Studies in animals and humans suggest the possibility of harmful health outcomes, particularly for the reproductive system, and in a less extent for the cardiovascular and thyroid functions. Thus, the purpose of this review was to analyse the possible implications of the massive use of triclosan, mainly on the reproductive and cardiovascular systems and on the thyroid function, both in animals and humans.Hygiene is essential to avoid diseases, and this is thanks to daily cleaning and disinfection habits. Currently, there are numerous commercial products containing antimicrobial agents, and although they are efficient in disinfecting, it is still not known the effect of the constant use of these products on human health. In fact, a massive use of disinfectants has been observed due to COVID-19, but the possible adverse effects are not yet known. Triclosan is one of the antimicrobial agents used in cosmetic products, toothpaste, and disinfectants. This compound is an endocrine disruptor, which means it can interfere with hormonal function, with its estrogenic and androgenic activity having already been stated. Even if the use of triclosan is well-regulated, with the maximum allowed concentration in the European Union of 0.3% ( Triclosan (TCS), 5-chloro-2- phenol, is an endocrine-disrupting chemical often used as an antiseptic, disinfectant, or preservative ,4,5,6.An endocrine disruptor is a chemical substance that causes adverse effects in an organism by disrupting its endocrine function . Many arw/w) of triclosan [In the European Union, 85% of triclosan is used in personal care products , and 10% in plastics and food contact materials (household utensils), while the remaining 5% is used in textiles  ,3,4,5,11riclosan . As for riclosan ,14, whilriclosan .Some studies presented by the European Chemicals Agency have concluded that triclosan can interfere with estrogenic (ER) and androgenic (AR) receptors and that it has adverse effects on the thyroid and cardiovascular systems. Specifically, these studies have led to the discovery of the vasorelaxant properties of triclosan, of the effects that this compound may have in miscarriages or malformations of a foetus  and its Currently, the literature is still unclear about the potential effects of TCS on organisms and humans, and the impact on human health of the passive use of these compounds, mainly during the COVID-19 season, is still completely unknown. Therefore, and based on this assumption, the aim of this review was to analyse in more detail the possible implications of the massive use of triclosan, mainly on the reproductive and cardiovascular systems and on the thyroid function in both animals and humans.Triclosan is a white, odourless, tasteless, crystalline powder with a molecular weight of 289.5 g/mol. It is commercially known as Irgasan DP 300 and Irgacare MP for oral applications ,24.ow = 4.8), which suggests that it bioaccumulates in adipose tissue [Structurally, TCS is a molecule with functional groups for phenol ether and it has a lipophilic nature . This inhibition will suppress the membrane phospholipids and the proliferation of microbial cells. TCS has been considered safe for humans and animals since ENR is absent in eukaryotes; however recent studies demonstrate that TCS is considered toxic for eukaryotes ,31. It aThis compound has a half-life of approximately 11 days at the water surface and is degraded in aerobic soil with a half-life of 18 days .TCS can be absorbed through several routes, including the mucous membrane of the oral cavity, dermal exposure, or the gastrointestinal tract. Considering that most products containing TCS are used for dermal application, its main route of absorption is the skin, through which it is rapidly absorbed due to its lipophilic properties  Table 3Table 3. Concentrations in the millimolar range have been detected in humans; therefore, considering the amount of TCS found in human consumer products, all exposure routes are important for the final exposure . In a stRegarding the distribution of this substance, the studies currently available only concern rodents. Kanetoshi et al. were the first to demonstrate the metabolization of this substance in the bile and the liver. The authors demonstrated that within 24 h of TCS administration, the highest levels were present in the bile (33.6 µg TCS/g tissue), followed by the liver which contained about 3.0 µg TCS/g tissue. Concentrations in these two tissues remained high for 24 h after administration when compared to other tissues .In another study performed using rats, this compound was detected in the 15 tissues analysed 12 h after a skin application, with the gallbladder and bladder containing the highest concentrations while the testes, thymus and brain had the lowest concentrations . SimilarRegarding metabolism itself, TCS can be metabolised by three pathways: cytochrome P450 (P450), UDP-glucoronyltransferase (UGT) and sulphotransferase (SULT). The latter two are part of the phase II metabolism of xenobiotics ,43.The main metabolite of TCS hydroxylation is triclosan monohydroxylate. This in turn can be cleaved to 2,4-dichlorophenol and 4-chlorocatechol. A fourth metabolite can also be formed from the hydroxylation of 2,4-dichlorophenol, forming 3,5-dichlorocatechol  Figure .The fact that there are two pathways of the phase II metabolism is not influenced by the route of administration; however, the ratio between the transformation of TCS by the two pathways is already influenced by the species ,45.Glucuronidation and the sulphation of TCS causes the addition of glucuronic acid and sulphate, respectively, to the hydroxyl group, thus destroying the proton translocating nature and adding a charged/polar group to the TCS. This in turn will enhance its hydrophilic properties and decrease TCS’s ability to accumulate in adipose tissue .The liver is the main site of TCS metabolism; however, it also occurs at minimal levels in the skin. It is primarily metabolized through the SULT pathway, yet 24 h after administration, both phase II metabolites are found. Despite undergoing metabolization, the unmetabolized form of TCS remains the predominant form .In the liver, the production of metabolites depends on the dose of TCS. For exposures of 1–5 µM of TCS for 30 min, there was an equal production of TCS-sulphate and TCS-glucuronyl, and for exposures greater than 20 µM the glucuronidation predominated while for doses below 1 µM it led to sulphation .In a 2014 study, 46 volunteers, namely, 26 men and 20 women aged between 4 and 80 years, provided urine samples which were tested and both TCS-sulphate and TCS-glucuronyl were detected. The main metabolite identified was TCS-glucuronyl .Regarding the elimination of TCS, the main route of excretion is through urine (57–87% of an administered dose) and faeces is the secondary route (5–33% of an administered dose). This compound is excreted mainly in its metabolised forms: TCS-glucuronic and TCS-sulphated; thus, during the first 4 days after an exposure, between 24% and 83% of the TCS consumed is excreted, and only after 8 days does it reach basal excretion levels ,48.Triclosan has already been detected in several matrices, such as in urine, blood, serum, and breast milk . For thiTCS has been widely studied in animals at the level of the reproductive system. It is also known that in this system, the effects are mainly due to the modulation of androgenic and estrogenic receptors.Oryzias latipes fish. At concentrations of 625, 1250 and 2500 µg/L, no embryos were born, and they found that at 313 µg/L the incubation time increased significantly; however, fertility was not affected during the study [In a study conducted by Ishibashi et al., several concentrations of triclosan were tested on embryos and adults of the he study .Danio rerio) to test the toxic effects of three concentrations of TCS  on the liver. The growth of the zebrafish showed no significant changes; however, there was an increase of 11.67–19.16% in the zebrafish weights and of 21.53–47.42% in the liver weights at exposures of 0.16 and 0.25 mg/L. At these concentrations, the superoxide dismutase (SOD) activity decreased while the malondialdehyde (MAD) levels gradually increased with different TCS concentrations. Thus, the authors demonstrated that exposure to TCS causes oxidative stress in the liver depending on the exposure time and concentration. This exposure created several hepatocellular changes, an increased hepatic plaque gap, necrosis, and atrophy, with the last two being concentration dependent. The same authors also observed a TCS concentration-dependent apoptosis and they quantified the expression of Bcl-2 and Bax to better understand which pathway of apoptosis was promoted by TCS. The expression of Bcl-2 at concentrations of 0.16 and 0.25 mg/L was significantly decreased while Bax was increased at concentrations of 0.08 and 0.25 mg/L. This relationship between Bax and Bcl-2 led the authors to hypothesise that hepatocyte apoptosis is directly related to the exposure of zebrafish to TCS [Liu et al. used zebrafish  levels increased in the 20 and 200 µg/L of TCS groups. On the other hand, in females, there was only a decrease in the VTG levels at 20 and 200 µg/L of TCS. These results indicate that TCS has an estrogenic influence in zebrafish, with males being more sensitive than females. There was also a significant decrease in mature spermatozoa and destruction of the testis structure at the concentration of 200 µg/L of TCS, and in females, an increase in the number of immature oocytes was observed at the same concentration .Another model widely used for in vivo studies are rats. Kumar et al. observed that the administration of 5 mg/kg/day to male Wistar rats did not induce a significant change in the testes weight, but higher doses of TCS induced a significant change in the testes and sexual tissues. The weight of the testes, epididymis, ventral prostate, vas deferens and seminal vesicles decreased by 20–50% with 10 mg/kg/day and 35–49% with 20 mg/kg/day. The authors also quantified several hormones and observed a decrease of 38.5% in the luteinizing hormone (LH), 17% in the follicle stimulating hormone (FSH), 35% in cholesterol, 31% in pregnenolone and 41% in testosterone after a dose of 20 mg/kg/day .Regarding the study of gender differences in reproduction, in the following year, 2010, Stoker et al. performed a daily administration of TCS  to female Wistar rats, between the Postnatal day (PND) 22 and the PND 42 , to examine the effects of TCS on female pubertal development. They analysed if the time until the opening of the vaginal canal was affected, as well as the histology of the uterus and ovaries, and some hormones including E2 and LH. The authors observed that this compound altered the reproductive development and response to exogenous oestrogens. They also observed a lower age for the opening of the vaginal canal with the administration of 150 mg/kg, which was an estrogenic response of the triclosan since stimulation through oestrogens is required for the canal to open. The serum oestradiol concentrations at PND 42 were decreased followed by administration of TCS at 37.5 mg/kg and 150 mg/kg, while the LH levels did not show significant changes. Thus, the study suggested that TCS increases oestrogen activity with a potential to alter the oestrogen-dependent functions. In summary, the authors concluded that TCS affects the reproductive development and uterine responses to exogenous oestrogens in developing females .Two years later, in 2012, Jung et al. analysed the estrogenic activity of TCS using well-established models, both in vivo and in vitro. For the in vivo approach, female Sprague-Dawley