family welfare policies are capable of compensating for the increase in household living expenses regarding childbirth, such that the country’s fertility situation can be optimized. In this study, the fertility promotion effects of family welfare policies in OECD(Organization for Economic Co-operation and Development) countries are investigated through regression analysis, grey correlation (GRA), and the fuzzy set qualitative comparative analysis fsQCA method. As indicated by the results: (1) Family welfare policies notably boost fertility, and the boosting effect is long-lasting. However, this boost will be weakened in countries where fertility rates remain below 1.5. (2) The contribution of welfare policy measures to the fertility-promotion effect varies by country. The contribution of cash benefits is highest in over half of the countries worldwide, the contribution of relevant services and in-kind expenditure is highest in 29% of the countries, and that of tax incentive expenditure is highest in 14% of the countries. (3) The policy mix to boost fertility also varies according to the social context, with three policy groups derived using the fsQCA method. To be specific, the core antecedent conditions comprise cash benefits, relevant services, and in-kind expenditure. On that basis, China should pay attention to the following three points when formulating family welfare policies to tackle their demographic challenges. First, a system of family welfare policies should be developed as early as possible in the context of increasingly severe demographic issues since the incentive effect of family welfare policies will be weakened in countries with chronically low fertility rates. Second, the effects of improvements vary by country, and China should comprehensively consider its national circumstances when formulating and dynamically adjusting the mix of government fertility support policies in accordance with its social development. Third, employment is the main means of securing family income and takes on critical significance to sustaining families. Unemployment exerts a significant disincentive effect, such that it is imperative to reduce youth unemployment and enhance the quality of youth employment. On that basis, the disincentive effect of unemployment on fertility can be reduced. As industrialization and urbanization have progressively deepened, the demand for labor tends to be reduced, and the cost of childbirth has risen, thus causing lower fertility rates. As a result, population issues have turned out to be a common issue in numerous countries. The global fertility rate will drop to 2.2 in 2050, and population growth will generally be decelerated in the decade ahead, as estimated by the United Nations World Population Outlook. Global population issues are becoming increasingly exacerbated. The OECD average fertility rates were already below the replacement level of 2.1 in 1984. The average fertility rate declined from 2.06 to 1.65 between 1984 and 2003, and it has rebounded since 2003 to roughly 1.69. However, the average fertility rate has been constantly below the population replacement level. As of 2019, fertility rates in 21 countries of the organization were lower than the international population threshold of 1.5 . To be sOECD countries have formulated and implemented a series of welfare policies to stimulate fertility, so as to cope with the long-term population decline. A complete family welfare policy system has been formed based on long-term practice . Family However, the family welfare policies developed and implemented in OECD countries have different policy priorities and vary in their effectiveness. As depicted in China, subjected to a declining birth rate, has begun to take measures to actively tackle the problem of low fertility. There are two main aspects of the measures: one is to lift birth restrictions, and the other is to provide tax incentives. The policy stipulating that couples who are both only children can have two children was rolled out in China in November 2011 to liberalize birth control. The implementation of the policy of allowing a couple to have two children if one of them is the only child in their family, i.e., the “separate two-child” policy, was launched on 15 November 2013, as clearly stated by the “Decision of the Central Committee of the Communist Party of China on Several Major Issues of Comprehensively Deepening Reform”. In 2015, the fifth plenary session of the 18th Central Committee decided to fully implement the policy that a couple can have two children, which is recognized as the aim to fully liberalize the “two-child policy”. Moreover, a couple of childbearing age can have three children, as stipulated by the “Decision on Optimizing the Fertility Policy for Long-term Balanced Population Development” of the political bureau meeting of the CPC Central Committee in 2021. In terms of tax benefits, the standing committee of the 13th National People’s Congress adopted a decision on amending the Individual Income Tax law in 2018, in which a deduction of CNY 1000 per person per month was stipulated for children’s education. Effective 1 January 2022, expenses regarding the care of taxpayers under the age of three would be deducted at a flat rate of CNY 1000 per month per taxpayer, as provided by the “Notice of the State Council on the Establishment of Special Additional Deductions for Personal Income Taxes for the Care of Infants and Children Under the Age of 3” on 28 March 2022.The implementation of the above-described policies has had some effect . After tIn brief, despite the similar welfare policies of OECD countries in response to fertility challenges, their policy effects vary considerably. Accordingly, when formulating future fertility support policies in China, the following issues should be considered.(1)Whether family welfare policies have a boosting effect on fertility and whether the effect is long-term.(2)From a holistic perspective, this study analyzes how the combination of fertility support policies in different countries works better under different economic development conditions.(3)The formulation of relevant fertility support policies considering China’s actual situation.Based on the above issues, the effect of family welfare policies on fertility is studied by regression analyses; the contribution of each policy to fertility is investigated through grey correlation, and then the optimal policy combination is found through a fuzzy set qualitative comparative analysis from a holistic perspective. In light of China’s actual situation, this provides experience and inspiration for the formulation and systematic construction of China’s welfare policy.The rest of this study is organized as follows. In There has been a wealth of research on the effects of family maternity support policies. The established literature can be divided into two categories based on the subject of the articles: the effect of family welfare policies on fertility rates; and how the effect of family welfare policies varies across countries.The cost of childbearing is an important factor in household decision-making, and the increase in household expenditure resulting from childbearing increases with the age of the children, with food and education expenditure accounting for the largest share of additional costs  [022 [2]).Jung, Kim, and Lim 2019)  analyzed  analyzeIn brief, an analysis of the existing literature reveals that family welfare policies can promote fertility, and the effect of family welfare policies on fertility varies considerably by country. Moreover, the analysis of the effects of family welfare policies on fertility is generally conducted using traditional econometric methods or through qualitative analysis. A traditional econometric analysis follows the independence of variables and the symmetry of cause and effect, thus ignoring the interrelationships between policies and failing to indicate the asymmetry of cause and effect. Furthermore, only the effect of individual welfare policies on fertility is analyzed, instead of the effect of policy groups on fertility. In contrast, a qualitative analysis is capable of analyzing the effects of individual policies and policy groups from a holistic perspective, whereas it has limited data handling for time series and