Naegleria ssp. Naegleria species are free‐living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so‐called “brain‐eating amoeba.” Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free‐living relatives.The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome‐mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including  NAEGLERIA gruberi is a free‐living, heterotrophic, microbial eukaryote, and a close relative of N. fowleri, the so‐called “brain‐eating amoeba”. It is a nonpathogenic member of the excavate supergroup, which contains key pathogens such as Kinetoplastids , Giardia and Trichomonas, and is evolutionarily distant from animals, fungi, and plants  form, but upon environmental stimuli can transform into a flagellate, synthesizing basal bodies and flagella de novo, or encyst to allow for dispersion , a thorough investigation on this important oxygen‐depended enzyme is lacking.Found in soils and freshwater worldwide, Candida albicans  is a terminal oxidase typically involved in bypassing the electron transport chain in plant mitochondria, even though it has also been identified and localized in the mitochondria and related organelles of many nonrelated microbial eukaryotes including trypanosomes  using the following accession numbers; GCA_008403515.1 (N. fowleri) and GCA_003324165.1 (N. lovaniensis). The amino acid sequence of the AOX from N. gruberi was obtained from uniprot (D2V4B2). The Phyre2 Web portal for protein modeling, prediction, and analysis was used in intensive mode , Cryptosporidium parvum (Q6W3R4), yeast; Candida albicans (A0A1D8PEM4), plant; Arabidopsis thaliana (Q39219), and, fungi; Neurospora crassa (Q01355).The predicted amino acid sequences of the alternative oxidase homologues of Three local databanks of proteomes, representative of all diversity from eukaryotes, bacteria, and proteobacteria were assembled from the National Center for Biotechnology Information (NCBI): 193 proteomes from Eukaryotes (one per genus); 1017 proteomes from Bacteria (3 per family), and 1082 proteomes from Proteobacteria (1 per genus). Homology searches were performed using HMMSEARCH, from the HMMER‐3.1b2 package  was cultured in M7 media at 28°C   was designed by Eurogentec based on the provide sequence of AOX of To separate organelles from cytosol, cell fractionation by centrifugation was carried out as previously described , diluted to 1:1000. Secondary antibody staining was carried out for 1 h in the dark using anti‐Rabbit‐IgG‐Alexa 488. Slides were washed and mounted using Prolong Gold Antifade with DAPI. Laser Confocal Microscopy was carried out using the LSM 880 Laser Confocal with Airyscan by Zeiss. Laser sets used were 405, 488, and 594, with airyscan detector plate imaging for high resolution. Images were captured and analyzed using Zen software suite by Zeiss.Samples were prepared as previously described . The experiment was then repeated with the drug additions in reverse order. Student's t‐test was used to determine significance.Real‐time respirometry was monitored using an OROBOROS Oxygraph‐2k with Clark polarographic oxygen electrodes and automatic titration‐injection micropump. The chambers of the oxygraph were calibrated using 2 ml of either M7 or PYNFH media without glucose for 20 min. Naegleria cells were seeded at a density of 2000 cells per well of a clear F‐bottom 96‐well plate and incubated in identical conditions as stated above. The plate was placed on a Juli™Stage live cell monitoring system, programmed to capture an image of each well every hour for 5 days. On day‐2, wells were inoculated with concentrations of SHAM at 1, 0.1, 0.001 and 0.001 mM. Plates were then incubated for a further 3 days. At the end of the experiment, the images were used to count cell numbers using ImageJ. Using an image with a scale bar, we used the grid function with a known area per square that was applied to all images taken at 0, 24, 48, 72, 96, and 120 h. Using the cell counter function, we counted the number of cells in a grid square and multiplied it to the surface area size of a F‐bottom 96‐well plate to estimate the total cell number per well. The experiment was completed with three biological replicates, whereby each biological replicate consisted of three technical replicates. Cell counts were graphed using GraphPad Prism 8.We carried out an exhaustive search for AOX homologs in updated eukaryotic and bacterial databanks. As bacterial hits were mainly from proteobacteria, we also searched for AOX homologs in a proteobacteria databank with more diversity within this phylum. We identified 323 AOX homologs: 265 from 193 eukaryotic taxa, 56 from 1,082 proteobacterial taxa, and 2 from 896 bacterial taxa (other than proteobacteria) Table .Regarding the eukaryotic distribution, in addition to previously described groups Alveolata, Euglenozoa, Metazoa, Choanoflagellates, Stramenopiles, Fungi, Rhodophya, Heterolobosea, and Viridiplantae , Chlorophyta (UFB = 82%) shows that, although the presence of AOX could not be inferred in the ancestor of Archaeaplastida, it can be inferred in the ancestors of both groups. Finally, regarding Fungi, the presence of three separate and monophyletic groups containing Ascomycota and Basidiomycota (UFB = 80%), Chytridiomycota (UFB = 100%) and a mix of Zoopagomycota, Chytridiomycota, Cryptomycota, Mucoromycota, Blastocladiomycota, and Microsporidia (UFB = 74%), suggests that AOX was acquired at least three times independently in this group.N. gruberi, N. fowleri, and N. lovaniensis were aligned against the AOX found in protists; Trypanosoma brucei brucei, Cryptosporidium parvum, yeast; Candida albicans, plant; Arabidopsis thaliana and, fungus; Neurospora crassa. The N‐termini displayed little conservation. However, key conserved residues were detected across all species from the middle of the sequences, continuing toward the C‐terminus  , including between the pathogenic N. fowleri, nonpathogenic N. lovaniensis and, N. gruberi species. It has previously been reported that a mutation from trp‐206 (trp‐151 on tbAOX) to ala‐206 resulted in a decrease in respiration of 95% , complex I and complex III of oxidative phosphorylation .Click here for additional data file.Figure S1. Sequence alignments of AOX reveals conserved domains.Figure S2. Sequence alignments of AOX reveals conserved domains.Figure S3. Confocal microscopy demonstrating mitochondrial localization of AOX.Figure S4. High‐resolution real‐time respirometry raw trace data reveals cyanide resistant respiration.Table S2. Universally conserved amino acids of AOX and their roles.Click here for additional data file.


CD276  is one of the most important immune checkpoints of the CD28 and B7 superfamily, and its abnormal expression is closely associated with various types of cancer. It has been shown that CD276 is able to inhibit the function of T cells, and that this gene may potentially be a promising immunotherapy target for different types of cancer.CD276 in cancer to date, the present study has employed single-cell sequencing and bioinformatics methods to analyze the expression patterns, clinical significance, prognostic value, epigenetic alterations, DNA methylation level, tumor immune cell infiltration and immune functions of CD276 in different types of cancer. In order to analyze the potential underlying mechanism of CD276 in glioblastoma (GBM) to assess its prognostic value, the LinkedOmics database was used to explore the biological function and co-expression pattern of CD276 in GBM, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. In addition, a simple validation of the above analyses was performed using reverse transcription-quantitative (RT-q)PCR assay.Since few systematic studies have been published on the role of CD276 was highly expressed, and was often associated with poorer survival and prognosis, in the majority of different types of cancer. In addition, CD276 expression was found to be closely associated with T cell infiltration, immune checkpoint genes and immunoregulatory interactions between lymphoid and a non-lymphoid cell. It was also shown that the CD276 expression network exerts a wide influence on the immune activation of GBM. The expression of CD276 was found to be positively correlated with neutrophil-mediated immunity, although it was negatively correlated with the level of neurotransmitters, neurotransmitter transport and the regulation of neuropeptide signaling pathways in GBM. It is noteworthy that CD276 expression was found to be significantly higher in GBM compared with normal controls according to the RT-qPCR analysis, and the co-expression network, biological function and chemotherapeutic drug sensitivity of CD276 in GBM were further explored. In conclusion, the findings of the present study have revealed that CD276 is strongly expressed and associated with poor prognosis in most types of cancer, including GBM, and its expression is strongly associated with T-cell infiltration, immune checkpoint genes, and immunomodulatory interactions between lymphocytes and non-lymphoid cells.The results revealed that CD276 in different types of cancers, especially GBM, and CD276 may potentially serve as a biomarker for cancer.Taken together, based on our systematic analysis, our findings have revealed important roles for The online version contains supplementary material available at 10.1186/s12885-023-10575-1. PD-1 (programmed cell death protein 1) and CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) immune checkpoints has been routinely used in tumor immunotherapy, although, as a recently discovered immune checkpoint in tumor immunotherapy, an increasing number of studies have focused on the role of CD276 (B7-H3) , RNF4 (recombinant human ring finger protein 4), LGALS8  and LGALS9   Fig. .Table 1GCD276 in cancer patients. The Kaplan-Meier database was employed, and the results obtained showed that CD276 expression was lower in the BLCA, HNSC, CESC, KIRC, KIRP, LIHC, LUAD, PAAD and PCPG cancer types, indicating was CD276 presented a hazard for patients  in BRCA, CHOL, GBM, LUSC, MESO, SARC, SKCM, THCA, UCEC and UCS , ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) and NT5E (ecto-5'-nucleotidase), in PCPG, KICH, LGG and THCA  receptor 1], TGFB1 (transforming growth factor beta 1), NECTIN2 (poliovirus receptor-related protein 2), IL10RB (interleukin 10 receptor beta) and CSF1R (colony-stimulating factor-1 receptor)  receptor 1], in the KICH, LGG, LIHC and THCA cancer types  ligand 7], CXCL16 (CXC chemokine ligand 16) and CXCL8 (CXC chemokine ligand 8), in THCA, KICH, PAAD and UVM  Fig. B. Furthepes Fig. C. FinallUVM Fig. D. IntereHOL Fig. A-D. In aCD276 in GBM, TCGA and GEO databases were used to reveal that the mRNA expression level of CD276 in GBM was significantly higher compared with that in the normal control group  was used to analyze the mutational landscape of CD276 in pan-cancer, and the associations among different CD276 expression levels and the gene mutational landscape in GBM. Missense