Pleurotus ostreatus, the yield and quality of fruiting bodies are easily affected by high temperatures in summer. Nitric oxide (NO) plays an important regulatory role in the response to abiotic stress, and previous studies have found that NO can induce alternative oxidase (aox) experssion in response to heat stress (HS) by regulating aconitase. However, the regulatory pathway of NO is complex, and the function and regulation of the aox gene in the response to HS remain unclear.In China, during the cultivation process of 2O2) and superoxide anion (O2−) contents, and slowed O2− production. Further RNA-Seq results showed that NO regulated the oxidation-reduction process and oxidoreductase activity, affected the cellular respiration pathway and activated aox gene expression. The function of aox was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the OE-aox strains exhibited obviously improved growth recovery after exposure to HS. During exposure to HS, the OE-aox strains exhibited reduced levels of NADH, the product of the tricarboxylic acid (TCA) cycle, and decreased synthesis of ATP, which reduced the production and accumulation of reactive oxygen species (ROS), whereas the RNAi-aox strains exhibited the opposite result. In addition, aox mediated the expression of antioxidant enzyme genes in the mycelia of P. ostreatus under HS through the retrograde signaling pathway.In this study, we found that NO affected nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) levels, reduced hydrogen peroxide (Haox gene in P. ostreatus mycelia can be induced by NO under HS, that it regulates the TCA cycle and cell respiration to reduce the production of ROS, and that it can mediate the retrograde signaling pathway involved in the mycelial response to HS.This study shows that the expression of the The online version contains supplementary material available at 10.1186/s12934-021-01626-y. Pleurotus ostreatus is one of the most widely cultivated mushroom species globally and is mainly cultivated in horticultural facilities in China [P. ostreatus, high temperatures in summer limit the growth of mycelia, the formation of primordia and the development of fruiting bodies, and thereby seriously affecting the yield. However, the mycelia response to changes in temperature is very complex and includes different physiological and metabolic changes that affect the overall development of edible fungi as well as interactions between cells and molecules. Metabolomics analyses have shown that heat stress (HS) promotes the degradation of unsaturated fatty acids and nucleotides, increases the contents of amino acids and vitamins, and accelerates glycolysis and the tricarboxylic acid (TCA) cycle [2+) as signaling molecules, also play an active protective role during exposure to HS [in China . During A) cycle . FurtherA) cycle . The synA) cycle . Moreovere to HS . In rece2+-mediated NO signal transduction is involved in the response of Brassica seedlings to metalloid stress [Prunus persica (L.) Batsch, treatment with a NO solution can protect the fruit from pathogen infection by inducing the activity of defense enzymes and the expression of pathogenesis-related genes [2O2) and malondialdehyde in calli and significantly inhibits the increase in ion leakage and growth inhibition and decrease in cell viability induced by HS [Ganoderma lucidum, NO is involved in the regulation of ganoderic acid synthesis [P. ostreatus, NO can alleviate the oxidative damage induced in mycelia by HS [NO, an essential endogenous signaling molecule that is involved in many biological processes in plants, animals, bacteria, and fungi, is considered a broad-spectrum antistress molecule –8. The rd stress , and in ed genes . In two ed by HS . Previoued by HS . In funged by HS  but it med by HS , 16. In ed by HS . In addied by HS , 19. Recynthesis , and in ia by HS . Howeveraox), an integral protein (32–36 kDa) of the inner mitochondrial membrane [Neurospora crassa, a model fungal organism, aox transcripts are undetectable or present at very low levels under normal growth conditions [aox gene is strongly induced and the AOX protein can be found in mitochondria [Medicago truncatula, NO can induce the expression of aox, and AOX can help regulate the accumulation of ROS, protect the photosystem, and enhance plant resistance to salt stress [Blastocladiella emersonii [Aspergillus fumigatus, silencing of the mitochondrial aox gene makes the strain more sensitive to ROS, and easier to kill by macrophages [P. ostreatus remains unclear.Mitochondria are the main site of ROS bursts and play a key role in cell energy metabolism. The electron transport chain (ETC) of mitochondria consists of two pathways: the main cytochrome c pathway and the alternative pathway. Alternative oxidase  technology was used to explore the possible regulatory pathways and key genes involved in the NO-mediated alleviation of HS-induced damage in P. ostreatus, and the regulatory effect of NO on the expression of the aox gene was studied in detail. Moreover, the relationships among aox, energy metabolism and ROS were explored using overexpression (OE) and RNA interference (RNAi) technology.The expression of the stresses , and pre tobacco . Our preia to HS . HoweverP. ostreatus to HS [P. ostreatus mycelial growth after HS, as shown in Fig. 2O2 and O2− are important components of ROS, and as shown in Fig. 2O2 in mycelia increased significantly after HS . Exogenous sodium nitroprusside  significantly reduced the accumulation of H2O2 in mycelia after HS , but the level was still significantly higher than that in the control group . As shown in Figs. 2− content in mycelia by 1.64-fold and the production rate of O2− by 60.74 %. In addition, compared with the levels found in the HS group, exogenous SNP decreased the O2− content and production rate by 12.85 and 9.34 %, respectively. 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxidec (cPTIO) is an NO scavenger. Research shows that the addition of 250 µM cPTIO can significantly increase the content and production rate of O2−. Our results showed that exogenous NO could affect not only the content of H2O2, but also the content and production rate of O2−.NO, an important signaling molecule, plays an important regulatory role in the growth and development of organisms and their responses to stress. Our previous study showed that NO plays an important role in the response of us to HS . In this+) and reduced (NADH). NADH is produced during glycolysis pathway and the TCA cycle, participates in material and energy metabolism in cells and serves as a control marker in the energy production chain of mitochondria. As shown in Fig. P = 1.500 × 10− 9), and this increase could be partially offset by treatment with SNP. HS also significantly affected the NAD+/NADH ratio , and the addition of exogenous SNP alleviated the imbalance in NAD caused by HS. In contrast, the addition of cPTIO significantly decreased the NAD+/NADH ratio compared with that observed in the HS group  , whereas the addition of exogenous cPTIO significantly increased the ATP content under HS   is a coenzyme that exists in all cells and is found in two forms: oxidized  identified from the SNP_HS vs. cPTIO_HS comparison were examined to elucidate the possible mechanism through which NO alleviates the oxidative damage to mycelia induced by HS Fig. . In the To understand the functions of the DEGs identified from the SNP_HS vs. cPTIO_HS comparison, a pathway enrichment analysis was performed. The results showed that the DEGs were mainly concentrated in the following pathways: oxidoreductase activity, oxidation-reduction process, cofactor binding, protein kinase activity, phosphotransferase activity, alcohol group as acceptor, and protein phosphorylation Fig. A. Previohttp://www.ebi.ac.uk/interpro/entry/InterPro/IPR027243/) [https://www.uniprot.org/uniprot/A1AZJ0). These results indicate that exogenous NO can inhibit the cytochrome pathway and activate the alternative oxidation pathway.The respiratory chain is closely related to ROS. To further explore whether NO can regulate the respiratory chain to alleviate mycelial damage under HS, six DEGs related to the respiratory chain were identified from 579 DEGs via functional enrichment Table . Two of 027243/) . The sucaox can be regulated by NO, participates in the oxidation-reduction process pathway and has oxidoreductase activity.ROS are mainly produced by the respiratory chain under high oxygen conditions and in a high reduction state during the transition of mitochondria from complex III to complex IV, which results in the leakage of a large number of electrons and the reduction of oxygen molecules. AOX prevents excessive reduction of downstream complexes (cytochrome pathway) by introducing a branch into the ETC at the ubiquinone pool. When AOX bypasses complexes III and IV of the cytochrome pathway, it significantly reduces ATP production and single electron leakage, which results in the reduction of ROS production . Moreoveaox under HS, the effects of exogenous NO donors and scavengers on aox gene expression were assessed. As shown in Fig. aox gene in P. ostreatus mycelia changed steadily with increases in the time of exposure to HS. During the first 24 h of exposure to HS, aox gene expression first increased and then decreased within a small range. An increase in the exposure time to 48 h significantly increased the relative expression of the aox gene, and the level detected after 48 h was approximately 8-fold higher than that at 0 h. As shown in Fig. aox gene was significantly increased after HS , whereas exogenous SNP treatment almost completely enhanced this effect , and cPTIO blocked the effect of SNP on aox gene expression . The results showed that NO can promote the expression of the aox gene in mycelia after HS. In conclusion, NO might participate in the response of P. ostreatus to HS by regulating the expression of the aox gene.To further verify the regulatory effect of NO on aox plasmid  to screen the RNAi-aox strains. The results are shown in Additional file aox gene in the response of P. ostreatus to HS, plates containing the various strains were cultured at 