panel data, such that analytical limitations are generated. In this study, quantitative and qualitative research are integrated to deepen the analysis of the long-term and heterogeneous nature of the effect of family welfare policies on fertility through the analysis of the effect of family welfare policies on fertility. Moreover, the contribution of individual national welfare policy programs is measured before categorizing countries through a fuzzy set qualitative comparative analysis to identify more effective family-benefit policy combinations in different contexts.The “New Family Economics”, which emerged in the 1950s, is an economic method for analyzing the costs and benefits of household decisions, with Harvey Leibenstein and Gary Stanley Becker as its main representatives. The theory takes the family as the object of study while providing an economic analysis of fertility, marriage, and other behaviors to maximize the realization of utility by allocating the limited resources of the family in a rational manner. When analyzing family reproductive decisions, the theory considers children as a family’s durable goods and analyzes changes in the family’s demand for children through the analysis of the family’s demand for durable goods (Becker (2004) . MaximizAs revealed by the above theory and the established literature, family welfare policies help to improve a country’s fertility situation, such that hypothesis 1 is formulated: public expenditure on family welfare has an elevating effect on fertility. Nevertheless, the implementation of family welfare policies differs from country to country in terms of the focus of government public expenditure, which is dependent on the specific measures covered in the policy, such that the resulting enhancement effect varies according to the country. Hypotheses 2 and 3 are thus formulated. Hypothesis 2: Each policy contributes differently to improving fertility. Hypothesis 3: The combination of family welfare policy measures to boost fertility varies across realities. The relevant variables in the text are shown in t is the year; In this study, the effect of family welfare policies on fertility enhancement is analyzed through empirical regression. Regressions have been extensively used to analyze the effects of various factors on fertility in accordance with the established literature such as Choi and Myungsuk (2013)  and JungRelevance analysis has been commonly used for data analysis in the grey system theory. The grey correlation analysis refers to a research method that describes the magnitude and order of the relationship between different indicators. If the correlation is higher, the factor more significantly affects the explanatory variable, and vice versa. The method requires fewer sample data and does not require the number of sample data, the pattern of data distribution, or the existence of a linear relationship. Thus, this method is adopted to measure the contribution of family welfare policy measures to fertility increases. The effects of cash welfare expenditure, relevant services and in-kind expenditure, and tax incentives on changes in fertility are examined using a grey correlation model. This reveals which individual country measure contributes most strongly to the fertility-raising effect. The analysis sequence is determined prior to the grey correlation calculation. The dependent variable sequence is expressed as the reference sequence A qualitative comparative analysis is conducted through the analysis of set theory and Boolean algebra, which investigates how the combination of antecedent conditions results in the occurrence or appearance of the outcome variable from the perspective of the set. This analysis method refers to an integrated research method that combines the advantages of qualitative and quantitative research . A qualQCA methods can fall into deterministic sets, fuzzy sets, and multi-valued sets, which is dependent on the form of the set. The fundamental difference between the above-mentioned three types is revealed in the different values taken for the division of the calibration affiliation of the variables. Conditional variables and outcome variables should be dichotomous variables, i.e., only 0 (no membership) or 1 (full membership), as required by the deterministic set qualitative analysis (CSQCA). A fuzzy set qualitative analysis (fsQCA) extends the deterministic set qualitative analysis to be able to cover affiliations in the middle of 0 and 1. Besides, a multi-valued set qualitative analysis (mvQCA) is extended from a deterministic set qualitative analysis between deterministic and fuzzy set states, which adds information to the variables by multi-segmenting their values based on the deterministic set. Thus, the qualitative analysis of fuzzy sets is more consistent with the reality of the situation. For instance, Bong, Jeon, and Seung (2021), Lin, and Kim (2016), and Yunkyu (2014) ,28,32 quThe total fertility rate of 21 OECD countries from 2001 to 2015 serves as the result variable in this study. The core explanatory variable was public expenditure on family benefits. The control variables were the unemployment rate, the mean age of women at childbirth, maternity leave, paternity leave, urbanization, the child-rearing ratio, medical expenditure, the GDP per capita, women’s education, as well as household income.The unemployment rate was measured using the unemployment rate of the population aged 15–64 in the selected OECD countries from 2001–2015. For the mean age of women at childbirth, Parental leave was measured using the number of weeks of maternity leave and paternity leave in the selected OECD countries from 2001 to 2015. Urbanization is the share of the urban population in the total population of the selected OECD countries from 2001 to 2015. The average urbanization rate of the selected OECD countries from 2001 to 2015 is depicted in The child-rearing ratio is the ratio of the population under 15 years to the working-age population in the selected OECD countries from 2001 to 2015. The average child dependency ratio in the selected OECD countries from 2001 to 2015 is depicted in Medical expenditure is an indicator of the total medical expenditure as a share of GDP in the selected OECD countries from 2001 to 2015. The average total expenditure on health care in the selected OECD countries from 2001 to 2015 is depicted in Gross domestic product (GDP) per capita for the selected OECD countries from 2001 to 2015 is used to represent the level of economic development of the respective country. Household income is defined as the income of a minimum wage worker for a couple without children whose partner is not working. The household income divided by the income of an otherwise identical family working at the average wage. Women’s education or female education refers to the female gross enrolment rate in tertiary education. In this chapter, the effects of family welfare policies on fertility, the long-term effects of family welfare policies on fertility, and the effects of heterogeneity are presented using a regression analysis. The correlation between specific programs of family welfare policies and fertility is analyzed using grey correlation. Subsequently, the fsQCA method is employed to analyze how specific programs of welfare policy can be combined in different contexts to help increase fertility.p < 0.01), suggesting that the instrumental scalar was not unidentifiable, i.e., the instrumental variables passed the unidentifiability test. The Cragg–Donald Wald F statistic of 18.2704 exceeds the Stock–Yogo weak instrumental variable threshold of 10% at 16.38 and passes the weak instrumental variable test. The results of the two-stage regression are shown in column (4) of Considering the endogeneity of the core explanatory variable household welfare policy, this study also employs the method of an instrumental variable regression with the home government expenditure as the instrumental variable. In this study, two-stage least squares (2SLS) were used to test the instrumental variables, and the Kleibergen–Paap rk LM statistic was 15.4312  in 2001–2015, with low-fertility countries being those with a total fertility rate lower than 1.5 for over 13 years; the remaining countries fall into the category of high-fertility countries. Public