rats were treated with 17α-ethinyl-oestradiol (1 mg/kg) and TCS , between PND 19 and 21. The TCS increased the uterine weight and mRNA expression for complement 3, which is an oestrogen-sensitive gene in the uterus, in immature mice after a treatment with TCS at 37.5 mg/kg and 187.5 mg/kg. This shows that TCS has estrogenic activity. In vitro, the estrogenic activity upon TCS exposure was analysed on the expression of calbindin-D9k (CaBP-9k), which is a biomarker for the detection of endocrine disruptors in GH3 cells . The expression of CaBP-9k was increased in the presence of TCS, which demonstrates the endocrine disrupting nature of TCS . In a siMus musculus rats’ gestation, Crawford et al. compared several doses of TCS  with BPA. Females receiving the TCS dose on gestational days 0 and 1 showed no significant differences in the implantation sites when compared with the control. On gestational day 2, significant differences were already observed for the 785 mg/kg triclosan dose and on day 3 for the 523 mg/kg triclosan dose. When exposed to 18 and 785 mg/kg of triclosan from gestational days 1–3, the number of implantation sites decreased significantly on gestational day 6. The number of implantation sites was also reduced after an injection of these two doses on gestational day 3 and only of the higher dose on gestational day 2 [Regarding the effects of TCS in al day 2 . Thus, tIn a different study, 3-month-old rats were administered with 1, 10 and 100 mg TCS/kg/day from gestational day 6. The TCS levels were measured up to gestational days 11 and 16. An exposure to TCS demonstrated a dose-dependency in increasing foetal death. The incidence of spontaneous abortion at doses 10 and 100 reached 60% and 80%, respectively. The level of oestrogen sulphotransferase protein (EST) was decreased at gestational day 16 at the highest dose, as were its plasma and placental activities. These abortions were most likely caused by the inhibition of EST activity leading to placental thrombosis and degeneration . Thus, tOne year later, in 2016, Feng et al. recorded the total and uterine weights of female mice throughout gestation after an administration of TCS , and noted a lower weight gain in the groups where 300 and 600 mg/kg of TCS was administered when compared to the control; however, significant differences were only found regarding the uterine weight in the 600 mg/kg group. The tissues presenting the highest concentration of TCS were the placenta, liver, and kidneys with 12.83, 9.52 and 8.74 µg/g, respectively. In the placenta, the mean TCS levels following 0, 30, 100 and 300 mg/kg of TCS administrations were 0.033, 13.05, 28.23 and 55.83 µg/g, respectively. The LH and FSH levels were identical to the control group, whereas the levels of human chorionic gonadotropin (hCG), prolactin, progesterone, oestradiol, and testosterone were significantly reduced in all the exposure groups when compared to the control. Thus, the placenta may be a target of TCS in rats during the gestation period .In the same way, a study performed by Montagnini et al. tested the effect of low doses of TCS  over the course of three generations of Wistar rats named F0, F1 (the offspring of F0) and F2 (the offspring of F1). The TCS was orally administered to the F0 generation only between PND 49 and 120 in females, while in males it continued until PND 140. Thus, weight changes, food consumption during the pre-mating period, sexual behaviour assessment, male organs of the F1 generation, plasma testosterone quantification, all sperm parameters, testicular histomorphometry, sexual and physical development of the F1 and F2 generations and neuronal behavioural tests were accounted for. They concluded that these concentrations of TCS had no significant effect other than a reduction in the viability and motility of spermatozoa from the F1 generation being administered with 2.4 mg/Kg at PND 140. Doses administered between PND 49 and PND 140 did not affect the sperm parameters; therefore, the authors concluded that TCS could have an impact on gametogenesis during the foetal period .More recently, Raj et al. analysed the effects of accumulated TCS on the histopathology and secretory functions of the epididymis, seminal vesicle, and sperm indices in adult Swiss-strain rats. Four different doses of triclosan  were administered for 42 consecutive days. All the concentrations significantly decreased the epididymal and seminal vesicle weights and led to a decrease in the sperm percentage and viability, and sperm count; furthermore, the percentage of non-viable spermatozoa increased. The histology of the epididymis was marked with changes throughout its length with disorganization and the appearance of vacuoles. The final chromatographic study showed an accumulation of TCS in the epididymis and seminal vesicle. Thus, Raj et al. concluded that TCS caused alterations in the epididymis and seminal vesicle, as well as in the sperm indices, epididymal sialic acid levels and fructose levels of the seminal vesicle .Concerning the effect of this compound on the placenta, in 2010, TCS was tested on the placental tissue from ewes with gestation periods ranging between 126–130 days. In this study it was demonstrated that the TCS was a potent inhibitor of placental EST activity. This inhibition was analysed, and the authors observed that it occurred mainly competitively; therefore, it can be assumed that TCS occupies the substrate binding sites. The role of placental EST is not yet fully known; however, alterations in its expression are thought to be associated with spontaneous abortions and foetal loss , as prevIn summary, TCS has a variety of reproductive effects in fish, rats and even sheep. The decreased fertility beyond the induction of abortion was the most observed consequence in any of the models. This may be due to the way the substance acts on the receptors or on oestradiol concentrations, as observed by Wang et al., where they demonstrated an inhibition of EST activity which caused a thrombosis in the placenta. Furthermore, TCS also appears to affect the serum concentration of several hormones such as LH, FSH, hCG, pregnenolone and cholesterol, which can lead to reproductive problems such as foetal loss and decreased fertility in subsequent generations.In humans, TCS has not been widely studied yet, with most of the existing studies being related to the hormonal levels of EST, FSH and the effects at the foetal-placental level, namely, spontaneous abortions.The first authors to demonstrate that TCS could be associated with adverse reproductive effects were Wang et al., finding an association between urinary TCS concentration levels and increased spontaneous abortions. These authors recruited a population of 452 women at 14 and 24 weeks of pregnancy with mean ages of 28 and 27 years, respectively. Their E2 levels at low-TCS concentrations were lower than the control; however, at high-TCS levels there were no changes. The plasma EST levels were reduced at both low- and high-TCS concentrations, while the EST activity at high-TCS concentrations was lower compared to the control and the low-TCS concentrations . This inMiscarriages may be linked to an inhibition of the placental 11β-HSD2 enzyme. In this sense, Zhang et al. observed that triclosan altered the expression of 11β-HSD2 in human syncytiotrophoblasts and examined the apoptotic mechanism induced by TCS. At concentrations of 0.1 µM and higher of TCS, the mRNA levels decreased significantly, and at the protein level there was a significant inhibition over a similar range of concentrations. There was also a decrease in the viability of syncytiotrophoblasts in a concentration dependent manner (0.001–10 µM), with statistical significance at concentrations above 0.1 µM. Moreover, the authors observed that triclosan induced the apoptosis of syncytiotrophoblasts and that the inhibition of 11β-HSD2 was explained by the induction of apoptosis .In 2017, an epidemiological study also demonstrated the association between the urinary TCS levels of 401 pregnant women (at 16 weeks of gestation) and birth weight, length, head circumference and length of gestation. Urine was collected at 16 and 27 weeks of gestation, and TCS was detected in 91% and 83% of the samples, respectively, with a mean concentration of 16 ng/mL. This study showed a positive association between the urine triclosan concentration and a moderate reduction in birth weight, length, head circumference and gestational length .Two years later, Jurewicz et al. analysed the association between triclosan concentration and ovarian reserve, which is a marker of female fertility. They counted the antral follicles and quantified the anti-müllerian hormone, follicle stimulating hormone and oestradiol. The authors recruited 511 menstruating women (25–39 years old) that provided blood and urine samples. The blood samples were taken at the beginning of the menstrual cycle, approximately between the second and fourth day, so that the hormones could be counted, while the antral follicle counts were performed in both ovaries during the beginning of the follicular phase of the menstrual cycle. Urine TCS concentrations (0.3–1677.68 ng/mL) were shown to be negatively associated with the antral follicle count, while the anti-müllerian hormone, follicle stimulating hormone and oestradiol did not show a statistic relevance. Thus, the authors concluded that TCS may be associated with decreased female fertility, although further studies are needed to demonstrate the mechanism involved in this effect .The following year, Yuan et al. compared the levels of TCS present in urine with the sperm quality in a Chinese population, between 2018 and 2019. From the 406 subjects (aged between 21 and 56), 57.1% had never smoked and 83.5% had never consumed alcoholic beverages. The parameters analysed included the sperm volume, concentration, count, total, progressive and non-progressive motility, immobility, percentage of hyperactivated sperm, mean velocity, curvilinear velocity, straight line velocity, lateral head deviation amplitude, linearity, sway, beat cross frequency and straightness. TCS was detected in 74.6% of the samples with a mean of 1.7 µg/L. None of the parameters studied were associated with TCS exposure, although a higher TCS exposure showed higher motility parameters indicating a potential non-linear influence of TCS on sperm quality [In summary, the effects caused by TCS in humans are very similar to those observed in animal models. Spontaneous abortion caused by chronic exposure to TCS is the most observed consequence, although the specific pathway by which it occurs is not yet known. Furthermore, sperm motility is also disrupted by TCS exposure. Thus, we can conclude that fecundity/fertility ends up being the most affected by exposure to TCS due to its influence on both sperm and eggs, which may be a cause of the increased infertility present in developed countries.Regarding the cardiovascular system, the effect of TCS has not yet been studied as much as the reproductive system; however, some effects on the heart rate and cardiovascular morphology are already known.In a study conducted by Saley et al., zebrafish were exposed to 1mL of 0, 0.4, 40 or 400 µg/L doses, between 8 and 120 h post fertilisation (hpf), and the dose was changed every 24 h. Doses below 40 µg/L showed minimal changes while a 400 µg/L dose resulted in a dilated auricle, smaller ventricles and unlooping of the heart. The same concentrations were used to assess the cardiac function and structure. The heart rate was reduced by 30% and the beat volume by 40%, which resulted in an output decrease of 60% at the highest dose. At the highest dose, the