mutations were found to occur in the vast majority of cancers, including GBM -receptor interaction, focal adhesion, the TNF signaling pathway, salivary secretion, maturity onset diabetes of the young and glycine, serine and threonine metabolism , apoptosis and inflammation, whereas it negatively regulated GBM cell DNA damage and repair, cell cycle and tumor cell stemness , PD-1 and CTLA-4. Therefore, identifying novel immunotherapy biomarkers is a critical aspect of tumor immunotherapy [CD276 expression was positively correlated with a number of immune checkpoint genes, including TGFBR1, TGFB1, NECTIN2, IL10RB and CSF1R, in most types of cancer. In general, patients with cancer lose their T-cell functions, and have declining numbers of T-cells. We discovered that CD276 expression was positively correlated with exhausted T-cell marker genes and immune checkpoint genes in tumors, including CTLA4, PDCDD1LG2, KDR, HAVCR2, TGFBR1, TGFB1, NECTIN2, IL10RB and CSF1R. However, further in vitro and in vivo studies are required to evaluate the usefulness of monoclonal antibodies against CD276 for future therapeutic strategies.The occurrence and development of tumors cannot be separated from the regulation of the tumor microenvironment (TME), and the TME has attracted an increasing amount of attention in tumor immunotherapy. Cancers can evade the immune response and influence immunotherapy by means of immune checkpoint genes, such as otherapy . In the CD276 was highly expressed in adult cerebellum and fetal brain tissue. Further functional analysis of GBM showed that high expression levels of CD276 were associated with the proliferation, invasion, migration and angiogenesis of tumor cells. RT-qPCR analysis subsequently confirmed the high proliferation level of GBM cells through the mRNA expression level of PCNA. In addition, the expression of CD276 was found to be correlated with the sensitivity of GBM to chemotherapy drugs. Patients with GBM with high expression levels of CD276 were resistant to lomustine and nitrosourea, whereas no sensitivity was identified with temozolomide treatment.Single-cell analysis revealed that CD276 has provided ample evidence to suggest that CD276 may act as a potential prognostic biomarker and immunotherapy target. However, although information was utilized from different databases for systematic bioinformatics analysis, there were still a number of limitations associated with the present study. First, we used different databases and analytical methods, and several deviations occurred when comparing the results from certain studies; therefore, further in vivo and in vitro experiments are required to confirm the results. In addition, although we have explored the possible function and value of CD276 in numerous types of cancers, these aspects need to be further verified. Finally, monoclonal antibodies against CD276 are currently in phase I clinical trials, and further optimization of cancer immunotherapy approaches are required in humans.In general, our comprehensive evaluation of CD276 is expressed differently in different cancers and at different stages of cancer development, suggesting that CD276 may exert different roles in different cancers, although, overall, high expression of CD276 in the majority of different types of cancer is associated with poor patient prognosis, poor survival and poor clinical outcomes. For GBM, the expression level of CD276 in adult cerebellum and fetal brain tissue may be relatively higher, and this high expression of CD276 may be associated with GBM tissue proliferation, migration, invasion, EMT and angiogenesis, although it was negatively correlated with DNA damage and repair. In addition, patients with GBM with high CD276 expression may be resistant to lomustine and nitrosourea. Through a comprehensive evaluation of CD276, we have also shown that CD276 expression is closely correlated with tumor immune regulation, immune cell infiltration and immune checkpoint responses, which has provided important insights into the future optimization of cancer immunotherapy utilizing the CD276 pathway.In conclusion, the present study has shown that Additional file 1: Supplementary Fig. S1. Pan-cancer paired CD276 expression. (A) The expression of CD276 in cancer and corresponding normal tissues. (B-P) TCGA database was applied to investigate the expression of CD276 in paired cancer tissues and corresponding adjacent normal tissues. The paired tissues with significant differences and statistical significance were listed in the figure. *p < 0.05; **p < 0.01 and ***p < 0.001. Supplementary Fig. S2. Analysis of the relationship between CD276 expression and clinicopathological parameters in cancers. (A-T) The clinical relevance of CD276 in different cancer stages was analyzed using UALCAN database. Supplementary Fig. S3. Analysis of the relevance between CD276 expression and clinicopathological parameters in different cancers. (A-F) The clinical correlation of CD276 in different cancer stages was analyzed using UALCAN database, and the expression of CD276 in different cancer stages shown in the Fig. was not statistically significant. Supplementary Fig. S4. Analysis of DNA methylation in pan-cancer. (A-I) UALCAN database was used to explore promoter methylation levels of CD276 in different cancers, and the cancers selected in the Fig. showed statistical differences between tumors and normal tissues. Supplementary Fig. S5. CD276 is highly expressed in many cancers and normal tissues. Immunohistochemical results of HPA dataset showed that protein expression of CD276 was high in various cancers, just as shown in Fig. A-E, and protein expression of CD276 was also high in various normal tissues, just as shown in Fig. F-I. Supplementary Fig. S6. After constructing PPI network, we obtained a series of proteins closely related to CD276. DAVID database was used to conduct GO and KEGG enrichment analysis on CD276 and its related genes, so as to explore the biological functions associated with CD276. Supplementary Fig. S7. Merged enrichment plots for CD276 on the basis of GSEA in pan-cancer. Fig. A-H shows the signaling pathways associated with CD276 by GO, KEGG and Reactome analysis, including those involved in neutrophil degranulation and signaling by interleukins. Supplementary Fig. S8. The relationship between CD276 and immunoregulation-related genes. The relevance between the expression of CD276 and immune-activating genes, immunosuppressive genes, chemokine receptors and chemokines were analyzed using TISDB database. Supplementary Fig. S9. The expression and prognostic value of CD276 in GBM. A-B. The mRNA expression of CD276 in GBM using TCGA, E-MATB and GEO databases; C. The protein expression of CD276 in GBM. D-E. The prognostic value of CD276 in GBM using CGGA database.


The primary cilium is a solitary, sensory organelle that extends from the surface of nearly every vertebrate cell, including craniofacial cells. This organelle converts chemical and physical external stimuli into intracellular signaling cascades and mediates several well-known signaling pathways simultaneously. Thus, the primary cilium is considered a cellular signaling nexus and amplifier. Primary cilia dysfunction directly results in a collection of diseases and syndromes that typically affect multiple organ systems, including the face and teeth. Despite this direct connection, primary cilia are largely unexplored in craniofacial research. In this review, I briefly summarize craniofacial abnormalities tied to the primary cilium and examine the existing information on primary cilia in craniofacial development and repair. I close with a discussion on preliminary studies that motivate future areas of exploration that are further supported by studies performed in long bone and kidney cells.  The axoneme contains nine microtubule doublets whose assembly and maintenance are dependent on intraflagellar transport (IFT). Motor proteins associate with complexes containing IFT proteins (IFT-A or B) and travel along the microtubule doublets, delivering or removing cargo necessary for axoneme maintenance. Kinesin-II is thought to attach to IFT-B to carry this complex, as well as IFT-A and inactive dynein-II, to the ciliary tip in anterograde transport [retrograde transport [The primary cilium is a solitary sensory organelle that projects from the cell surface of most vertebrate cells, including bone and dental cells. It operates as both a chemo- and mechanosensor to transduce external stimuli into intracellular signaling cascades. This nearly ubiquitous structure is comprised of an axoneme that extends from a basal body, which helps anchor the axoneme to the cell . The poiransport ,2. Near ransport ,2. The aciliopathies. Ciliopathies typically affect multiple organs simultaneously, including the craniofacial tissues and dentition.Proteins associated with various cell signaling pathways are present in the ciliary domain through a process tightly regulated by the transition zone. Some of these proteins are unique to the primary cilium and many are highly concentrated in the ciliary domain, especially near the axoneme base, compared to the rest of the cell. This focused assortment of cell signaling components enables the primary cilium to mediate and amplify a variety of signaling pathways important for bone and dental cell behavior. Ciliary signal transduction can be distinct from signaling orchestrated by the cell membrane, but in some instances it is complementary or additive. Based on these characteristics, the primary cilium is a cellular antenna that operates as a signaling nexus to direct cell behavior. The cilium is so critical that alterations in IFT machinery or transition zone utility which disrupt primary ciliary function cause an assortment of human diseases called In this review, I will first summarize the most prevalent ciliopathies affecting craniofacial structures. Next, I will discuss existing research that demonstrates the importance of primary cilia in dental cell behavior and development of craniofacial tissues. I will close with an exploration of primary cilia characteristics observed in other tissues that are worth further investigation in dental cells.Kif3a was once a common target for primary cilia disruption because it is important for axoneme maintenance and subsequent ciliary signaling. These mice exhibit classic symptoms of FND such as cleft palate, cleft skull, bifid nasal septum, and agenesis of corpus callosum [Ciliopathies are diseases that directly result from alterations in the primary cilium itself or components associated with the ciliary complex that render the primary cilium dysfunctional. These conditions typically affect multiple organ systems and approximately 30% of ciliopathies involve craniofacial and dental defects including cleft lip and palate, dental hypoplasia, midfacial hypoplasia, craniosynostosis, retrognathia/micrognathia, and irregularities in the number, size, or anatomical location of