expenditure on family benefits falls into three types, i.e., cash benefits, expenditure on goods and services, and expenditure on tax concessions. As indicated by the baseline regression analysis of 5.2, family welfare policies notably boost fertility. In this section, the effect of the three policy programs on fertility is further analyzed. The association between the three types of expenditure and the total fertility rate in the respective country, i.e., the contribution of the three types of expenditure to the improvement of the total fertility rate, is investigated using the grey correlation. Furthermore, how the combination of family welfare policy programs is more effective in boosting fertility in a variety of country contexts is analyzed using the fsQCA method.In the qualitative comparative analysis of fuzzy sets, the respective variable is considered a set with a different degree of affiliation. On that basis, the variables should be calibrated, such that they are transformed into a fuzzy affiliation score, i.e., between 0.0 and 1.0, prior to the group analysis. The qualitative comparative analysis of fuzzy sets requires three qualitative anchor points for variables in the calibration process . The percentile employed in most existing research is adopted in this study to determine the qualitative anchor points. Moreover, the 5th, 50th, and 95th percentiles of the sample data are identified to be fully unaffiliated, crossover, and fully affiliated in accordance with the characteristics of the data distribution. Furthermore, the sample data variables are calibrated . The varThe necessity test is a test of whether the respective condition variable is required for the outcome variable, i.e., the occurrence of the outcome should arise from that condition variable. The necessity of each condition variable is studied by fsQCA3.0, and the results are listed in The combination of policies that can boost fertility from the above-described family welfare policies is analyzed using the fsQCA method. In this study, the fuzzy set calibration is performed in the same way as studies in the established literature, where the values of the corresponding indicators in the 5th, 50th, and 95th percentiles serve as the calibration parameters when calibrating the indicators. Moreover, the built-in coefficients of the fsQCA software system are employed  for setting the consistency and case thresholds. The standard analysis is conducted to obtain three conformational solutions that are conducive to improving the low fertility situation . Furthermore, the most representative intermediate solution is adopted, and Ragin and Fiss’ presentation of the QCA results are drawn on, as listed in The respective column in C1: Higher burden of child support. This grouping states that “fertility rate = cash benefits * services and in-kind support * child-rearing ratio” and suggests that countries with a high child-support burden can increase their fertility rates by providing cash benefits and services and in-kind support to families. In this group, unemployment and family tax incentives do not affect the level of fertility rates, i.e., neither the level of unemployment nor the strength of family tax incentives affects fertility rates, provided the basic conditions of this group are satisfied. The United Kingdom, Belgium, Australia, France, and Ireland are involved as the countries in this grouping scenario.C2: Low unemployment and high child-support burden. This group states that “fertility rate = services and in-kind support * tax incentives * ~unemployment rate * child-rearing ratio”. This group suggests that high services and in-kind support and high tax incentives are sufficient conditions to raise fertility rates in countries with low unemployment. Cash welfare policies do not have a significant effect on fertility rates when the basic conditions of the histogram are met. The countries in this group of scenarios are the Netherlands and the United Kingdom.C3: Low unemployment rate and low burden of child support. This group states that “fertility rates = cash benefits*~services and in-kind support*tax incentives*~unemployment*~ child-rearing ratio”, suggesting that high cash benefits, high tax incentives, and low services and in-kind support can contribute to higher fertility rates when unemployment is low and the burden of child support is low. This group only involves one country, which is the Czech Republic.(1)Family welfare policies are capable of significantly enhancing fertility status see . The het(2)The correlation degree between three types of family welfare policy and the fertility rate is calculated using the method of grey correlation degree analysis. In other words, cash welfare expenditure, relevant services and in-kind expenditure, and tax incentives and the fertility rate. It was found that cash benefit expenditure made the largest contribution to fertility improvement in 12 of 21 countries, relevant services and in-kind expenditure made the largest contribution to fertility improvement in 6 of 21 countries, and tax concessions made the largest contribution to fertility improvement in 3 of 21 countries .(3)Through the qualitative analysis of fuzzy sets, three policy combinations are found to improve fertility status. As depicted in In this study, the effect of family welfare policies on boosting fertility is analyzed based on a systematic analysis of the panel data originating from OECD countries between 2001 and 2015 using several methods . The results of this study are presented as follows:In brief, increasing government subsidies to families can be conducive to increasing the total fertility rate. A grey correlation analysis and fsQCA qualitative analysis can be employed in the practice of family benefits policy to indicate the types of policies that public expenditure on family benefits should orientate towards. As indicated by the results of a grey relation analysis and fsQCA qualitative analysis, cash allowance expenditure, family service expenditure, and in-kind expenditure significantly increase the fertility rate, and they serve as the core conditions in the grouping of fertility rate enhancement as well. Accordingly, the support of cash benefits to families and that of family-associated services should be emphasized more considerably in practice. Furthermore, the policy mix should be adjusted dynamically in accordance with the macro-environment to curb the decline of the fertility rate.OECD countries had an earlier start to deal with low fertility, and their policy systems and response measures are more refined. Thus, the measures taken by OECD countries can bring great enlightenment to China to assist China in addressing the problem of low fertility, though there are some differences between China and OECD countries in terms of the macro-environment when China faces its population problem.(1)In the face of increasingly serious population issues, a family welfare policy system should be established as early as possible to improve the fertility situation. Because of the heterogeneity and long-term nature of the uplifting effects of family welfare policies, China’s early formulation and improvement of family welfare policies can have a greater effect in encouraging fertility.(2)The development of a family welfare policy requires an increase in expenditure on cash benefits and relevant services and in-kind expenditure. As indicated by the results of (3)According to the macro-environment, the amount of the three types of public expenditure on family welfare should be dynamically adjusted. From the results of Despite the certain practical significance of this study, there are still certain defects and limitations. First, the research subject of this study is OECD countries with certain specificity. Given the applicability of the results of this study, this study should be considered in the context of the national conditions of the respective country. Second, there are numerous factors involved in fertility decision-making. Lastly, the factors involved in family fertility decision-making change at different stages of development, such that the formulation and implementation of welfare policies should be dynamically adjusted. Accordingly, the above-described three aspects can be studied in depth in subsequent research.


