Introduction: As an interdisciplinary field, drug delivery relies on the developments of modern science and technology. Correspondingly, how to upgrade the traditional dosage forms for a more efficacious, safer, and convenient drug delivery poses a continuous challenge to researchers.Methods, results and discussion: In this study, a proof-of-concept demonstration was conducted to convert a popular traditional liquid dosage form  into a solid dosage form. The oral commercial solution was successfully encapsulated into the core–shell nanohybrids, and the ethanol in the oral solution was removed. The SEM and TEM evaluations showed that the prepared nanofibers had linear morphologies without any discerned spindles or beads and an obvious core–shell nanostructure. The FTIR and XRD results verified that the active ingredients in the commercial solution were compatible with the polymeric matrices and were presented in the core section in an amorphous state. Three different types of methods were developed, and the fast dissolution of the electrospun core–shell nanofibers was verified.Conclusion: Coaxial electrospinning can act as a nano pharmaceutical technique to upgrade the traditional oral solution into fast-dissolving solid drug delivery films to retain the advantages of the liquid dosage forms and the solid dosage forms. It contToday, electrospinning, as a useful nanofiber fabrication method, has spread into almost all scientific applied fields , includiBased on the abovementioned knowledge, we hypothesize that a coaxial electrospinning process can be developed to convert the unspinnable commercial compound licorice oral solutions (prepared from the intermediate licorice fluidextracts) into solid nanofibers. The active ingredients from licorice can be sealed into the core sections of structural nanofibers with a polyvinylpyrrolidone (PVP) shell protection, which is a frequently used polymer . MeanwhiMw = 1,300,000) and K30  were purchased from Sigma-Aldrich Corp . Analytical grade ethanol was obtained from the No. 3 Reagent Factory . Compound Glycyrrhiza Oral Solution  was purchased from Shanghai Laobaixing Big Pharmacy, which was fabricated by Shanghai Mei You Pharmaceutical Co., Ltd. . The production date, product batch number, and expiry date were 2022-06-23, 220606, and 2025-06-22, respectively. Water was double distilled just before usage.Polyvinylpyrrolidone K90 , a power supply , a collector formed by wrapping aluminum foil around cardboard, and a homemade concentric spinneret.First, 50 mL of the commercial oral solution was measured in triplicate. Weights of 0.0, 5.0, and 10.0 g of PVP K30 were added into the solutions, respectively. The electrospraying processes used these three brown solutions to prepare the S1, S2, and S3 samples, respectively . The sheTwo kinds of core–shell nanofibers, i.e., the S5 and S6 samples were prepared using the coaxial electrospinning processes. The shell fluid flow rate was kept at 2.0 mL/h, and the core fluid flow rates were adjusted to 0.5 and 1.0 mL/h for S5 and S6, respectively. The distance between the tip of the spinneret and the collector was kept at a fixed value of 20 cm. The applied voltages were adjusted under the criterion that the EHDA processes would be continuously and robustly carried out, and the values are included in The morphologies of the solid EHDA products including samples of the S4 monoaxial nanofibers and the S5 and S6 core–shell nanofibers were assessed using a field-emission scanning electron microscope . The SEM pictures were used to estimate the average diameters of the nanofibers in about 100 places using the ImageJ software . The sampling processes included cutting a small piece of fibrous mat, which was fixed on a sample table using a double-sided conductive adhesive, and a thin layer of Pt was sprayed for 60 s before the sample was placed into the vacuum chamber for assessment.The inner structures of the S4 monolithic nanofibers and the S5 core–shell nanofibers were evaluated using a transmission electron microscope . The samples were prepared by placing a carbon film supported by a 200 mesh copper mesh around the fiber collector for about 2 min; then, the collected nanofibers were put into the vacuum chamber for detection.X-ray diffraction (XRD) tests were carried out using the Bruker X-ray Powder diffractometer . The raw materials and their fiber mats were measured within a 2θ angle range of 5°–60°. The applied voltage and working current were 40 kV and 30 mA, respectively. The scanning 2θ range was from 10° to 60° with a rotation speed of 5° per minute).−1 with a resolution of 2 cm−1. The sampling for the solid materials included weighing 0.2 g of potassium bromide powder, grinding it with about 10 mg of the sample, pressing the mixture into solid tablets, and placing the tablets into the instrument for scanning. As for the commercial oral solution, a drop of its liquid was dripped onto a blank potassium bromide tablet for measurement.Fourier transform infrared (FTIR) analyses were implemented using the PerkinElmer FTIR Spectrometer . The experiments were performed in range 500–4,000 cmNew methods for human health are always highly desired. In this study , three mThe third experiment was an artificial tongue experiment. A piece of paper was wetted on the laboratory desk as an artificial tongue. A piece of the collected core–shell fibrous mat S5 was cut and placed on the surface of the wet paper. The process of the film’s disappearance was recorded using a digital camera.The EHDA processes mainly take advantage of the quick and simple interactions between the electrostatic energy and working fluids, by which the solvents can be repelled . Thus, tIn an electrospinning system, there are typically four parts, i.e., a power supply for furnishing the high voltage, a collector for the nanofiber deposition, a spinneret to guide the fluid , and a pump  . Among tin situ spraying processes are interesting. The change trend is sketched in In the multiple-fluid processes, one major issue is the compatibility between the different working fluids ; 2023g. When the core fluid stopped pumping to the spinneret’s nozzle, a traditional single-fluid blending electrospinning of the PVP/sucralose solution was conducted to produce the S4 sample. The whole process is shown in Based on the investigations of the electrospraying of the core licorice solution  and the The SEM images of the S4 and S5 nanofibers are included in Although similar in morphology, the S4 and S5 nanofibers had obvious differences in the deposition state. A comparison of The TEM images of the prepared S4 and S5 nanofibers are given in A reasonable selection of a drug carrier must meet several conditions, which at a minimum include good compatibility, manipulation of the drug release behaviors, and good processability . PVP has-1), and also from sucralose . These results suggested the following speculations, which showed the good compatibility between the licorice extracts and the PVP: 1) the commercial oral solution was successfully encapsulated in the PVP nanofibers; 2) no chemical reactions occurred between them, as no new peaks appeared, i.e., a good chemical stability was ensured; 3) the sucralose was completely converted into amorphous nanocomposites, most probably on a molecular scale. These results could also be deduced from the molecular formulas. For example, there are numerous -C=O groups within the molecules of the PVP, whether K90 or K30, and abundant -OH groups present in all other functional molecules ; thus, the hydrogen bonds should be broadly present between these molecules for high compatibility and good physical stability. Meanwhile, hydrophobic interactions should also exist between the carbon skeletons of the PVP and the molecules of glycyrrhizic acid and liquiritin from licorice, further enhancing their compatibility.Compared with the FTIR spectra of the PVP K30, PVP K90, sucralose, and the commercial oral solution, the S5 electrospun core–shell nanofibers contained characteristic peaks both from the PVP , from the commercial oral solution  and the resultant S5 nanofibers are exhibited in Compared to the solid dosage forms, the liquid dosage forms have the advantages of simple administration and often a fast dissolution and delivery process after oral administration. However, their disadvantages are also obvious, i.e., poor stability and difficulty in terms of encapsulation and shipping. Provided the solid dosage forms can be dissolved all at once, they should hold the advantages of both the solid dosage forms and the liquid dosage forms. Thus, three different methods were developed to assess the functional performance of the fast dissolution of the resulting S5 core–shell nanofibers.Shown in To be further observe the fast dissolution process of the S5 nanofibers, a water contact angle instrument was used to record the processes. Shown in In traditional pharmaceutical methods, there are several different kinds of solid dosage forms, such as fast disintegration tablets, buccal films, sublingual tablets, and mini tablets. These dosage forms are very useful for oral administration to the elderly, children, people with dysphagia, and persons in the case of water shortage. When they are placed in the mouth, the limited moisture in the oral fluid is enough for dissolving the loaded drugs. Thus, an additional experiment was designed to mimic this case, in which the fibrous S5 mat was placed on a sheet of wet paper  on the laboratory desk. The fast disappearance processes recorded by a camera are shown in The abovementioned three methods showed that there was no problem with the fast dissolution of the S5 electrospun fibrous mats, i.e., they