teeth ,5,6. Cercallosum , providiSeveral diseases primarily impacting the craniofacial tissues have long been considered ciliopathies. The most prevalent or well-characterized are Joubert syndrome and its allelic counterpart Meckel syndrome, Oral–facial–digital syndrome, Ellis-van Creveld (EVC) syndrome, and Weyers acrofacial dysostosis. These syndromes are directly linked to mutations in components of the primary cilia complex, especially IFT proteins and proteins associated with trafficking in the ciliary pocket  ,11,12,13Increased awareness of the primary cilium’s involvement in these craniofacial diseases has sparked investigation of dental abnormalities in classic ciliopathies, such as Bardet–Biedl Syndrome (BBS), autosomal dominant polycystic kidney disease (ADPKD), and primary cilia dyskinesia (PCD). BBS is perhaps one of the most severe ciliopathies due to the wide range of organs affected, so it is not surprising that craniofacial tissues exhibit abnormalities. ADPKD patients develop fluid-filled cysts in the kidneys that often result in complete renal failure by the age of 60. One clinical report identified a dentigerous cyst in the lower jaw of a 42-year-old ADPKD patient . The cysMore ciliopathies potentially have secondary clinical features in the dentition that remain undetected or poorly characterized simply because they are overshadowed by disastrous impairments in other parts of the body. One study determined that polycystin-1 (PC1), a ciliary protein associated with ADPKD, is expressed in the dental follicle cells that give rise to dentigerous cysts . Furtherin vivo and in vitro [Before examining the available research investigating craniofacial defects, it is important to understand the presence and structure of primary cilia in relevant cell populations. Primary cilia participate in every stage of tooth development and project from various dental cells in mice and humans, both in vitro ,17,18,19in vitro ,21,22,23In some skeletal tissues, such as the growth plate, primary cilia are intentionally oriented to facilitate signaling interactions. Indeed, primary cilia in odontoblasts project into the dental pulp parallel to the dentin wall and ameloblast primary cilia emanate toward the outer enamel epithelium ,24,25. Tin vitro, as well as account for general observations that primary cilia are longer in regions with active cell differentiation and matrix production [In skeletal and kidney cells, the ciliary axoneme can adjust its length to tune primary cilia sensitivity. This could explain why primary cilium incidence and length were found to increase during DPSC odontogenic differentiation oduction ,23,26. Poduction . Dental oduction ,27,28. Building evidence suggests that primary cilia operate as signaling hubs in various dental cells. The Hedgehog (Hh) and Wnt signaling pathways have been prioritized since their roles in craniofacial development are well-established and the primary cilium is known to coordinate these pathways in other skeletal cells. In this review, I focus on signaling in the dental epithelium and mesenchyme and neural crest cells, odontoblasts, and DPSCs because this information is available in detail. It is important to note that preliminary studies implicate a role for ciliary Hh signaling in periodontal ligament stem cells and this pathway has been speculated in other dental cells that will not be covered in this review .Considering primary cilia are prevalent in every craniofacial tissue, it is not surprising that disrupting this organelle has disastrous consequences in craniofacial structures. The vast majority of craniofacial primary cilia research has been conducted in the context of development or early postnatal growth. This is logical given that most craniofacial defects manifest neonatally, but the lack of investigation in standard maintenance, repair, and aging may also be indicative of technical limitations. Since primary cilium function can be disrupted by altering various components of the ciliary complex, I will examine craniofacial phenotypes resulting from different genetic mutations in this section. The symptoms associated with each phenotype are also summarized in Wnt1Cre;fl/flKif3a mice were first generated to determine whether disrupting the primary cilium in neural crest cells could serve as a model for FND [Wnt1Cre;fl/flKif3a mice die at birth so tooth buds were implanted in renal capsules to evaluate dentin and enamel production [ for FND . Indeed, for FND ,29. The oduction . ImplantWnt1Cre;fl/flKif3a mutants. The loss of primary cilia resulted in decreased Hh signaling and increased Wnt signaling in the dental mesenchyme without changes in signaling domains in the epithelium [Additional studies were conducted to elucidate specific signaling mechanisms contributing to FND ,32. Motiithelium ,32. Howeithelium . The autithelium ,32. Moreithelium . OverallKif3a was found to have non-ciliary effects on Wnt signaling in some tissues [Ift88, a subunit of the IFT-B complex orchestrating anterograde transport from the ciliary base to the distal tip of the axoneme, is a popular tool for reducing primary cilium incidence and inhibiting its function. Neural crest primary cilia have also been disrupted using a Wnt1Cre;fl/flIft88 knockout model [in vivo. The authors also observed downregulated Hh signaling and upregulated Wnt signaling specifically in the dental mesenchyme, which is consistent with observations in the aforementioned Wnt1Cre;fl/flKif3a mouse model. Interestingly, another group identified expanded Hh signaling where an extra molar was formed mesial to the first molar in Wnt1Cre;fl/flIft88 and Oak Ridge Polycystic Kidney mice, which contain a hypomorphic mutation of Ift88 [Removing IFT proteins has proven to be a more specific mechanism for disrupting primary cilia since  tissues ,35. Geneut model ,25. IFT8of Ift88 ,36. ThesIft88 in epithelial cells  [K14;fl/flIft88 mice had chalky incisors and roughened enamel surfaces on all molars due to abrasions that became more severe with time. The rough, chalky appearance resulted from thinner enamel layers that were less mineralized compared to control mice. Without functional cilia, enamel rod patterning was disorganized and fewer ameloblasts were present at the secretory stage, indicating primary cilia are important for enamel production. Signaling pathways known to be important for enamel formation—Hh, Wnt, FGF, and BMP—were evaluated but only Hh signaling was downregulated. Mutant mice treated with a Hh agonist demonstrated partial rescue of enamel thickness, indicating functional primary cilia are required for complete Hh signaling. K14;fl/flSmo mice lacking a core component of the Hh signaling pathway were then generated to better examine the extent to which primary cilia mediate Hh signaling. The phenotype in these mutants, which did not survive birth, was much more severe compared to K14;fl/flIft88 mice. This indicates Hh signaling operates independently of the primary cilium in certain developmental stages. The phenotypic disparity, combined with the observation that agonizing Hh signaling partially recovered enamel production, is evidence that the primary cilium may serve as a Hh signaling amplifier in this context. The role of primary cilia in enamel production has also been evaluated using a conditional deletion of 88fl/fl) . K14;IftOsxCre;fl/flIft80 mouse model to disrupt primary cilia function in odontoblasts and dental pulp cells differentiating into odontoblast-like cells [in vivo. Mutant incisors erupted much later than controls during postnatal growth and later became malocclusioned. Molar eruption and crown formation appeared normal, but mutant roots were noticeably shorter. Cervical loop and odontoblast layers were thinner due to compromised cell proliferation and odontogenesis. Without functional cilia, odontoblasts lacked polarity and did not assemble into their usual alignment. Consequently, dentinal tubules were disorganized and secreted less calcified matrix. Mutants also exhibited decreased bone mass in the calvaria and alveolar bone, which is not surprising since Osx is expressed in osteoblasts. To study signaling mechanisms underlying the OsxCre;fl/flIft80 phenotype, incisor dental pulp cells were isolated from these mice and primary cilia were disrupted using an adenoviral Cre to knockout IFT80. Hh signaling was reduced in knockout cells and decreased proliferation was found to be caused by attenuated FGF2–FGFR signaling. Interestingly, dental pulp cell primary cilia length was reduced with the knockout in vitro, suggesting the niche and/or mechanism of disruption can influence primary cilia length in certain cells. IFT80 is another component of the IFT-B complex. Removal of IFT80 renders the primary cilium dysfunctional because key proteins are not trafficked along the axoneme to maintain its structure. Fibroblast growth factor 2 (FGF2) promotes proliferation, migration, and differentiation in dental pulp cells. One group hypothesized that FGF2-mediated FGF signaling is similarly important in odontoblasts and could be regulated by primary cilia. To test their hypothesis, they generated an ke cells . Primaryfl/flIft80 mice and treated with adenoviral Cre [These results were recapitulated in DPSCs isolated from iral Cre . PrimaryOsxCre;fl/flIft140 model was generated to investigate the role of primary cilia in dentinogenesis due to IFT140′s direct connection to Joubert syndrome, Meckel syndrome, and CED [OsxCre;fl/flIft80 mice, where a component of the IFT complex B was removed. The phenotypic overlap suggests primary cilium function is the driving factor in these disease states, rather than mutation of specific IFTs.IFT140 is part of the IFT-A complex, which directs retrograde transport from the axoneme tip to the ciliary base. An  and CED . At two Wnt1Cre;fl/flIft20 mice die shortly after birth due to breathing and feeding complications resulting from missing or malformed craniofacial structures [Wnt1Cre;fl/flKif3a and Wnt1Cre;fl/flIft88 mice, but the exact role in primary cilia function is complicated by IFT20′s non-ciliary role in collagen synthesis. The aforementioned genes  were highlighted primarily because they are utilized in recent mouse models to study specific mechanisms underlying primary cilia dysfunction in craniofacial development. However, there are a wealth of other ciliary proteins linked to craniofacial abnormalities including, but not limited to, intraflagellar transport protein 20 (IFT20), centrosomal protein 290 (CEP290), Meckel syndrome type 1 (MKS1), oral–facial–digital syndrome 1 (OFD1), and ADP-ribosylation factor-like protein 13B (ARL13B). IFT20 is another IFT-B complex component and has been shown to be critical for collagen synthesis that facilitates craniofacial bone formation . Wnt1Creructures . This phIn contrast to the IFT proteins, CEP290, MKS1, OFD1, and ARL13B are examples of how primary cilia function is compromised when components of the ciliary complex other than IFT