diastolic end volume was reduced by 17% whilst the systolic volume was unaffected, with 80% of the embryos showing regurgitation . Thus, tOnce more in zebrafish, Wang et al. studied the influence of TCS and its derivatives, 2,4,6-trichlorocatechol and 2,4-dichlorophenol, in cardiotoxicity. The authors used the three compounds in a mixture named TCS-DT at 0.28, 0.56 and 0.84 mg/L, during 6 to 120 hpf in larvae, and they also used 0.14, 0.28 and 0.56 mg/L between 30 and 90 days after fertilisation in adult zebrafish. At 48 hpf, the heart rates of the larvae were significantly reduced in the 0.56 and 0.84 mg/L treatments and the ejection fraction showed a concentration-dependent decrease. At 120 hpf, the looping distance from the atrium to the ventricle increased significantly with the concentration; thus, the TCS-DT exposure affected the cardiac looping and led to abnormal ventricles influencing the cardiac function. Moreover, the creatinine kinase MB and triglyceride levels in the adult hearts increased at all concentrations, the SOD levels increased at 0.14 and 0.28 mg/L, but decreased at 0.56 mg/L, while the lactate dehydrogenase (LDH) levels increased at 0.14 mg/L and decreased with the remaining concentrations. The authors concluded that changes in these activities led to damage in the cardiomyocytes, suggestive of myocarditis. Overall, this demonstrates that exposure to TCS-DT affects normal cardiovascular growth and development in zebrafish .Ma et al. studied the relationship between a TCS-induced abnormal expression of miR-181a-5p and vascular development. The study began by analysing which miRNAs expressions were affected by TCS exposure, which was miR-181a-5p. Zebrafish embryos were treated with 62.5, 125 and 250 µg/L of TCS between 4 and 120 hpf. At the lowest concentration, there was only a small impact on the vascular development and distribution; however, at the other concentrations, a gradual decrease in the blood flow was noted. For 250 µg/L of TCS, the blood flow was mostly confined in the head; however, there was no distribution of the blood vessels or blood flow in the forebrain and diencephalon. The distribution of red blood cells decreased by 60% in the group treated with 250 µg/L. Additionally, exposure to TCS led to an increase in the miR-181a-5p expression. When miR-181a-5p inhibitors were injected, the number of blood cells increased, while an injection of an analogue of miR-181a-5p decreased the number of blood cells. Thus, the authors concluded that the increased expression of miR-181a-5p was due to exposure to TCS, causing atrophy of the blood vessel development in zebrafish .Cherednichenko et al. analysed the effect of TCS on ryanodine 1 and 2 receptors in rats. After an administration of 6.25, 12.5, or 25 mg/kg of TCS, the authors observed several cardiovascular deficits in the rats including a reduced cardiac output, reduced diastolic volume in the left ventricle, and a reduced maximum time derived from the left ventricular pressure development. The highest dose group exhibited a 25.3 ± 15.7% decrease of the cardiac output, implying that TCS had severe cardiovascular effects. There was an 18% decrease of the exerted force which was significantly lower than the vehicle and placebo groups after a TCS dose of 40 mg/kg. These deficits were reversible as the rats regained full strength 24 h after the TCS administration .More recently, the effect of TCS on vascular contractility was evaluated using the aorta and mesenteric arteries of Sprague-Dawley rats. In mesenteric arteries contracted with phenylephrine, triclosan induced a concentration-dependent relaxation for arteries with and without endothelium. Similarly, when vasoconstriction was induced by high concentrations of potassium, a treatment with triclosan induced relaxation in the mesenteric arteries with and without endothelium. It was concluded that the mesenteric artery relaxation induced by the triclosan was independent of the endothelium. Moreover, when examining the arteries incubated with triclosan 20 min before a vasoconstriction with phenylephrine and KPSS , the authors determined that triclosan significantly inhibited the contraction. In the aorta, similar results were observed as in the mesenteric arteries; however, a higher concentration of triclosan was needed for the relaxation to occur .In summary, it can be stated that TCS affects the cardiovascular system in fish and rats. The effects range from vascular relaxation to an alteration of the cardiac morphology and heart rate. Furthermore, these studies also show that not all changes caused by TCS are irreversible, as presented by Cherednichenko et al., as 24 h after TCS administration, the rats recovered from some of the deficits presented. However, the molecular mechanisms underlying these cardiovascular changes remain unclear.Regarding the effects of TCS in humans at the cardiovascular level, the literature is still very scarce, with only two epidemiological studies and two in vitro studies with human cells.m/m) was analysed over a 5 year period in 438 patients aged up to 75 years with cardiovascular diseases . The authors observed that the prolonged use of toothpaste did not lead to an increase in adverse events or secondary cardiovascular events. Only a small decrease in time to the first serious adverse event was noted between men in the triclosan group and the placebo group [The first study to demonstrate this association between cardiovascular diseases and TCS was performed by Cullinan et al. . In thisbo group .m/m) for 5 years on the inflammatory biomarkers of cardiovascular diseases. These biomarkers included the total cholesterol, C-reactive protein, low density lipoprotein and endothelial dysfunction. The authors observed no differences between the placebo group and the TCS group, with only slight decreases or increases in the serum levels of each of the biomarkers, except for low density lipoprotein, which decreased in the TCS group compared to the placebo group. Thus, the authors concluded that the use of toothpaste with 0.3% TCS has a minimal influence on systemic markers in patients with cardiovascular disease, showing no clinical significance in terms of reducing the risk of cardiovascular disease [Resorting to the same population sample, the same research group demonstrated the effects of using toothpaste with TCS  were used, as they possess the ability to differentiate into cardiomyocytes (CMs). In this study, exposure to TCS (1 µM) inhibited the differentiation of hESCs into cardiomyocytes and their spontaneous beating. This inhibition occurred through the interference of triclosan with the gene expression  and with excessive methylations in the DNA .A study conducted by Zhang et al., resorting to the use of HUVECs , was also performed. In the first group, the cells were treated with several doses of TCS for 24 h , and in the second group the cells were first treated with SC79 (PI3K/Akt activator) or MHY1485 (mTOR activator) and then treated with TCS at 50 µM for 24 h. After treatment with the activators and TCS for 24 h, the authors demonstrated that these activators were not affected by the dysfunction caused in HUVECs. In order to verify if TCS induced apoptosis in the HUVECs, a LDH test was performed demonstrating an increase in the membrane permeability, and in the caspases 3/7 activity. The levels of BAX and Bcl-2 were also calculated, where the Bcl-2 and BAX levels decreased and increased, respectively. These data demonstrate that TCS does indeed promote endothelial cell apoptosis. Then, the authors also analysed the mechanism by which TCS induced reactive oxygen species production in HUVECs and the results showed that the phosphorylation of PI3k, Akt, mTORC1 and mTORC2 was suppressed. The final test analysed the migration ability of HUVECs after exposure to TCS by wounding the culture at confluence, showing that TCS significantly reduced the regeneration ability compared to the control .m/m)) does not appear to have significant effects when ingested; however, in in vitro studies that have already been conducted, this compound at much higher concentrations, was found to affect the cell viability, to induce apoptosis and to decrease considerably the capacity for cell regeneration. Thus, we can conclude that studies in humans are still too scarce to infer an effect of this compound at the cardiovascular level, despite epidemiological studies pointing in that direction; therefore, it is of paramount importance to see if this compound bioaccumulates and if the doses allowed by law are really safe for human health.In short, TCS at the maximum concentrations currently allowed  aged 21–23 days to 0, 10, 30, 100, 300 and 1000 mg/kg/day of TCS for 4 days to test the hypothesis that exposure to TCS altered T4 (thyroxine) levels. The authors found that the serum T4 concentration decreased by 28, 34 and 53% in the 100, 300 and 1000 mg/kg/day triclosan treatments, respectively. The study demonstrated that triclosan decreased the circulating T4 concentration in female rats .In another study of triclosan toxicity was tested in two different ways. First, Wistar female rats were exposed to TCS  from GD7 to PND16, and second, the offspring of unexposed females were exposed to TCS  from PND 3. The T4 levels were analysed in all four groups  at gestational day 15 (8 days after the beginning of exposure), showing a decrease by 59%, 72% and 72%, respectively, for the three concentrations of TCS, and again at postnatal day 16 (the last day of exposure), where the levels had decreased by 38%, 55% and 58%, respectively, in the females, while in the offspring no significant changes were observed. The authors concluded that TCS can decrease maternal T4 levels during gestation and lactation .Resorting to male Wistar rats, Taha et al. tested the effects of compounds with an oestrogen-like structure, such as TCS, on the thyroid function. The animals were treated with two concentrations of TCS, namely, 10 and 50 mg/kg, for 60 days. The levels of the thyroid hormones, FT3 (free-triiodothyronine), FT4 (free-thyroxine), TSH (thyroid stimulating hormone), T3 (triiodothyronine) and T4, were quantified by the ELISA method. The concentration of 10 mg/kg showed no significant effect for any of the thyroid hormones; however, the concentration of 50 mg/kg led to symptoms of hypothyroidism due to changes in the T3, T4 and TSH levels .Zhang et al. used Sprague-Dawley rats to better understand the mechanism by which triclosan interfered with thyroid homeostasis. After 31 days of treatment with TCS, thyroid hormones were quantified in plasma, and the results showed a decrease in all hormones inversely proportional to the concentration of the TCS administered. At the highest concentration (200 mg/kg/d), TT4 , FT4, TT3  and FT3 were decreased by 23.1%, 20.5%, 22.0% and 19.1%, respectively. Only the TSH and TRH (thyrotropin releasing hormone) levels were unaffected. The authors also observed histological changes in the thyroid, dependent of the concentration of TCS used. Subsequently, they analysed by which pathway the TCS affected the thyroid and concluded that besides activating the JNK  and p38 pathways in vivo and inducing the p38 pathway from ROS in vitro, there was also an induction of the TRHr (thyrotropin releasing hormone receptor) expression through the p38 pathway, which contributed to the decrease in TPO (thyroid peroxidase). Thus, with this study it was concluded that TCS affected the thyroid homeostasis and histology through the p38/TRHr pathway mediated by TPO .Cyprinodon variegatus larvae. The total thyroid hormone concentrations were measured at 9, 12, 15 and 18 days post-hatching (dph). The results showed that the