28 °C for 5 d, transferred to 40 °C for 48 h, and then incubated at 28 °C to allow growth recovery. After 3 d of growth recovery after HS, mycelial germination was observed in the wild type (WT), OE-aox and RNAi-aox strains, as shown in Fig. aox strains exhibited a faster recovery rate and a complete colony edge, whereas RNAi-aox strains presented a slower mycelial recovery rate and showed defects on the edge of the colony. In conclusion, the aox gene plays an active role in the recovery of P. ostreatus mycelia after HS. Figure 2O2 content, the O2− content and the production rate of the aox-transformed strains under HS. Under HS, the accumulation of H2O2 in the OE-aox 47 and OE-aox 71 strains was significantly lower  than that in the WT strain. In addition, the H2O2 content in the OE-aox 34 strain was 4.29 % lower than that in the WT strain. In contrast, the H2O2 contents in the RNAi-aox 12, RNAi-aox 29 and RNAi-aox 7 strains were 4.59 %, 17.71 and 21.11 % higher, respectively, than that in the WT strain. As shown in Figs. 2− production rate and content of the OE-aox strains under HS were significantly lower than those of the WT strain, whereas those of the RNAi -aox strains increased significantly. These results indicated that the aox gene can regulate the production and accumulation of ROS. ROS are mainly caused by electron leakage in the respiratory chain. As shown in Figs. aox strains were decreased by 26.47 and 9.82 %, respectively, and the average NAD+/NADH ratio in these strains was 1.55-fold higher. In contrast, the average NADH and ATP contents of the RNAi-aox strains were 66.86 and 29.79 % higher, respectively, than those in the WT strain, and the NAD+/NADH ratio was decreased by 26.52 %. Therefore, it can be speculated that the aox gene plays an important role in regulating the mitochondrial ETC and energy metabolism and in maintaining mitochondrial homeostasis.Using previously reported methods , we succaox gene can affect the production and accumulation of ROS by regulating mitochondrial respiration, and it participates in the mycelial response to HS.In conclusion, the aox serves as a marker gene for mitochondrial retrograde regulation [P. ostreatus, two genes (cat1 and cat2) encode CAT, four genes  encode SOD, one gene encodes TrxR, and one gene encodes GSH-PX [aox in P. ostreatus can alleviate ROS stress by regulating the antioxidant enzyme system, we measured the expression of these key antioxidant enzyme genes in aox-transformed strains exposed to HS at 40 °C for 48 h. As shown in Fig. cat1 in the OE-aox 47-, OE-aox 71- and OE-aox 34-transformed strains was significantly downregulated to 26.5 %, 25.96 and 35.30 % respectively, of the level found in the WT strain. In the RNAi-aox 12 and RNAi-aox 29 strains, the relative expression of cat1 was significantly increased by 1.57-fold and 7.28-fold, respectively, compared with that found in the WT strain, but no significant change in the expression of this gene was detected in the RNAi-aox 7 strain. As shown in Fig. cat2 in the OE-aox and RNAi-aox strains was significantly downregulated and upregulated, respectively. In conclusion, aox can negatively regulate cat gene expression under HS. As shown in Figs. trxr and gsh-px genes was significantly downregulated in the OE-aox-transformed strains. In addition, the trxr gene was significantly upregulated in all RNAi-aox strains, and the relative expression of the gsh-px gene was significantly upregulated in RNAi-aox12 and RNAi-aox 29 and slightly upregulated in RNAi-aox 7. SOD is one of the major defense systems used to remove O2−. The relative expression levels of the four SOD-encoding genes in the aox-transformed strains under HS are shown in Figs. sod1 gene expression was significantly increased in the OE-aox strains and decreased to 72.66 %, 67.71 and 79.93 % in RNAi-aox 12, RNAi-aox 29 and RNAi-aox 7, respectively. In addition, as shown in Figs. sod2, sod3 and sod4 in the OE-aox strains were significantly downregulated, to 34.93 %, 60.33 and 32.16 %, respectively, of the level found in the WT strain. However, sod2 and sod3 gene expression was not significantly changed in the RNAi-aox strains, and the expression of sod4 was slightly downregulated in these strains compared with the WT strain. Because the sod1 gene encodes Gu-SOD, which mainly exists in the cytoplasm, it can be concluded that the OE of aox during HS can regulate the expression of SOD-encoding genes.The signaling from organelles that controls nuclear gene expression is called retrograde signaling, and previous studies have shown that gulation . AOX alsgulation . Moreovegulation . We meass GSH-PX . To studaox strains exhibited downregulated expression of key antioxidant enzyme coding genes under HS (40 °C for 48 h), and the RNAi-aox strains exhibited upregulated expression of CAT-, GSH-PX- and TrxR-encoding genes. It can thus be hypothesized that the significant upregulation of the aox gene after HS (40 °C for 48 h) can further affect the expression of key antioxidant enzyme-encoding genes.In conclusion, in addition to SOD1, the OE-aox interference can regulate the expression of key antioxidant enzyme-encoding genes, an experiment in which different concentrations of an AOX inhibitor (BHAM) were added was then performed. The results showed that the growth rate of P. ostreatus mycelia at 28 °C was affected by exogenous BHAM. As shown in P. ostreatus, but the addition of exogenous BHAM at a concentration higher than 200 µM affected overall mycelial growth and significantly reduced the mycelial growth rate. Furthermore, whether exogenous BHAM could regulate the expression of the aox gene was assessed in the experiment. As shown in Additional file aox gene compared with the control (CK) level, which indicated that low concentrations of BHAM can inhibit the expression of the aox gene. However, a BHAM concentration of 400 µM upregulated the expression of the aox gene, and because this concentration also significantly inhibited the mycelial growth rate, it can be speculated that 400 µM BHAM might affect the growth environment of mycelia and induce abiotic stress, which eventually leads to the induction of aox gene expression. In addition, because the mycelial growth of the RNAi-aox strains did not significantly differ from the normal growth of P. ostreatus mycelia, 50–200 µM was used as the BHAM concentration in the subsequent experiments.AOX is insensitive to inhibitors of the cytochrome pathway such as cyanide, antimycin A, and miyxothiazol, but is inhibited by primary hydroxamic acids, such as BHAM , 45. To aox strains. The effects of exogenous BHAM on the H2O2 content and the O2− content and production rate were then assessed. The results showed that compared with the CK-HS group, the H2O2 content and O2− content and production rate of mycelia under HS were significantly increased by different concentrations of BHAM  in response to HS. This finding provides a new perspective on the fungal regulatory mechanism of NO in response to abiotic stress.NO is a type of free radical involved in many types of stress and physiological processes and serves as a key regulator of physiological processes . In this2−, which can be used as a substrate to produce H2O2 and hydroxyl radicals [P. ostreatus is often exposed to high temperature. The results obtained in this study show that exogenous NO can reduce the content of NADH and the production of ATP under HS. Previous studies have shown that NO can regulate the NAD+/NADH ratio in Ganoderma lucidum under HS [+/NADH ratio is mainly regulated by the TCA cycle, and NADH serves as a substrate for the generation of ATP in the respiratory chain [2O2-induced apoptosis [Saccharomyces cerevisiae, the activation of Mac1 transcription factors by NO produced under a high temperature-stress conditions is important for the activation of the copper-dependent SOD1 [The regulatory effect of NO on metabolism during stress relief is not unusual. Recently, an increasing number of studies have shown that the application of exogenous NO is useful for allaying oxidative stresses caused by drought, high temperature and salinity –49. HS rradicals . In addiradicals , 53. Durunder HS , which iry chain . Therefory chain . In ricery chain . In yeaspoptosis . For exaent SOD1 . The resCamellia sinensis, a total of 89 putative AP2/ERF transcription factors related to the temperature response were identified by RNA-Seq [Populus trichocarpa leaves were obtained by RNA-Seq analysis, including genes related to the drought stress response and involved in photosynthesis, cell wall organization, and osmoprotectant metabolism [P. ostreatus mycelia to HS by regulating cell metabolism, affecting the composition and structure of the cell membrane, or modifying the catalytic activity of proteins. Interestingly, the functional enrichment analysis identified 6 DEGs among the 579 DEGs that were not only regulated by NO but also closely related to the respiratory chain. Among these 6 DEGs, mitochondrial chaperone BCS1 (g12952) and the succinate dehydrogenase iron-sulfur subunit (g3097) are necessary for complexes II and III, respectively, which are closely related to the cytochrome pathway, and AOX (g11376) is involved in the alternative oxidation pathway. Further analysis showed that only AOX was upregulated after SNP addition, whereas the other DEGs were downregulated. In P. ostreatus, NO can induce aox gene expression by inhibiting the expression of aconitase gene and protein, which results in citric acid accumulation [aox gene. For instance, Fu et al. showed that NO is an inducer of aox in tobacco plants infected with tobacco mosaic virus [aox is induced in Arabidopsis cell suspensions treated with NO [aox gene expression in both fungi and plants.Stress-induced gene expression changes are key components of the molecular mechanism underlying plant adaptation to environmental challenges . In rece RNA-Seq . Under dtabolism . Therefomulation . This fiic virus , and ano with NO . TherefoA. fumigatus [Histoplasma capsulatum [Paracoccidioides brasiliensis [aox plays important roles in countering abiotic stresses, including drought, light, temperature and salinity [aox