are disrupted . Bardet–OsxCre;fl/flIft140 mouse model [Ift140 was targeted due to its high level of expression in the early stages of healing. Following tooth extraction, primary cilia were prevalent in cells within the defect site and the cilium incidence gradually decreased over time with healing. Mutant mice exhibited significant reductions in bone volume and expression of osteogenic genes compared to control mice, suggesting an impaired healing response. Alveolar bone marrow stromal cells (BMSCs) isolated from these mice demonstrated decreased cell viability, proliferation, osteogenesis, and mineral deposition in addition to reduced cilium incidence compared to control cells. Osteoclast activity did not appear to change, indicating the diminished socket healing was due to impaired osteoblast and BMSC activity. Compared to its investigation in craniofacial development, the role of the primary cilium in dental repair is largely unexplored. One study investigated primary cilia in tooth socket healing using the aforementioned se model . Ift140 OsxCre;fl/flIft140 model [Osx. Dentin formation in the injury site and dentin matrix protein expression were significantly reduced in mutants. The authors interestingly noted that primary cilia were difficult to visualize near the injury site without treating the cavity. When treated, primary cilia were prevalent in cells near the exposure site in control mice, but cilium incidence was markedly reduced in mutants. This work complements a recent study showing Hh signaling is upregulated and Gli1-expressing dental pulp cells localized near the injury site in a similar dentin exposure model [Another group interrogated the role of primary cilia in reparative dentinogenesis using the fl model . A shallre model . Considere model ,45,46,47in vitro and in vivo [The innate healing potential of human teeth is very limited largely due to the slow rate of repair and lack of enamel regeneration. Primary cilia are important for mineralized tissue regeneration in bone and growing evidence suggests dental cell primary cilia have functions similar to those found in better-studied cells. Further investigation of this sensory organelle is required to better understand how dental cells can be manipulated, especially considering that primary cilia can be sensitized. For example, fenoldopam, an approved blood pressure medication, has been shown to lengthen osteocyte primary cilia such that osteogenesis is enhanced  in vivo ,49. Over in vivo ,51. Thismechanotransduction. The primary cilium is a well-established mechanosensor in kidney and bone cells, and mechanosensory roles have been preliminarily demonstrated or speculated in other tissues [Thus far, I have examined the chemosensory role of the primary cilium, but this organelle is also critical for sensing and converting physical external stimuli into intracellular signaling cascades, a process termed  tissues ,53,54. C tissues ,56. A siDental pulp cells and odontoblasts are also thought to be mechanosensitive. Pulp cells extracted from human exfoliated deciduous teeth (SHED) demonstrated enhanced mineralization and lengthening of the primary cilium when exposed to hydrostatic pressure . When prin vitro, resulting in enhanced odontogenesis [Calcium signaling is a well-established mechanism that triggers a variety of intracellular signaling cascades that promote tissue formation and maintenance. The primary cilium has been shown to mediate intracellular calcium signaling responses distinct from those that occur at the cellular membrane . Mechanoogenesis . Closer ogenesis . ElucidaNexCre;fl/flArl13b model [in vitro encouraged or prevented axonal growth, respectively, indicating the primary cilium rapidly controls neural growth. Axon projections are critical for neural signaling that facilitates tissue formation and maintenance, as well as pain. This study reveals an important role for neural primary cilia in craniofacial tissue development, but an important future direction is to determine the extent to which primary cilia also direct intercellular signaling in neural networks. It is speculated that the primary cilium does mediate signal transduction in neurons, but how this process perhaps regulates dental cell behavior and pain remains unknown [Primary cilia have been implicated in neuronal migration, patterning, and axon guidance during development . Nerve ffl model . Tissues unknown .Teeth are densely innervated to facilitate dental pulp viability and injury response. Sensory nerves secrete factors that promote mineralized tissue formation for standard tooth maintenance or in response to injury ,64. It iin vitro and in vivo. The lack of primary cilia knowledge is therefore not a consequence of technical limitations, and significant advances could be made in dental cell signaling if more time and resources are allocated to interrogating primary cilia.Another proposed topic for further exploration is determining if and how dental cell primary cilia mediate various signaling pathways. The cilium operates as a signaling hub for the cell due to the dense collection of signaling proteins located in the ciliary complex. This trait also enables the primary cilium to detect low-magnitude stimuli and initiate signaling cascades in response, qualifying it as signaling amplifier. I previously covered development studies showing that primary cilia tune Hh and Wnt signaling to direct communication between the dental mesenchyme and epithelium ,29,32. TThe primary cilium is a unique sensory organelle that has been observed in nearly all craniofacial cells. It plays a key role in the development, maintenance, and repair of bones and teeth, and its dysfunction directly results in many craniofacial abnormalities. Although the genetic links between primary cilium function and craniofacial disease states are well established, very little is known regarding the role and behavior of dental cell primary cilia. In this review, I summarized existing information that demonstrates the importance of primary cilia in craniofacial research and motivates further investigation of this organelle. There is sufficient evidence to suggest primary cilia play a role in mineralized tissue formation, mechanotransduction, and dental and neural cell signaling, and these studies should be prioritized. In this review, I propose four future research directions of particular interest: (1) odontoblast fluid shear, (2) odontoblast intracellular calcium signaling dynamics, (3) signaling between nerves and dental cells, and (4) the simultaneous coordination of multiple signaling pathways in dental cells . This or


Male infertility is an important issue that causes low production in the animal industry. To solve the male fertility crisis in the animal industry, the prediction of sperm quality is the most important step. Sperm RNA is the potential marker for male fertility prediction. We hypothesized that the expression of functional genes related to fertilization will be the best target for male fertility prediction markers. To investigate optimum male fertility prediction marker, we compared target genes expression level and a wide range of field data acquired from artificial insemination of boar semen.EQTN), zona pellucida sperm-binding protein 4 (ZP4), and sperm acrosome membrane-associated protein 3 exhibited high accuracy  as markers to evaluate male fertility. Combinations of EQTN-ZP4, ZP4-protein unc-13 homolog B, and ZP4-regulating synaptic membrane exocytosis protein 1 (RIMS1) showed the highest prediction value, and all these markers are involved in the acrosome reaction.Among the genes related to acrosomal vesicle exocytosis and sperm–oocyte fusion, equatorin (EQTN-ZP4 model was efficient in clustering the high-fertility group and may be useful for selection of animal that has superior fertility in the livestock industry. Compared to the EQTN-ZP4 model, the ZP4-RIMS1 model was more efficient in clustering the low-fertility group and may be useful in the diagnosis of male infertility in humans and other animals. The appointed translational animal model and established biomarker combination can be widely used in various scientific fields such as biomedical science.The The online version contains supplementary material available at 10.1186/s40104-022-00729-9. Male infertility is a severe problem that extends across all animals. In humans, increasing male infertility threatens the deterioration of next-generation and eventually, the entire human race. The livestock industry comprising cow, pig, and chicken also suffers from male fertility issues. In cows and pigs, male infertility is directly correlated with the economics of the livestock industry and world food production. Concerning this issue, many scientists point out that a decrease in sperm quality causes male infertility. Therefore, the precise assessment of sperm quality is the first step to treat male infertility. Traditionally, sperm quality is evaluated employing laboratory techniques assessing sperm motility and viability , 2. HoweThere is an emphasis on the importance of an omics approach to predict male fertility, instead of conventional laboratory assays –7. Many Pig, as an animal model, contributes greatly to biomedical research, particularly studies on obesity, arthritis, cardiovascular disease, skin and eye conditions, and xenotransplantation , 26. ComThe sperm-oocyte interaction is the starting point of embryonic development, and for successful interaction, all molecular mechanisms must work in harmony . Before Firstly, ‘acrosomal vesicle exocytosis’ and ‘sperm-egg fusion’ related genes were listed to investigate relevant boar fertility prediction marker models.n = 3) were chosen based on total litter size data after AI trials. The differentially expressed genes in high- and low-fertility groups were progressed to further process.Secondly, to narrow down target genes, high (average litter size = 13.97 ± 0.07)- and low (average litter size = 11.17 ± 0.13)-fertility representative samples  curve analysis.Finally, each evaluated markers were combined to improve boar fertility prediction value. Multiple marker model predictability of litter size  was evaluated with ROC curve analysis. Multiple marker models which exhibited outstanding prediction value in the previous analysis were subjected to further statistical analysis. The predictability of marker models to distinguish boars into three groups  was evaluated to develop an optimum combination.All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Chung-Ang University . All boars and sows used in the experiment were reared at 20 ± 5 °C under a 2:1 light/dark cycle, continuous air circulation, and appropriate feed and water.2 and at 37 °C for 30 min [A total of 26 Yorkshire boar (body weight over 90 kg and age between 9 to 24 months) semen was collected using the gloved-hand technique  from Sunr 30 min . The speSperm motility and motion kinematics were analyzed by a computer-assisted sperm