had the advantages of the commercial compound solution for simple oral administration. Meanwhile, the active ingredients should be stabler when they are sealed into the core sections of the core–shell nanofibers and present in a solid state, which will be further investigated. In addition, the ethanol in the licorice fluidextract commercial solution was totally removed by the electrospinning procedure; thus, the electrospun S5 mats should be safer. Any residue of ethanol in the S4 and S5 samples and its contents in the commercial solution will be detected in the near future. Thus, compared with the solution, on one hand, the S5 electrospun fibers are more convenient for encapsulation and shipping and are more stable for long-term storage. On the other hand, they can ensure a safer and more convenient oral administration .Based on the above investigations, the fast conversion of the S5 electrospun core–shell nanofibers to a liquid state to deliver the contained active ingredients can be ensured. In addition to maintaining the advantages of liquid dosage forms, the electrospun core–shell nanofibers provide other benefits, including simple aftertreatment for commercial applications, convenient administration, and enhanced stability. Meanwhile, the mechanism for the rapid dissolution processes is clear, as diagrammed in Three key elements played important roles in the materials’ conversions and applications. Firstly, the PVP K90, as a shell polymeric matrix, was soluble in water and had a good electrospinnability, by which the unspinnable core licorice solution was effectively solidified into the S5 core–shell nanofibers. During the dissolution processes, the PVP K90 shell contacted the water first. It experienced a typical dissolution process of the macromolecules, i.e., absorbing water, swelling, disintegration, and dissolving into the bulk solution. Secondly, we used the PVP K30 as an additive in the commercial oral solution, which increased the viscosity of the core fluid to ensure a successful coaxial working process during preparation. During storage, PVP K30 can effectively keep the active ingredients in an amorphous state, preventing the possible recrystallization. When the fibrous S5 were utilized for oral administration, the PVP K30 quickly absorbed the water and released the loaded active ingredients in an erosion manner, quickly. The third element in the core–shell structure was the intentional arrangements of the components within it. If the shell and core section were to exchange places, the resultant core–shell nanofibers might still show fast dissolution; however, the stability might be reduced because the active ingredients would be located in the shell section; meanwhile, the PVP K30 has a stronger hygroscopicity than PVP K90. The proposed mechanism was demonstrated by a simple experiment. Shown in the top-right inset of With the gradual expansion of electrospinning in pharmaceutics, its ability to convert dosage forms is drawing increasing attention . The welvia coaxial electrospinning. Certainly, some other investigations such as the residue ethanol in the core-shell hybrids, the stability and the toxicity assessments should be further conducted for real commercial applications.In this study, modified coaxial electrospinning was developed through a combination of a spinnable blended solution containing PVP K90 and sucralose as a shell fluid and an unspinnable commercial oral licorice solution as a core fluid. Some PVP K30 was added to the commercial oral solution. Its EHDA-treating properties were investigated to convert it into solid core–shell nanofibers through the coaxial working process. The SEM and TEM assessments verified that the resultant nanofibers had a linear morphology, and the designed core–shell nanostructures encapsulated the active ingredients in the commercial oral solution. The FTIR spectra and XRD patterns indicated that the active ingredients of licorice (such as glycyrrhizic acid and liquiritin) had good compatibility with the polymeric matrix PVP K30, and they were kept in an amorphous state in the nanofibers. Three different types of methods (including water dripped on the fibrous mats and fibrous mats placed on a surface wet with water) were developed, and the fast dissolution properties of the electrospun core–shell nanofibers were demonstrated. A mechanism for a fast dissolution process was proposed. The present study pioneered a proof-of-concept demonstration of a method by which traditional liquid dosage forms can be upgraded into solid dosage forms and retain the drug delivery advantages of both the traditional liquid dosage forms and the solid dosage forms. Based on the reported protocols, there would be more dosage conversion possibilities for the safer and more convenient drug delivery through core–shell nanofibers


The problem of mental health in academia is increasingly discussed in literature, and to extract meaningful insights from the growing amount of scientific publications, text mining approaches are used. In this study, BERTopic, an advanced method of topic modeling, was applied to abstracts of 2,846 PubMed articles on depression, anxiety, and burnout in academia published in years 1975–2023. BERTopic is a modular technique comprising a text embedding method, a dimensionality reduction procedure, a clustering algorithm, and a weighing scheme for topic representation. A model was selected based on the proportion of outliers, the topic interpretability considerations, topic coherence and topic diversity metrics, and the inevitable subjectivity of the criteria was discussed. The selected model with 27 topics was explored and visualized. The topics evolved differently with time: research papers on students' pandemic-related anxiety and medical residents' burnout peaked in recent years, while publications on psychometric research or internet-related problems are yet to be presented more amply. The study demonstrates the use of BERTopic for analyzing literature on mental health in academia and sheds light on areas in the field to be addressed by further research. Mental health disorders are common in the general population, and in academia their prevalence might be even higher , information-theoretical topic modeling CorEx  is suggested in the BERTopic approach, although other techniques can be integrated into this stage as well. UMAP is an algorithm based on manifold learning techniques and ideas from topological data analysis . HDBSCAN extends a density-based clustering algorithm DBSCAN with hierarchical clustering, is able to deal with noisy data, and does not assume predefined shape or size of the clusters :For word representations of topics, the class-based term frequency inverse document frequency (c-TF-IDF) weighting scheme is applied in BERTopic. This scheme is a modified TD-IDF scheme: the latter assesses representativeness of terms for an individual document, and the former for a topic, which is a cluster of documents. Grootendorst  defines A model can be fine-tuned with the Maximal Marginal Relevance (MMR) algorithm. MMR diversifies words within documents and thus influences topic interpretability. The diversity parameter of MMR  refers to the document and needs to be distinguished from the topic diversity (TD) metric, which is described below.v, which was shown to be the best performing metric in the comprehensive study comparing 237,912 measures on six benchmarks for topic coherence  moves one word token per step over the documents to create virtual documents to compute word probabilities. For an indirect confirmation measure, Cv combines the cosine similarity measure with the normalized pointwise mutual information (NPMI).Topic models can be assessed and compared. Such metrics as topic coherence (TC) and topic diversity (TD) are widely applied for model evaluation  are calculated between each word vector and the topic vector, which is the average of the word vectors. The arithmetic mean of the similarity values is taken as the final TC (Cv) score. The score ranges from 0 to 1, with higher values indicating more coherent topics  through topic embeddings. The second is the hierarchical clustering dengrogram. It presents agglomeration levels of the topics, which are constructed by hierarchical clustering. The clustering is based on the Ward linkage function applied to the cosine distance matrix between topic embeddings. The Ward approach consists in minimizing the merging cost of combining clusters Due to the different mathematical underpinnings of the heatmap and the dendrogram, resulting similarities between the topics might differ.i:Evolution of topics with time can be explored and visualized in BERTopic. Dynamic topic modeling assumes that topics exist on the whole timespan but their representations change. Therefore, BERTopic first generates the global topic representation by fitting the model on the entirety of documents. Then, the global IDF values are multiplied by term frequency of documents at a given time  or Beck Depression Inventory (BDI), are known to domain specialists; some of them are explained in the Results section. For embeddings, the author resorted to all-MiniLM-L6-v2 model, the recommended BERTopic option. This sentence-transformers model maps sentences and paragraphs to a 384-dimensional dense vector space and is capable of capturing semantic similarities between documents  with a few changes. Firstly, the document embeddings are not presented here, as the plot of insufficient resolution would not give full credit to the interactive visualization created by the BERTopic function (see the Python code). Secondly, a simple visualization is added related to associations between a search term