knockdown plants is strongly compromised [aox is upregulated under mitochondrial respiratory chain inhibition or oxidative stress conditions [P. brasiliensis, aox expression is also upregulated by ROS generation and mitochondrial respiratory chain inhibitors, and heterologous expression of aox in S. cerevisiae decreases intracellular ROS generation [2−, aox in A. fumigatus may play a role in antioxidant defense mechanisms [aox strains rapidly resumed their growth after HS, and aox OE could reduce the contents of NADH and ATP and the content and production rate of O2−. In contrast, the opposite results were obtained with the RNAi-aox strains. Previous studies with Arabidopsis thaliana have shown that under hypoxic stress, aox can regulate energy metabolism, affect ROS production and enhance resistance [aox gene might reduce the production of O2− by regulating the respiratory pathway and maintaining mitochondrial homeostasis and function in P. ostreatus.AOXs have been documented to occur in plants , algae , yeasts umigatus , Histoplpsulatum , and Pariliensis . AOX is iliensis , 36. In salinity , 69, 70.promised . In fungnditions , 72. Forneration . In the chanisms . In thissistance , and we sistance . Therefoaox strains was downregulated after HS, whereas the expression of these genes in the RNAi-aox strains was upregulated, and these findings might be related to the regulation of ROS production by aox. BHAM is an inhibitor of AOX, and exogenous BHAM also promoted the expression of antioxidant enzyme-encoding genes under HS, which was very similar to the results obtained with the RNAi-aox strains. Therefore, it can be speculated that in response to HS, the aox gene can mediate the retrograde signaling pathway in P. ostreatus mycelia to regulate the expression of antioxidant enzyme-encoding genes.A large number of studies have shown that AOX mediates a retrograde signaling pathway that in turn regulates gene expression both transcriptionally and post transcriptionally in response to stress. AOX serves as a link between metabolic activities (mitochondria) and signaling (nucleus) , 76. In P. ostreatus mycelia and can induce aox gene expression. The construction of OE-aox and RNAi-aox strains showed that high aox gene expression can affect cell respiration by reducing the content of NADH, which is a product of the TCA cycle, thereby reducing the production and accumulation of ROS. Further studies showed that under HS, aox can mediate the reverse signaling pathway to regulate the expression of antioxidant enzyme genes in P. ostreatus, thereby regulating the mycelial response to ROS. Based on these findings, we proposed a potential cascade of cellular events comprising the NO-mediated alleviation of ROS production via aox gene expression  was grown in Luria-Bertani (LB) medium  containing 100 µg/mL kanamycin  and 50 µg/mL rifampin  and was used to transform P. ostreatus. Trans 1-T1 phage-resistant chemically competent cells  were used for plasmid construction and were grown in LB broth containing kanamycin (50 µg/mL).The aox 47, OE-aox 71, OE-aox 34, RNAi-aox 12, RNAi-aox 29 and RNAi-aox 7 strains were used in this study. According to the research methods used in our previous study [The WT, OE-For the exogenous BHAM addition experiment described in this paper, the WT strain was cultured at 28 ℃ for 5 d on PDA plates with different concentrations of BHAM . Then, the PDA plates of different treatment groups were transferred to 40 ℃ for 48 h and finally placed at 28 ℃ to resume growth for 3 d.2O2 content was determined using an H2O2 Quantitative Assay Kit  according to the manufacturer’s instructions. In an acidic environment, H2O2 can oxidize Fe2+ to Fe3+, and then Fe3+ combines with dye molecules to form a complex, which has the maximum absorption wavelength at 595 nm. The absorbance at 405 nm was detected by a microplate reader  to determine the amount of complex formation, and then the content of H2O2 was calculated.Protein concentrations were determined using the Bradford Protein Quantification Kit , with bovine serum albumin as the standard, following the manufacturer’s instructions. The intracellular H2− will destroy the cell membrane. The content and production rate of O2− in the mycelia of the different strains subjected to the different treatments were detected using an O2− Content Detection Kit  according to the manufacturer’s instructions. O2− reacts with hydroxylamine hydrochloride to form nitrite ions, which form a red compound under the action of p-aminobenzenesulfonic acid and naphthalene ethylenediamine hydrochloride, and this compound has a characteristic absorption peak at 530 nm. The content and production rate of O2− in mycelia were then calculated based on the absorbance value at 530 nm obtained using a microplate reader .Excessive accumulation of O+) and reduced (NADH). In this study, the changes in the NADH content and the NAD+/NADH ratio in the WT and aox-transformed strains under HS were determined using an NAD+/NADH Assay Kit  according to the manufacturer’s instructions. Ethanol is oxidized to acetaldehyde in the presence of alcohol dehydrogenase, and during this process, NAD+ is reduced to NADH. Subsequently, NADH reduces 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2 H-tetrazole to orange formazan in the presence of 1-methoxy-5-methylpenazinium methyl sulfate, and the product has a characteristic absorption peak at 450 nm. The samples from the different treatment groups were ground up with liquid nitrogen, and 30 mg was added to 400 µL of extraction solution, homogenized, incubated on ice for 10 min, and centrifuged at 12,000 g and 4 °C for 10 min. The supernatant was then collected. The total amounts of NAD+ and NADH were determined according to the instructions provided with the kit, and the NAD+/NADH ratio was calculated.Nicotinamide adenine dinucleotide (NAD) is a coenzyme that exists in all cells and is found in two forms: oxidized .Respiration is the core process of mitochondrial metabolism, and a large amount of free energy is released by oxidative phosphorylation for the production of ATP . In thisP. ostreatus induced by HS, four different treatment groups  were established for RNA-Seq analysis, with 3 replicates in each group and 12 samples in total. For the preparation of RNA-Seq samples described in this paper, the WT strains were cultured at 28 °C on PDA plates for 5 d as the CK group and then transferred to new PDA plates with 100 µM SNP (SNP_HS) or 250 µM cPTIO (cPTIO_HS) treatment according to a previous study [SNP is a donor of NO, and cPTIO is a scavenger of NO. To understand the regulatory mechanism through which NO alleviates the mycelial damage in us study , after wP. ostreatus using the TRIzol® Reagent , according to the manufacturer’s instructions, and genomic DNA was removed using DNase I . The RNA quality was determined using a 2100 Bioanalyzer . The RNA-Seq transcriptome library was prepared using the TruSeq™ RNA sample preparation kit from Illumina  and 1 µg of total RNA. Briefly, messenger RNA was isolated according to the polyA selection method using oligo (dT) beads and then fragmented using fragmentation buffer. Double-stranded cDNA was then synthesized using a SuperScript double-stranded cDNA synthesis kit  with random hexamer primers (Illumina). The synthesized cDNA was then subjected to end repair, phosphorylation and ‘A’ base addition according to the library construction protocol established by Illumina. The libraries were subjected to size selection to obtain cDNA target fragments of 200–300 bp using 2 % Low Range Ultra Agarose followed by 15 cycles of PCR amplification with Phusion DNA polymerase (NEB). After quantification by TBS380, the paired-end RNA-Seq library was sequenced with an Illumina NovaSeq 6000 sequencer (2 × 150-bp read length), and the sequencing data were deposited into the Sequence Read Archive of the National Centre for Biotechnology Information (NCBI) with the accession number SRP277542.Total RNA was extracted from the mycelia of http://deweylab.github.io/RSEM/) was used to quantify the gene abundances [To identify the DEGs between the CK and HS, SNP_HS and cPTIO_HS groups, the expression level of each transcript was calculated using the fragments per kilobase of exon per million mapped reads (FRKM) method. RSEM (undances . Differeundances . In addiaox strains were successfully obtained in our previous study [hyp), which was expressed under the control of the upstream lac promoter. The RNAi-aox plasmid was constructed as follows, the P. ostreatus gpd promoter was PCR amplified, after which the aox-sense and aox-antisence were amplified using primers with homologous arms, as shown in Additional file aox-JC-F/R primers  following an extraction method for fungal samples. First-strand cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. The ChamQ SYBR RT-qPCR Master Mix Kit  and the ABI 7500 real-time PCR amplifier  were used for RT-qPCR. According to our previous study , the RT-P value < 0.05 was considered significant by SPSS software  [In this study, at least three biological replications were performed in each group. All data were recorded using Excel 2010 software . Duncan’s multiple range test was used for all significance analysis, and a IL, USA) . All figAdditional file 1: Fig. S1. Structure of the RNAi-aox vector. Fig. S2 Validation and selection of RNAi-aox strains. A Preliminary selection of RNAi-aox transformed strains by amplification of the hyg gene. B RT-qPCR analysis of the expression of aox in the tested strains. Fig. S3 Regulation of mycelial growth and aox gene expression by different concentrations of BHAM. A Effects of different concentrations of BHAM on colony morphology. B Effects of different concentrations of BHAM on the mycelial growth rate C Effects of different concentrations of BHAM on aox gene expression in mycelia. The values are presented as the means ± SEs from three independent experiments. Different letters indicate significant differences among the samples (P < 0.05 according to Duncan’s test).Table S1 Primers used in this study. Table S2 Reads mapped to the reference P. ostreatus CCMSSC 00389 genome.