TT4 levels varied significantly in all conditions while the control had a gradual decrease. The T4 concentrations increased up to 15 dph, which was not observed for the T3 levels or the T3:T4 ratio. The peak of TT3 observed at the 20 µg/L of TCS concentration occurred 2 days after the control, being indicative of the metamorphosis climax in Cyprinodon variegatus [In a different approach, Schnitzler et al. tested whether TCS  affected the ontogenetic variation of thyroid hormones in developing riegatus .In summary, TCS decreases the serum levels of T4, T3 and TSH in rats, affecting the thyroid homeostasis and histology and leading to symptoms of hypothyroidism. The same was not found in larvae where the T4 levels increased and there were no significant changes in the other hormones; thus, further studies are needed in this area as the effects of TCS on the thyroid function in animals are not yet fully defined.As for the TCS effects on human thyroid function, the few studies performed so far seem to show no adverse effects.m/m) for 4 years was analysed and the thyroid effects were observed. All 132 patients, aged up to 75 years old, had a history of hospital admission for myocardial infarction, unstable angina, and abnormal electrocardiogram. The authors measured the TSH, FT4, FT3, anti-TGb and anti-thyroid peroxidase antibody levels; however, no significant association was observed [In 2012, the use of toothpastes containing triclosan  study 2003–2006, Braun et al. analysed the relationship between maternal (202 women), neonatal (274 infants) and childhood (153 children) urinary TCS concentration and the corresponding serum thyroid hormone levels. The concentration of TCS in urine at 16 weeks of gestation decreased slightly until birth; however, the opposite occurred in children aged between 1–3 years. At 16 weeks’ gestation, there was no significant association between the TCS levels and thyroid hormone concentration. At the time of delivery, there was an inverse relationship between TCS and the TT4 and FT3 levels; however, due to the size of the population that was used during the study, the authors could not state that an exposure to TCS would affect the TT4 and FT3 levels .Koeppe et al., using NHANES  data from 2007–2008, counted the serum levels of both free and total T3 and T4, thyroglobulin and TSH in 352 adolescents (aged 12–19 years) and 1479 adults (aged 20 years and older), and compared them with TCS concentrations measured in the urine samples collected. A positive and significant relationship between TCS and the T3 concentrations was observed only in the adolescents .Berger et al. analysed the relationship between the presence of different chemicals, including TCS, with the serum levels of thyroid hormones in 338 pregnant women (aged 18–45 years) and 364 new-borns. The levels of TT4, FT4 and TSH were measured in the pregnant women while in the new-borns, only the TSH was counted. The results showed an inverse relationship between the urinary TCS concentration and the serum TT4 levels; however, no relationship was found between the urinary TCS concentrations and the TSH levels in the new-borns. It was, therefore, concluded that exposure to TCS may influence thyroid hormone levels during pregnancy .More recently in 2019, the TSH, FT4, TT4, FT3, TT3, thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TgAb) levels were measured in pregnant women at week 10 of gestation. There was no association between triclosan with the absolute TSH, FT4 or FT3 concentrations. There was no significant association between TCS and thyroid function throughout pregnancy. The association of TCS with the TSH or FT4 also had no changes with a variation of TPOAb. The association of FT4 with the TSH did not change with the various concentrations of TCS . Thus, tIn a study conducted by the Korean National Environmental Health Survey (KoNEHS), the relationship between TCS exposure and thyroid hormones  was tested. The study population consisted of 2630 men and 3360 women, who provided blood and urine samples. There was a positive association between TCS exposure and the serum TSH levels in the women; however, in the men, no significant changes were observed. As for the serum levels of TT3 and TT4, no changes were noted in either the women or men. These results lead to postulating that TCS may interfere with thyroid function; however, more studies are needed to complement the limitations of this study .In a similar study, using data from the environment and reproductive (EARTH) study, Skarha et al. analysed the interaction between urinary TCS levels with the serum levels of thyroid function biomarkers  in 317 women. They reached the same conclusions as Koeppe et al., with the observed urinary TCS concentrations having had no significant association with the biomarkers of the thyroid function .In summary, most of the studies reviewed did not observe any changes in the thyroid function upon exposure to TCS, with only one study showing an inverse association between the TCS concentration and T4 levels; thus, considering that the lack of associations may be related to a low daily exposure to TCS, more studies are needed to determine the effects of TCS, since the studies performed so far are contradictory.From all the studies presented in this review, we can conclude that in animals, TCS has clear effects; however, the same is not verified in humans, which may be due to a greater complexity, a lower TCS exposure or simply due to the difficulty in conducting in vivo studies.Regarding the effects on the reproductive system, TCS similarly affects both animals and humans. In animals, TCS causes morphological changes in the male rat sexual organs, an increased fertilization time in fish and gestation time in rats and causes spontaneous abortions in sheep by altering the oestrogen sulphotransferase activity. While in humans, of the most common effects, alterations of the sex hormone levels stand out, and also the incidence of spontaneous abortion due to the alteration of oestrogen sulphotransferase levels as described in animals. It is also possible to observe that TCS affects both the ovarian reserve and sperm morphology and motility.In the cardiovascular studies, as far as animals were concerned, TCS caused cardiac deficits in fish and mice; however, these were reversible in mice after 24 h. It also induced the relaxation of rat arteries and a long exposure to TCS inhibited their contraction. In humans, more specific conclusions were only drawn in vitro, in which TCS inhibited the differentiation of hESCs and had the ability to induce apoptosis in HUVECs.Finally, from the studies analysing the thyroid function, various concentrations of TCS were tested in animals, leading to the conclusion that TCS decreased the circulating levels of T4, T3 and TSH and altered the morphology of the thyroid gland; however, in humans, the effects were inconclusive, since the studies analysed had a very small population sample with contradictory results, which reveals the need for further studies relating the TCS effects for the thyroid gland.Thus, we can conclude that triclosan continues to be poorly studied, especially at the cardiovascular and thyroid levels. In this sense, it is essential to carry out more studies in vitro, ex vivo and in vivo on the effects of TCS on reproduction, especially on male and female fertility, on cardiovascular effects and on the relationship of thyroid hormone levels with physiological concentrations of TCS. Even at a low percentage, humans are still exposed to this endocrine disruptor on a daily basis; therefore, more epidemiological studies with larger population samples, and in different age groups and different countries are needed to really understand what effects TCS has on human health.In summary, we will only know the true danger, or lack thereof, after this endocrine disruptor is further studied, especially in humans, as it is still a highly used chemical in cosmetic products and disinfectants in the European Union.


Deficits in speech understanding constitute one of the most severe consequences of hearing loss. Here we investigate the clinical and genetic risk factors for symmetric deterioration of speech recognition thresholds (SRT) among cancer survivors treated with cisplatin.SRT was measured using spondaic words and calculating the mean of measurements for both ears with symmetric SRT values. For clinical associations, SRT‐based hearing disability (SHD) was defined as SRT≥15 dB hearing loss and clinical variables were derived from the study dataset. Genotyped blood samples were used for GWAS with rank‐based inverse normal transformed SRT values as the response variable. Age was used as a covariate in association analyses.p = 0.004). Current smoking (p = 0.002), years of smoking (p = 0.02), BMI (p < 0.001), and peripheral motor neuropathy (p = 0.003) were positively associated with SHD, while physical activity was inversely associated with SHD (p = 0.005). In contrast, cumulative cisplatin dose, peripheral sensory neuropathy, hypertension, and hypercholesterolemia were not associated with SHD. Although no genetic variants had an association p value < 5 × 10−8, 22 genetic variants were suggestively associated (p < 10−5) with SRT deterioration. Three of the top variants in 10 respective linkage disequilibrium regions were either positioned within the coding sequence or were eQTLs for genes involved in neuronal development .SHD was inversely associated with self‐reported health (Current results improve our understanding of risk factors for SRT deterioration in cancer survivors. Higher BMI, lower physical activity, and smoking are associated with SHD. Larger samples would allow for expansion of the current findings on the genetic architecture of SRT. We investigated the risk factors associated with decline in understanding speech as measured by speech recognition threshold among testicular cancer survivors. Associated factors were higher BMI, lower physical activity and smoking, and peripheral motor neuropathy alongside genetic variants with suggestive associations including those related to three genes involved in neuronal development. Chemotherapy consisted of cisplatin‐based regimens; survivors did not have prior chemotherapy for other cancers and completed chemotherapy more than a year prior to the enrollment. Enrolled survivors completed comprehensive health questionnaires, underwent brief physical examination and were audiometrically evaluated. The Platinum Study involves eight medical centers in the United States, Canada, and United Kingdom.2.2Hearing was evaluated by a licensed audiologist conducted in a clinical setting, inside a sound‐treated booth using calibrated equipment. Pure‐tone and speech audiometry was obtained for each ear following professional standards.Figure For each subject, SRT values for left and right ears were compared Figure  and subj2.32 with body surface area (m2). When not available, body surface area or chemotherapy cycle details were imputed to meet the expected standard cisplatin cycle dosage (100 mg/m2 per cycle).To obtain the dose of administered cisplatin, chemotherapy treatment regimens and/or individual chemotherapy cycles were abstracted from the subjects' medical records. Cumulative cisplatin dosages were calculated for standard treatment regimens or from individual chemotherapy cycles for nonstandard treatment regimens. If necessary and depending on data availability, dosages were converted to mg/mFor clinical associations besides age and dose of cisplatin, subjects were excluded from the analysis if evaluations and audiometry were done more than 1.2 years (438 days) apart. Age was calculated as the difference