The aging of an organism is hallmarked by systemic loss of functional tissue, resulting in increased fragility and eventual development of age-related neurodegenerative, musculoskeletal, cardiovascular, and neoplastic diseases. Growing scientific evidence points to mitochondrial dysfunction as a key contributor in the aging process and subsequent development of age-related pathologies. Under normal physiologic conditions, the body removes dysfunctional mitochondria via an autophagic process known as mitophagy. Urolithin A (UA), a metabolite produced when gut microflora digests the polyphenol compounds ellagitannin and ellagic acid, is a known inducer of mitophagy via several identified mechanisms of action. The primary objective of this scoping review is to identify and summarize the clinical relevance of UA supplementation in the prevention of age-related pathology and diseases. A computer-assisted literature review was performed using PubMed and EMBASE for primary source research articles examining UA supplementation and aging-related pathologies. A total of 293 articles were initially identified from a database search, and 15 articles remained for inclusion in this review, based on predetermined criteria. Analysis of the 15 identified publications demonstrated that UA holds potential as a dietary intervention for slowing the progression of aging and preventing the development of age-related disease. This review also illustrates the potential role that mitochondrial health and inflammation play in the progression of age-related pathology. Identifying the clinical relevance of UA supplementation in the prevention of age-related pathology and diseases will help further the focus of research on treatments that may improve the longevity and quality of life in patients at risk for these comorbidities. Mitochondrial dysfunction and inflammation in age-related disease The aging of an organism is hallmarked by cellular senescence and the accumulation of cellular dysfunction, resulting in the systemic deterioration of tissue . This syMitophagy and urolithin A Under normal physiologic conditions, the body removes dysfunctional mitochondria via an autophagic process called mitophagy . ImpairePotential implications for UA in age-related diseaseNutritional intervention in various diseases and ailments has gained traction in recent years due to its minimally invasive nature, affordability, and wide accessibility. UA supplementation stands among these interventions as a potential protective agent against the development of age-related disease . UA has Objectives Age-related pathology and diseases, such as Alzheimer’s disease and various musculoskeletal, cardiovascular, and neoplastic diseases, remain as the major causes of mortality and reduced quality of life and currently have no cures. Additionally, current antineoplastic drug regimens are not always efficacious and come with many unwanted side effects. Given the progressive interest in natural remedies and health-conscious practices in disease prevention, further analysis of current literature that assesses UA supplementation in human test subjects or human cell lines was warranted to provide practicing clinicians with organized evidence for utilizing UA within their patient populations. In summary, identifying the clinical relevance of UA supplementation in preventing aging and developing age-related disease will help further focus research on treatments that may improve longevity and quality of life in patients at risk for age-related pathology.Search strategy A computer-assisted scoping review was performed using PubMed and EMBASE for primary source research articles examining UA supplementation in the prevention or reversal of aging and age-related disease, such as neurodegenerative, musculoskeletal, and cardiovascular disease. The database key search terms included “Urolithin A” and “aging” or “age related disease” or “mitophagy” or “autophagy” or “sarcopenia” or “cardiovascular disease” or “joint degeneration” or “neurodegenerative disease.” Articles were filtered to include those published after 2017 to improve the efficacy of the review and capture the most recent and prominent research. The reference list of retrieved articles was also considered when found to be relevant and if those additional articles fit the search criteria but were not discovered through the initial search. The relevance of these articles was assessed through a hierarchical approach, first with an evaluation of the title, then the abstract, followed by the full manuscript. For any articles not freely available, access was gained through the Nova Southeastern University library when available.Selection criteria Studies were eligible if they examined UA supplementation in age-related processes or diseases and provided clinically relevant intervention measures. During the article screening process, the writers determined that studies investigating the impact of UA on neoplastic disease were also relevant to the objectives of this review. The decision was made to include articles investigating neoplastic disease as an age-related pathology even though it was not explicitly mentioned in the key search terms. Eligible study designs included randomized control trials (RCT), prospective and retrospective studies, cross-sectional studies, case studies, and other studies on human subjects and/or human cell lines. Exclusion criteria included meta-analyses, systematic reviews, editorials, non-human studies, and limited access to the available text. Due to the novel nature of the investigation of this topic, specific study populations were not considered as a factor in determining inclusion eligibility for this review. Additionally, because studies completed in animal models only provide theoretical data regarding the safety and efficacy of a substance, these studies cannot confirm these qualities within humans and thus were omitted from this review. A flow diagram Figure  was deveResultsA total of 293 articles were identified from the initial electronic database search. Fifty-two articles were excluded based on lack of relevance found during the title and abstract screening, and 114 duplicates were removed. Of the remaining 179 articles, 109 were excluded for containing minimal or no mention of UA, focusing on an intervention that utilized UA in tandem with another agent, or testing in animal models. Three articles were excluded due to a lack of access to the full text. A total of 15 articles remained for inclusion in this review (Table DiscussionFrom the analyzed publications, five main age-related disease pathologies have been investigated in studies conducted in humans or human cell lines: neurodegenerative disease -17; muscNeurodegenerative disease Four of the identified articles focused on neurodegenerative disease -17. The The fourth neurodegenerative disease-based study in this review examined high glucose-induced neurodegeneration similar to what is seen in advanced diabetes mellitus . UA pretMusculoskeletal-related disease Three randomized controlled trials investigating the utility of UA as a preventive agent in the development of sarcopenia were examined in this review -20. PrimThe final three musculoskeletal-related studies focus on age-related bone and joint pathology ,22. UA sCardiovascular diseasesThough the impacts of UA in cardiovascular diseases have been largely studied in animal models, there is limited research conducted in humans or using human cell lines on the effects of UA in cardiovascular diseases. However, one study by Dirimanov and Högger looked at the impact of UA on the Akt kinase pathway in human cell lines . This paIntegumentary diseaseOne of the most visible signs of aging is the impact that cellular senescence and subsequent degradation of cellular components have on the appearance of our skin. Two studies included in this review investigated the effects of UA on the aging of the skin ,23. HumaAnother study investigated the protective effects of UA against UVA-induced photoaging . This stNeoplastic diseaseA few studies focused on the antineoplastic impacts of UA supplementation ,9,24. ZhThe final study analyzed in this review was a human cell line study completed by Sahashi et al. . This stFinal considerationsThough the publications examined in this review largely lean in support of the use of UA as a potential preventive measure in the mitigation of aging and age-related disease, the strength of this study is limited by the scarcity of published human trials. No studies concluded that UA had a negative effect on any of the studied populations. Three of the 15 studies included in this review were randomized controlled trials measuring UA’s impact on sarcopenia in humans. Thus, current evidence supporting the supplement’s use is strongest for this application. However, more longitudinal studies and human trials across all applications are needed to further explore the utility of UA as a dietary intervention in slowing and preventing the development of various age-related pathologies.Analysis of the 15 identified current publications demonstrates that UA holds strong potential as a dietary intervention for slowing the progression of aging and preventing the development of age-related diseases. None of these studies found UA to exhibit overall negative effects on the studied populations, further supporting its potential as an impactful dietary intervention in at-risk populations. This review also illustrates the potential role that mitochondrial health and inflammation play in the progression of age-related pathology. Further, this review suggests that the utilization of UA may offer benefits to patients actively combating the development of sarcopenia. More longitudinal studies and further analysis measuring the efficacy of UA in human trials would provide more robust suggestions on potential nutritional and dietary interventions for patients concerned with age-related pathology.