Medial opening wedge high tibial osteotomy (MOWHTO) is a widely used surgical treatment option for medial compartmental osteoarthritis with varus deformity. It is important that proper lower limb alignment is achieved. However, there has been no consensus about an optimal alignment in MOWHTO. Most studies suggest that achieving valgus alignment is necessary, and recent studies support slight valgus mechanical alignment of less than 3° of mechanical femorotibial angle. Overcorrection and undercorrection is not recommended for achieving good surgical outcomes. To prevent undercorrection and overcorrection in MOWHTO, the method of placing the weight-bearing line in the target range must be precise. There are several ways to place a weight-bearing line within the target range. While the most important factor for a successful MOWHTO is achieving an ideal mechanical axis correction, there are a few other factors to consider, including joint line obliquity, posterior tibial slope, ligament balancing, and patellar height. Several factors exist that lead to undercorrection and overcorrection. Preoperative amount of varus deformity, lateral hinge fracture, and fixation failure can result in undercorrection, while medial soft tissue laxity and the amount of correction angle and target point beyond hypomochlion can result in overcorrection. This study aimed to review the literature on optimal alignment in MOWHTO and report on the factors to be considered to prevent correction errors and how to achieve an optimal alignment. Medial opening wedge high tibial osteotomy (MOWHTO) is a widely used surgical treatment option for medial compartmental osteoarthritis with varus deformity in relatively young and active patients –4. The mAlthough numerous surgical techniques have been proposed, unintended correction errors still occur after MOWHTO –12. SeveThis study aimed to review the literature on an optimal alignment in MOWHTO and report on the factors to be considered to prevent correction errors and how to achieve an optimal alignment.What is an optimal alignment in MOWHTO?There has been no consensus about an optimal alignment in MOWHTO, and most studies suggest that achieving valgus alignment is necessary to acquire long-term clinical success and prevent recurrence of varus deformity . FujisawRecent studies have different opinions from studies that emphasized valgus alignment, which is more than 3° of valgus mFTA. In a recent biomechanical study , beyond Jakob et al.  suggesteMost studies recommended avoiding undercorrection  or excessive overcorrection to obtain good clinical outcomes , 25. UndIn addition, overcorrection leads to unsatisfactory clinical outcomes. Although there is no clear determination for overcorrection, it could result in cosmetic problems that cause patients to feel dissatisfied and lead to poor clinical outcomes . ExcessiTo prevent undercorrection and overcorrection in MOWHTO, the method of placing the weight-bearing line in the target range must be precise and detail oriented. There are several ways to place a weight-bearing line within the target range. These methods are classified into conventional and navigation-assisted methods, and conventional methods are further classified into preoperative and intraoperative methods according to the timing of calculation of the correction angle.Preoperative methods involve calculating the correction angle using a weight-bearing scanogram with calibration before operations.Line 1 is a planned weight-bearing line that runs from the center of the hip joint to the predicted new center of the ankle joint and passes through the predetermined target point of the tibial plateau. Line 2 runs from the hinge point of the osteotomy to the center of the ankle joint. Line 3 runs from the hinge point of the osteotomy to the predicted new center of the ankle joint. The angle between lines 2 and 3 is the planned correction angle α . Line 2 runs from the center of the ankle joint to the predetermined target point of the tibial plateau. The angle between lines 1 and 2 is the planned correction angle β  was introduced, paying attention to the differences in the anatomical structures of each patient. After making a simulative bone through 3-dimensional printing by taking a computed tomography, the manufacturer makes the instruments, and the surgeon performs the operation using the instruments. Yang et al.  obtainedAn intraoperative method under the guidance of fluoroscopy with a Bovie cable or radiopaque rod is widely used because it is easy and allows real-time monitoring Fig. . HoweverRecently, navigation systems have been introduced for MOWHTO. Several meta-analysis review studies –47 reporDespite these advantages, it has several disadvantages. Navigation-assisted MOWHTO requires additional operation time compared with conventional surgery, and there is an additional cost of purchasing a navigation device. Moreover, an infection may occur through a stab wound made by the pin used for the navigation system . Nha et Among the various planning methods, it is difficult to conclude which method is superior. However, it should be noted that when a surgeon selects an intraoperative method (cable method or navigation-assisted method), the alignment can change after the operation because they do not reflect the weight-bearing state.While the most important factor for a successful MOWHTO is achieving an optimal mechanical axis correction, there are a few other factors to consider Table . In genuThe purpose of HTO is to correct the mechanical axis in the coronal plane, but it unintentionally causes a change in the sagittal plane as well. El-Azab et al.  performeMOWHTO inadvertently causes a change in the position of the patella. In a meta-analysis conducted by Bin et al. , when MOSeveral factors exist that lead to undercorrection and overcorrection, and avoiding them is closely related to the success of MOWHTO, as mentioned earlier Table .Table 2PThe occurrence of undercorrection is related to the preoperative amount of varus deformity. The more severe the varus, the more likely is occurrence of undercorrection. Kamada et al.  divided Depending on the type of plate to be fixed after osteotomy, undercorrection or correction loss may occur. Given that the osteotomy gap must support body weight, it is affected by the stability of the plate fixation. Therefore, stable fixation is an important factor in maintaining the osteotomy gap. Hernigou et al.  reportedLateral hinge fractures can seriously affect the maintenance of the osteotomy gap and lead to delayed union or nonunion and correction loss , 67. AssRecently, researchers have been interested in the discrepancy between the planned mechanical axis and the actual postoperative mechanical axis. For navigation-assisted MOWHTO, which showed better accuracy in bony correction than did conventional MOWHTO, some studies , 71 repoFor successful MOWHTO outcomes, it is important to achieve an optimal alignment through accurate preoperative surgical planning. In addition, it is essential to recognize and pay attention to the correction amount, medial soft tissue laxity, risk factors for lateral hinge fracture, and other factors that can lead to correction errors. Moreover, the characteristics of each patient, such as JLO, PTS, ligament insufficiency, cartilage state, and patellofemoral degeneration, should be considered.


Based on an analysis of the life trajectories of 2510 conscripts and their families from a Spanish rural area in the period 1835–1977, this paper studies the development of the fertility transition in relation to height using bivariate analyses. The use of heights is an innovative perspective of delving into the fertility transition and social transformation entailed. The results confirm that the men with a low level of biological well-being (related to low socio-economic groups) were those who started to control their fertility, perhaps due to the effect that increased average family size had on their budget. The children of individuals who controlled their fertility were taller than the children of other families. Therefore, the children of parents who controlled their fertility experienced the largest intergenerational increase in height (approximately 50% higher). This increase could be due to the consequence of a greater investment in children (Becker’s hypothesis) or a greater availability of resources for the whole family (resource dilution hypothesis). The demographic transition refers to the process where the population of the majority of countries has shifted from a context of high fertility and mortality to a new state of low fertility and mortality . The fercoitus interruptus, abstinence and, to a lesser extent, vaginal douching, pessaries and sponges [This article focuses on the fertility transition in rural Spain, analysing how the reduction of marital fertility developed. Contraception was legally forbidden in Spain for almost the entire period of analysis and was finally decriminalised in 1978. As a result, Spanish women, especially rural women, found it difficult to access any type of synthetic contraceptive ,14. Ther sponges ,16. With sponges . There i sponges ,22,23,24For this article, we understand biological well-being at the individual level as one of the dimensions of well-being linked to nutrition and health. Thus, most anthropometric, such as height or body mass index, and demographic, such as mortality, indicators are linked to biological well-being. When approaching biological well-being in this study from height, we are taking into account the net nutritional status resulting from food consumption, but also the wear and tear caused by the diseases experienced, the workload and the basal metabolism during the years of growth. In other words, it would be the stock of health during the development/growth period. In the study area, historically, height was largely affected by environmental and nutritional factors in the uterus and during childhood.Several studies suggest that fertility control was started by groups with a higher socio-economic status ,26. ThisWe use height as a proxy for biological well-being. Numerous studies have shown that height is a good indicator of nutrition and epidemiological context during childhood and adolescence ,30,31,32This article uses a sample of 2510 conscripts born in 14 rural Spanish villages during the fertility transition and the previous decades. In the area of study, the fertility transition began at the turn of the twentieth century. There was a continuous fall in fertility levels, with the decline in the number of births during the 1930s being particularly important. Fertility continued to fall to below replacement levels in the last third of the twentieth century . FamilieThe analysis uses height data corresponding to military conscripts at approximately 21 years of age and for whom we also have information about family trajectory. Unlike selection biases found in other countries, Spanish military recruitment records include all individuals of that generation. The existence of a universal recruitment system from the 1830s ensured that most recruits were measured, except for fugitives, some migrants and those who had died. Although several legal mechanisms existed between 1837 and 1936 to avoid compulsory military service, it is important to note that all of them were implemented after measurement ,49,50,51The family reconstitution method  was usedThere is extensive knowledge about the evolution of height over time as an indicator of biological well-being. However, we know little about the relationship between this variable and the processes of demographic modernisation that have conditioned the development of living standards and inequality . The populations concerned undergo a process of social and economic change at both national and household levels. These processes can provide useful clues and ideas for identifying patterns in developing country populations and for designing appropriate public health and child nutrition policies. The study takes a long-term perspective of the rural context, allowing us to examine various in-depth stages of generational cohort analysis and the intergenerational transmission of well-being, taking into account the family socio-economic status. Hatton and Martin , in theiThis article is innovative because, for the first time, it connects the process of fertility transition in rural Spain, through an analysis of fertility control strategies, with the study of the biological well-being of parents and children as measured by stature using longitudinal demographic data. This perspective allows us to rethink the transformation of society during the twentieth century and to understand the importance of fertility control for families with low standards of living. The results obtained help us to understand the importance of the fertility transition process in improving the living standards of the poorest groups, shedding new light on why the fertility transition process was a key pillar of per capita economic growth in the twentieth century. This study is ground-breaking in that it allows us to understand the importance of the fertility transition at the individual and family levels.The area of study is composed of 14 rural villages and is located in the region of Aragon, in the northeast of the Iberian Peninsula see . The vilThe population mostly