and the topics. BERTopic allows finding topics relevant to a search term and gives the similarity score between the term and the topic. The author used NetworkX library in Python to visualize the results of this search as a graph with three relevant topics as nodes. Similarity scores on the edges are cosine similarities between the search term embedding and the topics embeddings. In the study, examples for two search terms are given, and the interested researcher can use the Python code to depict these relationships for any search term.Web scrapping from PubMed database was conducted with Python . It resulted in the sample of 2,846 abstracts of papers published in 903 journals in years 1975–2023. Prior to the analysis, the data was checked for quality . The histogram of papers published per year is presented in BERTopic models with different sets of parameters were compared in terms of topic interpretability, the number of outliers, TC, and TD. Each of the models was run three times  was tested. The default settings in HDBSCAN were minimal cluster size of 15 and minimal sample size of 15; for MMR, the default keyword diversity value was 0.8. Model 1 treated maximum 28% of abstracts as noise. With this proportion of outliers, the use of a different clustering model  as mixed or uninterpretable outliers, and the rest were assigned to one of the 27 topics.In this section, the final model is described. The identified 27 topics were further explored. Two largest topics had 238 and 223 articles, and two smallest topics had 31 articles each; these smallest topics were not stable in previous runs of Model 3. The topics were labeled based on their representative terms and documents. For instance, the automatically produced label for topic 5 is “depression_bdi_depressive” (BDI is Beck Depression Inventory). The first ten terms for topic 5, with their c-TF-IDF scores, are “depression” (0.035), “bdi” (0.026), “depressive” (0.019), “inventory” (0.018), “psychometric” (0.014), “scales” (0.014), “scores” (0.013), “affective” (0.013), “depressed” (0.012), and “temperament” (0.012). It can be concluded from these terms that the topic is related to psychometric studies exploring measures of depression. The classification score of the user-defined label “psychometrics of depression” (0.995) was high; in comparison, such a label as “depression and anxiety” scored 0.296. Hereinafter, the automatically produced labels are kept for BERTopic visualizations, and a few topics mentioned in the text of the paper are described by their user-defined labels, which were tested with the classifier and obtained scores above 0.990.For a specific topic, the most representative documents can be found. For instance, for topic 20 (“internet addiction”), the following abstract from the paper by Ni et al.  is one op < 0.01). There were significant positive correlations between SDS and SAS scores and Internet addiction (p < 0.001). Multiple logistic regression analyses showed that a single-parent family, the age of first exposure to Internet use, the age of the student, city residence, and homesickness were significantly associated with Internet addiction (p < 0.01). Special and closer attention should be paid to these factors, and a risk-focus approach should be implemented in university freshmen with depression, anxiety, and other influential factors associated with Internet addiction at the beginning of their university life to guarantee the fulfillment of their academic study and graduation.”“The prevalence of Internet addiction and influential factors associated with Internet addiction among freshmen college students were investigated in this study. A total of 3,557 first-year university students from a university in northwest China were surveyed with Young's 20-item Internet Addiction Test (IAT) questionnaire, a Self-Rating Depression scale (SDS), a Self-Rating Anxiety scale (SAS), and a basic information questionnaire. A rate of 6.44% of the participants surveyed showed Internet addiction. The students with Internet addiction had higher scores of SDS and SAS compared with those without Internet addiction , the heatmap, and the cluster dendrogram. In In Some topics have relatively high cosine similarity according to the heatmap and are agglomerated at the early stage of clustering in the dendrogram, such as topic 0  and topic 17 . Other topics are close according to the dendrogram but not to the heatmap. For example, topic 5 (“psychometrics of depression”) forms a cluster with topic 2 (“OCD and anxiety”) in the dendrogram, but in the heatmap has the highest similarity score with topic 11 .Dynamic topic modeling in the frame of BERTopic allows tracing evolution of topics with time. Finally, the BERTopic function that finds topics most similar to a search term was used, and the results were visualized with the custom graph. In With the term “internet addiction”, the researcher can see how topic 20 (“internet addiction”) is related to other topics. It appeared that topic 2 (“OCD and anxiety”) and topic 3 (“substance abuse”) are also relevant to this term, though with lower similarity scores. With the term “burnout measures”, the researcher can find topics related to psychometric instruments for burnout. These are topics 0 , topic 14 (“burnout in nursing”), and topic 17 . These results are discussed in the next section.Mental health in academia is a problem that requires attention from the research community. Depression, anxiety, and burnout are distinct but interrelated conditions highly prevalent in academic environments, and they are increasingly addressed by scientific publications. With growing number of these papers, methods of extracting meaningful insights from previous findings should be used that can process large amounts of data and employ human expertise. In this study, BERTopic, an advanced topic modeling technique, was applied to analyzing the abstracts of PubMed articles on depression, anxiety, and burnout in academia. Comparing to literature reviews that are conducted by human experts, topic modeling as a machine learning method gives a possibility to deal with a substantially larger number of sources: in this study, 2,846 scientific papers published from 1975 to 2023 were explored.The study illustrated the use of the modular structure of BERTopic, which provides researchers with sufficient flexibility for building models that satisfy their requirements. In this study, the model was selected based on topic interpretability, the proportion of outliers, and TC and TD as supplementary criteria. The undeniable subjectivity of the topic interpretability criterion can be understood, from the methodological point of view, as the need to be aware of multiple perspectives on topic selection depending on the specific research context . These results are presented in In addition to the general overview of the field, in which regard topic modeling is similar to a literature review, though on a substantially larger scale, the researcher can focus on specific topics and explore their relevant documents, similarities to other topics, and evolution with time. This focus is also subjective to some degree; the researcher studying the results of the model might be interested in topics related to OCD or borderline disorder. In this study, the author's attention was drawn to psychometric measurement and internet-related problems. Topic 5 (“psychometrics of depression”) contains abstracts of papers, many of which discuss psychometric measures for depression, validation of new scales, or translation and adaptation of the most frequently used scale, BDI, into different languages. The importance of valid and reliable psychometric instruments for depression, anxiety, and burnout should not be underestimated. For instance, in a meta-analysis .Publicly available datasets were analyzed in this study. This data can be found here: PubMed database (OL: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing—original draft, Writing—review & editing.


Aptamers that undergo conformational changes upon small-moleculerecognition have been shown to gate the ionic flux through nanoporesby rearranging the charge density within the aptamer-occluded orifice.However, mechanistic insight into such systems where biomolecularinteractions are confined in nanoscale spaces is limited. To understandthe fundamental mechanisms that facilitate the detection of small-moleculeanalytes inside structure-switching aptamer-modified nanopores, wecorrelated experimental observations to theoretical models. We developeda dopamine aptamer-functionalized nanopore sensor with femtomolardetection limits and compared the sensing behavior with that of aserotonin sensor fabricated with the same methodology. When thesetwo neurotransmitters with comparable mass and equal charge were detected,the sensors showed an opposite electronic behavior. This distinctivephenomenon was extensively studied using complementary experimentaltechniques such as quartz crystal microbalance with dissipation monitoring,in combination with theoretical assessment by the finite element methodand molecular dynamic simulations. Taken together, our studies demonstratethat the sensing behavior of aptamer-modified nanopores in detectingspecific small-molecule analytes correlates with the structure-switchingmechanisms of individual aptamers. We believe that such investigationsnot only improve our understanding of the complex interactions occurringin confined nanoscale environments but will also drive further innovationsin biomimetic nanopore technologies. Aptamers are artificial,single-stranded oligonucleotides isolated through an iterative evolutionarymethod to interact specifically with analytes