in days between date of birth and date of data collection, divided by 365 and rounded to the nearest integer. The association of age with hearing loss is well documented.Distribution and preparation of clinical variables used for clinical association analyses of “self‐reported hearing loss,” “problem hearing in crowds,” tinnitus, peripheral motor neuropathy, peripheral sensory neuropathy, heavy drinking, ever smoking, current smoking, hypertension and hypercholesterolemia are outlined in Figures https://CRAN.R‐project.org/package=MASS; RRID:SCR_019125) was used for association of SRT‐based hearing disability with self‐reported health. Logistic regression was used for other clinical association analyses with SRT‐based hearing disability. For the number of subjects in clinical associations refer to Figure For clinical association analyses, calculated SRTs were transformed to binary with values equal to or greater than 15 dB hearing level placed in the hearing disability group. Ordinal logistic regression from MASS package2.4During the clinical evaluation, peripheral blood samples were collected from subjects and were used for DNA extraction. Genotyping was performed by Regeneron Pharmaceuticals using Infinium Global Screening Array v1.0 (Illumina Inc.).ftp://ftp‐trace.ncbi.nih.gov/1000genomes/ftp/release/20100804/ALL.2of4intersection.20100804.genotypes.vcf.gz) to select for subjects with European ancestry. Variants with minor allele frequency (MAF) lower than 0.05 were filtered out  were excluded. Subjects that deviated from the mean heterozygosity rate by more than three standard deviations were excluded. For filtration based on related genotypes, pi‐hat threshold of 0.0625 was used to select related subjects. Related pairs were determined based on mean identity‐by‐descent  and in each related pair, the subject with higher missing call rate was excluded . bgzip from HTSlibhttps://www.htslib.org, RRID:SCR_002105) and 7‐Zip  were used for compression and decompression of genotype data prior and post imputation. Imputed genotypes were filtered to keep variants with an imputation R2 above 0.8 and to remove variants with MAF below 0.05 or with deviation from HWE (p < 10−6)  Figure .2.5https://CRAN.R‐project.org/package=GenABEL, RRID:SCR_001842). The transformed SRT data were used for linear regression‐based genome‐wide association analysis. Inclusion of the 10 genetic principal components is a common practice to control for population stratification.www.cog‐genomics.org/plink/1.9/, RRID:SCR_021271) which includes implementations of earlier methodhttps://www.ncbi.nlm.nih.gov/snp/) was used to check the details of target variants. LocusZoom webtool,https://my.locuszoom.org, RRID:SCR_021374) was used to generate regional Manhattan plots with LD population set to European. GTEx portal  was used to check if identified SNPs are known expression quantitative trait loci (eQTLs) or splicing quantitative trait loci (sQTLs).To prepare the SRT data for subjects with matching quality‐controlled genotype, the arithmetic mean of SRT measurements for both ears was subjected to rank‐based inverse normal transformation Figure  using Ge2.6https://www.gnu.org/software/bash/, SCR_021268), R , and RStudio  provided the analysis environment. Packages dplyr , tidyrhttps://CRAN.R‐project.org/package=tidyr, RRID:SCR_017102), purrr , sas7bdat (https://CRAN.R‐project.org/package=sas7bdat) were used for data handling in the analysis pipeline. ggplot2https://ggplot2.tidyverse.org, RRID:SCR_014601) was used for plot generation. Rcartocolor  provided the color palette used for genetic ancestry plot.Bash , 1.09; 95% confidence interval (CI), 1.07–1.11; 3.2p < 0.001; “problem hearing in crowds”: OR, 2.68; 95% CI, 1.77–4.06; p < 0.001; Figure p < 0.001; Figure Subjects who reported hearing loss or ‘problem hearing in crowds’ had significantly higher odds of having SRT‐based hearing disability  blocks with p values lower than 10−5, a suggestive threshold for genome‐wide associations in humans  did not identify any variants with s Figure . LocusZos Figure . Among ts Figure . The dis4ATE1, ZFHX3, ENAH, and SRP9) that play important roles in neural development and function.We studied a well‐established cohort of cisplatin‐treated testicular cancer survivors to investigate risk factors for impaired speech recognition threshold. As shown previously in the general population,4.1Consistent with age‐related hearing declines, we observed a significant association between SRT‐based hearing disability and age. Cumulative dose of cisplatin was not associated with SRT‐based hearing disability, suggesting the possibility that SRT deterioration is independent of the ototoxic effect of cisplatin. SRT is highly correlated with low frequency hearing sensitivity measured by PTA of 0.5, 1, and 2 kHz. Thus, our lack of association between SRT and cisplatin dose is consistent with results by others demonstrating cisplatin‐induced hearing loss in the high frequency range,The observed association between self‐reported health and SRT‐based hearing disability could indicate an impact of hearing disability on the subjects' perception of health. However, association does not imply causation, and other underlying conditions influencing health perception could contribute to SRT deterioration and, therefore, to the observed association.4.2p = 0.007; tinnitus: 38% cases/10% controls, p < 0.001).We have previously reported associations of both tinnitus and “self‐reported hearing loss” with SRT deterioration in a subset of The Platinum Study cohort4.3A 2005 meta‐analysis concluded a suggestive positive association between smoking and hearing loss.We did not find a significant association between heavy alcohol consumption and SRT‐based hearing disability, consistent with the reported lack of association between decline of SRT (in noise) and alcohol use in a longitudinal study.We observed significant associations between increasing BMI as well as lower physical activity with SRT‐based hearing disability. Associations between BMI or ‘obesity defined by BMI’ with hearing loss have been reported in both cross sectional4.4We did not observe a significant association between peripheral sensory neuropathy and SRT‐based hearing disability. However, there was a significant association between peripheral motor neuropathy and SRT‐based hearing disability. There is a wide array of identified factors that disproportionately contribute to the development of either peripheral sensory neuropathy or peripheral motor neuropathy.4.5We did not observe an association of hypertension or hypercholesterolemia (defined by taking prescription medications) with SRT‐based hearing disability. There are conflicting reports about the association of hypertension and hearing loss, with presence or lack of association being reported.4.6ATE1 and is an eQTL for it. ATE1 regulated neurite formation during development of the brainZFHX3(ATBF1) which encodes a transcription factor involved in neuronal differentiationZFHX3 is expressed in otic placodeZFHX3 is known to be regulated during development of inner ear in mouse.ENAH, a gene that is highly expressed in the nervous system and encodes a protein that regulates local translation in developing axons.SRP9 and a protein heterodimer that includes SRP9 potentially regulates protein biosynthesis in dendrites.TMEM63A which is expressed in oligodendrocytes and heterozygous missenses mutations in TMEM63A lead to transient hypomyelination during infancy.Suggestively implicated variants were assessed for location  and we used the GTEx portal to check whether the leading variants in each LD block were eQTLs or sQTLs. rs4752613 is located within the intronic sequence of SH2D4B gene which has a noticeable expression in mouse hair cells.We also compared our suggestive SNPs with the list of 51 identified SNPs associated with age‐related hearing loss.4.7A potential limitation for the impact and generalization of our findings to other populations is that 82.3% of the subjects in clinical associations and all of the subjects in genome‐wide association are white.Another limitation of the current study is reliance on questionnaire‐based evaluation of most of the clinical variables. Some of these variables could intrinsically be obtained through self‐assessment . Other life style variables such as smoking and drinking habits are usually self‐reported considering the limitations of real‐life tracking of these behaviors. Biases are intrinsic limitations of cross sectional studies.Lack of information on other ototoxic drugs  is another limitation of the study; however, we do not expect a systematic exposure of the participants (primarily young adults) to other major ototoxic medication.−8 and lack of a replication dataset are limitations of our genome‐wide association study. To the best of our knowledge, there are no datasets currently available that would allow for an accurate replication of our genome‐wide associations. Emergence of new datasets and increased sample size would provide the means for expansion of the current findings on the genetic architecture of SRT decline in cancer survivors.Lack of identification of SNPs reaching significance cutoff of 5 × 104.8Our results improve the current understanding of risk factors for the decline of speech recognition in cancer survivors. Higher BMI, lower physical activity, and smoking are associated with SRT decline.Image Integrity: LocusZoom images are modified to increase legibility and to remove redundant annotations.Mohammad Shahbazi: Conceptualization (lead); formal analysis (lead); investigation ; validation (lead); writing – original draft (lead); writing – review and editing . Xindi Zhang: Conceptualization ; investigation ; methodology ; writing – review and editing . Paul C Dinh: Data curation ; investigation ; methodology ; writing – review and editing . Victoria A Sanchez: Conceptualization ; investigation ; writing – review and editing . Matthew R Trendowski: Investigation ; methodology ; writing – review and editing . Megan Shuey: Formal analysis ; investigation ; writing – review and editing . Tessa Nguyen: Investigation ; writing – review and editing . Darren R. Feldman: Investigation ; writing – review and editing . David J. Vaughn: Investigation ; writing – review and editing . Chunkit Fung: Investigation ; writing – review and editing . Christian K Kollmannsberger: Investigation ; writing – review and editing . Neil E. Martin: Investigation ; writing – review and editing . Lawrence H. Einhorn: Investigation ; writing – review and editing . Nancy J Cox: Investigation ; methodology ; writing – review and editing . Robert D Frisina: Conceptualization ; investigation ; writing – review and editing . Lois B Travis: Conceptualization ; funding acquisition ; investigation ; project administration ; resources ; supervision ; writing – review and editing . Mary Eileen Dolan: Conceptualization ; investigation ; project administration ; writing – original draft ; writing – review and editing .Darren R. Feldman has a relationship with Gilead, Decibel, Astellas, Seattle Genetics, Novartis, and UpToDate. Victoria A. Sanchez reports paid research support and/or consultancy stipends from Autifony Therapeutics Ltd., Oticon Medical, Otonomy Inc., and Boehringer Ingelheim International GmbH (Unrelated to the project). All other authors declare no conflicts of interest.The study was conducted in line with the U.S. Common Rule and was approved by respective Institutional Review Boards. All subjects were >18 years old during enrollment and provided written consent for participation including access to medical records and genetic analysis.Appendix S1Click here for additional data file.