lived in nuclear households and was essentially devoted to agriculture  and sheep grazing. Until the mid-twentieth century, 80 per cent of the male working population was engaged in the agricultural sector , where mAll the agriculture in the area was in non-irrigated areas except for the land near the river Huerva, where fruit and vegetables could be cultivated. In 1947, ‘Las Torcas’ Reservoir was built in the municipality of Tosos and, from the 1970s onwards, irrigation infrastructures began to be developed, allowing agricultural production to be increased ,59.The average fertility was relatively stable at around 6–7 children per family; this lasted up to 1900 and declined rapidly thereafter, following the fertility transition. The study area was a high mortality region . Mortality rates began to decline in the second half of the nineteenth century due to increasing living standards. Anthropometric evidence indicates that standards of living were low–the average male height was around 160 centimetres in mid-nineteenth-century, well below that of their European counterparts or their fellow Spaniards in other regions ,62.Aragon (the region) underwent a process of economic modernisation from the second half of the nineteenth century, which continued for most of the twentieth century despite economic and social shocks ,64. ThisThe following three types of data have been used: 1. height data drawn from military conscription records; 2. individual demographic data drawn from parish registers (up to 1950), surveys (from 1950); 3. socio-economic data on occupation and literacy drawn from censuses, population lists and parish registers. We used height data for the military conscripts enlisted in the 14 villages mentioned in During the period of study, the conscription age varied over time. During the period 1856–1885, the age of military conscription was 20 years old; between 1885 and 1899, it was 19 years; between 1901 and 1905, it was 20; and between 1907–1939, it was 21 years old. Thus, we have standardised the average height to the age of 21 years. To do this, we have used the same strategy as Ayuda and Puche-Gil  based onThe demographic event analysis is based on the complete church registers of these 14 villages. These records provide high-quality information on all baptisms, marriages and deaths that occurred between the sixteenth century and 1950, although the starting date varies by location. For more details about the ‘Alfamén and Middle Huerva Database’, see ,76,77. TThe occupation of the individuals analysed and their fathers were taken from population lists (1857 and 1860), electoral censuses , and parish registers. Information on profession, and height was linked to population records for each individual. We have used bivariate analysis to determine the height-group to which the pioneers of the fertility transition belonged, how fertility control affected the average height of the children and whether the number of family members living in the same village affected the possibilities of controlling fertility and the biological well-being of the children. The results of these analyses reveal the origins of the fertility transition and whether there was a relationship between height and the different stages of the transition. Furthermore, in the This study uses descriptive statistics and statistical tests, such as confidence intervals, to approach the main question of the article on the relationship between fertility control and height. Therefore, most figures and tables have, as the main variable studied, the average height of a given group of individuals according to the birth cohort and their individual characteristics. Other variables studied are the average number of children and the average age at a given event. This simple methodology allows us to make a first approximation to the main question taking into account the small sample size available and the available variables. However, this sample size (together with the variability of heights between human beings due to genetic and other factors) is also sometimes a problem. t-test approximation allowed us to validate that our approximation is not erroneous overall, although unfortunately (possibly due to the sample size in some periods), the p-values are higher than 0.05 in the early decades, so the results for these decades should be taken with extra caution. In any case, with this methodology, we mainly want to study the temporal evolution (by decades) and trends of some groups with others. In our case, the trends are clear and consistent with what would be expected from the evolution of height and the fertility transition. If we take into account the 95% confidence intervals of the control families and the rest, most results (with means) are not statistically significant. However, a In addition, we performed six multivariate linear regression models OLS with heteroskedasticity-robust estimation linking height and fertility control in which the dependent variable is the height of the individual (in millimetres). The objective is to confirm whether there is a relationship between height and parental fertility control, controlling for various socio-economic, health and family factors. Model (1) includes fertility control, locality of residence and decade of birth as independent variables. Model (3) builds on model (1) and also includes parental socio-economic status and individual literacy. Model (4) also includes the claims made by the individual not to participate in compulsory military service. Model (6) includes all of the above variables plus the number of living siblings . Model (2) is similar to model (3) but without controlling for decade of birth and village. In the same way, model (5) is similar to model (6) but without controlling for decade of birth and village. Model (6), which is the most complete, can be expressed as follows:Stopping was the most common fertility control strategy during the fertility transition in Western countries, and the strategy most clearly associated with fertility control ,81,82,83Because of its characteristic of abruptly detaining fertility, stopping is easier to identify than other fertility control strategies such as spacing . MoreoveTherefore, from this point onwards, and for the purpose of this study, we will use the imperfect criterion that families who stopped having children before the woman reached the age of 36  were the families that voluntarily controlled their fertility.First, we will briefly examine the period before the fertility transition and compare it with the transitional period. This requires an in-depth analysis of the intergenerational transmission of height between fathers and sons periods and a comparison of whether there was any modification during the fertility transition. In First, we will analyse who the pioneers in controlling fertility by using the stopping strategy were in relation to their biological well-being. In this way, we can determine whether they were the local elite individuals . In These results seem to suggest that it was mostly individuals from poorer households  who had more incentives to control their fertility. As we will discuss in the next section, these results seem to contradict the perception that it was local elites who were the pioneers of the fertility transition. In fact, in Stopping proved to be a very useful strategy for reducing marital fertility. Demographic factors such as the fertility rate, infant mortality or the type of household , decisively condition the availability of women for paid employment: the number of hours, the times of their life cycle and the type of employment in which they can occupy. And it also explains the relationship between the massive diffusion of contraceptive means in the second half of the 20th century and the fall in fertility and the continued increase in the female activity rate in all developed countries.Moreover, we know that the differences in the decision to control fertility were not due to different ages of entry into marriage since, as Without seeking to carry out an exhaustive analysis, we can refer to different authors who have conducted research on the issue for the period of study. For the nineteenth century, studies have been carried out by Camps , BorderíThis subsection addresses the relationship between height and fertility control from the perspective of children and the influence that the fertility decisions had on them. As we can observe in What effect did stopping have on the biological well-being of children? To answer this question, The results are particularly interesting if we take into account that short individuals could be the pioneers of the fertility transition in the study area. As we have shown, before the fertility transition, parents of the lowest tertile of biological well-being mostly passed this condition on to their children. However, once the transition began, parents with a low level of biological well-being were those who controlled their fertility and started having children of above-average stature. That is, the fertility transition served as a mechanism to break the cycle that had condemned parents and children to remain at similar levels of biological well-being from one generation to the next. The children of controllers achieved mean heights above those obtained by their peers of other parents, regardless of whether socio-economic status is controlled for. The results in To ensure that there was a relationship between fertility control and height, we have developed six multivariate (OLS) linear regression models with heteroskedasticity-robustness estimations controlling for various socio-economic, health and family factors see . The depThis last subsection examines whether the number of immediate living family members  residing in the same village as the children (not necessarily in the same house) had any influence on the likelihood of their parents reducing their fertility using the stopping strategy during the fertility transition. Several studies have demonstrated the importance of kin, and most especially grandmothers, in children’s well-being ,92,93. THowever, when we studied whether the number of relatives had any impact on the decision to control, as we can see in The results obtained in this study help us understand the effect of fertility control on the biological well-being of those born during the twentieth century in the study area. Research carried out for different Western countries, and other large areas tend to confirm that it was families belonging to the urban elite classes who began to control their fertility ,25. MoreClassical fertility theory suggests that it was the increase in family size and its pressure on the family budget that led families to control their fertility ,27. Due The results obtained here also show that before the fertility transition, individuals with a lower level of biological well-being tended to mostly have children with a low level of biological well-being, and vice versa. Therefore, a vicious circle ensued in which levels of biological well-being were transmitted from parents to children . However, the trends in the results are clear and permanent, which was our objective in using this methodology in the study. Therefore, further studies with larger sample sizes are needed to confirm or refute our tentative findings.Additionally, the small sample size (and data availability) conditioned our decisions on methodology, analyses performed and depth achieved. In fact, as we mentioned when discussing methodology, and would like to point out again, the descriptive results in several cases have problems of non-significance due to the small sample size see . UnfortuTo complement the analyses with descriptive statistics (and the associated problems described), we developed an analysis with six multivariate (OLS) linear regression models that, in all cases, confirm a strong correlation between fertility control and height in the study area. Individuals who controlled for fertility had taller children than their contemporaries from families in the area who were not included in the fertility control group. However, more research is needed to further investigate this relationship.This paper analyses the fertility transition at the individual level in a small Spanish rural area using height data  at age 21. The objective is to determine the pioneers of the fertility transition and the effect that controlling fertility had on the biological well-being of the next generation. In other words, rethinking the fertility transition in relation to height (as an indicator of living standards).Traditionally, prior to the fertility transition, there was a positive relationship between height and socio-economic status ,39,40,41Stopping fertility had positive effects on the next generation. The children of families that controlled their fertility were, on average taller than the children of the rest of the children. In fact, the intergenerational increase in height during this period of economic modernisation was 50% higher among parents who controlled their fertility. That is, fertility control was strongly beneficial at the level of biological well-being for children, especially for the children of individuals from the lowest socio-economic groups. In the area of study, the fertility transition could have constituted a way to break the traditional poverty circles.