analysis system  . Washed The spermatozoa were stained with chlortetracycline (Sigma-Aldrich Co.) and Hoechst 33258 (Sigma-Aldrich Co.) and their capacitation status were measured in approximately 400 spermatozoa per slide , 37. Eac6 cells/mL. Sperm pellets were suspended in non-toxic guanidine-isothiocyanate lysis buffer containing β-mercaptoethanol  and homogenized using a 20G syringe. TRIzol (500 μL)  and chloroform (200 μL) were added to the homogenized sample and centrifuged at 12,000 × g for 25 min. After centrifugation, the upper layer (500 μL) was moved to a fresh tube and mixed with pure ethanol (500 μL) (Sigma-Aldrich Co.). Sperm RNA was attached to a spin cartridge and washed with the PureLink RNA Mini Kit (Invitrogen) wash buffer 1 and 2. The isolated RNA was immersed in 20 μL of nuclease-free water (60 °C). The quality (260/280 ratio) and quantity of isolated RNA were measured using an Epoch Microplate Spectrophotometer .Sperm RNA was isolated according to a previous study . Briefly-∆∆Cq method. All primers used for qPCR were designed based on Sscrofa11.1 Genome Assembly from The Swine Genome Sequencing Consortium  according to the manufacturer’s protocol. Total 400 ng of sperm RNA from each sample was reverse transcribed and 8–11 μg of cDNAs were yielded. The relative expression of target genes was quantified using the 7500 fast real-time PCR system . SYBR Green PCR master mix (Applied Biosystems) was used. The total reaction volume was 20 μL (100 ng of cDNA), and the cycling conditions were set to initial denaturation at 95 °C (10 min) followed by 40 cycles at 95 °C for denaturation (15 s) and 60 °C for annealing (60 s). The results were analyzed using the 7500 Software v2.3  using the 26 sperm cells/mL) [Male fertility status of randomly selected 20 Yorkshire boars was evaluated from the results obtained after AI using their semen. Boar semen was diluted with 100 mL of the Beltsville thawing solution (30 × 10ells/mL) . AI was ells/mL)  in an avThe Gene Ontology database was used to categorize target genes related with the functional classes “acrosomal vesicle exocytosis” and “sperm–egg fusion.” The listed genes were applied to the Pathway Studio program (Elsevier) to visualize their biological functions.t-test, linear and multiple regression test, and ROC curve analysis were performed using the SPSS v1.8 software . principal component analysis (PCA), k-medoids clustering, silhouette plotting, and heatmap analysis were conducted using the R software . P < 0.05 was considered to indicate significantly different results and was correlated with fertility. Standard error of the mean was used to show sampling distribution.All data obtained were tested for normality and homogeneity of variance. The normality test (Shapiro–Wilk test), homogeneity of variances test (Levene’s test), Student’s The functional parameters of spermatozoa, including sperm motility, hypermotility, motion kinetics, and capacitation status were analyzed from all boar spermatozoa. No functional parameter showed a significant difference between the high- and low-fertility groups Table . The speEQTN), zona pellucida sperm-binding protein 4 (ZP4), regulating synaptic membrane exocytosis protein 1 (RIMS1), Ras-related protein Rab-3A (RAB3A), protein unc-13 homolog B (UNC13B), and synaptotagmin-6 (SYT6) are involved in acrosomal vesicle exocytosis. Moreover, EQTN, Izumo sperm–egg fusion protein 1 (IZUMO1), sperm acrosome membrane-associated protein 3 (SPACA3), CD9, lysozyme like protein 6 (LYZL6), spermatogenesis-associated protein 46 (SPATA46), and IZUMO1 receptor (IZUMO1R) are involved in sperm-oocyte fusion. All genes associated with acrosomal vesicle exocytosis are differentially expressed in the high and low litter size groups . Of the genes categorized in “sperm–egg fusion” EQTN, IZUMO1, SPACA3, CD9, and IZUMO1R were differentially expressed in the high and low litter size groups . LYZL6 and SPATA46 were not differentially expressed according to litter size . Of the twelve genes investigated, ten differentially expressed genes were selected to be further evaluated as candidates for male fertility markers in validation trials.We found that equatorin . The expression pattern of the genes encoding these five proteins in 20 randomly selected boars was the same as the expression pattern of the high- and low-fertility groups. The male fertility prediction value was assessed with ROC curve analysis [ZP4 showed the highest values of sensitivity (SN), specificity (SP), negative predictive value (NPV), positive predictive value (PPV), and OA, all of which were 90% , ZP4-UNC13B , and ZP4- RIMS1  showed outstanding predictive value (Table P < 0.05). The combinations of UNC13B-SPACA3-CD9  and RIMS1-SPACA3-CD9  showed the highest predictive values among three-marker predictive models (Table P < 0.05). The multiple regression model was established . To identify the predictive model most efficient in diagnosing male fertility in the high-, medium-, and low-fertility groups, PCA, k-medoids clustering, and silhouette plotting were employed. The average litter size of high-, medium-, and low-fertility groups after clustering with each prediction model is shown in Additional file EQTN-ZP4 model is the most efficient in diagnosing the high-fertility group  was used, and the following gene sets were applied to investigate the changing patterns of marker-related diseases and cell processes: all 12 genes related to “acrosomal vesicle exocytosis” and “sperm–egg fusion,” 10 differentially expressed genes in the high- and low-fertility groups, and 5 genes passed the validation trial. The percentage of fertility-related diseases and cell processes changed according to the validation level of each gene set (Fig. EQTN, ZP4, and SPACA3 exhibited high accuracy as markers to evaluate male fertility. Combinations of EQTN-ZP4, ZP4-UNC13B, and ZP4-RIMS1 showed the highest prediction value. The EQTN-ZP4 model was efficient for clustering the high-fertility group and may be useful for selection of animal that has superior fertility. Compared to the EQTN-ZP4 model, the ZP4-RIMS1 model was more efficient in clustering the low-fertility group.To the best of our knowledge, no systemic empirical research exists addressing the question of how to translate basic research findings into new diagnoses in male infertility. This is the first translational study to predict male infertility using pigs with sperm RNA markers. In this study, we found that Evaluation of sperm quality is critical in the timely diagnosis and prevention of male infertility. Most diagnostic tools analyze the phenotype and morphology of spermatozoa. However, the clinical value of such current sperm evaluation methods is still debatable  because Large-scale transcriptomic analysis in many mammalian species has revealed fertility-related differential gene expressions –24. AlthZP4, EQTN, and SPACA3 showed high accuracy in male fertility prediction. ZP4 is known to play a critical role in the taxon-specific binding of the spermatozoa to the oocyte [ZP4 mRNA expression is associated with fertilization [Arabidopsis, short suspensor transcripts are expressed during male gametogenesis and conveyed to female gamete for zygotic translation. Paternally derived ZP4 transcripts might affect fertilization by aiding in detecting transcripts in the zygote.The marker genes e oocyte  and indue oocyte . Althouge oocyte . Evaluatlization . In ArabEQTN and SPACA3 transcripts in spermatozoa. mRNA levels cannot directly be correlated with protein expression as post-transcriptional processes are crucial in the final synthesis of the native protein [EQTN and SPACA3 transcripts may be associated with male fertility through the regulation of complex processes including acrosome reaction, sperm–oocyte fusion, fertilization, and beyond. Although, the mRNA expression of EQTN and SPACA3 are prominent markers for determining male fertility, the elucidation of the functional roles of mRNA in sperm warrants further research.EQTN is expressed in the equatorial segment of spermatozoa and induces an acrosome reaction to help sperm–oocyte fusion –57. EQTN protein . TherefoEQTN-ZP4, ZP4-UNC13B, and ZP4- RIMS1 prediction models that showed 100% accuracy in the prediction of the fertility status in the validation trial data. All genes in the three-gene prediction models were related to acrosome reaction. This indicates the importance of acrosome reaction in fertilization success. Interestingly, ZP4 and UNC13B showed “immune response” and “cell differentiation” as related cell processes. Thus, ZP4 and UNC13B might be potential targets for future studies on unraveling the connection between immune response and fertility.After identifying single fertility markers, we conducted multiple regression tests with all possible gene combinations to establish an optimum prediction model. Multiple markers for the diagnosis of various diseases, such as cancer and neurological diseases have been developed , 64. We EQTN-ZP4 model will be useful in this field because of its efficiency in clustering high-fertility groups. Although we only focused on the reproductive traits of pigs, other traits such as immune resistance, heat stress resistance, and growth rate could be improved by adopting the same approach. Compared to the EQTN-ZP4 model, the ZP4- RIMS1 and RIMS1-SPACA3-CD9 models were more efficient in clustering the low-fertility group by excluding the high- and medium-fertility groups. Therefore, we suggest that these models may be useful in biomedical studies to detect male infertility/sterility.For further evaluation of the multiple-marker prediction models, PCA and clustering were conducted. PCA is a powerful tool to combine multiple factors that affect the main variable and has been used to identify biomarkers of various diseases –68. In tZP4 gene is highly predictive of male fertility. ZP4 in combination with EQTN has a higher predictive value and may be useful in the livestock industry. The EQTN-ZP4 male fertility prediction model demonstrated outstanding predictive value in detecting the low-fertility group. Therefore, we postulate that this pig as a translational model and biomarker combination can be utilized to diagnose male infertility/sterility. However, as our study was conducted in a single pig stud, validation of our proposed prediction model requires further investigation and trans-species evaluation.In conclusion, our study established an efficient male fertility prediction model using the pig model and multiple gene combinations. These genes were involved in biological events critical to male fertility. The Additional file 1: Table S1. Primers designed for RT-qPCR. Table S2. Sperm motility, motion kinematics, and capacitation status of 20 randomly selected boar spermatozoa. Table S3. Average litter size of high-, medium-, and low-fertility groups after clustering.Additional file 2: Table S4. All fertility-related diseases and cell processes related with markers.


Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from  The mitochondrial electron transport chain (ETC) is a central component of cellular metabolism, consisting of four respiratory complexes (cI-cIV) that link proton translocation across the inner mitochondrial membrane to the step-wise transfer of electrons. Impaired electron flux in the ETC therefore affects both upstream and downstream processes such as the tricarboxylic acid cycle and mitochondrial ATP production, respectively. In consequence, ETC deficiencies are the underlying cause of a large number of severe metabolic disorders , 2, whicCiona intestinalis, a di-iron protein, that like all alternative enzymes acts in a non-proton-motive reaction [Nevertheless, naturally evolved by-pass mechanisms exist in plants and lower organisms that can overcome ETC inhibition. Alternative respiratory chain enzymes have two main representatives. First, there are alternative dehydrogenases , which transfer electrons from NADH to ubiquinone thereby generating its reduced form ubiquinol. Second, there are alternative oxidases (AOX), which complete the electron transfer from ubiquinol to oxygen , 5. Herereaction , 7. Notareaction , 7, and reaction . Notwithreaction  or mousereaction , in drosreaction –15, and reaction  by usingreaction , 17. Furreaction , attenuareaction , and bloreaction , illustrreaction  and promPossible routes for administering AOX therapeutically include AOX protein or AOX-encoding nucleic acid constructs. The former is inherently problematic, due to its likely immunogenicity , 23, as In the present study, we therefore set out to test whether an AOX-encoding cmRNA, codon-optimized for use in mammalian cells, and the introduction of alternative targeting information would suffice to produce an enzymatically active protein. Such a validation is a prerequisite for any future therapeutic use in vivo.AOX-encoding constructs engineered for cmRNA production were custom-synthesized by GenScript . The GeneOptimizer software (GeneArt)  was applRosa26) [Primary mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) and AOX-transgenic (AOXRosa26)  embryos Rosa26) , 36. MEFRosa26) . PASMCs 5 iMEFs or A549, or 6 × 105 PASMCs were used for transfection using Lipofectamine 2000 Transfection Reagent  following the manufacturer’s instructions in growth medium without antibiotics. To test for transfection efficiency, we measured fluorescence intensities of upon cell transfection in 4–5 areas using CellProfiler, an open-source software for quantitative analysis of biological images. This revealed approximately 36% AOX positive cells/total number of cells (AOX/Hoechst) when using 0.6 µg of AOX cmRNA/ml cell culture medium 24 h after transfection.2.5 × 106 MEFs were plated in a T175 flask  and cultured overnight, then incubated for 3 h with 10 µg/ml mitomycin C  in DMEM-glucose, without FBS and antibiotics. Mitomycin C was washed out and cells were detached with trypsin. After counting, 7.5 × 105 mitomycin C-treated cells were plated in a T25 flask  and cultured overnight. Finally, cells were transfected with AOX cmRNA for 24 h, then grown in fresh media and collected every day over a period of 7 days.5 × 10Protein extraction, SDS-PAGE and protein transfer were carried out as previous described . MembranFollowing transfection, iMEFs, cultured for 24 h on 35 mm Nunc glass bottom dishes (ThermoFisher Scientific), were fixed using 2% paraformaldehyde  for 20 min at 4 °C, permeabilized using 0.3% Triton X-100  in 2% paraformaldehyde for 20 min, and blocked using 5% BSA for 30 min at room temperature. Cells were stained with primary antibodies diluted in 5% BSA overnight at 4 °C as follows: custom-raised rabbit anti-AOX  [6 iMEFs or A549 cells were plated in a T175 flask , or 6.5 × 105 mouse PASMCs in a 10 cm dish , and transfected 24 h before the experiments. Growth medium was replaced 1 h before the assay. Cells were detached with 0.05% trypsin and counted by trypan blue exclusion. After centrifugation at 200 gmax for 5 min at 25 °C, medium was removed and cells were suspended in PBS and aliquoted for respirometry and protein extraction. Mitochondrial respiration was assayed using an O2k oxygraph . 2 × 106 iMEFs or A549 cells, or 1 × 106 PASMCs were directly resuspended in the oxygraph chamber in 2 ml of respiration medium B [Whole-cell and permeabilized cell respiration was measured as described previously  with minmedium B , 38. Aft5 iMEFs were plated in a six-well plate , transfected with cmRNA for 24 h, and incubated for 48 h in DMEM lacking glucose, glutamine and phenol red  supplemented with 10 mM galactose , 10% FBS , 100 U/ml penicillin and 100 μg/ml streptomycin , 2 mM L-glutamine , 1 mM sodium pyruvate , and 5 µM antimycin A . The numbers of total and dead cells were counted using a hematocytometer by trypan blue exclusion. Representative images per well were acquired using an Axio Vert.A1 inverted microscope, processed by ZEN 2 imaging software (Zeiss) and color-inverted  by Fiji ImageJ software.2.5 × 10p value ≤ 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism software  with analyses and post hoc tests as indicated.All data are shown as mean ± standard deviation. To ensure adequate power and reproducibility, all experiments were replicated a minimum of three times, except where stated in figure legends. The number of samples per experiment was limited by the number that could be conveniently co-analyzed, such as on a single gel or in a single respirometry or microscopy session. No samples were excluded. Samples were not randomized. The variance was similar between the groups that have been statistically compared. A Blot and immunocytochemistry images were adjusted for brightness and contrast, cropped, resized and annotated for presentation, but not manipulated in any other way.Ciona intestinalis AOX cDNA the native mitochondrial targeting sequence , so as I–v1.101 . We nextI–v1.101  to prediI–v1.101 . FinallyI–v1.101 , 42. AllI–v1.101 . Three 5I–v1.101 ; a 102 nRosa26) [To evaluate the stability and utility of the designed cmRNA constructs we tested them in different mammalian cells. We first transfected iMEFs with each of the three AOX cmRNA pairs . Semiquantitative analysis by Western blot revealed that transfected iMEFs showed AOX protein expression levels similar to those seen in iMEFs derived from AOX transgenic mice (AOXRosa26) . ImportaConsidering a potential use of AOX cmRNA species specifically in the rapid treatment of acute pathologies, the earliest detection of AOX protein is of great importance. Western blot analysis of iMEFs transfected with cmRNA using the ATP5F1B 5′ UTR revealed detectable AOX protein as early as 3 h after transfection, reaching a plateau level by ~12 h Fig. . EquallyUsing respirometry, we next tested whether the cmRNA-encoded AOX expressed in iMEFs is catalytically functional. In trial experiments, we transfected iMEFs with increasing amounts of AOX cmRNA and tested both oxygen consumption of digitonin-treated cells (Supplementary Fig. Transfection with each of the constructs had no effect on respiration via the standard ETC, whereas oxygen consumption was resistant to antimycin A only after transfection with those predicted to be catalytically active Fig. . We alsoWe recently demonstrated an essential role for mitochondrial respiratory chain function in acute pulmonary oxygen sensing . FurtherIn this study we successfully generated cmRNAs coding for a humanized version of AOX, which were introduced into three different human and mouse cell-lines by lipofection. AOX cmRNA constructs incorporating different 5′ UTR elements were expressed at comparable levels, and expression persisted for at least six days in iMEFs that were prevented from proliferating, without evident off-target effects. The expressed protein exhibited enzymatic activity, whilst a version engineered to abolish such activity did not.AOX has been postulated as a therapeutic agent for use in diseases characterized by mitochondrial dysfunction. The characterized AOX cmRNA constructs should now ideally be tested in a collection of cell-lines with primary respiratory chain defects, as have been used previously to demonstrate the effects of conventional transfection using plasmids and lentivectors . FollowiSupplemental Material


Giardia duodenalis is subdivided into at least eight genetic clusters that have been named “assemblages” by specialists in the field. Some of these assemblages have been given the status of species, with Linnean binames. In the framework of the predominant clonal evolution model (PCE), we have shown that, from an evolutionary point of view, G. duodenalis assemblages are equatable to “near-clades”, that is to say: clades whose discreteness is somewhat clouded by occasional genetic exchange, but remain discrete and stable in space and time. The implications of this evolutionary status for the species described within G. duodenalis are discussed in light of the most recent genetic and genomic studies. The pattern of this species’ subspecific genetic variability and genetic clustering appears to be very similar to the ones of various parasitic, fungal and bacteria species. This underlines the relevance of a compared population genomics of pathogenic species allowed by the broad framework of the PCE model.Genetic and genomic data have long recognized that the species  Giardia duodenalis is a cosmopolitan parasite that causes waterborne infections of the digestive tract called Giardiasis. It infects humans and many other mammalian species, including dogs, cats, rats, beavers and cattle [d cattle . The medG. duodenalis is complicated by the fact that this parasite features two nuclei. Their genomes are similar but not identical. This leads to a phenomenon termed “allelic sequence heterozygosity” (ASH).Genetic analysis of G. duodenalis has been the target of isoenzyme*/Multilocus Enzyme Electrophoresis* (MLEE*) pioneering studies [G. duodenalis is a complex of species [Bg), Glutamate Dehydrogenase (Gdh) and Triose Phosphate Isomerase (Tpi). They all have shown that this species is subdivided into at least eight genetic clusters, labeled A to H, that have been named “assemblages”. These assemblages feature some host specificity [G. duodenalis assemblages and have revealed additional genetic clusters within them, termed subassemblages [Since the 80s,  studies . These s species . Furthercificity , althougcificity ,4. More cificity  have conG. duodenalis has a clonal population structure, like many other parasitic species. The evidence was based on the observation of repeated ubiquitous Multilocus Genotypes* (MLGs*) at frequencies that do not fit panmictic* expectations, the presence of statistically significant linkage disequilibrium* and the lack of recombinant genotypes. This clonal pattern has been corroborated by other authors [In the framework of the “clonal theory of parasitic protozoa”, we have proposed ,7 that G authors , with coThe definition of clonality proposed by us ,7 limitsG. duodenalis assemblages perfectly fit the definition of NC [G. duodenalis is concerned, discrepancies between phylogenies inferred from different genes have been considered as evidence for genetic exchange [The advent of high-resolution genetic and genomic surveys  has allowed us to refine the clonal evolution model . We haveon of NC . One shoon of NC ,17,18,19on of NC . When G.exchange ,21. Howeexchange , for exaexchange , or undeexchange .G.duodenalis, but it does not affect the interpretation of phylogenetic data.The existence of gene flow among and within the assemblages still is in debate. Ankarklev et al.  postulatG. duodenalis, it does not affect either the discreteness or stability of the assemblages, in accordance with the statement by Tsui et al. [However, if genetic exchange obtains in i et al. . These pi et al. ,29,30,31Ramírez and Llewellyn have claimed ,33 that We have designed  the RussSuch Russian doll patterns (RDPs) have been observed in many species of pathogenic bacteria, yeasts and fungi, and parasitic protozoa . It, thG. duodenalis is concerned, subclustering patterns (=lesser NCs), that is to say: RDPs have been repeatedly recorded within the assemblages (=main NCs). Subassemblages or subtypes (=lesser NCs) within assemblage A have been corroborated by several studies and have been termed A I, II and III [When  and III ,29,37. R and III ,29. Howe and III ,37. Figu and III  also ide and III , with ge and III , with th and III , fully cG. duodenalis assemblages have been therefore fully corroborated by many authors relying on various techniques and different sampling strategies. This ubiquity of MGBTs down to low evolutionary levels does not rule out the possibility of limited genetic exchange within the assemblages. However, it does indicate that PCE is verified within them, which makes it possible to reject for this species the pseudo-speciation model proposed in [RDPs within posed in ,33.This is a long-open debate among the specialists in the field. The need to design a robust terminology that permits scientific exchanges is compelling . HoweverG. duodenalis and G. enterica [G. canis, assemblage E, G. bovis, assemblage F, G. cati, and assemblage G, G. simondi [Several authors have proposed to consider assemblages A and B as different species, respectively termed enterica ,26. It h simondi .Giardia and other parasites will make it possible to explore possible signatures of sexual recombination and to describe distinct biological species. In other words, if sex occurs among assemblages, they would not deserve the status of distinct species, which deals with the BSC [G.duodenalis. Capewell et al. [However, the specialists in the field seem to be undecided about the use of the biological species concept* (BSC*), which states that different species are defined by the lack of genetic exchange among them . More ge the BSC . Other a the BSC ,21,23,26 the BSC  is that l et al.  propose G. duodenalis assemblages are genetically discrete and highly stable in space and time. These properties are more than enough to apply the phylogenetic species concept*  to themathogens , the PSCG.duodenalis, like for other pathogen species, may be virtually unlimited. Available data for G. duodenalis is not sufficient to confirm it, but for other species, RDPs and tiny NCs are observed down to extreme microevolutionary* scales . This is aureus . It woulG.duodenalis, as is the case for the parasitic species Leishmania [Trypanosoma cruzi [G. duodenalis. Indeed, some tests proposed to detect genetic exchange in parasites’ natural populations are based on the hypothesis that these parasites are diploid [G. duodenalis. In relation to this, it can be recalled that the BSC [It has been inferred  that aneishmania  and Trypma cruzi . This fa diploid . Aneuplo diploid . Such te the BSC , based oG. duodenalis molecular epidemiology, population genetics and evolution has made significant progress. Extensive comparison with various other pathogen species, including bacteria, parasitic protozoa and fungi [G. duodenalis population structure is not a special case, and, on the contrary, exhibits striking similarities with that of other pathogens. This underlines the relevance of a compared population genomics of pathogenic species allowed by the broad framework of the PCE model.In the last 20 years, our knowledge of nd fungi  shows thThe existence of at least eight “assemblages” that can be equated to NCs is firmly established, as well as their stability in space and time. The question of genetic exchange among and within the assemblages needs further exploration, however, it is well ascertained that such possible gene flow does not erase the integrity of the assemblages/NCs in the long run.G. duodenalis, such as G. enterica, G. bovis, G. simondi, etc., [Giardia species such as G. microti or G. muris.It is important to insist on the fact that the new species described within i, etc.,  are not i, etc.,  like forG. duodenalis population structure at a microevolutionary level, for knowing whether G. duodenalis pertains to the category of the so-called “measurably evolving pathogens” [In the near future, genomic studies should be actively developed to better address the problems of this parasite’s drug resistance and pathogenicity and to further explore the thogens” .