Mycoplasma hyopneumoniae is a bacterium that causes pneumonia in pigs and can facilitate the establishment of other respiratory diseases. The microbiota, which comprises the microorganisms found in a specific environment , can be altered by the presence of several pathogens. Therefore, knowing how M. hyopneumoniae can affect the microbiota of the lower respiratory tract and nasal turbinates in experimentally infected pigs is important to understand the interaction between microorganisms that could lead to the development of disease. Thus, we investigated the bacterial composition of the lungs and noses of infected and non-infected pigs. The results showed that the lungs of infected pigs were mostly colonized (growth and multiplication of a microorganism) by M. hyopneumoniae, and there were not many other species in the lungs. In contrast, in the non-infected pigs, a more diverse lower respiratory microbiota was observed, meaning that there were more species of bacteria in the non-infected pigs than in the infected ones. No differences were observed between the nose microbiota in infected and non-infected pigs. In conclusion, this pathogen can alter the number of bacterial species in the lungs, which could result in more respiratory problems in swine production.Mycoplasma (M.) hyopneumoniae, the etiological agent of swine enzootic pneumonia, has been reported to increase the susceptibility to secondary infections and modulate the respiratory microbiota in infected pigs. However, no studies have assessed the influence of M. hyopneumoniae on the respiratory microbiota diversity under experimental conditions. Therefore, this study evaluated the impact of M. hyopneumoniae infection on the respiratory microbiota of experimentally infected swine over time. To accomplish this, 12 weaned pigs from a M. hyopneumoniae-free farm were divided into two groups: M. hyopneumoniae strain 232 infected (n = 8) and non-infected (n = 4). The first group received 10 mL of Friis medium containing 107 CCU/mL of M. hyopneumoniae while the control group received 10 mL of sterile Friis medium. Inoculation of both groups was performed intratracheally when the animals were 35 days old (d0). At 28 days post-inoculation (dpi) and 56 dpi, 4 infected animals plus 2 controls were humanely euthanized, and biopsy samples of nasal turbinates (NT) and bronchus-alveolar lavage fluid (BALF) samples were collected. The DNA was extracted from the individual samples, and each group had the samples pooled and submitted to next-generation sequencing. Taxonomic analysis, alpha and beta diversity indexes, weighted unifrac, and unweighted unifrac distances were calculated. A high relative frequency (99%) of M. hyopneumoniae in BALF samples from infected animals was observed with no significant variation between time points. The infection did not seem to alter the diversity and evenness of bacterial communities in NT, thus, M. hyopneumoniae relative frequency was low in NT pools from infected animals . PCoA diagrams showed that BALF samples from infected pigs were grouped and far from the control samples, whereas NT from infected animals were not separated from the control. Under the present coditions, M. hyopneumoniae infection influenced the lower respiratory microbiota, which could contribute to the increased susceptibility of infected animals to respiratory infections. Mycoplasma (M.) hyopneumoniae is a respiratory pathogen of swine, with a worldwide distribution. Infections cause chronic pneumonia, dry, non-productive cough, and a reduction in average daily weight gain [ght gain . The ageght gain . The intM. hyopneumoniae in the composition of lung and nasal microbiota under experimental conditions remains to be investigated, and additional studies are required for a better understanding [The microbiome is the community of microorganisms that live on the mucosal surface and/or the skin of animals . The prestanding .Streptococcus, Lactobacillus and Actinobacillus are the main components of the microbiome in healthy pigs, whereas Moraxella, Veillonella and Porphyromonas are suggested to play a potential role in porcine respiratory diseases [M. hyopneumoniae infection under field conditions [M. hyopneumoniae infection in the lower respiratory tract and nasal microbiota of swine under experimental conditions, this study focused on assessing the abovementioned conditions in experimentally infected swine.Infections caused by respiratory pathogens were shown to greatly affect the diversity and composition of the oropharyngeal microbiota in pigs, where the results indicate that diseases . As repodiseases , the divnditions ,9. HowevM. hyopneumoniae-free farm, were randomly allocated into two different groups, four in the non-infected, or control group (CG), and eight in the infected group (IG). Both groups were kept in separate, but similar facilities, with feed and water ad libitum, and were able to acclimatize for a week. During the acclimation period, at dpi −7, serum (ELISA) and laryngeal swab samples (qPCR) were collected from all animals and tested, confirming their M. hyopneumoniae-free status.A group of 12 male Large White pigs, weaned at 28 days of age and originating from a certified 7 CCU/mL of M. hyopneumoniae, strain 232 [At the day of inoculation, when the piglets were 35 days of age ), the eight animals in the IG group were inoculated with 10 mL of Friis medium containing 10rain 232 ,12,13. BThis study was approved by the Ethical Committee in Animal Use of the School of Agricultural and Veterinary Sciences, São Paulo State University, Campus Jaboticabal, under protocol # 9.952/16.At 28 dpi and 56 dpi, 2 CG and 4 IG animals were humanely euthanized according to the guidelines of the Brazilian Federal Veterinary Medicine council. Immediately after death, the noses of the animals were transversally sawed using a disinfected saw and both nasal turbinates (NT) were exposed. Then, 1 cm of tissue from each turbinate was collected with the aid of sterilized forceps and scalpel blades. Samples were stored in sterile DNase and RNase-free cryovials , flash-frozen in liquid nitrogen and stored at −80 °C until processing.M. hyopneumoniae-like lung lesions were evaluated, as described by Straw et al. [After NT collection, the trachea and lungs were removed for bronchus alveolar lavage fluid (BALF) collection and macroscopic lung lesion evaluation. For BALF sampling, an incision was made in the trachea, approximately 5 cm before the trachea bifurcation, and with the aid of a sterilized plastic pipette and automatic pipettor, 20 mL of sterilized PBS  were dispensed into the bronchus. The lung was slightly massaged for 10 s, and the liquid was aspirated back using the pipette. Then, BALF samples were stored in duplicates in 2.0 mL DNase and RNase-free cryovials , flash-frozen in liquid nitrogen and kept at −80 °C until they were used. Macroscopic w et al. . The sco2 lung samples comprising both affected and non-affected tissue were collected and stored in 10% buffered formalin for histopathological evaluation and scoring, as described by Livingston et al. [Afterward, 2 cmn et al. . Lastly,M. hyopneumoniae, M. hyorhinis and M. flocculare bacterial loads in the individual BALF and NT samples, total DNA was extracted following an in-house protocol, as described by Kuramae-Izioka et al. [® 2000 spectrophotometer . DNA samples were stored at −20 °C until they were used. The qPCR assay was performed as previously described by Fourour et al. [To evaluate a et al. , with moa et al. . Purity r et al.  and Ferrr et al. , followir et al. .g for 20 min. The supernatant was discarded, and the pellet that was formed was used for DNA extraction. Total DNA was extracted from 0.05 g of the NT and the BALF pellet using the DNeasy Blood and Tissue  commercial extraction kit, according to the manufacturer’s instructions. The purity and quantification of the extracts were assessed using a Nanodrop® 2000c spectrophotometer .BALF samples were thawed and centrifuged at 4 °C  at 12,000× ®  before being submitted to Illumina sequencing.The samples from control and infected pigs of each dpi (28 and 56) were pooled in equimolar amounts according to the concentration (ng/µL) obtained from spectrophotometry. The control pools were named: non-infected nasal turbinate 28 dpi (NINT28); non-infected nasal turbinate 56 dpi (NINT56); non-infected BALF 28 dpi (NIBALF28); and non-infected BALF 56 dpi (NIBALF56). The pools from infected animals were identified as infected nasal turbinate 28 dpi (INT28); infected nasal turbinate 56 dpi (INT56); infected BALF 28 dpi (IBALF28); and infected BALF 56 dpi (IBALF56). Finally, the integrity and quality of the pooled DNA samples were assessed using a ByoanalizerSample libraries were prepared using reagents and procedures according to the manufacturer’s instructions . Previously published primers targeting the hypervariable region V3–V4 of the 16s bacterial rDNA [The library validation was performed in a Bioanalyzer 2100 , and quantified using the Kappa Library Quant Kit for Illumina , according to the manufacturer’s instructions .The sequencing data that were generated were demultiplexed using the Illumina bcl2fastq (v2.19.1.403) software, and the sequencing quality assessment was performed using the DADA2 software . BrieflyPrincipal coordinates analysis of weighted unifrac and unweighted unifrac were performed to detect similarities between treatment (infected and non-infected) and time (28 dpi and 56 dpi), and based on the metrics, plots were generated using QIIME2 (Version 2019.10). Only the taxa present at >0.1% of all 16S rRNA sequences in each pool were considered. Taxonomic analysis was performed using the SILVA (release 132) database. Sequences were classified using the Naive Bayes classification of reference sequences, which were clustered with 99% similarity and trimmed to include only the V3–V4 region, limited by the primer pair used in the sequencing. For features classification, QIIME2 software was used.No macroscopic lung lesions were observed in the control group, while the average in the IG at 28 dpi was 15.8, reducing to 6.3 at 56 dpi. Regarding the microscopic evaluation, higher score values were seen in the IG when compared to the CG. The ADWG values in the IG were similar at both time points, whereas a numerically higher value was observed in the CG at 56 dpi. Detailed results are shown in M. hyopneumoniae was not detected in BALF or NT samples from individual animals in the CG, nor in the NT samples from the IG. Not surprisingly, 1.3 × 106 and 4.9 × 105 copies/µL of M. hyopneumoniae DNA were detected in the BALF samples from the IG at 28 and 56 dpi, respectively. M. hyorhinis was detected in the BALF samples of both groups (IG and CG) only at 28 dpi, whereas for NT samples, the pathogen was detected at both time points but only in the IG. Lastly, M. flocculare was detected only in the NT samples at 56 dpi. Average quantification values are presented in Results from NGS identified a total of 3621 features  with a sequence size of 465 bp. One pool (NINT28) presented a lower reads number  when compared to others. The OTUs and reads obtained for each pool are described in Although the samples IBALF28 and IBALF56 presented a high number of reads, there was a low number of OTUs after rarefication , indicating a low species diversity in both samples. The nasal turbinate pools  presented an acceptable OTUs quantity and variation, except for sample NINT28, which had a low OTUs count (29), likely due to the low yield of the sequencing .Regarding the alpha diversity metrics calculated for each pool, the Faith, Shannon, Pielou and Simpson indexes for IBALF28 and IBALF56 presented low values, influenced by the low number of OTUs. The values obtained in the calculation of each Alpha diversity metrics are also shown in The unweighted unifrac distance-based PCoA resulted in two main separate clusters. The first one was composed of IBALF28 and IBALF56, while the other one was composed of NIBALF 28 dpi, NIBALF 56 dpi, NINT56, INT28 and INT56 samples, indicating a qualitative similarity between bacterial types in the grouped samples. NINT28 was isolated from the abovementioned