of interest.17 Similarly, other conventional methods such as microdialysis or fastscan cyclic voltammetry (FSCV) are limited in probe dimensions: thesmallest probes to date are in the range of 100 μm 18 to hundreds of nm 20 in diameter. Further, existing implantable neural probes or FSCVelectrodes have exposed surfaces, which are prone to biofouling inbiological environments.21 By confiningthe sensing area within an aptamer-filled nanopore that occludes nonspecificbinders, we minimized surface biofouling and demonstrated the potentialto measure dopamine in undiluted biofluids such as human serum.Herein, we havedeveloped a specific and selective dopamine aptamer-modifiednanopipette sensor with a nanoscale sensing area (10 nm nanopore)and femtomolar detection limits. Such nanoscale sensors for neurotransmitterdetection approach synaptic dimensions, facilitating localized measurementswith nanoscale spatial resolution. Implantable aptamer-based neuralprobes, while comparably sensitive and selective, are on the microscale.decrease was observedupon dopamine detection while an increase was recordedfor serotonin sensing. While prior works implicated structure-switchingaptamers to modulate the opening and closing of aptamer-modified nanopores,27 in-depth characterization of analyte-specific aptamer structuralrearrangement in the context of nanopores has been lacking.Beyond developing highly sensitive nanoscale dopamine biosensorsfor neuroscience, our goal was to glean mechanistic insight into thefunction of small-molecule nanopore sensors driven by structure-switching.Detection of comparably sized and equally charged small molecules using identical nanopore dimensions,allowed investigations into the influence of target-specific aptamerconformational dynamics on the measured sensor response. We observeda contrasting phenomenon when comparing the dopamine and serotoninnanopipette sensors using the same measurement system. The ionic currentchanged in opposite directions: a 32 were performed to simulate the aptamers in their free vs target-boundstates, enabling visualization of the conformational dynamics of theserotonin and dopamine aptamers upon capturing their respective targets.Correlations between experimental and theoretical findings demonstratethe potential of MD simulations as an in silico toolto predict aptamer structure-switching mechanisms. Further, valuesof aptamer height change extracted from the simulations enabled calculationsvia the finite element method to correlate the electrochemical behaviorof the sensor to aptamer-specific conformational dynamics inside thenanopore.Thus, to understand the driving mechanisms for signal transductionin aptamer-modified nanopores, the conformational dynamics of thedopamine and serotonin aptamers were studied and compared experimentallyand theoretically. The divergent directionality of structure switchingfor the two neurochemical aptamers on surfaces was corroborated byquartzcrystal microbalance with dissipation monitoring (QCM-D). Further,molecular dynamics (MD) simulations, previously shown to predict oligonucleotidetertiary structures accurately,27 Further, finite element method simulations of the ionicflow through the nanopore corroborated this pore size; the experimentalelectrical resistance value of 367 MΩ suggests an opening of9.2 nm .The current flux through the nanopore is measured upon applicationof a bias between two Ag/AgCl quasi-reference electrodes: one insidethe nanopipette and one in the bulk solution 1a. Dopam33 This nonlinear, diode-like effect observed inthe current–voltage curves is called ion current rectification(ICR), which is influenced by the surface charge inside the nanoporewhen the pore size and geometry are kept constant.36 The rectificationcoefficient (r) can be extracted from the ICR bytaking the logarithm of the ratio of the absolute values of the currentmeasured at a positively applied voltage, by the current at the correspondingnegative voltage:r value changed from−0.2 ± 0.03 for bare quartz surfaces due to deprotonatedhydroxyl groups to +0.3 ± 0.02 upon assembly of positively chargedaminosilanes  in undiluted (1×) PBS, the sensor showeda decrease in the current response from baseline 2a. AlterKd) has been reported as 150 nM.37 The observednonlinear behavior and detection limit orders of magnitude below the Kd suggest that aptamer-modified nanopipettesare nonequilibrium sensors. Shift from conventional equilibrium behaviormay arise from the conical geometry and nanoconfinement of the sensors.The frequency of target rebinding events may be higher at the mostconfined tip region compared to the unbinding and release of targetsoccurring more prominently further away from the tip that tapers tolarger volumes. Single molecules captured from a larger volume andconcentrated at the nanopipette tip have been shown to enable fM detectionlimits.38 Inducing mass transport of chargedmolecules like dopamine through the nanopore by applied voltages drivesthe analyte trajectory through an aptamer-filled column, thus augmentingmolecular interactions. Macromolecular crowding has been shown toenhance the sensitivity through cooperative effects between electrolytesand polymers.40The dopamine aptamer binding affinity , aligning with finite elementmodels that correlate nanopore size with optimized sensor response(see Supporting Information). Limitingthe size of the nanopore and ensuring aptamer confinement have furtheradvantages for biosensing in complex biological media. The DNA aptamerspreclog the pore to prevent nonspecific molecules from entering, whichenabled measurements in undiluted human serum. Dopamine detectionin human serum has important implications in neurodegenerative disorderssuch as Parkinson’s disease and Alzheimer’s disease.41 A current decrease of comparable magnitude tomeasurements in 1× PBS was observed for dopamine aptamer-modifiednanopipette sensors with increasing amounts of dopamine in human serum and l-3,4-dihydroxyphenylalanine(L-DOPA) as well as serotonin with statistical significancein this complex medium .54 For the dopamine aptamer-modified sensor, the r value becomes increasingly negative, while the r value of the serotonin aptamer-modified sensor increases in valueupon serotonin capture. These results indicate that dopamine vs serotoninaptamers alter the surface charge density in opposite directions upontarget recognition within the nanopore.We hypothesized that the opposite directionality observed in thenanopipette sensors is driven by the divergent structure-switchingof aptamers inside the confined nanopore. The 4 aptamer conformational dynamics that modify the hydration layerat the surface of the sensor can be harnessed to amplify the signalof small-molecule binding.55 The QCM-Dprovides qualitative insight into both the directionality and magnitudeof conformational change for aptamers tethered to surfaces with restricteddegrees of freedom, thereby mimicking the dynamics inside the nanoporesensors. Dopamine aptamer assembly on the QCM-D substrate was confirmedby a decrease in frequency of 23.7 Hz  indicative of an assembled rigid monolayer with a height of 4.1nm, calculated via the Sauerbrey equation  and over N = 6 measurementsconducted with three different chips . When this procedure was repeated on a chip functionalized withthe scrambled control DNA, negligible change in frequency was observedupon exposure to equal amounts of dopamine, indicating target-specificstructural rearrangement .Further, injection of the PBS buffer as well as incubation of structurallysimilar molecules (L-DOPA and norepinephrine) resultedin a stable baseline , QCM-D is an ensemble method that monitors the dynamics of aptamermonolayers, which may not represent the behavior of individual aptamerswithin the nanoscale tip. To interrogate single molecule aptamer structure-switchingmechanisms, MD simulations were conducted.The compression of the dopamine aptamerlayer upon target recognitionobserved at the surface of the QCM-D sensor is in the opposite directionto what was observed for the serotonin aptamers upon binding serotonin(1.2 nm elongation of aptamer backbone upon target capture).Figure S4).The structure-switching dynamics of the dopamine and serotoninaptamers upon interactions with respective targets were conductedwith a constraint on the 5′ end  to mimic covalently tetheredstates with reduced degrees of molecular freedom. The conformationalstability of the aptamers can be determined by the deviations in theroot-mean-square deviation (RMSD) within the simulation time interval;smaller deviations indicate higher structural stability. The conformationdynamics were simulated for an interval of 100 ns, which guaranteedobservation of the system in a stable state based on monitoring theRMSD . Specifically, hydrogen bonds formed between the hydrogens of dopamineand the aptamer nucleobases A28, A30, and G33 at distances of 3.124.42, and 3.08 Å, respectively, constitute the binding pocket,and hydrophobic interactions S5a. SpecFrom these intermolecular interactions, where thebases A28, A30,G33, A35, A27, and G29 interact strongly with the dopamine molecule,it can be inferred that the binding takes place inside the asymmetricinterior loop of the dopamine aptamer. The 3D structure of the dopamineaptamer in the absence and presence of dopamine was extracted at theend of the MD simulation 4c. AccorFigure S5b). Explicitly, thebinding pocket was composed of hydrogen bonds formed between the hydrogensof the serotonin and the G19, G20, T26, and G27 aptamer nucleotideswith distances of 2.12 2.59, 1.89, and 2.79 Å, respectively (S6b). The 3D structuresof the free serotonin aptamer and the target-bound state with theserotonin molecule