Increased circulating uric acid (UA) concentration may disrupt cardiac function in heart failure patients, but the specific mechanism remains unclear. Here, we postulate that hyperuremia induces sterol regulatory element binding protein 1 (SREBP1), which in turn activate hepatic fatty acid biosynthesis response, leading to cardiac dysfunction.Increased circulating uric acid was observed in heart failure patients and inversely correlated to cardiac function. Besides, uric acid correlated to circulating lipids profile based on metabolomics in heart failure patients. Using cultured human hepatoellular carcinomas (HepG2) and Tg(myl7:egfp) zebrafish, we demonstrated that UA regulated fatty acid synthase (FASN) via SREBP1 signaling pathway, leading to FFA accumulation and impaired energy metabolism, which could be rescued via SREBP1 knockdown. In ISO treated zebrafish, UA aggravated heart failure via increased cardiovascular cavity size, decreased heart beats, pericardial edema and long-stretched heart deformation.Our findings suggest that UA-SREBP1-FASN signaling exacerbates cardiac dysfunction during FFA accumulation. Identification of this mechanism may help in treatment and prevention of heart failure.The online version contains supplementary material available at 10.1186/s12967-023-04050-5. Serum uric acid (SUA) is the end product of both dietary and endogenous purine metabolism . In clinInterestingly, several studies reported that elevated UA levels regulate the oxidative stress, inflammation and enzymes associated with free fatty acids (FFA) metabolism –9. FFA aAccordingly, the aims of this study were (i) to assess whether SUA levels would be elevated in HF patients compared with control subjects; (ii) to evaluate the association between SUA, FFA levels and cardiac remodelling and dysfunction; (iii) to determine whether SUA would regulate FFA in vitro and vivo; and (iv) to elucidate the molecular mechanism regarding SUA induced lipid metabolic impairment during HF progression.All patients had been recruited from the First Affiliated Hospital of Xi’an Jiaotong University. Written informed consent was obtained from all study participants, with ethnic committee approval at the First Affiliated Hospital of Xi’an Jiaotong University and was conducted according to the Declaration of Helsinki.All experimental procedures on animals were approved by Medical Faculty Mannheim (license no: I-19/02) and carried out in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.Tg(fli1:EGFP) [Tg(myl7:egfp) [Zebrafish lines, i1:EGFP)  and Tg(ml7:egfp) , were ral7:egfp)  under stTg(myl7:egfp) larvae were anaesthetized in 0.0003% tricaine at 72 hpf, and mounted head up in 0.8% low-melt agarose and placed on a coverslip bottom dish in wells made from a layer of 3% agarose. Images were collected using a Zeiss LSM510 confocal microscope equipped with a 40× water objective. In each stack, 60 confocal slices spanning the expression of Tg(myl7:egfp) were collected at 1–1.5 µm intervals and merged by ImageJ.For in vivo imaging of the zebrafish heart structure, Fertilized zebrafish embryos were transferred into 6-well plate, around 30 embryos per well with 5 mL eggwater. At 24 hpf the chorion of zebrafish embryos was removed using sharp forceps and 0.003% PTU was added to the eggwater. Uric Acid , Isopropenrol, Hydrochloride , Palmitic Acid  and Stearic Acid  treatments were started at 3 hpf and continued until the end; Medium was changed every day.Human hepatocellular carcinoma cells (HepG2) were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin and streptomycin at 37 °C in 95% air and 5% CO2. HepG2 cells at 50–70% confluence were transfected with 40 nM SREBP1 siRNA or scramble control siRNA using Lipofectamine 2000 (Invitrogen) for 24 h, and then treated with 1 mM uric acid (Sigma-Aldrich) for another 48 h. Human SREBP1 siRNA and scrambled control siRNA were purchased from Santa Cruz Biotechnology.TG(fli1:EGFP) zebrafish larvae at different time points using the RNeasy Mini Kit following the manufacturer’s protocol (Qiagen). First-strand cDNA was generated from 1 μg RNA using the Maxima First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Thermo Scientific). Primer design for zebrafish was done using NCBI or by Roche Universal Probe Library Assay Design Center and primers are listed in Additional file Total RNA was isolated from TGfli:EGFP zebFor cell culture, RNA was isolated from cultured cells by using TRIzol (Invitrogen). The mRNA level was measured by using SYBR Green (Bio-Rad) with β-actin–actin as internal control. Primer sequences used for qPCR are in Additional file For zebrafish, detection was done in cooperation with the Metabolomics Core Technology Platform from the Centre of Organismal Studies Heidelberg. At 96 hpf, around 50 zebrafish larvae per measurement were anaesthetized with 0.003% tricaine and snap frozen. Adenosine compounds, thiols, free amino acids, fatty acids and primary metabolites were measured as previously described .For cell culture and human serum FFA measurement, targeted metabolomics were applied for FFA detection. The sample preparation, instrumentation, metabolic annotation and data analysis procedure are referred in our previously published methods with minor modification –28.For Western blot analysis, HepG2 cells were lysed in the RIPA buffer supplemented with protease inhibitors (Thermo Scientific). Lysates were separated by SDS-PAGE and immunoblotted with antibodies as indicated. Western blot analysis was performed with the following antibodies: SERBP1 , Fatty acid synthase , β-actin .4772 consecutive patients admitted to the cardiology department of the First Affiliated Hospital of Xi’an Jiaotong University for AMI between January 2016 and December 2020 were enrolled. The inclusion criteria were confirmed admission diagnosis of metabolic syndrome. The exclusion criteria were: (1) severe nonmetabolic disease with an expected survival of less than 1 year and unwillingness to participate; (2) patients over the age of 80 years or living far away from the hospital’s catchment area. A patient could only be included once. The medical records of the patients were collected from the Biobank of the First Affiliated Hospital of Xi’an Jiaotong University, which contains the identified data derived from raw medical records, information about patients’ detailed medical histories, present medication, biochemical and echocardiography results. Written informed consent was obtained from all study participants, with ethnic committee approval at the First Affiliated Hospital of Xi’an Jiaotong University.t-test or one-way ANOVA . For clinical results, simple t-test was used to compare continuous variables after normality test and χ2 test was used to compare categorical variables. The simple linear analysis was used to calculate the correlation. GraphPad Prism 8.3.0 was used for analyses and p values of 0.05 were considered as significant: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bioinformatic analysis was performed using the OmicStudio tools at