OsFAD3 gene encodes fatty acid desaturase, catalyzing the conversion of linoleic acid (LA) to alpha-linolenic acid (ALA) in rice. However, the genetic characterization of OsFAD3 and its role in the conversion of LA to ALA remains elusive. Here, we validated the effects of two homologous genes, OsFAD3-1 and OsFAD3-2, on the ALA and LA/ALA ratio in rice grains using near-isogenic lines. Two major haplotypes of OsFAD3-1 are identified with different effects on the ALA and LA/ALA ratio in rice germplasm. High expression of OsFAD3-1 is associated with high ALA accumulation and eating quality of rice grains. Overexpression of OsFAD3-1 driven by a seed-specific promoter increases the ALA content up to 16-fold in the endosperm. A diagnostic marker is designed based on an 8-bp insertion/deletion in the OsFAD3-1 promoter, which can recognize OsFAD3-1 alleles in rice. These results indicate that OsFAD3-1 is a useful target gene in marker-assisted breeding programs to improve varieties with high ALA and appropriate LA/ALA ratio in brown rice.Rice, as one of the main food crops, provides a vital source of dietary energy for over half the world’s population. The  Oryza sativa L.) is one of the most important staple foods and provides the source of dietary carbohydrates and energy for billions of people worldwide. It is usually consumed after removing and polishing the outer layers and the embryo, which will cause considerable loss of nutrients, dietary fiber, and beneficial compounds [Rice , linoleic (18:2), and alpha-linolenic (C18:3) acids, which have nutritional and functional significance ,7. It ha:1, linolFAD2 genes. Among them, OsFAD2-1 is located on rice chromosome 2, and the other three FAD2 genes are clustered on chromosome 7. OsFAD2-1 is highly expressed in rice seeds and plays a pivotal role in the conversion of oleic acid to linoleic acid in rice grains [FAD2 genes resulted in an increase of oleic acid and a decrease of linoleic acid in rice [OsFAD2-1 mutants increased the oleic acid more than two times and decreased LA dramatically to an undetectable level in rice grains compared to the wild type [FAD3 genes encoding ω-3 fatty acid desaturase, i.e., OsFAD3-1, OsFAD3-2, OsFAD7, and OsFAD8 [OsFAD3-1 and OsFAD3-2 are orthologous genes localized on the endoplasmic reticulum (ER) membrane, which catalyze LA to ALA in rice seeds [OsFAD3-2 led to a 27.9-fold increase of ALA content in the rice seeds, higher than that of the wild type [OsFAD7 and OsFAD8 had less effect on increasing ALA content in rice seeds [PAL6, LIN6, MYR2, and ARA6) have been identified to contribute to natural variation in unsaturated fatty acid compositions in rice germplasm [OsFAD3-1 and its role in the conversion of LA to ALA in rice remains elusive.Fatty acid desaturase (FAD) is a key enzyme in the biosynthesis of polyunsaturated fatty acids . The chae grains ,17. RNA  in rice . CRISPR/ild type . The ricd OsFAD8 ,21. Of tce seeds . Overexpild type . Moreovece seeds ,22. Receermplasm . HoweverOsFAD3-1 on the ALA and LA/ALA ratio in rice seeds and to explore the allelic variation of OsFAD3 in rice germplasm for designing diagnostic markers. The results can be deployed in genomic breeding programs to develop nutritional rice varieties.In this study, we used the near-isogenic line and its derived segregating population and overexpression transgenic experiment with aims to validate the effect of FAD3 genes (OsFAD3-1 and OsFAD3-2) in rice [http://rapdb.dna.afrc.go.jp/, accessed on 22 July 2022). To compare their effects on unsaturated fatty acid compositions, we developed two substitution lines (named IL3.1 and IL3.2) that contain a particular Nipponbare (NIP) allele (OsFAD3-1NIP or OsFAD3-2NIP) within the background of an indica variety Zhengshan97 (ZS97)  . The two7 (ZS97) A–C. The 7 (ZS97) D–F compa7 (ZS97) G,H. ThesOsFAD3-1 effect, an IL3.1-derived F2 population was generated for further genetic analysis. The population comprised of 110 genotyped individuals, using seven polymorphic markers to target the corresponding three introduced segments, as IL3.1 contains one OsFAD3-1 region on chromosome 11 and two other introduced NIP segments on chromosomes 5 and 9  around 10 days after flowering . The transcript level of OsFAD3-1 in the embryo is significantly higher than that of the endosperm during early seed development. qRT-PCR analysis confirms that OsFAD3-1 is expressed in the spikelets at 3–12 days after flowering, with a higher expression level of OsFAD3-1ZS97 than OsFAD3-1NIP throughout the reproductive stage, as revealed by using near isogenic lines (NILs) that harbor contrasting alleles in the background of ZS97  and NIL(OsFAD3-1NIP) (OsFAD3-1ZS97) contains higher contents of OA and ALA, but lower content of LA than NIL(OsFAD3-1NIP) (OsFAD3-1ZS97) reduces the LA/ALA ratio by 35.7% compared to NIL(OsFAD3-1NIP). These results demonstrate that highly expressed OsFAD3-1ZS97 may cause high ALA content and low LA/ALA ratio in rice embryos.Expression profile analysis reveals that  of ZS97 B. The saobserved . Consistobserved C. Moreovobserved . The unsD3-1NIP) C–G. NIL(D3-1NIP) H,I. NIL overexpressing OsFAD3-1 were evaluated in fatty acid content in the seeds. OE1 and OE2 significantly decrease LA and increase ALA in the embryo and endosperm compared to the wild type (WT), leading to a low ratio of LA/ALA  reveals a total of 29 single nucleotide polymorphisms (SNPs) and insertion/deletions (Indels) in the germplasm  as compared to NIL(OsFAD3-1NIP). Consistently, overexpression of OsFAD3-1ZS97 decreased HP, CP, SB, and CS, and increased BD  of fatty acids and most unsaturated fatty acids are mainly present in the embryo  or other chemical methods ,33,34. DOsFAD gene from the variety Nipponbare (NIP) introduced into the indica variety Zhengshan97 (ZS97) were developed using a marker-assisted backcross scheme [2 population and paired near isogenic lines (NILs) for the validation of the gene effect. All the rice materials were grown at the experimental stations of Huazhong Agricultural University in Wuhan  or Lingshui .Chromosome segment substitution lines (ILs) each carrying a particular s scheme . The ILshttp://redb.ncpgr.cn/modules/redbtools/, accessed on 10 June 2018) according to sequence variation. PCR amplification was carried out with a 20 μL system as described previously [Total DNA was extracted following the CTAB method . Primerseviously . The priOsFAD3-1 were used for haplotype analysis in a panel of 501 rice accessions. The genomic sequences of OsFAD3-1 (LOC_Os11g01340) and OsFAD3-2 (LOC_Os12g01370) were obtained from the Rice Genome Browser . The haplotypes were classified according to all SNPs according to the method described [The sequence variations of escribed .2 population using the IciMapping 4.2 software . Significant differences were determined among rice accessions or between pairwise lines using one-way analysis of variance (ANOVA) or Student’s t-test.QTL analysis was performed in an FUbiquitin gene (LOC_Os03g13170) was used as an internal reference. Gene expression quantity was analyzed as described previously [Total RNA in the rice spikelets at 3, 5, 8, and 10 days after flowering (DAF) were extracted separately, using the TRIzol Reagent Kit . The first-strand cDNA was synthesized using a Star Script II First-strand cDNA Synthesis Kit . Reverse transcription PCR (qRT-PCR) was performed with SYBR Green Master  on an ABI ViiA7 Real-Time PCR System  as described previously . The riceviously . Three bOsFAD3-1 and a seed-specific promoter (named Gt13) of GluA-1 (LOC_Os01g55690) were cloned into linearized pCAMBIA1301 (CAMBIA) with XbaI and HindIII successively using the Seamless Assembly and Cloning Kit . OsFAD3-1 was amplified from the ZS97 cDNA, and the seed-specific promoter Gt13 was obtained from the japonica rice NIP using specific primers (Gt13 and XFAD3FL1) [To construct the seed-specific overexpression vector, FAD3FL1) ,43. The Fatty acids of the embryo and milled endosperm were extracted and measured by a gas chromatography-mass spectrometry instrument (GC-MS) as described previously . BrieflyThe milled rice was ground into flour and passed through an 80-mesh sieve for the analysis of pasting properties. The starch paste characteristics of the flour samples were evaluated by employing a Rapid Visco Analyser  . Six parOsFAD3-1 encoding fatty acid desaturase catalyzes the conversion of LA to ALA and affects the eating quality of rice grains. Highly expressed OsFAD3-1 increases ALA and reduces the LA/ALA ratio in rice grains. A gene-specific marker of OsFAD3-1 developed can accurately distinguish OsFAD3-1 alleles. The genetic characterization of OsFAD3 facilitates a better understanding of unsaturated fatty acid metabolism, which is useful in brown rice breeding programs to improve varieties with high ALA and appropriate LA/ALA ratio for health benefits.