Drug-induced changes in urine color induced by drugs may have clinical significance. Pink urine syndrome (PUS), which has been associated with urinary uric acid (UA) disorders, is most frequently reported in patients with morbid obesity undergoing gastric bypass surgery and/or from propofol anesthesia use in those who potentially have preexisting UA metabolism disorders. However, PUS has rarely occurred following exposure to propofol in non-obese patients, and literature on long-term follow-up after PUS is scarce. We report a case of PUS induced by propofol in a previously healthy non-obese woman after undergoing thoracoscopic wedge resection of pulmonary nodules under general anesthesia using propofol. The patient suddenly developed pink urine 4 h after surgery. A pink sediment rapidly precipitated at the bottom of the test tube following centrifugation of the urine. Amorphous, colorless UA-like crystals were identified under a polarizing microscope. The diagnosis of PUS was confirmed by examining the urinary UA concentration. The patient recovered and as followed-up for 1 month, during which she did not experience any urinary complications. To our knowledge, this is the first report to describe in detail a case of PUS caused by propofol in a non-obese patient with follow-up. PUS is usually benign and can resolve by rapidly on administering lactated Ringer’s solution; however, the potential risk of urinary complications, particularly UA lithiasis, should be fully realized. Urine discoloration caused by chemicals or drugs is an important clinical symptom that indicates a potentially dangerous condition, which may also be disturbing for patients and clinicians . PropofoA 41-year-old woman with suddenly excreted pink urine after undergoing thoracoscopic wedge resection of pulmonary nodules under general anesthesia. Her medical history otherwise was not significant. She reported that the pink substance was initially seen on the walls of the indwelling urethral catheter 4 h post-operatively. It gradually increased and ultimately stabilized. She was otherwise well and experienced no significant urinary irritation. The following morning, we observed pink urine in the urinary drainage bag and numerous pink crystals on the walls of the catheter . The pat−2 and her temperature was normal. Renal percussion revealed no pain and no other abnormalities were found. Preoperative laboratory investigations showed a serum uric acid (UA) concentration of 218 μmol L−1 , morning urinary pH of 6.5  and UA concentration of 3,320 μmol L−1 . On postoperative day 1, the serum UA concentration was 166 μmol L−1, morning urinary UA concentration was 4,825 μmol L−1 and urinalysis revealed a pH of 6. Preoperative and postoperative urinalyses and renal function tests showed no other abnormalities.On examination, the patient had a body mass index of 25.71 kg m−1, lower than the real value due to UA precipitation on the catheter walls) and decreased in pH (5)  .The patient had received no premedication, and general anesthesia had been performed using propofol, dexmedetomidine, sufentanil, cisatracurium, sevoflurane, and remifentanil. Postoperative analgesia was achieved using oxycodone, sufentanil and parecoxib sodium. The patient did not receive any other medications or treatment up to the point of excretion of pink urine, apart from the routine fluid therapy. The concentration of UA in the patient’s morning urine, 4 days post-operatively, was within normal limits . The patPUS is a rare condition with uric acid metabolism disorders characterized by strawberry-red urine and the formation of massive pink sediment . Pure UAA receptor, and cause sedative and hypnotic effects  receptor agonist. It can potentiate the response to the inhibitory neurotransmitter GABA at the GABA effects . Propofo effects . Propofo effects . The exa effects . Therefo effects .via its V1 receptor, favors renal UA clearance and causes UA crystalluria (Other risk factors, including V1 receptor activation, low urinary pH and high urine osmolarity, have been implicated in PUS . Stress-talluria . A low utalluria . The redtalluria . Althougtalluria .To our knowledge, this is the first case report to describe in detail the development of PUS induced by propofol with follow-up in a non-obese patient. Understanding the nature of PUS allays distress among patients and clinicians, prevents unnecessary testing such as urinalysis, urine cultures and blood cultures, and enables prompt treatment . ClinicaIn conclusion, PUS can occur in non-obese patients with no preexisting UA metabolic disorders. Propofol is a key risk factor for PUS in such patients, which directly increases colorless UA excretion in urine and promotes pink urinary pigment production through bilirubin metabolism. High urinary excretion of UA crystals may be transient with no toxicity to the human body. Therefore, unnecessary testing should be avoided. Despite its benign nature, prompt intravenous fluid therapy is still necessary to prevent urinary complications.


Although 37.5–51% of transgender adults state they would’ve considered freezing gametes before gender-affirming therapy if offered and 24–25.8% of transgender adolescents express interest in having biological children, less than 5% of transgender adolescents have opted for fertility preservation. We sought to assess fertility preservation utilization in our multidisciplinary adolescent gender clinic. We also aimed to identify fertility preservation utilization and interest among non-binary adolescents and young adults. A retrospective review was conducted of patients seen in the Stanford Pediatric & Adolescent Gender Clinic from October 2015 through March 2019 who were >10 years of age at initial visit. All individuals with documented discussion of fertility preservation were offered referral for formal fertility preservation consultation but only 24% of patients accepted. Only 6.8% of individuals subsequently underwent fertility preservation (n = 9). Transfeminine adolescents are more likely to pursue fertility preservation than transmasculine adolescents (p = 0.01). The rate of fertility preservation in non-binary adolescents did not significantly differ from those in transfeminine adolescents (p = 1.00) or transmasculine adolescents (p = 0.31). Although only one non-binary individual underwent fertility preservation, several more expressed interest with 36% accepting referral (n = 4) and 27% being seen in consultation (n = 3). Despite offering fertility preservation with designated members of a gender clinic team, utilization remains low in transgender adolescents. Additionally, non-binary adolescents and their families are interested in fertility preservation and referrals should be offered to these individuals. Further studies and advocacy are required to continue to address fertility needs of transgender adolescents. Gender variance, also referred to as gender expansiveness, is a situation in which a person’s sex assigned at birth is incongruous with the gender with which they identify , 2. GendAs gender-affirming hormone therapy in adolescents increases, fertility preservation has become an area of increasing concern for parents, adolescents, and their gender healthcare providers due to the potential for irreversible changes in fertility. Long-term use of these gender-affirming sex hormones may result in infertility though it remains somewhat uncertain what duration of hormonal treatment produces irreversible effects –11. ReseAlthough prior studies assessing fertility preservation utilization in adolescents and young adults have primarily focused on transfeminine and/or transmasculine individuals, there are some data in non-binary populations which demonstrate a strong interest in fertility discussions. In a survey conducted of Australian non-binary and transgender adults, 95% felt that fertility preservation should be offered to all transgender and non-binary people and 7% reported having undergone a fertility preservation procedure . BelgianThis study sought to assess the fertility preservation utilization rate when services were offered as part of a multidisciplinary pediatric gender clinic team, compared to previous studies when external referrals were provided. In addition, we aimed to investigate how non-binary individuals utilize fertility preservation as no study to date has assessed fertility preservation practices in non-binary adolescents and young adults.This review of electronic medical records was conducted of all patients seen in the Stanford Pediatric & Adolescent Gender Clinic from October 2015 through March 2019 who were >10 years of age at time of initial visit. Prior to initiation of data collection, the protocol was submitted to the Institutional Review Board and granted exemption for formal protocol review and approval. Individuals were excluded from the review if they were pre-pubertal , not interested in beginning gender-affirming therapy, or had previously initiated gender-affirming therapy with another gender healthcare provider. Gender-affirming therapy was defined as the use of GnRHa and/or gender-affirming sex hormones. Information obtained from chart review included patient age at clinic visit, sex assigned at birth, affirmed gender, outcome of fertility preservation counselling, and reasons for declining fertility preservation if documented.The Stanford Pediatric & Adolescent Gender Clinic is a single, multidisciplinary clinic team which consists of on-site members specializing in pediatric endocrinology, adolescent medicine, gynecology, urology, psychology, and social work. In addition to these on-site members, there are also designated team members, including a Urologist specializing in male infertility and a Reproductive Endocrinology and Infertility (REI) specialist, located one floor down from the Gender clinic. If patients express interest in fertility preservation, a referral is placed to this specific adolescent fertility and REI specialist or our designated infertility urologist whose clinics are located in the same building.Descriptive statistics were used to summarize the included patients and the subgroups , with frequencies for categorical variables and mean plus range for continuous variables. The comparisons among subgroups were performed using Fisher’s exact tests with a significant level as p < 0.05. All statistical analyses were conducted using SAS 9.4 .Of the 184 eligible patients seen during the study period, 132 met inclusion criteria . Of the All patients received fertility counseling as part of the written and verbal consent for gender-affirming hormone therapy though only 89% of the study patients (n = 117) had documentation of this discussion in their medical records, including documented offer of referral to fertility specialist. Only 31% of patients (n = 41) accepted the referral for formal consultation and of these individuals, only 36.6% of them (n = 15) were seen for an appointment. A total of 9 patients ultimately underwent fertility preservation (2 oocyte and 7 sperm), making the overall rate 6.8%. Only 12 individuals had documentation of reasons for declining fertility preservation which included lack of interest in having children and/or biological children , concern about delay in treatment or need for further pubertal progression prior to fertility preservation , possible plans for future discontinuation of gender-affirming hormones , and discomfort with the idea of masturbation for semen sample collection . Two individuals also expressed a potential desire to stop sex hormone therapy in the future for fertility preservation or to have children later in life.Fertility preservation by gender identity is depicted in A total of 11 non-binary patients met inclusion criteria for this study. All of these individuals had documented discussion of fertility preservation but only 4 accepted referral for fertility preservation. Three non-binary patients were seen in formal consultation for fertility preservation and only 1 patient (~9.1%) ultimately underwent a fertility preservation procedure. Due to difficulty producing a sample through masturbation, this patient underwent electroejaculation under anesthesia to obtain a sample for sperm cryopreservation.In this study, transfeminine adolescents and young adults were significantly more likely to pursue fertility preservation than transmasculine adolescents (p = 0.01). The rate of fertility preservation in non-binary individuals did not significantly differ from those in transfeminine individuals or transmasculine individuals .Despite the inclusion of designated gender care team members who specialize in fertility preservation, utilization among transgender adolescents in the US remains low at 6.8% in this study population . Only 24The relatively low overall rate of fertility preservation in our study, 6.8%, is consistent with prior studies in adolescents from US and Canada-based studies –19, despAs in prior studies, the rates of fertility preservation were higher in transfeminine adolescents compared to transmasculine adolescents  , 18. ThiDespite the creation of a multidisciplinary gender clinic with designated fertility preservation providers, challenges navigating the system remain a barrier to fertility preservation in our transgender population. One patient had their referral to fertility preservation denied as they had already updated their name and gender marker  and the referral was inappropriately flagged as in error. Although this error was remedied with resubmission of the referral, it created a delay in approval. Continued education in workflow and streamlining of the referral process is required to ensure effective and complete healthcare is provided to transgender and gender non-conforming individuals.Limitations of this study include its retrospective design and relatively small and unevenly distributed study population. Additionally, if patients independently sought fertility preservation and did not inform their gender healthcare provider, they may not have been included in total numbers. These reported data also do not include 1 patient who had already undergone fertility preservation prior to initial clinic visit. Additionally, the reasons for declining fertility preservation were obtained through chart review, rather than directed interview, and thus likely represent incomplete data.The majority of currently available fertility preservation techniques require the child undergo a substantial amount of pubertal progression of the “noncongruent” gender, which includes some irreversible changes, before gamete preservation is possible. Whole gonad cryopreservation with in-vitro activation is an area of ongoing research which may potentially allow fertility preservation without requiring pubertal progression though this is not currently a clinically viable option –32. FurtDespite increased attention in recent years, the field of pediatric and adolescent transgender fertility remains an understudied and under-discussed area of medicine. Fertility preservation utilization in adolescents seeking gender-affirming hormone therapy is low across all gender identities despite prior reports of high levels of interest in adult transgender individuals. Non-binary adolescents, like their transfeminine and transmasculine counterparts, have an interest in fertility preservation and should be offered the same counseling and opportunities. Continued work in the field is required to increase fertility preservation options in this population. Ongoing nationwide advocacy is also required to promote insurance coverage of fertility preservation to reduce the impact of cost on decisions about fertility.S1 Data(XLSX)Click here for additional data file.