https://www.omicstudio.cn/tool and MetaboAnalyst at https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml. Schematic diagram was performed via Figdraw and Powerpoint.Experimental results are expressed as mean with standard deviation (mean ± SD). Statistical significance between different groups was analysed using Student’s p = 0.043)  patients, 259 heart failure with mid-range ejection fraction (HFmrEF) patients and 4323 normal ejection fraction (EF) patients. Baseline information were shown in Table p < 0.001). Both non-essential and essential FA profiles were increased in heart failure patients  level, but no alteration in cardiovascular size compared to control larvae  zebrafish larvae at 72, 96 and 120 hpf .To further understand the specific mechanism how UA regulated FFA levels and heart fuction, zebrafish experiments were performed, as zebrafish embryo can tolerate the absence of blood flow for few days because its oxygen is delivered by diffusion rather than by the cardiovascular system, making it an excellent model for studying heart failure . 1 mM UAvae Fig. a–d occurhpf Fig. a, quantiTg(myl7:egfp) zebrafish larvae were applied, in which cardiomyocytes were marked with green fluorescence protein [To investigate the exact cardiovascular structure alteration after UA treatment,  protein . 0.5 mM nppb expression , fatty acid desaturase 2 (fads2), fatty acid elongase 2 (elov2), and stearoyl-CoA desaturase (scd) were performed , and transfected it with sterol regulatory element binding protein 1 (SREBP1) siRNA to check the rescue effects Fig. a. First,In the present study, we have identified that UA correlates to FFA levels and cardiac function among patients with metabolic syndrome. UA correlated to FFA levels in HF patients and zebrafish model. High UA lead to impaired fatty acid metabolism and aggravate the lipotoxic heart injury. In addition, we demonstrate that targeting the UA-SREBP1-FASN pathway could be a possible therapeutic approach for HF. Our study provides new insights into heart failure with potentially important therapeutic implications.The influence of serum uric acid level on cardiovascular disease is still controversial. Previous studies have explored the effect of uric acid on cardiovascular disease, but the results are inconsistent, and there are bias and other confounding factors , 34. OurIt has previously been reported that patients with metabolic syndrome are prone to suffer from HF , 36. Of Metabolomics approach has enabled us to observe metabolic changes of specific disease from a more mechanistic view. In the present study, by exploring the FFA profile in HF patients and UA treated zebrafish and hepatic cells, we further clarify the change of fatty acid metabolism spectrum induced by uric acid treatment. In HF patients, we have observed increased circulating FFA levels including SFA, MUFA and PUFA. Correlation analysis has identified a positive correlation between UA levels and FFA, and a negative correlation between FFA and cardiac function as evaluated by EF and proBNP. In zebrafish, it is further clarified that UA itself could not causes HF but could aggrevate HF, accompanied by dyregulated FFA metabolism pathways. At last, it is testified in vitro that UA result in FFA increase in HepG2 cell line, which could be contereacted by SREBP1 silencing. The above result further establish the interaction of UA and FFA during HF progression.The major novelty of the present study is that we point out that targeting UA-SREBP1-FASN axis might be a therapeutic approach for HF. SREBP1 has previously been identified as an intracellular cholesterol sensors located in the endoplasmic reticulum that provide feedback regulation of intracellular cholesterol. Recently it has been reported that UA could cause endothelial dysfunction via SREBP2 transactivation of YAP . But it Our study has several limitations. Firstly, the regulatory function of SREBP1 has not been tested in vitro, further validation studies of SREBP1 KO animal model are needed. Secondy, the function of SREBP1 has only been tested in HepG2 cell line, bot not in human liver sample. Third, the correlation study could not explain why UA is increased in HF patients, although the mechanism may involve many factors, and needs to be further explored. Last, due to a retrospective study, several clinical parameters such as diuretic related therapy were not provided. As diuretic treatment  was tightly associated with hyperuricemia, it would be quite interesting to investigate in HF patients in the future.To sum, our findings suggest that UA-SREBP1-FASN signaling exacerbates cardiac dysfunction during FFA accumulation. Identification of this mechanism may help in treatment and prevention of heart failure.Additional file 1. Fig. S1: Correlations between FFA levels and clinical parameters in HF and control patients. Network analysis between fatty acid and clinical parameters. The color in each cell represents value of correlation calculated by Spearman‘s rank correlation coefficient. *p<0.05, **p<0.01, ***p<0.001. Fig. S2: UA aggravates ISO induced heart failure leads to impaired energy metabolism in zebrafish. A Heatmap of several metabolites in UA, ISO and co-treatment induced heart failure zebrafish larvae at 96 hpf. The color in each cell represents the expression of each zebrafish cluster sample as the scale bar showed, each zebrafish cluster includes 50 zebrafish larvae. B PCA analysis of every sample among each group. C VIP scores of the top metabolites among each group. Fig. S3: Expression analyses of elov2, scd and fads2 in zebrafish after UA and ISO treatment. 1 mM UA treatment and 0.5 mM ISO induced elov2, scd and fads2 mRNA expression in Tg(fli1:EGFP) zebrafish larvae at 72, 96 and 120 hpf. Expression of mRNA was analysed by RT-qPCR and was normalized to both b-actin and b2m, each single dot represents one zebrafish cluster’s data, which includes 20–30 zebrafish larvae. For statistical analysis one-way ANOVA followed by Sidak’s multiple comparison test was applied, *p < 0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. Fads2, fatty acid desaturase 2; elov2. fatty acid elongase 2; scd, stearoyl-CoA desaturase. Fig. S4: Heatmap of fatty acids in HepG2 after UA treatment and Srebp1 knockdown. Heatmap of fatty acids in HepG2 after UA treatment and Srebp1 knockdown. The color in each cell represents expression of each cell culture sample as the scale bar showed, n = 3–4 per group. Table S1: Primers. Table S2: Multiple linear regression analysis in the metabolic syndrome cohort (big cohort). Dependent variable: EF value. UA, uric acid; ALT, alanine transaminase; AST, aspartate transaminase; Cre, creatinine; LDL-C, high density lipoprotein; *p < 0.05. Table S3: Baseline information of the heart failure cohort . EF, ejection fraction; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; UA, uric acid; ALT, alanine transaminase; AST, aspartate transaminase; Cre, creatinine; Chol, cholesterol; TG, triglycerides; HDL-C, high density lipoprotein; LDL-C, high density lipoprotein.