in the distal stem loop were extracted  S5b. Expl6 such ensemble measurements are limited. Circulardichroism can infer the formation of specific structural motifs butcannot indicate the location of rearrangement. The change in distanceof certain bases in the aptamer backbone can be monitored locallyvia FRET. However, mapping the entire structural dynamics from FRETis challenging; the attachment of fluorophores in certain locationsof the DNA backbone can interfere with structure switching and targetrecognition. To this point, MD simulations of aptamers and their respectivetargets contribute structural information at the single molecule level.The MD simulations enabled the visualizationof the 3D structuresof individual dopamine and serotonin aptamers. Further, the bindingpockets to respective targets were identified based on the energeticsof intermolecular interactions. While methods such as circular dichroismspectroscopy and Förster resonance energy transfer (FRET) enabletracking aptamer structural rearrangements upon target binding,Experimental results by QCM-D and theoretical analysis by MD simulationsboth indicated that the conformational changes of aptamer moleculesmodulate the measured ion current changes in nanopipette sensors.To connect our findings on the aptamer structure-switching dynamicsto the ionic current modulation through nanopores, a finite elementmodel was designed. The model consisted of a solid nanopore sensor(geometry determined from electron microscopy images) modified withan ion-permeable charged layer with a variable thickness, representingthe aptamers. We aimed to interrogate the effect of altering the aptamerlayer charge density on the rectifying behavior of the nanopore sensors.We expected the signal transduction through the nanopore to be determinedby two phenomena: (i) variation of the stored charge density in thelayer of the aptamer molecules at the nanopore walls and (ii) changesin the aptamer layer permeability for ions carrying the current. Theformer occurs as the expansion/contraction of the aptamer layer cancause a proportional decrease/increase in the number of charges perunit volume, respectively, even when assuming no overall change inthe total number of charges.Supporting Information section SI-5). The simulationfor the dopamine aptamer-modified nanopipettes indicated that thecontraction of the charged aptamer layer on nanopore walls resultedin stronger rectification and a slight decrease of the ionic currentat positive voltages. This trend matches what was observed in theexperiments upon sensor exposure to dopamine.Both the QCM-D and MD simulationsindicated that the dopamine aptamer layer compresses upon target recognition.The values extracted from the simulation for the free aptamer (5.5nm) and dopamine-bound aptamer (4.9 nm) were used to approximate thechange in the charge density in the dopamine aptamer layer. An increasein the charge density by ∼11%  was used as a parameter in the finite element model . In this case, the total change of the ionic current for the freedopamine aptamer vs dopamine aptamer–target complex approachesthe experimentally observed 11% current decrease for 1 nM analyte.However, the difference in curve shapes observed in the model comparedto the experimental I–V characteristicssuggests a qualitative agreement rather than quantitative comparison.However, the chargedensity variation alone did not account forthe experimentally observed changes in the current magnitude. Forthe dopamine sensors, we hypothesize that the contraction of the dopamineaptamer layer leads to a more compact and hence less permeable medium,through which ions are transported with a smaller diffusion constant.I–V curve for the negatively charged aptamers (regardless of sequence).Alternatively, at negative biases, the effect of changing the thicknessof the charged layer and the influence of altering the ion diffusivitytend to cancel each other out, resulting in a lack of observable trendfor sensing.The finite element model appears to be generalizable for differentaptamers when the sequence-specific conformational dynamics is wellunderstood. While the behavior and the measured signals follow anopposing trend for the dopamine vs serotonin aptamers, the mechanismbehind the signal transduction bears close similarities. Conformationalchange upon analyte binding appears to cause a contraction (dopamine)or expansion (serotonin) of the charged aptamer layer accompaniedby a change in mass transport  throughthis medium. Interestingly, these factors are most pronounced onlyon the positive side of the Supporting Information section SI-5).Two factors play major roles: (i) the initial thickness of the aptamerlayer  and (ii) the total changeof the aptamer layer height upon target addition. According to thesecalculations, the optimal initial aptamer layer should be around 5nm (for a nanopore with 9.2 nm opening). Our dopamine aptamer layerthickness of ∼5.5 nm determined through MD simulations , which identifies the ratio of the surfacevs bulk conductivity occurring within the aptamer-modified nanopore.59 The approximated Du value of8.4 and 4.0 for the dopamine and serotonin aptamer-functionalizednanopores, respectively , impliesthat surface conductivity dominates vs the bulk conductivity  and the currentthat flows through the assembled aptamer layer (Iaptamer). These calculations support our experimentaland theoretical findings (Supporting Information section SI-8).60 By interrogatingaptamer structure-switching dynamics extensively through differentmodels and connecting these findings to the observed sensor response,we have improved our understanding of the distinctive behavior ofaptamer-modified nanopore sensors.An alternative way to interrogate the influenceof the aptamerconformational dynamics on the ionic flux through the nanopore isby calculating the different factors that contribute toward the changein current. The specific conformation of the aptamers influences thecharge density of mobile counterions in the nanopores that neutralizethe negative DNA backbone. Thus, the experimentally measured currentresponse has contributions from the current excluded by the volumeof the 3D DNA  and real-time sensing capabilities.Our approach represents an advancement over existing detection systems,which require analyte separation and sample pretreatment. However,it is important to note that for clinical assessments, benchmarkingagainst established gold standard methods will be imperative.In parallel with the development of nanoscale dopamine sensors,we investigated the fundamental mechanisms governing the modulationof ionic flux through nanopores. In particular, our goal was to understandthe opposite directionality of the measured current when comparingaptamer-modified nanopipette sensors targeting two neurotransmittersof comparable mass and equal charge: dopamine and serotonin. ThroughQCM-D, a complementary surface-based piezoelectric sensor, we confirmeddivergent aptamer conformational dynamics upon target binding.To corroborate ensemble measurements from QCM-D with computationalanalyses at the single molecule level, MD simulations were performed.Correlations between the theoretical and experimental findings validatedthe MD simulation as a potential layer of control for establishingdesign rules for aptamer-based nanopore sensors. For example, visualizationof the aptamer–target interactions may provide insight intohow to optimize aptamer surface densities to minimize steric hindranceor what size nanopores to employ based on the magnitude of structureswitching. We envision that integration of MD simulation within theaptamer selection process could create an intermediate feedback loopto generate aptamers endowed with optimal structure-switching capabilities,which would accelerate the development of next-generation aptamer-modifiedsensors. Further, values extracted from the simulations can be incorporatedinto finite element models to understand the combined influence ofchanges in the charge density and ion permeability of the aptamerlayer. Such investigations improve our understanding of complex interactionsoccurring in confined nanoscale environments.The central roleof aptamer conformational change within nanoscaleconfinement as the driving mechanism for small-molecule target detectionendows the sensors with inherent selectivity. The structure-switchingresponse is target specific, resulting in minimal effects from nonspecificmolecules on the measured current response. Gaining insights intothe fundamental mechanisms underpinning aptamer-modified nanoporebiosensors expands the versatility of this technology for detectingsmall molecules. Beyond the development of innovative nanotools thatbring us closer to unraveling the complexity of brain chemistry, ourfindings will drive further innovations in biomimetic nanopore technologies.2HPO4, 1.8 mM KH2PO4) and pH 7.4 or in human serum(Sigma-Aldrich) as received. Deionized water  was used for the preparation of all solutions.All aptamers were purchased and HPLC-purified by Microsynth AG . Aptamer stock solutions of 100 μM were aliquotedand stored at −20 °C until use.Sigma-Aldrich Chemie GmbH was the main supplier for the chemicals used in this work unless otherwisenoted. All measurements utilized phosphate buffer saline (PBS) at1× concentration .The capillaries were transformed into nanopipettes using a laser puller. For reproducible nanopipettes, the laserpuller was preheated for at least 1 h prior to use, and a pull wasactivated without fastening a capillary prior to nanopipette fabrication.To achieve ∼10 nm diameter orifices, the following parameterswere used: (line 1) heat 750, filament 4, velocity 40, delay 150,and pull 