clusters, however, considering axis 1, which explained the majority of the variation, it was close to IBALF28 and IBALF56, as observed in The weighted unifrac distance-based PCoA presented the axis with the highest value (71.21%), which explained the majority of the variance compared to the others. Two main clusters were noted, the first being composed of IBALF28 and IBALF56, and the second composed of NIBALF 56 dpi and NIBALF 28 dpi, indicating a quantitative similarity in the bacterial communities between samples. The other samples were dispersed along axis 1 of the diagram, as shown in Mycoplasma hyopneumoniae in IBALF28 e IBALF56, with a relative frequency of 99% in both time point samples . The nasal turbinate pools INT28 and INT56 presented relative frequencies of M. hyopneumoniae of 0.83% and 0.90%, respectively. There was no detection of M. hyopneumoniae in NINT28, NINT56, NIBALF 28 dpi and NI BALF 56 dpi. M. hyorhinis was reported in INT28, NIBALF28 and NIBALF56, with a relative frequency of 37.56%, 0.29% and 0.67%, respectively. M. flocculare had a frequency of 0.79% in NIBALF28 and 0.10% in NIBALF56. The main taxonomic results of each pool, with genera and species (only for Mycoplasmas spp.), are shown in The taxonomic analysis showed a significant predominance of Lactobacillus sp. was not detected in BALF samples of the IG, while it was detected at a relative frequency of 2.68% and 2.21% in NIBALF28 and NIBALF56, respectively. Similarly, the relative frequencies of Actinobacillus and Streptococcus in NIBALF28 and NIBALF56 was 15.80% and 1.18%, and 3.91% and 3.26%, respectively.At the phylum level, differences in the relative abundance were observed between infected and non-infected samples, despite sample type. In the CG, the most abundant phylum were Proteobacteria, Firmicutes and Actinobacteria. In contrast, in the IG the phylum Tenericutes was by far the most abundant (>90% in BALF), followed by Proteobacteria (41.86% in NT) and Bacteroidetes (12.71% in NT) .M. hyopneumoniae experimental infection considerably reduced the number of OTUs detected in BALF at both time points, when compared to control animals. This result is reinforced by the low Faith indexes found in both inoculated group pools , compared to the control pools , showing a sharp reduction of microbial diversity in experimentally inoculated animals. In this study, M. hyopneumoniae, which was also confirmed by the closeness to 0 values found by Pielou’s and Simpson’s indexes. Furthermore, it is likely that the high relative frequency observed in the BALF samples from infected animals was mostly due to the experimental infection model and the high virulence of the challenge strain (232). Similarly, a previous study using shotgun genome sequencing showed similar results of over 90% relative frequency of M. hyopneumoniae in pooled samples of affected lungs from field animals [M. hyopneumoniae over other bacteria in swine lungs with swine enzootic pneumonia (SEP)-like lesions; these studies assessed the microbiota of naturally infected pigs during the finishing phase and at slaughter [M. hyopneumoniae-infected pigs [M. hyopneumoniae infection has a negative impact on lung health due to losses in microbiota diversity. In our study, the genera Lactobacillus, Streptococcus and Actinobacillus, which have been associated with a healthy oropharyngeal microbiota [M. hyopneumoniae infection may have played a role in the colonization of the abovementioned genera in the infected lungs.The taxonomic analysis of experimentally infected animal BALF pools at both time points (28 dpi and 56 dpi) resulted in a high relative frequency of laughter ,26. In cM. hyopneumoniae is a fastidious growing bacterium and a poor competitor for nutrients and adhesion sites in comparison with other bacteria [M. hyopneumoniae to establish and grow. Because this study is part of a greater project and focused only on the respiratory microbiota diversity under experimental conditions, complementary and detailed data on the immune responses upon challenge are described elsewhere [Due to its small genome and low metabolic capacity, bacteria ,27. Furtbacteria ,29. In abacteria . Moreovebacteria , playinglsewhere . M. hyopneumoniae infection showed little influence on nasal microbiota diversity in this study, as demonstrated by the alpha diversity indexes, the OTUs number of NT pools and DNA quantification by qPCR. This could be related to the low relative frequency of the agent detected in such pools with little variation over time, as shown by the taxonomic analysis. However, it is fairly documented in the scientific literature that M. hyopneumoniae shows a tropism for lower parts of the respiratory tract of swine [M. hyopneumoniae in NT samples as challenge infection happened in the trachea. Consequently, it is unlikely that M. hyopneumoniae would exert a significant impact on the microbiota of the upper respiratory tract in this study. In contrast with BALF results, of swine , being pof swine . FurtherM. hyopneumoniae in both BALF-infected pools severely affected the diversity and abundance of the microorganisms in the community. However, this was not observed in nasal turbinate pools, as shown in the PCoA using weighted unifrac distances. A similar abundance of microorganisms was noted in non-infected animals’ pools and in infected nasal turbinate pools, indicating that in the latter, the presence of M. hyopneumoniae did not impact microorganism abundance.The PCoA diagram based on the unweighted unifrac distance showed that there were no critical differences among NT pools, of either infected or not, and the non-infected BALF pools. However, despite being separated from other samples, infected BALF pools from 28 dpi and 56 dpi were close to each other along the main axis, indicating a high similarity of bacterial composition between samples, therefore confirming that for BALF samples, infection status was the main variable involved in the dissimilarity of bacterial communities. The predominance of M. hyopneumoniae infection is 28 dpi [It has been documented that the peak of lesions and clinical signs after s 28 dpi ,33. HoweM. hyopneumoniae DNA in NT samples of the IG, contradicting the results of the taxonomic analysis, where M. hyopneumoniae was detected at a frequency of 0.8% and 0.9%. However, it is known that any PCR test may produce false-negative results if the pathogen load is below the detection limit [M. hyorhinis was detected in BALF of both groups at 28 dpi and in NT samples of the infected group at both time points, while M. flocculare was detected only in NT samples at 56 dpi. These results also diverge from the relative frequency observed in the taxonomic analysis, which could be due to the different DNA extraction methods used for the different analyses. Furthermore, other limitations of the applied molecular techniques, such as the initial abundance of DNA and the presence of PCR inhibitors, could have played a role [Regarding the multiplex qPCR analysis, the results indicate no presence of on limit  and thaton limit ,34. For d a role ,19,34. M. hyopneumoniae 232 experimental infections in the respiratory tract microbiota, and consequently, the extrapolation of the results to animals raised under field conditions should be done with caution. Additionally, the results of non-infected nasal turbinate at 28 dpi were significantly affected by the low number of reads in the sequencing, which could have influenced the outcome. Considering that the M. hyopneumoniae 232 strain used was previously characterized as moderate virulence [The present research showed the influence of irulence , the resIn our study, pooling samples were also necessary to limit the cost of the analysis. In addition, the results regarding the microbiome analysis should be interpreted with caution as we evaluated the lung composition under experimental conditions, and therefore, other infection doses might lead to different disease outcomes and other bacteria genera. Lastly, background contamination in the microbiome analysis should be considered as control samples were not sequenced.M. hyopneumoniae experimentally-infected pigs using next-generation sequencing. However, further research investigating the susceptibility of lung microbiota to M. hyopneumoniae infection would bring relevant information about disease establishment in relation to lung microbiome conditions.To the best of our knowledge, this is the first report of respiratory microbiota diversity in Mycoplasma hyopneumoniae (232) experimental infection seemed to alter the microbial diversity and evenness in the lower respiratory tract of the pigs when compared to the non-infected animals, whereas the nasal microbiota did not seem to be affected by the infection, which may be due to the fact that M. hyopneumoniae shows tropism for the lower respiratory tract of pigs.


Congenital hearing loss is often caused by an inner ear malformation, in such cases, the presence of other anomalies, such as microtia, and venous anomalies of the temporal bone and laryngomalacia makes it challenging to perform cochlear implantation surgery.This study reports the case of a 28-month-old girl with congenital profound hearing loss, laryngomalacia, and malformed inner ear, who received cochlear implantation surgery. The bony structure, vessels and nerves were first assessed through magnetic resonance imaging and computed tomography before exploring the genetic basis of the condition using trio-based whole exome sequencing. Perioperative evaluation and management of the airway was then performed by experienced anesthesiologist, with the surgical challenges as well as problems encountered fully evaluated.Cochlear implantation was eventually performed using a trans-mastoid approach under uneventful general anesthesia.Due to the small size of the cochlea, a short electrode FLEX24 was inserted through the cochleostomy.Considering the high risk of facial nerve injury and limited access to the cochlea when patients present significant bony and venous anomalies, cochlear implantation in such patients require careful preoperative evaluation and thoughtful planning. In these cases, airway assessment, magnetic resonance venography, magnetic resonance arteriography, and magnetic resonance imaging and computed tomography can be useful to minimize the risks. Intraoperative facial nerve monitoring is also recommended to assist in the safe location of facial nerve. However, this treatment was subsequently shown to be effective in most cases of inner ear malformation.,3 This study presents the case of a 28-month-old with multiple anomalies who underwent CI before discussing the main surgical implications of the intervention.Being a well-established form of treatment, cochlear implantation (CI) is often performed in patients with bilateral severe to profound sensorineural hearing loss. Yet, while congenital hearing loss is commonly caused by inner ear malformation, At the age of 6 months, the patient was diagnosed with profound sensorineural hearing loss in both ears  was fully inserted without difficulty.Secondly, it is important to prepare multiple methods of identifying facial nerve and Scala tympani of the cochlea. Standard CI is carried out on the posterior tympanum through a trans-mastoid facial recess approach that opens the facial nerve recess to expose the round window niche and round window membrane. However, according to Hoffman et alThis case report of CI presents a patient with profound sensorineural hearing loss, along with laryngomalacia, vascular anomalies and severe middle and inner ear malformation. With the procedure being challenging, the main issues encountered preoperatively, intraoperatively and postoperatively as a result of the middle and inner ear malformation are discussed.Conceptualization: Xue Gao, Pu Dai.Data curation: Juan Zhao, Xi Wang, Xing Liu, Min Liu, Meng-Meng Guo.Formal analysis: Zhen-Dong Wang, Ya-Yan Lu, Jia Li, Yong Feng, Kun Yang.Investigation: Xue Gao, Pu Dai.Resources: Jin-Cao Xu.Supervision: Xue Gao, Jin-Cao Xu, Pu Dai.Validation: Xue Gao, Guan-Hua Li, Xi Wang, Wei Wang.Writing – original draft: Xue Gao, Juan Zhao.Writing – review & editing: Pu Dai.