Human T-lymphotropic virus (HTLV) infection has a worldwide distribution and currently, more than 2.5 million individuals have been infected in Brazil. The study aimed to investigate HTLV infection prevalence among blood donors in Mato Grosso do Sul, characterizing seroepidemiological profiles of HTLV-1/2 positive individuals and evaluating the blood bank's HTLV screening system. A cross-sectional survey was conducted among blood donors from Mato Grosso do Sul state (MS)—Central Brazil, between January to December 2021. The information was obtained from databases, samples from the collection of HEMOSUL, and active searching, with the completion of laboratory analyses. 35,278 blood donors were screened for anti-HTLV-1/2 by chemiluminescence immunoassay (CMIA). Among them, 78 were initially reactive for anti-HTLV-1/2 (2.21/1000). Out of 78, 67 returned to the blood center to collect a second sample for retesting with a second screening with CMIA. After confirmation, 8 samples were indeterminate, and 8 were confirmed as positive for HTLV antibodies. New tests were performed for the 8 positive samples, and 6 were confirmed as HTLV-1 infection , one as negative, and one as indeterminate. The present study describes the low prevalence of HTLV infection in blood donors from MS and contributes to the definition of the regional infection profile. The prevalence found in this study (0.017%–0.17/1000) shows to be a much lower value than the rates reported in other states in Brazil. We highlight the need for confirmatory testing for those seropositive donors in screening assays and the need for adequate counseling and patient management for those confirmed HTLV individuals. HTLV-1 is mostly associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM) but is also associated with other inflammatory diseases6. HTLV-1 has a worldwide distribution, with South Japan, sub-Saharan Africa, Caribbean Islands, Melanesia, and South America considered the most prevalent regions7.Human T-lymphotropic virus (HTLV) is a retrovirus with a tropism for T lymphocytes. HTLV-1 and HTLV-2 were identified in 1980 and 1982, respectively, besides HTLV-3 and HTLV-4 in 20058. From the data obtained, several seroprevalence studies were carried out. It is estimated that 2.5 million individuals are infected with HTLV-1 in Brazil, and a heterogeneous prevalence was observed according to the region and specific populations9. Previous studies conducted in the state of Mato Grosso do Sul  have reported rates varying from 0.1%—10.0% in specific groups, such as pregnant women10, indigenous population11, prisoners12, Japanese community14, men who have sex with men15 and remnant quilombos16.Considering that blood transfusion can be a route for HTLV transmission, screening of blood donors is important to prevent it. Thereat, screening for HTLV-1/2 infection in Brazilian blood banks has become mandatory since 1993This cross-sectional study aimed to describe the HTLV infection prevalence in blood donors in Mato Grosso do Sul state and the seroepidemiological profile of the positive HTLV-1/2 individuals, and also evaluate the screening system for HTLV of the blood bank.A total of 45,751 blood donations were screened between January 2021 to December 2021, corresponding to 35,278 blood donors. The general profile of blood donor candidates based on self-report before the donation was in majority men (55.48%), with ages ranging from 16 to 69 years (average 35 years). Most donors reported being widows (47.62%), 85.03% identify as Brazilian caucasian, and most donations were realized in the capital—Campo Grande (77.7%).Seventy-eight candidates were considered inapt due to being positively screened for anti-HTLV-1/2 , 67 returned to the blood center to collect a second sample for retesting with a second screening with CMIA. The spread of signal/cut-off ratio (S/CO) values of the second collection samples is presented in Fig. .21/1000,Of all 67 samples, 8 were indeterminate, and 8 were positive in confirmatory tests HTLV-1/2 INNO-LIA) realized in HEMOSUL  from HEMOSUL were retested by our group and were confirmed negative for HTLV infection. New tests were performed for the 8 positive samples using stored blood samples from HEMOSUL or through new blood collection of the 3 individuals who agreed to participate in the study. Of these 8 samples, 6 were confirmed as HTLV-1 , one as negative  (DMS-01: S/CO ratio 5.93), and one as indeterminate (S/CO ratio 50.56).10. The youngest child (DMS-02.01) was born in 2008 and breastfed for 2 years. Both DMS-02 and DMS-02.01 had blood collected by our research group. DMS-02 was confirmed for anti-HTLV-1, and DMS-02-01 had a negative result for anti-HTLV-1/2. The participant was referred to the medical service but did not attend and was going to a city change.One of the individuals who agreed to participate in the study (DMS-02) was a 46-year-old female and never donated blood before. She reported being married once when she had 2 children, and 2 cesarean surgeries were performed. Also, she reported never having a blood transfusion, having piercings or tattoos, having an accident with others’ blood, being arrested, or using drugs. She was unaware of having other IST. No information was available on the HTLV-1 status of her ex-husband. The first child was born in 2006. At that time, MS state had already been testing anti-HTLV on antenatal since 2002The other positive (DMS-04) was already a blood donor, a 40-year-old female, and married twice. No information was available on the HTLV-1 status of the first husband with whom she had her first child in 2002, and she reported breastfeeding for 9 months. Her mother (DMS-04.02), sister (DMS-04.03), current husband (DMS-04.05), and one child of the couple (DMS-04.01) accepted to participate in the study and the serology for HTLV-1/2 were negative for all them. DMS-04 reported receiving one bag of blood transfusion in 2020 due to blood loss in childbirth. She was also reported having an accident with others’ blood, having a tattoo, being diagnosed with herpes, and sharing personal objects like pliers. DMS-04.01 was born in 2008 and was not breastfed. The couple’s youngest daughter was born in 2020 and breastfed for 8 months. She tested for anti-HTLV-1/2 in the private health system and reported that the result was negative. Participant DMS-04 was referred to the medical service. During the consultation, the doctor consulted the previous result of the last antenatal screening and reported that she already had serology positive for anti-HTLV-1, and she reported being unaware of this result.A third blood donor candidate agreed to participate in the study (DMS-01), a 20-year-old female blood donor who had never donated blood before. The value obtained on the screening test in CMIA was a S/CO ratio value of 5.93. She reported as single in the research interview, but she reported in the blood bank questionnaire that was married. Her mother also agreed to participate in the study (DMS-01.01). Both DMS-01 and DMS-01.01 blood samples were negative HTLV-1/2 in our study. DMS-01 was referred to the medical service. She reported having rheumatologic symptoms, and other tests were ordered by the doctor.Some inconsistencies were found comparing the answers given to HEMOSUL before the donation and to research group during the interview, such as marital status and ethnicity. Regarding the other three confirmed HTLV blood donor candidates, the information available is based on the institutional database only Table . Of thes10, the indigenous population11, prisoners12, the Japanese community14, men who have sex with men15 and remnant quilombos16. The present study describes the seroepidemiological profile of blood donors from Mato Grosso do Sul state, Central Brazil. A study of blood donors from 27 Brazilian state capitals centers assessed the geographic distribution of HTLV screening prevalence rates between 1995 and 2000. It was observed a heterogeneous distribution of HTLV prevalence among the Brazilian regions, with higher rates in the Northern/Northeastern. In the state of Mato Grosso do Sul the prevalence estimated was 2.1/1,000, not considering confirmatory tests17. The prevalence found in the present study considering the initial screening tests (2.21/1000) indicates maintenance of the prevalence over more than 20 years between these studies. However, using confirmatory tests, the prevalence found in this study (0.017%—0.17/1000) shows to be a much lower value. Low prevalence was also observed in the pregnant population from Mato Grosso do Sul (0.13%)10. Other blood donor