80; (line 2) heat 700, filament 3, velocity 60, delay 135,pull 180. We note that these parameters may vary from instrument toinstrument27 Briefly, vapor phase depositionwas conducted under vacuum at 40 °C for 1 h to assemble monolayersof (3-aminopropyl)trimethoxysilane (APTMS) on the nanopipettesurfaces. Maintaining ambient humidity below 40% is crucial for monolayerassembly, and silane clogging has been observed if this parameterwas disregarded. To increase the yield of functional sensors, silanizednanopipettes were first characterized in 1× PBS to ensure propersurface assembly prior to subsequent steps. Then, nanopipettes werefilled for 1 h with 1 mM solutions of 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) dissolved in a 1:9 (v/v)mixture of dimethyl sulfoxide and PBS. Aptamer disulfide bonds werereduced for 1 h at room temperature using a 50-fold excess of tris(2-carboxyethyl)phosphine(TCEP) relative to DNA aptamer concentration. The DNA solution wasdiluted to 5 μM in 1× PBS and cleaned to remove unreactedTCEP and cleaved protective groups using Zeba spin desalting columns.The purified DNA solution was then heated up to 95 °C for 5 minand then renatured by cooling to room temperature to ensure unhybridizedsequences in optimal conformations for surface assembly. The MBS solutionwas removed from the nanopipettes, and the sensors were rinsed with1× PBS. Then, the prepared aptamer solution was incubated fora minimum of 2 h to ensure functionalization to the nanopipette surface.Prior to storage or experimental use, the aptamer solution was removedand the sensors were rinsed 3-fold with PBS. Nanopipettes were storedfilled with Milli-Q water to reduce etching of the quartz65 and were stored at 4 °C in high humidityenvironments to prevent solution evaporation, which may lead to nanoscaletip breakage upon salt crystal formation. Filling and emptying ofthe nanopipettes were enabled by MicroFil syringe tips .DNA sequences were functionalizedon the inside of the quartz nanopipette using a previously reportedprotocol.–1 voltage sweep rate.Current measurement was enabled by two Ag/AgCl quasi-reference counterelectrodes fabricated in house. One electrode was positioned insidethe nanopipette and another in the bulk solution. The current wasmeasured via a custom-built high gain current amplifier, and the datawere recorded using a custom written LabVIEW interface , based on WEC-SPM package provided by Warwick Electrochemistryand Interfaces Group. Data were collected using an FPGA card PCIe-7852R. The current magnitudes and potentials reportedin the paper are denoted with respect to the electrode in the bulksolution. Cyclic voltammograms were acquired by sweeping voltage at0.2 V sF areavailable in the Supporting Information.The QSense E4 (Biolin Scientific) was used for QCM-D measurements,and thiolated aptamers were assembled on QSense gold chips (QSX 301).The chips underwent a rigorous cleaning procedure consisting of 2min sonication cycles in the following solutions chronologically:2-propanol, acetone, and Milli-Q water. The chips were dried usingpressurized nitrogen and then ozone-cleaned for 30 min. The QCM-Dchip was then sandwiched between two electrodes that apply a voltageto excite the piezoelectric material at its resonance frequency. Awinding channel at the flow cell inlet ensures a stable liquid temperatureof 24 °C upon contact with the chip surface. The signal fromthe third harmonic is shown in all graphs. Further details of themeasurement protocols and mathematical calculations for extractingthe change in monolayer height from the Δ66 The final state of these resulting structureswas used as a starting point for the MD simulations. The simulationof the dopamine and serotonin aptamers and their interactions withtheir respective targets was performed in Gromacs using the AMBER99SB-ildnforce field. The aptamers were placed in the center of a water boxwith suitable dimensions according to the size of the different aptamers.Then, the respective analyte, dopamine or serotonin, was insertedinto the box with a 1 Å distance between the box surface andthe aptamer. Afterward, ions and water molecules represented usingthe transferable intermolecular potential with 3 points (TIP3P) modelwere inserted into the box.The single-stranded dopamine and serotonin DNA aptamers were firstmodeled with the MacroMoleculeBuilder command line tool v2.17.NVT (constant number (N), volume (V), and temperature (T), respectively) and NPT (constant number(N), pressure (P), and temperature(T), respectively) and assembled with a time stepof 2 fs for 100 ps. Finally, the simulation was performed at roomtemperature and pressure, 294 K and 1.01 atm, for 200 ns. The MD simulationswere performed using high performance computing (CINECA). Gromacstools were used to analyze the trajectories of conformational change of the MD simulation. Further detailsof the RMSD calculations and intermolecular interactions between theaptamer and analyte can be found in the Supporting Information.AmberTools in Gromacs was used togenerate the analyte topology files. By application of the steepestdescent algorithm and considering periodic boundary conditions (PBC)in all directions, energy minimization was performed. The systemswere equilibrated in Supporting Information.Rectifying behaviorof the ionic current through nanopipettes was modeled using the finiteelement method software package Comsol Multiphysics (version 6.0)with Transport of Diluted Species and Electrostatics modules. Furtherdetails of the model geometry, equation formulation, mesh, etc. areavailable in the P < 0.05 considered statistically significant. Comparativedata were evaluated by one-way analysis of variance followed by Tukey’smultiple group comparisons.All statistics were carried out using GraphPadPrism 9 . Data are reported asmean values ± standard errors of the mean values with probabilities


Klinefelter syndrome (KS), caused by the presence of an extra X chromosome, is the most prevalent chromosomal sexual anomaly, with an estimated incidence of 1:500/1000 per male live birth . High stature, tiny testicles, small penis, gynecomastia, feminine body proportions and hair, visceral obesity, and testicular failure are all symptoms of KS. Endocrine , musculoskeletal, cardiovascular, autoimmune disorders, cancer, neurocognitive disabilities, and infertility are also outcomes of KS. Causal theories are discussed in addition to hormonal characteristics and testicular histology. The retrieval of spermatozoa from the testicles for subsequent use in assisted reproduction treatments is discussed in the final sections. Despite testicular atrophy, reproductive treatments allow excellent results, with rates of 40–60% of spermatozoa recovery, 60% of clinical pregnancy, and 50% of newborns. This is followed by a review on the predictive factors for successful sperm retrieval. The risks of passing on the genetic defect to children are also discussed. Although the risk is low (0.63%) when compared to the general population (0.5–1%), patients should be informed about embryo selection through pre-implantation genetic testing . Finally, readers are directed to a number of reviews where they can enhance their understanding of comprehensive diagnosis, clinical care, and fertility preservation. Klinefelter syndrome (KS) is a congenital trisomy of male sex chromosomes, characterized by the presence of one or more extra X chromosomes ,2, phenoAbout 80–90% of KS cases present a non-mosaic karyotype 47,XXY), while the remaining 10–20% evidence a mosaic karyotype , higher grade aneuploidies  or a structurally abnormal X chromosome , with mosaic cases originating a less severe KS phenotype 7,XXY, wh,12.KS is the most common chromosomal sexual anomaly and the most common chromosomal anomaly in men . In a DaIn its typical clinical picture, KS is phenotypically characterized by high stature (long limbs), small and hard testicles, small penis, gynecomastia (in late puberty), absence of body, pubic and facial hair, visceral obesity (feminine distribution of adipose tissue), eunuchoid body proportions (wide hips), and signs of androgen deficiency and infertility (mainly azoospermia) ,16,17,18In KS, hypogonadism is caused by a primary testicular disease. Hypergonadotropic hypogonadism is derived from structural and functional dysfunction of testicular Leydig cells (LCs) and Sertoli cells (SCs), and the hypothalamic–pituitary–gonad (HPG) axis. LCs present luteinizing hormone receptor (LHR) alterations, leading to luteinizing hormone (LH) resistance. This causes a subsequent decrease in testosterone (T) synthesis and secretion. Failure to respond to LH induces a state of elevated serum LH levels, which are thought to induce a compensatory LC hyperplasia; however, due to the small size of the testes and LC incompetence, LC hyperplasia does not compensate for the loss of androgen production. Normally, T exerts negative feedback over the hypothalamus, eliciting a decrease in gonadotropin-releasing hormone (GnRH) release. As GnRH acts over the adenohypophysis/anterior pituitary to induce the release of follicular stimulating hormone (FSH) and LH, this release is diminished. Additionally, T also exerts negative feedback on LH release from the adenohypophysis. These negative feedbacks are not observed in KS, which indicates the presence of a disrupted HPG axis, thus being also responsible for the rise in serum LH levels. Regarding the SC, these usually present receptors for androgens and FSH. By binding to the SCs, they elicit the synthesis and release of several molecules that act on the germ cells to induce and stimulate spermatogenesis. In KS, SCs do not respond to FSH, inducing a compensatory state