The gender diverse and transgender community is a minor patient group that is encountered with increasing frequency in the clinical setting, attributed to the improved awareness and access to medical facilities. Partial impairment to permanent elimination of fertility potential and outcomes depending on the treatment modality usually is a result of gender-affirming therapy, which includes both hormone therapy and surgical intervention. Although seldom conducted in the clinical field, transgender patients should be counseled on their fertility preservation options prior to medical and surgical gender transition. There is relatively limited data and clinical information regarding fertility preservation for transgender individuals. Current treatment regimens are based on protocols from fertility preservation after oncological treatments. Major barriers for the transgender population exist due to the lack of information provided and clinical narrative that is not familiar to the physician or health care provider, although there are various options for fertility preservation. A deeper understanding of this clinical agenda and the mandatory processes will ultimately result in a much more comprehensive and specific care for transgender individuals who are in great need for fertility counseling or treatment options that concern fertility preservation. In this review, current clinical approaches will be summarized and fertility preservation options along with ongoing and future clinical trials in fertility preservation for transgender individuals will be thoroughly reviewed. The transgender community represents a minor but growing patient population in the field of fertility and reproductive medicine. Transgender and nonbinary individuals comprise approximately 2% of the total population, with an estimated total number of 1.4 million transgender and nonbinary people residing in the United States, alone according to a 2016 survey . DefinitAccess to medical treatment and hormonal interventions has become less challenging to this minority population with an increased cognizance of gender variance and social acceptance to transgender and nonbinary people. Gender-affirming hormonal therapies include puberty-suppressing gonadotropin-releasing hormone agonists (GnRH) and sex hormone treatments, such as estrogen administration for those desiring feminine secondary sex characteristics and testosterone hormone therapy for male secondary sex characteristics. Surgical interventions of gender-affirming therapy consist of breast augmentation or mastectomy and gonadectomy. Although treatment modalities are potentially available to all gender fluid and transgender individuals, the patient may undergo some or all of these aforementioned treatment modalities, depending on their specific needs .With the increase in gender-affirming therapy, counseling transgender patients about fertility and fertility preservation has become an area of increasing concern. One of the main adverse effects in the long-term use of gender-affirming hormone therapy is infertility, although further clinical studies are required to elucidate the exact duration of hormonal treatment that causes irreversible effects ,7. RecenFor this specific patient population, physicians and medical providers may be less accustomed to the treatment modalities that are available. Results from a 2019 survey led by Tishelman et al. showed that medical providers from different nationalities acknowledged the need for further data and research based on the available options for transgender fertility preservation . There iIn this review, current clinical approaches and options on fertility preservation for the transgender population will be reviewed and provide insight along with an in-depth discussion on ongoing trials and future studies on fertility preservation for transgender individuals.The spectrum of an individual’s specific gender identity varies considerably in the transgender population. Often described as nonbinary, the gender of the transgender population falls outside the traditional binary system of man/woman. Commonly used terms to define an individual who does not fit the gender binary classification include gender fluid, gender neutral, and gender diverse. Gender identity is a person’s initial experience of gender or the internal being of self from a gender perspective ,16. GendMany patients have shown their interest in having biological children, contrary to the prevalent assumption that transgender people are not interested in parenthood. In some studies, more than half of the patients showed a strong desire to become parents; one study showed that 54% of transmen desired to have children of their own , whereasTestosterone is the treatment of choice for transmen and nonbinary persons undergoing masculinizing therapy. Testosterone is administered to transmen who are receiving masculinizing gender-affirming medical treatment via intramuscular/subcutaneous injection or through a transdermal route. Testosterone administration causes amenorrhea in the majority of the patients, as shown in one study where 82% of the patients became amenorrheic within 3 months . HoweverHistopathologically, testosterone administration affects folliculogenesis and the ovaries. Grimstad and colleagues showed that testosterone therapy for a mean exposure time of 36 months resulted in persistent ovarian function with follicular cysts in approximately half of the study population (49%), polycystic ovaries in 6%, and ovarian atrophy in 4% of the cohort, whereas 39% of the patients had normal ovarian histology. Patients with prolonged testosterone treatment showed an overall decrease in ovarian volume, although the mean volume of the ovaries in the study population remained within the normal range . In a reFertility preservation options for transgender men are oocyte cryopreservation, embryo cryopreservation, and ovarian tissue cryopreservation with in vitro maturation (IVM) . Oocyte There are few studies and limited data concerning COS while on gender-affirming hormonal treatment. Theoretically, a prolonged testosterone discontinuation may result in the reoccurrence of intrinsic ovarian stimulation and a healthier resting follicular pool compared to those on hormone therapy. However, the majority of the transmen community is reluctant to the discontinuation of hormonal treatment, resulting in a lower participation rate in fertility preservation. One study confirmed that a patient with a 1-week cessation of testosterone treatment was able to undergo ovarian stimulation and ended up with successful cryopreservation of 11 mature oocytes , whereasAs an alternative fertility preservation method, transmen patients have the option of embryo cryopreservation. Patients may use cryopreserved oocytes with sperm from either a partner or a donor for embryo fertilization. One of the advantages of cryopreserved embryos when compared to oocyte cryopreservation is the confirmation of higher quality blastocysts developed from the retrieved oocytes. Furthermore, several studies have reported a higher survival rate of the embryos (95–98%) in comparison to the 80% survival rate for oocytes ,35. EmbrIn 2019, the ASRM practice committee announced that ovarian tissue cryopreservation with IVM is no longer considered experimental . This feHaving female secondary sex characteristics and serum hormonal levels of estrogen and testosterone similar to cisgender women is the main objective of gender-affirming hormonal treatment in transwomen. Feminizing hormone therapy for transwomen usually involves a combination of androgen-lowering medications, which include GnRH agonists, spironolactone, or cyproterone acetate along with estradiol . Feminizp < 0.01). The authors of this study concluded that feminizing hormone effects on spermatogenesis may not necessarily be irreversible [Semen cryopreservation or testicular tissue cryopreservation is one of the fertility preservation options for transwomen . Sperm cversible . A recenversible . Anotherversible . The decMany transwomen patients find masturbation or ejaculation distressing, resulting in worsening of gender dysphoria, although it may seem relatively less invasive compared to transmen who have to undergo ovarian stimulation and oocyte retrieval. Some patients may have erectile and/or ejaculatory dysfunctions due to hypoandrogenism and electroejaculation, and penile vibratory stimulation may be helpful in such cases ,56,57. TTranswomen who have completed gender-affirming reconstruction are irreversibly sterile, and there are no fertility preservation methods available at this final stage of gender-affirming treatment. Alternative options such as surrogacy or uterine transplantation can be considered for this group of patients. Due to the diverse nature of elected partners in the transgender and gender fluid population, it is necessary to consider the fact that a gestational carrier may be required for a transgender couple to build a family . UterineThe Micro RNA Profile in the Ovarian Follicle Fluid of Transgender Men Treated with Testosterone and the Association with Fertility Potential (NCT03725280)  is a proA Belgian observation retrospective study entitled “Fertility preservation in Transgender Persons: A Retrospective Look at the Decision and a Survey of the Desire for Children” (NCT05120245)  is curre”Fertility preservation and Reproductive Needs of Transgender People: Desires, Attitude, and Knowledge of Subjects with Gender Dysphoria” (PaFer) (NCT03836027) is a prospective cohort observational study that has completed recruitment . The aimThe recently completed Fertility Decision Making in Youth and Young Adults (AFFRMED) (NCT05175170)  is a sinAlthough there is a strong desire for parenting among transgender individuals, various studies have reported the low usage of available reproductive and fertility services. Riggs et al. conducted a survey with a study population of 409 transgender and nonbinary individuals and results showed that of the people who were not parenting, 33% of the cohort had a strong desire to be parents . MoreoveThere is still an inconsistent and a small percentage of patients utilizing fertility preservation counseling and procedures, despite recommendations for fertility preservation counseling prior to gender-affirming medical treatments. Chen and colleagues showed that with transgender adolescents initiating gender-affirming hormone therapy and were referred for fertility preservation counseling, approximately 12% had preservation counseling, and only 5% of the study group underwent gamete cryopreservation . Only 45The transgender community experiences various limitations to achieving parenthood such as cost of treatment or lack of insurance coverage, gender dysphoria, and the invasive nature of gender-assigning procedures, although there are established recommendations for fertility counseling prior to gender-assigning therapies . A KoreaThe invasive nature of fertility preservation procedures is another barrier that causes reluctance for transgender patients to continue treatment, in addition to the high medical costs. Ovarian stimulation may take several weeks with multiple injections of gonadotropins, which results in the increase of serum estradiol levels, and the transgender patient is exposed to multiple transvaginal ultrasounds which may trigger gender dysphoria . The amoThere are currently no guidelines regarding fertility preservation for transgender individuals. As the parenting desire and demand of fertility counseling of transgender people is increasing, counseling and medical services should be readily available . Since tResults from various studies and clinical trials based on gender-affirming therapy have shown a detrimental impact on the potential for future fertility. Transgender patients undergoing gender-affirming treatment are at a risk for decreased reproductive capacity, and there is a growing awareness in the medical community focusing on the need for fertility preservation counseling prior to initiation of gender-affirming care. However, numerous studies have reported a limited standard of care with many transgender patients who are experiencing inadequate fertility counseling. Further prospective multicenter studies are needed to address the aforementioned shortcomings and advocate for better quality fertility services and parenting options for transgender and nonbinary individuals.