studies conducted in low prevalence regions in Brazil found rates of prevalence of 0.10%, 0.08%, 0.11%, 0.08%, 0.13%, and 0.15% in Araçatuba, Presidente Prudente, Serrana, Ribeirão Preto (São Paulo state), Manaus (Amazonas state) and in the state of Maranhão, respectively20.Previous studies on HTLV infection prevalence in the state of Mato Grosso do Sul have focused on specific groups, such as pregnant womenThe profile of eligible and inapt blood donor candidates for HTLV based on self-report is similar Table . In the The three positive individuals who agreed to participate in the study show some particularities. When accompanied in the medical service by the group, DMS-01 related some autoimmune symptoms, and her rheumatoid factor showed a positive result, which could have caused interference with HTLV screening, generating a false positive result in the confirmatory test realized on the blood bank. Fujirebio INNO-LIA HTLV I/II Score kit manufacturer’s instructions provide information that some infections may interfere with the analysis, as well as immunological changes such as autoimmune conditions.DMS-02 reported during the interview that, despite the referral to schedule an appointment received from HEMOSUL, she was unable to access the health service. She had been trying to schedule for almost a year. Since forwarding had no effect, this gap shows the urgent need to organize a referral system between the blood bank managed by the reference centers since the individual already presents laboratory results, a possible diagnosis, and the clinical condition of the same must be investigated.21. At the time, the child had 2 years old and did not present a seroconversion. The need for an improvement of maternal counseling about breastfeeding was already reported in the state11, demonstrating that infection patterns are not so well understood by health workers22 when they are expected that they should be able to properly provide counseling for positive patients about HTLV care and prevention.Participant DMS-06 was followed up throughout her entire antenatal period, but she related that she was not informed about her positive serology for anti-HTLV-1 during her last pregnancy. Her daughter was breastfed for 8 months, which could lead to mother-to-child transmission of HTLV through breastfeeding22. The default in marital status found in this study may be due to the omission of risk factors, such as possibly multiple partners. Regarding ethnicity, most of them reported as Brazilian caucasian. Other studies that used data from HEMOVIDA bring the information that Brazilian caucasian include brown and mixed race23, and there is no clear difference between them24. Brazilian Institute of Geography and Statistics (IBGE) has 5 options for self-declared race/color: white, black, brown, indigenous, or yellow25. We suggest a standardization following the IBGE pattern applied for the blood donor candidates.Regarding inconsistencies found in the answers given to HEMOSUL and the research group, it may be that some blood donors see the blood banks as a convenient place for free testing27. Then, the blood bags with S/CO < 4 may have had unnecessary disposal. Blood banks have no diagnostic purpose since the main objective is to avoid false negatives, then a high rate of false-positive results is expected in the blood bank screening tests due to low positive predictive value28, this implies an overestimation of the prevalence of HTLV infection if only a screening test is considered. Therefore, the algorithm of the tests influences the prevalence rate. In this study, we considered positive HTLV-confirmed result donors only. During the study period, the HEMOSUL blood bank performed confirmatory tests for HTLV on the positive samples in the screening. However, since May 2022, HEMOSUL has not performed confirmatory tests for HTLV. Furthermore, we must consider the impact of a false-positive result on the quality of life of a person without the confirmatory test to show him that he is uninfected. However, conducting confirmatory tests for HTLV is not mandatory for Brazilian blood bank services29.All individuals whose initial sample was anti-HTLV positive with a S/CO ratio < 11.79 was subsequently found to be HTLV negative. Although the manufacturer’s instructions of Abbott Architect rHTLV I/II assay indicate that samples with S/CO ratio ≥ 1 are considered reactive, previous studies suggest that the S/CO ratio < 4 excludes HTLV infection and the risk of blood transmissionConsidering that our data showed that the HTLV-confirmed samples were only 7.69% (6/78) of the initially positive samples and that there is no specific treatment for HTLV, identifying an infected blood donor candidate represents a great opportunity to block the transmission, consequently, the associated diseases, but this requires adequate counseling and patient management. This fact highlights the importance of retesting positive blood donors and/or submitting to a confirmatory test.We identified some limitations related to the HTLV infection prevalence in blood donors in the review literature. Most studies used initial blood bank screening data, which may result in an overestimation of prevalence due to the low cutoff value of the S/CO ratio. Another limitation of our study was the limited availability of behaviour pieces of information in the HEMOSUL database for each blood donor.In conclusion, our findings provide additional blood donors data for the prevalence of HTLV-1/2 in Brazil and highlight the need for a closer look at HTLV infection results in Brazilian blood banks, proposing confirmatory testing for those seropositive donors in screening assays and the need for adequate counseling and patient management for those confirmed HTLV infected individuals. We recommend implementing confirmatory tests at the blood bank or centralizing them within the public health system. Additionally, there is a need for a systematic approach to referring seropositive individuals to medical care, as access can be challenging.This study was approved by the Ethical Committee on Human Research of the Universidade Federal de Mato Grosso do Sul (CEP/UFMS) by protocol number CAAE: 50,022,721.6.0000.0021. All subjects or their legal guardians, in the case of individuals under age 18, gave their written informed consent to participate in the study at the time of sampling. All research was performed following relevant guidelines and regulations.A cross-sectional study was conducted among blood donors from Mato Grosso do Sul state (MS)—Central Brazil, between January 2021 to December 2021. Firstly, the data was obtained from databases, and samples from the blood collection from Mato Grosso do Sul Centro de Hematologia e Hemoterapia (HEMOSUL), which has 12 units in 10 cities . The inclusion criteria were to be a blood donor in the follow-up year. Then, the screened anti-HTLV seropositive individuals were invited to participate in the study with their family members to confirm the anti-HTLV seropositivity and to invite the family members of the case to participate. Inclusion criteria were: (i) a screening diagnosis for HTLV-1 infection; (ii) being a blood donor´s family member; (iii) having signed the informed consent form.Participants underwent an interview with a standardized questionnaire containing sociodemographic and sexual behavior information and provide blood samples. All subjects gave their written informed consent to participate in the study. In the case of individuals under age 18, informed written consent was obtained from themselves and their legal guardians.i2000SR equipment) for the presence of anti-HTLV-1/2 antibodies. It was considered positive when samples had a cut-off ≥ 1, following the manufacturer’s instructions. The grey zone considering a cut-off ranging from 0.9 to 1.0, is configured in the equipment by HEMOSUL. Positive samples were confirmed by HTLV-1/2 INNO-LIA assay (Fujirebio INNO-LIA HTLV I/II Score).In the follow-up year, the coordinating center of HEMOSUL screened all serum samples using chemiluminescent microparticle immunoassay (CMIA)  commercial kit for the presence of anti-HTLV-1/2 antibodies (Murex HTLV I + II—DiaSorin), following the manufacturer’s instructions. Positive samples were repeatedly tested and confirmed by HTLV-1/2 Western Blot (WB) assay (MP Diagnostics HTLV BLOT 2.4—Singapore). HTLV infection was defined as a repeatedly positive ELISA and positive WB . Categorical variables were presented with absolute and percentage frequency. Continuous variables were expressed as mean, standard deviation, median, and range.