of high serum FSH levels. FSH serum levels also remain high due to the SC decrease in the synthesis and release of inhibin-B, which would act on the adenohypophysis, where, by a negative feedback mechanism, it would lead to a decrease in the synthesis and release of FSH ,21,22,23Low androgen levels are responsible for the observed androgen insufficiency (hypogonadism) in KS, with clinical features also dependent on other effects caused by an extra X chromosome ,24. SignNewborn characteristics  are within normal limits, albeit slightly lower ,33,34,35KS has multisystemic consequences, with higher morbidity and mortality than the general population, especially if not diagnosed and treated with T as early as possible . EndocriSeveral metabolic disturbances are related to KS hypogonadism. Hypogonadism causes loss of muscle synthesis (causing a decrease in muscle mass), loss of glucose turnover (causing hyperglycemia), loss of fat turnover , and insulin resistance. Increased insulin resistance also produces LCs, aggravating the negative impact on T production ,30. DecrThis metabolic state promotes the development of cardiovascular diseases, with a higher prevalence of atherosclerosis, arterial hypertension, myocardial infarction, stroke, arrhythmia, and heart failure. As T acts as a vasodilator, tissues tend towards a vasoconstrictor state due to decreased T levels, aggravating cardiovascular diseases. Low T levels and the metabolic syndrome, on the other hand, cause changes in hemostasis (clotting factors), which favor the development of thrombophlebitis, pulmonary thrombosis, and lower limb ulcers. The main hypothesis proposes that insulin resistance, obesity, and the metabolic syndrome increase plasminogen activator inhibitor (PAI) levels, conducting to a decreased fibrinolytic state (blockage of thrombus formation) ,52,53. PEndocrine disorders also lead to abnormalities in bone metabolism, resulting in osteoporosis and tooth defects. In addition to hypogonadism, decreased lean body mass  and increased adiposity have been associated with low bone mass. Low bone mass is also associated with low levels of vitamin D  in KS cases. Patients also evidence increased bone resorption. Bone resorption is mediated by the hormone insulin-like peptide 3 (INSL3), released by LCs in response to LH stimulation. INSL3 stimulates bone anabolism by acting on osteoblasts. However, individuals with KS have low serum levels of INSL3 due to LC insufficiency, which stimulates the secretion of sclerostin, which will exert catabolic effects on osteoblasts, favoring osteoporosis .Patients with endocrine disorders and the metabolic syndrome are more likely to develop sexual dysfunction (erectile dysfunction and premature ejaculation), which can result in libido loss, anxiety, and depression ,14.Patients with KS may have neurocognitive deficits, including a decrease in intelligence quotient without loss of intellectual capacity, a delay in the development of complex learning and experience skills, and verbal deficits , which include language receptive skills  and expressive language skills , deficits in reading and writing (dyslexia leads to comprehension and communication difficulties), and deficits in cognitive control processes . Patients may have reduced attention, working memory, and cognitive flexibility, as well as a reduced capacity to evaluate facial expressions and form interpersonal relationships. Intriguingly, brain structure research demonstrated a reduced brain volume due to gray and white matter loss ,30,57.In general, patients report that the disease has a negative impact on their quality of life since childhood , pointing to decreased vitality and general health, insomnia, lower-paying jobs, greater difficulty in living with a partner, increased medication needs, and infertility .In KS patients, various psychiatric illnesses such as schizophrenia, bipolar disorder, autism, attention deficit hyperactivity disorder, depression, and anxiety are more commonly documented ,58,59.KS is also associated with an increased prevalence of autoimmune diseases  and cancer . Although no gene or genetic mechanism has been identified, this increase is most likely due to additional X chromosomal material, ,30.The extra X chromosome originates from nondisjunction errors of the sex chromosomes, being 50% of paternal origin and 50% of maternal origin. Paternal nondisjunction of sex chromosomes occurs during meiosis I, whereas maternal nondisjunction can occur during meiosis I or II, or during early post-zygotic mitotic divisions ,10,60. IThe presence of an extra X chromosome, androgen deficiency, and mosaicism are thought to influence the KS phenotype. Several mechanisms, including non-inactivation of specific genes, overexpression of somatic genes, altered methylation status, and altered androgen receptor sensitivity, have been proposed to explain the variability (severity) of the KS phenotype ,62. The SHOX gene escapes X inactivation, is located in the pseudoautosomal region of the X and Y chromosomes, and is involved in skeletal development (the growth and maturation of bones in the upper and lower limbs) [SHOX gene effect can be enhanced since there are more copy number variants in regions expressing genes that also escaped X inactivation in KS patients [Specifically, several factors are associated with the KS phenotype. KS frequency rises with maternal and paternal age . Patientr limbs) ,67. The patients ,65,68,69Other mechanisms have been proposed, including the involvement of CAG (cytosine-adenine-guanine) repeats in the androgen receptor (AR) gene, FSH polymorphisms, and imprinting defects. The AR gene is located on the X chromosome and contains a highly polymorphic trinucleotide repeat known as the CAG region , which has been linked to physiological androgen effects ,71. WhenKS is the most frequent chromosomal abnormality in infertile individuals 3–4%) and represents 8–12% of patients with secretory azoospermia ,81,82,83–4% and rBecause KS patients are not a homogeneous group, the peripheral karyotype may not predict testicular cell chromosomal constitution or the presence or absence of spermatogenesis ,95. In aSpontaneous pregnancies with birth of chromosomal normal children have been observed in mosaic KS patients  and non-In chromosomal normal men, 46,XY germ cells originate a similar proportion of 23,X and 23,Y spermatozoa. When 47,XXY spermatogonia can produce spermatozoa, the XX pairing will originate 24,XY and 23X spermatozoa, and the XY pairing will originate 24,XX and 23,Y spermatozoa in the same proportion. However, scientists discovered that the rate of 23,X spermatozoa in ejaculated spermatozoa from non-mosaic KS patients was higher than the rate of 23,Y spermatozoa, indicating a preferential pairing of homologous sex chromosomes (X favoring). These findings provided evidence that the majority of spermatozoa did not originate from 46,XY spermatogonia, a hypothesis that was further supported by the presence of a high proportion of spermatozoa showing numerical sex chromosome abnormalities, indicating that a large number of 47,XXY germ cells are capable of completing the spermatogenic process . The autRegarding the mechanisms underlying the presence of testicular mosaicism in focal spermatogenesis, the authors elaborated several hypotheses.In non-mosaic KS, the authors suggested that in a subset of spermatogonia the extra X-chromosome was eliminated, allowing the development of 46,XY primary spermatocytes while the SC maintained a 47,XXY chromosome constitution ,102,103.Despite the presence of severe oligozoospermia or cryptozoospermia, spontaneous pregnancies and deliveries have been reported in mosaic KS patients . PregnanThe testis is a gland with two major functions: spermatogenesis and hormone synthesis. The integrity of the testicular cells and of the HPG axis is required for proper testicular function. FSH and T receptors are not expressed in germ cells; hence, hormone effects are mediated through SC. Under FSH and T stimulation, SCs produce and secrete metabolites, hormones, and stimulating factors that act on germ cells to promote germ cell proliferation, meiosis, differentiation, survival, and apoptosis. However, in KS patients, spermatogenesis and hormone production functions are disrupted in consequence of LC, SC, and HPG axis dysfunction, with alterations in the pituitary feedback inhibition threshold being identified ,125.At the hormonal level, patients show high serum levels of estradiol (E2), FSH, and LH, decreased or normal levels of T, high levels of sex hormone-binding globulin (SHBG), formerly known as androgen-binding protein, low levels of INSL3, an LC product, low levels of inhibin B, a SC product, and low levels of anti-Müllerian hormone (AMH), a SC product ,129,130.INSL3 receptors can be found on both LCs and germ cells. The synthesis of INSL3 by LCs is constitutive and thus independent of the cyclic LH stimulation. INSL3 interacts with specific receptors on LCs to modulate steroidogenesis, and the synthesis of INSL3 is modulated by T and LH (stimulate) and E2 (inhibits). Serum levels of INSL3 are consequently reduced in KS patients ,132,133.AMH induces Müllerian duct regression in males, preventing the development of the uterus and fallopian tubes, whereas T stimulates Wolffian ducts’ differentiation into vas deferens, epididymis, and seminal vesicles. The synthesis of AMH in immature SCs is stimulated by FSH and E2. FSH also stimulates SCs’ aromatase conversion of T to E2. Immature SCs have E2 and FSH receptors but not androgen receptors, therefore AMH levels remain high because T inhibits AMH synthesis. SCs mature and express androgen receptors after puberty, and