Stropharia rugosoannulata (S. rugosoannulata) is a fungus with great edible and nutritional values; however, the development mechanism of its fruiting body has not been studied. Thus, this study aimed to analyze the differentially expressed genes (DEGs) in four stages; primordia stage (Sra1), young mushroom stage (Sra2), picking stage (Sra3), and opening umbrella stage (Sra4). Therefore, total RNA was extracted for further RNA-sequencing analysis. In three pairwise comparison groups (PCGs), Sra1 vs. Sra2, Sra2 vs. Sra3, and Sra3 vs. Sra4, a total of 3,112 DEGs were identified among the three PCGs. A GO analysis of the DEGs showed that there were 21 terms significantly enriched in Sra1 vs. Sra2 PCG. There was no significantly enriched GO term in the other two PCGs. Furthermore, KEGG pathway analysis showed that these DEGs were mainly enriched in glucose and amino acid metabolisms. Moreover we found that intron retention (IR) and the alternative 3′ splice site (A3SS) accounted for more than 80%. The development of the S. rugosoannulata fruiting body mainly involved glucose and amino acid metabolisms. IR and A3SS were the two main types of ASE, which played an important role in the development and maturation of the S. rugosoannulata fruiting body. Stropharia rugosoannulata , is an edible fungus of Stropharia in Strophariaceae with high medicinal and nutritional values  and plays an important role in soil remediation  were mainly involved in purine and fatty acid metabolism during the growth and development of the fruiting bodies . S. rugosoannulata also belongs to Basidiomycetes. The fruiting body development of Basidiomycetes was generally divided into the primordia stage (Sra1), young mushroom stage (Sra2), picking stage (Sra3), and opening umbrella stage (Sra3). The development differentiation of these four stages was obvious and easy to distinguish. Therefore, the fresh S. rugosoannulata fruiting body tissues from the four developmental stages were collected, and the same part of the fungus cap was collected from each tissue, which was frozen using liquid nitrogen and then stored in a −80°C refrigerator, and three samples were collected at each stage as replicates.In this study, the mushroom production experiment of S. rugosoannulata fruiting body samples was extracted using TRIzol®Reagent (Invitrogen) according to the manufacturer’s instructions. The purity and concentration of total RNA were examined by NanoDrop 2000, and the RNA integrity number value of total RNA was detected by Agilent2100. The qualified total RNA was sequenced through the Illumina next-generation high-throughput sequencing platform.Total RNA of For qualified RNA samples, mRNA was enriched with magnetic beads containing Oligo (dT) and then synthesized cDNA by reverse transcription. Under the action of reverse transcriptase, the first cDNA strand was synthesized with random six base primers. The second cDNA strand was synthesized by adding a buffer, DNA polymerase I, and dNTPs. Then, double-stranded cDNA was purified using AMPure XP Beads. The purified double-stranded cDNA was treated with end repaired mix; A-bases and splice were also added. The AMPure XP Beads were used to select the fragment size of the double-strand cDNA, and finally PCR amplification was performed to construct cDNA libraries. The Illumina sequencing platform was used for library sequencing. The library preparations were sequenced on an Illumina Novaseq 6000platform by the Beijing Allwegene Technology Company Limited  and paired-end 150bp reads were generated.Trimmomatic software (version 0.33) was used to conduct quality control on the raw data to ensure the accuracy of the analysis. Reads containing adapter, N ratio greater than 10% (N indicated that base could not be determined), and having low quality were removed to obtain high-quality sequences for the subsequent analysis. Sequence reads were mapped to the reference genome GCA_003314255.1 using Spliced Transcripts Alignments to a Reference  software.2 (foldchange)| > 1 and p < 0.05.HTSeq software (version 0.6.1p1) was used to analyze the gene expression levels, using the union mode. The expression levels of individual genes and the number of genes at different expression levels were presented in the results. Then we used the DESeq2 package in R software (version 3.5.2) to analyze the DEGs between different groups. The threshold of DEG screening was |logp value < 0.05 after correction by the Benjamini and Hochberg (BH) method.For the DEGs, we used “clusterProfiler” function package in R software (version 3.5.2) to conduct the enrichment analysis on GO terms and KEGG pathways. The significantly enriched GO term and KEGG pathway were screened with p value was calculated using the likelihood-ratio test to represent the difference of the inclusion level between the two groups of samples. There were five types of differential alternative splicing events detected by rMATS, including skipped exon (SE), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE), and intron retention (IR) . And then, ion (IR) .p < 0.05 was regarded as statistically.All statistical analysis was completed using R software (version 3.5.2). After removing the low-quality sequences with Trimmomatic software, the sequencing amount of each sample was counted, and the results are shown in STAR software was used to map the processed data with the reference genome GCA_003314255.1, and a statistical analysis was performed on the exon, intron, and intergenic regions of the reference sequence according to reads . As showS. rugosoannulata fruiting bodies. We divided the four stages of Sra1–4 into three pairwise comparison groups (PCGs) according to the two successive development periods as follows: Sra1 vs. Sra2 group, Sra2 vs. Sra3 group, and Sra3 vs. Sra4 group. A total of 3,112 DEGs were identified among the three PCGs  transporters, and metabolic pathways . DEGs incharides , and actS. rugosoannulata samples, evaluate the change of the corresponding inclusion level in the two samples, and obtain the distribution map of the differential alternative splicing events in each comparison group (Alternative splicing was one of the main mechanisms for protein diversity . We usedon group . In the on group . In the on group . In the on group .S. rugosoannulata fruiting body, and the proportion of IR from the young mushroom stage to the picking stage was the highest and then showed a downward trend, while A3SS increased in the later stage of fruiting body maturation, indicating that these two alternative splicing events were likely to be mainly involved in the regulation of S. rugosoannulata fruiting body development and maturation.We found that IR and A3SS accounted for more than 80% from the primordia stage to the picking stage of S. rugosoannulata fruiting bodies by comparing the transcriptome data of S. rugosoannulata fruiting bodies at the primordia stage, young mushroom stage, picking stage, and opening umbrella stage, providing clues for further understanding the regulation mechanism of S. rugosoannulata development.In this study, we analyzed DEGs and differential alternative splicing events related to the growth and development of S. rugosoannulata is a popular edible fungus with beneficial healthcare value, so it is of great significance to analyze the functional genes in its growth and development process for quality improvement. To our knowledge, this study was the first systematic transcriptome study of S. rugosoannulata fruiting bodies at different developmental stages. At different stages of fruiting body development, there were significant differences in gene transcription levels. We noticed that the number of DEGs was the largest from the primordia stage to the young mushroom stage, while it was significantly reduced from the picking stage to the opening umbrella stage, which was consistent with the study of S. rugosoannulata fruiting body was vigorous in growth and development with an active metabolism and increased demand for energy and protein (S. rugosoannulata fruiting bodies rich in polysaccharides and amino acids (Ganoderma lucidum (S. rugosoannulata fruiting bodies by regulating the antioxidant enzyme activity and ROS level. protein . The act protein , thereby protein , and DEGno acids . The DEG lucidum . This evPhyscomitrella patens by Chang et al. showed that high temperature caused nearly 50% of the expressed genes to develop alternative splicing, in which the proportion of IR decreased and SE dominated, indicating that alternative splicing had differential regulation in response to heat shock (Ophiocordyceps sinensis (Volvariella volvacea, and consequently several potential Cu-Znsod1 and Mnsod1/2 amino acid sequences with different lengths and sequence motifs could be obtained by random combinations of alternative splicing events (S. rugosoannulata fruiting body development were not revealed in this study, which will be focused on in future studies.Alternative splicing referred to the transcription process in which two or more mature mRNAs were generated from the same pre-mRNA through different splicing methods, which play an important role in regulating the growth and development of plants as well as responses to environmental stress . For exaat shock . Tong etsinensis . Yan et g events . These sg events . HoweverS. rugosoannulata fruiting body, we systematically identified the DEGs at each stage and analyzed their biological functions. The pre-mRNA alternative splicing atlas of the four stages was further constructed, and the characteristics of alternative splicing events among different stages were also identified. We found that the development of the S. rugosoannulata fruiting body was mainly involved in biosynthesis, glucose metabolism, and amino acid metabolism. IR and A3SS were the two most important types of alternative splicing, which played an important role in the development and maturation of S. rugosoannulata fruiting bodies. This study was the first to describe the transcriptome dynamics during the development of S. rugosoannulata fruiting bodies from a gene and pre-mRNA splicing aspect. Our work provided clues for further exploring the molecular mechanism of S. rugosoannulata fruiting body development.In this study, based on the RNA-sequencing data of four growth and development stages of the