A unique form of melanosomes contributing to brilliant iridescent colors in modern bird feathers, previously unknown in fossil birds, is identified in the Early Cretaceous bird Eoconfuciusornis. The discovery highlights the complexity of plumage color nanostructures utilized early in bird evolution as far back as 130 million years ago. Various plumage colors are roughly produced by pigment-based colors and structural colors ,2. BrillStudies of fossil melanosomes give us a unique opportunity to explore potential patterns of melanosome diversification, and help to reconstruct the evolution of avian plumage color. Fossil melanosomes were first detected in preserved feathers more than a decade ago , which hEoconfuciusornis specimen with Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) analyses . The fossil melanosomes are clearly visible as hollow rods with air holes that are roughly circular in cross section  exhibit numerous morphotypes relating to sexual display (e.g. [Eoconfuciusornis and Confuciusornis, the pennaceous rectrices in Caudipteryx, the ribbon-like tail feathers in Epidexipteryx and the rectricial fan combined with elongated central paired pennaceous plumes in Yuanchuornis. The available fossil evidence indicates that birds had diversified globally by the Early Cretaceous [Eoconfuciusornis may have also contributed to evolution through sexual selection, with enhanced visual signals afforded by diverse plumage colors.It is probable that the hollow melanosomes in ay (e.g.  and refeetaceous . In addietaceous , the occnwab227_Supplemental_FileClick here for additional data file.


Assessment of male fertility is based on the evaluation of sperm. Semen evaluation measures various sperm quality parameters as fertility indicators. However, semen evaluation has limitations, and it requires the advancement and application of strict quality control methods to interpret the results. This article reviews the recent advances in evaluating various sperm-specific quality characteristics and methodologies, with the help of different assays to assess sperm-fertility status. Sperm evaluation methods that include conventional microscopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis, provide precise information related to sperm morphology and function. Moreover, profiling fertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility. Identification of different sperm proteins and diagnosis of DNA damage has positively contributed to the existing pool of knowledge about sperm physiology and molecular anomalies associated with different infertility issues in males. Advances in methods and sperm-specific evaluation has subsequently resulted in a better understanding of sperm biology that has improved the diagnosis and clinical management of male factor infertility. Accurate sperm evaluation is of paramount importance in the application of artificial insemination and assisted reproductive technology. However, no single test can precisely determine fertility; the selection of an appropriate test or a set of tests and parameters is required to accurately determine the fertility of specific animal species. Therefore, a need to further calibrate the CASA and advance the gene expression tests is recommended for faster and field-level applications. Fertiliitations . Semen aitations  and variitations  are wideSemen primarily consists of two main components: the sperm and seminal fluids; sperm are produced in the seminiferous tubules of the testicles, and the seminal fluids, in the accessory sex glands and excurrent ducts. Both of these components are examined in semen analysis in terms of sperm count and semen volume . Semen aFor a long time, semen evaluation was believed to be the single most important laboratory test for assessing male fertility  however,The structure of sperm is peculiar, and functions to transfer the male gametes and required genetic information , equippeIn bulls, semen quantity, quality, and/or health status are responsible for a significant percentage of reproductive failure in cattle production . As AI iIn domestic animals, fertility indicators are affected by several factors, including the host environment and nutritional factors; however, in this review, we have focused solely on semen evaluation methods. Semen evaluation can be conducted at different levels, that is semen and sperm, or by assessing the mechanical, physical, or functional characteristics of sperm , which dThis article reviews the developments in current semen analysis methods and the knowledge obtained from determining the fertility of sperm. Therefore, we aimed to review the progress in semen evaluation techniques and to make conclusive remarks regarding recent advances in semen evaluation of domestic animals. This review focuses on the reliability and limitations of semen analysis for the diagnosis of fertility.Determination of sperm concentration, together with the assessment of motility and morphology, is an important method to determine fertility. Sperm concentration per mL of semen can be determined by counting the sperm in the chamber of a hemocytometer, a microcell, or a photometer. However, there are possibilities of underestimation and overestimation of sperm numbers depending on the types of products used . The calA study comparing various methods used for the determination of sperm concentration revealed that flow cytometry was the most precise method . Howeverin vivo and in vitro, and it is an integral part of sperm functional test [Morphometric characteristics of sperm are one of the most important indicators of fertility. It is universally accepted that sperm with normal morphology have a significant effect on fertility both nal test –40. Abnonal test . Differenal test . This syThe advancements in sperm motility assessment include the use of the light microscope and phase-contrast microscope, using 20 and 40× objectives, which are reported to yield substantially good results . The micPhase-contrast microscope is generally used to determine mass motility or progressive sperm motility . Mass moPR is usually expressed as a percentage of sperm motility, which is suggestive of sperm fertility, indicating proper spermatogenesis and maturation of sperm during epididymal transit . In humaVarious factors can affect the results of phase-contrast microscopy, such as, magnification, working conditions , personal experience, and environmental factors ,59. SomeThe CASA is the first major system to analyze motion characteristics of sperm. It provides instant quantification of sperm motility and concentration. CASA is relatively better than phase-contrast microscopy as it can generate accurate and highly reproducible data of different kinetic parameters of sperm ,61. FurtIn domestic animals, commercial use of a fully automated CASA system was introduced in 1985 . In the in vivo [in vitro [CASA generated motility data of sperm, (percentage and concentration of motile sperm) which is very helpful for the prediction of fertility in humans in vivo  and in vin vitro . The devin vitro ,71.6 sperm/mL has been recorded in some CASA studies [However, the CASA system has some limitations, affected by the higher sperm concentration, which interferes with progressive motility ,72,73. D studies –77.In addition to sperm motility and concentration, the CASA system can analyze different kinematic parameters including curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), wobble (WOB), beat cross frequency (BCF), and amplitude of lateral head displacement (ALH). VCL analyzes the velocity of a sperm head on time-average basis along its actual curvilinear path (measured in in two dimensions). VSL measures the velocity of a sperm head on a time-average basis along the straight line between its first and last detected position. VAP measures the velocity of a sperm head along its average path on a time-average basis. This path is computed by smoothing the actual path according to algorithms in the CASA instrument, these algorithms vary among instruments. ALH measures the magnitude of the lateral displacement of a sperm head about its average path. This can be expressed as the maximum or an average of such displacement . LIN estALH and BCF are measurements derived from VAP, where ALH is the amplitude of variations of the current path of the sperm head in its relationship with VAP, and BCF is the average rate at which the actual sperm trajectory crosses the VAP (a derivation of the true frequency of flagellar beat and frequency of rotation of the head). Although, CASA is a new technology and most of the progression of the sperm measurements are derived from the speed/velocity measurement, we did not find studies showing their correlation with fertility. Despite this, we recommend further studies should be performed on how these measurements can be utilized for assessing sperm fertility, unless there is no need to measure.Rather than the PR, VAP is preferred to evaluate and predict the fertilizing potential of fresh or post-thawed bull semen . The evaThe FAST method analyzes the movement of sperm by using the FAST program. FAST measures the flagellar beat frequency and the tangent and curvature of the flagellar wave to determine the arc-length (true length of the track). The analysis is usually conducted at higher frame rates, and tracks are represented quantitatively in three dimensions . The FASD) system analyzes sperm tracks in three dimensions ; Z-axis is reconstructed as the sperm of most animal species are swimming in a spherical helix [D method has the disadvantage that the Z-axis is presumed to be harmonic, but in most cases, it is not possible. In the 3D patterns, there are clear differences, such as, in a bull sperm with a much higher speed and ALH, the helix diameter is almost twice that of boar sperm. A sperm of Saanen goat has similar VCL as that of a bull sperm, however, the BCF is an important factor here, showing small stepwise increases in the Z plane in Saanen goat sperm, in comparison to the larger Z-plane increases observed in bull sperm [D method is useful in describing qualitative differences, it is not necessarily useful in describing quantitative differences. However, there is a need for further investigations to uncover the correlation between 3D measurements and fertility indicators.The three dimensions  over the first 30 μm length of the flagellum. There is a decrease in sperm flagellar beat frequency as it becomes capacitated and