an increase in T levels cause a decrease in AMH synthesis, which coincides with the establishment of the blood–testicular barrier (BTB) and meiosis, both of which are dependent on androgen action and SC maturity for the synthesis of cell junction proteins ,133. TheSHBG is primarily synthesized in the liver and serves as a transporter of androgens and estrogens in blood  as well as a regulator of hormone availability to target tissues . In KS, The inadequate testicular environment favors the loss of SCs and germ cells, resulting in a reduction in spermatozoa production . DespiteAt the histological level, a progressive testicular insufficiency is observed due to replacement of the interstitium by fibrosis and hyalinization of the seminiferous tubules. These alterations are responsible for the development of small and hard testes, as well as azoospermia. In the testicular interstitium, LC hyperplasia is observed, whereas in the seminiferous tubules, a gradual loss of spermatogonia is observed due to SCs’ massive phagocytosis and degeneration. Tubular cell degeneration leads to the sequential replacement of the normal testicular architecture by tubular atrophy (collapse), sclerosis (deposition of collagen components), fibrosis (stiffening caused by connective tissue replacement), and cell hyalinization  over time ,132,143.Several mechanisms have been proposed for the increasing depletion of germ cells, including insufficient extra X chromosome inactivation, LC deficiency, disrupted apoptosis control, and imprinting aberrations ,145,146.Male infertile individuals with severe oligozoospermia or secretory azoospermia may be able to father children with the development of ICSI ,153,154 The existence of focal spermatogenesis in KS patients also has allowed for successful spermatozoa recovery (SSR) from the seminiferous tubules ,161,162.Several markers were described in non-mosaic KS cases to characterize their androgen insufficiency and spermatogenesis dysfunction in comparison to the healthy population ,173,174.There are currently no clinical or biological parameters that can predict an SSR with certainty in these KS patients. However, several patient characteristics were shown to be related to SSR cases as compared to unsuccessful sperm retrieval cases.Several studies found a significant increase in SSR with lower age ,182,183,Other studies were conducted to identify potential spermatogenesis markers, but without comparing cases with SSR versus cases with unsuccessful sperm retrieval. These included the findings of decreased testicular volume , decreasThe goal of T replacement therapy in young boys (early-to-mid-puberty) with KS is to promote linear growth, increase muscle mass, preserve bone density, and allow for the development of secondary sexual characteristics . AlthougMany KS patients have sought infertility treatment as adults, with no other symptoms or signs besides infertility. These patients had decreased testicular volume, high FSH and LH mean serum levels, and normal or slightly lower T concentrations. This group of patients did not belong to the main group (65–85%) of KS patients reported as having low T levels .For patients, the testicular SRR is critical. Previous reviews showed a mean SRR of 44% , with a According to studies, non-mosaic KS patients with low T levels should be treated with aromatase inhibitors first to decrease E2 levels and therefore enhance T intratesticular availability. This was suggested to potentially improve spermatogenesis in KS cases with foci of spermatogenesis . When apThe mTESE  is a verThese extreme rates were not anticipated. As a result, we conducted a thorough literature review that included 63 reports . The oveIn cTESE (37 reports), the SRR was 44% (228/516), with a range of 16–100%. There were 3 reports with ≥50 KS cases  ,31,165, In mTESE (18 reports), the SRR was 43% (427/991), with an SRR range of 17–100%. There were 7 reports with ≥50 KS cases  ,182,185,The mTESE requires a surgical unit and specialized equipment, is aggressive , complex, time-consuming, and expensive. Many authors perform cTESE in a surgical unit, and the procedure is also time-consuming and expensive, as multiple testicular openings are performed if spermatozoa are not found . A very Recently, comparisons of both methods, cTESE and mTESE, were conducted. A meta-analysis involving 1248 KS patients found no statistically significant differences in SSR between the two methods, with a mean SRR of 44%  . A more Several previous studies reported the use of cryopreserved testicular spermatozoa ,208,215,Some studies provided larger numbers of patients enabling comparisons between fresh and cryopreserved spermatozoa treatment cycles. There were 10 fresh and 16 cryopreserved testicular spermatozoa treatment cycles in a report of 38 KS patients with an SRR of 39% using cTESE and mTESE. In ICSI cases with cryopreserved testicular spermatozoa, the authors observed a significantly higher embryo cleavage rate (ECR) . There wThe authors of a meta-analysis of cases with secretory azoospermia first concluded that there were no significant differences in embryological and clinical outcomes using ICSI either with cryopreserved testicular spermatozoa or with fresh testicular spermatozoa. However, the authors also confirmed that when different pathologies, such in KS cases, were individually examined, fresh testicular sperm produced better outcomes . Thus, iIn terms of NB, the current updated literature analysis  reveals There has been some concern about the possibility of an increased risk of chromosome abnormalities in the offspring of KS patients, after increased rates of both autosomal aneuploidies ,226,227 Up to the present, all children born after using testicular sperm from KS patients present a normal karyotype. This may be explained by previous observations that indicated that the majority of the 47,XXY germ cells are not meiotically competent, being thus unable to originate chromosomal abnormal sperm , and by Until the present day, all published data have revealed that, with the exception of two cases, none of the children born from KS patients exhibited an abnormal chromosomal constitution ,221,229.Population studies are not entirely consistent when it comes to the frequency of KS diagnosis per age groups. In an English population research, Abramsky and Chapple  found thSwerdlow et al.  found inIn an Australian population study, Herlihy et al.  reportedDuring the prepubertal period, reproductive counseling is usually not provided, and children with physical–cognitive deficits must be properly supported like any other child with the same problems and without a Klinefelter karyotype. In the absence of signs of masculinization  and the presence of androgen deficit , from the age of puberty, the andrologist may consider introducing corrective treatment for the androgen deficit towards the end of puberty (16–18 years) depending on the clinical picture.The adolescent should be informed that the vast majority of KS patients are infertile, with spontaneous pregnancies occurring quite infrequently ,96. If tSome evidence indicates that fertility preservation should perhaps not be offered to KS patients under the age of 16 , and thaThere are several techniques for retrieving epididymal and testicular sperm, including percutaneous epididymal sperm aspiration (PESA), microscopic epididymal sperm aspiration (MESA), fine needle testicular aspiration (TESA), testicular sperm extraction through open biopsy procedure (cTESE), and microsurgical seminiferous tubule identification with microsurgical testicular sperm extraction through wide opening of the testis (mTESE). PESA, MESA, and TESA can be used to treat cases of obstructive azoospermia, with TESA being the most commonly used technique due to the better clinical outcomes. It is necessary to employ cTESE or mTESE for secretory azoospermia (non-obstructive azoospermia), which occurs in KS. The advantages and disadvantages of cTESE and mTESE techniques have already been highlighted. In KS patients, testicular attainment causes secretory azoospermia rather than obstructive azoospermia, with cTESE or mTESE employed for fertility treatments. Patients with decreased serum T levels are additionally indicated for aromatase inhibitor treatment before testicular sperm extraction, and any testicular sperm collection should take place at least six months after terminating T medication.All of these issues are delicate and require multidisciplinary specialized guidance, which is why some physicians prefer to discuss these options at the age of 18.We believe that this study will serve as a clinical guide, allowing children, adolescents and adults with the Klinefelter karyotype to be diagnosed earlier, properly referred and treated, avoiding significant systemic complications, and preserving fertility in a timely way.We hope that this review will encourage each country’s health system to publish population-based studies on KS in order to improve data on incidence and prevalence, as well as to uncover the reality of systemic complications, indicating the usefulness of better including KS in the differential diagnosis of several conditions, and thus for them to suggest karyotyping.The outstanding clinical results obtained with testicular spermatozoa and the absence of babies born with the Klinefelter karyotype documented here confer the TESE technique a high level of confidence. Although the option for cTESE or mTESE depends on the experience of the clinical team and the resources available, and although large systematic reviews indicate proximity in clinical results, it is evident that more comparative research with a large number of cases is needed.