hyperactivated, resulting in tumbling sperm which shows swimming star spin patterns [In combination, the findings of FAST and 3patterns . In genepatterns .The CASA analyzes sperm motility, which is directly correlated with fertility reported for bovine , equine D sperm evaluation.The results of CASA should be defined in terms of the measurement conditions that are correlated with the outcome figures. In particular, the rate of image acquisition, time for tracking sample, smoothing algorithm, sperm concentration per sample, chamber’s type and depth, model and software version, microscope optic and magnification should be clearly defined to interpret CASA results to determine fertility in different species and conditions –97. The Over the decades, the CASA system has developed protocols to efficiently analyze the kinematic parameters of sperm in animals ,99. HoweD reconstruction, and tail analysis. This will further enhance our understanding of sperm functionality.Sperm morphology needs to be better elaborated by including the entire cell and especially understanding the tail characteristics by polychromatic staining in CASA. There seems to be a need for agreement in the selection of stains for use in conjunction with CASA for tail-characteristic studies. In future, developments could lead to no further need for staining and looking at the possibilities of more detailed analysis by using phase-contrast and Nomarski differential interference contrast optics. It is also expected that the CASA system will develop a new automatic analysis for new tests such as reactive oxygen species (ROS), mitochondrial assay, sperm maturity, sperm chromatin packaging, 3The integrity of the plasma membrane is essential for proper sperm function and fertility, pertaining to its function to maintain homeostasis ,102, proPlasma membrane integrity is usually analyzed for a viability test, as it is pivotal for communication with other cells and the environment . MethodsSperm viability is a key factor for quality analysis and a prerequisite for success in fertilization, particularly in cases, such as AI, where low sperm numbers are used . Eosin–nPisum sativum agglutinin (FITC-PSA) [Acrosomal integrity is one of the determining factors for fertility because, for successful fertilization, sperm must have an intact acrosome and must react on time when they reach the site of fertilization –124. TheITC-PSA) . FITC-PNITC-PSA) , servingITC-PSA) . The limITC-PSA) . Later, 6(3) probe can also be used, which has increased fluorescence compared to the JC-1 probe but lower specificity toward mitochondria [Abnormal mitochondrial integrity or functionality in the supra structure or mitochondrial genome and low membrane potential of mitochondria or the change in level of oxygen consumption, are important for sperm function, affecting motility  and matuchondria .The most common analytical tests used to assess sperm-DNA integrity include DNA fragmentation tests, measured by DNA fragmentation index (DFI) or sperm chromatin structure assay, and protamination, measured by sperm protamine deficiency assay . These tThe chromatin integrity of sperm is vital not only for fertility, but also for subsequent maintenance of gestation and offspring fitness . The speMoreover, sperm chromatin integrity can be analyzed by detection of protamine-deficient sperm. During spermatogenesis, histone proteins are replaced by protamine to acquire a tightly packaged chromatin structure . Sperm wThe ROS has both physiological and pathological roles, and an optimal amount is required for normal sperm function and fertilization . OxidateThe ROS levels can be determined directly either by measuring the ROS levels or oxidation-reduction potential, or indirectly by measuring molecules, peroxidation, or DNA damage . In huma2+) to the sperm perinuclear and neck regions and the flagellum, and the generation of controlled amounts of ROS [2+ levels, and Indo-1 acetoxymethylester (Indo-1 AM) has been used to measure intracellular Ca2+ in sperm with the help of flow cytometry [Sperm maturation and capacitation are prerequisites for successful fertilization and are regulated by protein phosphorylation , the infs of ROS . Capacitytometry  or fluo-ytometry ,165,166.Flow cytometry is a high-throughput technique that enables the analysis of thousands of sperm within seconds, capturing many physiological features of each sperm and can analyze sperm viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, OS markers, DNA damage, sperm count, and sperm size ,167. In The viability and acrosomal status of sperm are vital in terms of determining sperm fertility. Cytometry can be used to assess the viability and acrosomal status of sperm. Viability of sperm can be assessed using PI and ethidium homodimer stains  or a comSeminal plasma is a biological fluid contributed by the different accessory sex glands of male individuals. Seminal plasma is the supernatant recovered after centrifugation of liquefied semen, which constitutes >90% of semen . It servAs with the proteomic analysis of blood plasma, owing to the wide dynamic range of protein concentrations, identification of low-abundance proteins in seminal plasma by mass spectrometry is challenging. Particular obstacles are the high-abundance proteins expressed in the seminal vesicles and prostate . PolyacrLiquid chromatography analyzes the presence of specific metabolites associated with sperm fertility . MetabolAssessing metabolites could be helpful in estimating a bull's fertility even without manipulating sperm . This teThe seminal plasma examination in bulls can be conducted using a proton nuclear magnetic resonance spectrometer. This method showed that citrate and isoleucine were low, whereas tryptamine/taurine and leucine were greater in high fertility bulls than in low-fertility bulls, which can be identified by peaks in seminal plasma . In seruGene expression pattern of the sperm, in terms of defined fertility indicators, was found to be an important indicator of fertility. The transcriptomic profiling of human sperm assumes significance as it carries information about spermatogenesis, sperm function, and paternal roles in post-fertilization events . In boviPRM1), insulin like growth factor 1 (IGF1), bone morphogenetic protein 2 (BMP2); sperm function are, testis specific serine kinase 6 (TSSK6), cysteine-rich secretory protein (CRISP), heat shock transcription factor Y-linked (HSFY2); fertility are, ubiquitin-conjugating enzyme E2 D3 (UBE2D3), integrin-β, leucine decarboxylase 1 (LDC-1); and embryonic development are, miR34c-5p, B-cell lymphoma-2-like protein 11 (BCL2L11), breast cancer type 1 (BRCA1). The most abundant translated bovine transcripts are binder of sperm 3 (BSP3) and spermatogenesis associated 18 (SPATA18), which are involved in the regulation of germ cell development and the maintenance of chromatin integrity during spermatogenesis, respectively [In bovines, the genes related to spermatogenesis are protamine 1 , pregnancy-associated glycoprotein 5 (PAG5), PAG7, and PAG10, were abundantly transcribed in sperm, suggesting their possible role in implantation and placental development [TEKT1) is essential for oocyte genome activation leading to successful fertilization, and any defects in this process might lead to male infertility [MAP7, PTK2, PLK1S1, microtubule associated protein 7 (MYH9), and protein kinase C zeta type (PRKCZ) are essential for spermatogenesis and sperm function [Improved gene expression test, in lieu of the conventional and CASA assessments, is associated with phenotypic traits of developing embryos and offspring, and can identify idiopathic infertile males ,197. Forin axons . Furtherelopment . The proertility . MAP7, Pfunction .Polymerase chain reaction (PCR)-based techniques are best due to their more quantitative nature, but their major limitation is the need for prior knowledge of the gene sequences to be assessed ,200. MosCryopreserved semen can be assessed for its general characteristics by CASA, but to assess the phenotypic normality of sperm, we propose that further gene expression assessment be done, particularly if the genetic materials in the same cycle are going to be used in wider scales –204. GenSemen analysis remains the only choice for accurate prediction of fertility in males. The advancement in semen evaluation methods still requires refinement, species-specific standards, and quality control. The prediction of fertility in interspecies and/or intraspecies by a single analytical method is not yet possible due to the complexity of different tests. Various methods can be used to estimate sperm quality parameters, even though few techniques require shorter time. Various techniques have their own pitfalls and the possibility of variability; a combination of tests is therefore required to achieve high correlations of test results with fertility. The CASA system mainly assesses motion characteristics of sperm and despite the addition of morphology and concentration parameters, it is impossible to evaluate all sperm quality parameters for species with relatively small head size at the same time. Moreover, the major problem with the automatic CASA system is the inability to differentiate sperm from cells, cytoplasmic droplets, and cellular debris of similar size.The flow cytometry method shows high sensitivity and reproducibility for DNA analysis but requires expensive equipment and proper training. Similarly, seminal plasma spectrometry reveals the results of the actual metabolism and is more accurate in fresh semen, but it was not well determined for most species. Gene expression demonstrated a marked improvement in fertility prediction, enabling the measurement of DNA fragmentation and the normality of acrosome, particularly in post-thaw sperm. However, the requirement of molecular laboratories, capital inputs, and more skilled personnel limits its use for research purposes only. We recommend the use of seminal plasma metabolite analysis for different animal species as the last level of fertility assessment.Finally, we can conclude that the conventional semen analysis must undergo quality control measures, and reference values should be followed for clinical interpretation of results. Similarly, the primary basis of semen assessment requires constant visual supervision, calibration by trained personnel, and objective evaluation. Therefore, it is recommended that novel tests should be incorporated into the clinical andrology.