CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report  CSMD1 with early idiopathic menopause in women and show that loss of Csmd1 leads to gonadal dysfunction in both male and female mice.Many molecular and physiological mechanisms in the regulation of fertility are shared between female and male mammals. Here, Lee et al. report an association of CNVs in  However, numerous molecular and physiological mechanisms of fertility regulation are shared between male and female mammals5. For instance, programmed germ cell degeneration is a pervasive part of gonadal biology in both sexes7. In human males, roughly 80% of the meiotic descendants of spermatogonial stem cells undergo apoptosis prior to ever becoming mature spermatozoa8. Likewise, human females are born with ~500,000 oocytes and ovulate roughly 400 times after menarche. However, only roughly 1000 oocytes survive the sojourn to menopause, representing colossal germ cell loss not attributable to ovulation9. This ratio of surviving:apoptotic germ cells differs between species, but is narrowly maintained within species10. The principles that govern the loss versus survival of given germ cells are poorly understood.The human genetic study of infertility has historically classified male infertility and female infertility as distinct diseases, leading to the assembly of many separate cohorts for the study of sex-specific reproductive processes12. We previously identified a reproducible association between rare copy number variant (CNV) burden and oligo-spermia and azoo-spermia in men14. One of several candidate loci from this study included the complement regulatory CUB and Sushi multiple domains 1 (CSMD1) gene.Defects in germ cell development intrinsic to the gonad are defined as primary gonadal dysfunction. Primary gonadal dysfunction — which manifests as early idiopathic menopause in women and oligo-/azoo-spermia in men — is a subset of the infertility phenotype that is tractable for human genetic analysis and has a prevalence of at least 1% in adult males and femalesCSMD1 shares homology with complement-interacting proteins15. Complement acts as an inflammatory/phagocytic signal in the innate immune system16, and previous work has shown that classical complement components C1q and C3 are also responsible for microglia-mediated phagocytosis of excess neuronal cells in a normal post-natal developmental process known as synaptic pruning17. Common variants in CSMD1 (refs. 19) and complement C4 (ref. 20) have also been associated with schizophrenia in independent, well-powered human association studies. Furthermore, some of the most significantly associated variants previously associated with azoospermia include the greater MHC locus, which encompasses complement C2, C4, and factor B22. CSMD1 is also known to inhibit the classical complement pathway in vitro23.The primary protein sequence of 24 to search for novel, shared factors required for normal gonadal function in both sexes, identifying an association with the gene CSMD1 in male and female gonadal dysfunction, and replicate our findings with samples from the UK Biobank25. We characterize the expression patterns of CSMD1 in female and male gonads and measure morphological and reproductive traits in Csmd1-knockout mice, thereby adding a level of functional support to our genetic findings. Finally, to consolidate the putative roles of complement with Csmd1-mediated pathology, we investigate the in situ abundance of macrophages and complement component C3 in Csmd1-wildtype versus Csmd1-null tissues. Our findings highlight the value of human genetic analysis as an entry point into surprising and previously unappreciated pathways in disease processes — in this case, a novel connection between of complement-related processes and gonadal function.In this study, we incorporate array and exome sequencing data from a large cohort of early idiopathic menopause from the Women’s Health Initiative26, Supplementary Fig. Due to the strong purifying selection against infertility mutations, we hypothesized that male and female gonadal dysfunction are driven largely by rare mutation events. To test this hypothesis, we obtained SNP array and phenotype data from 12,002 women  and 2,072 men  with documented reproductive health history. Due to reduced linkage disequilibrium between common SNPs and rare variants, we instead leveraged the SNP log R ratios and B-allele frequencies to perform ab initio discovery of CNVs that occupy the entire allele frequency spectrum . This CNV association is driven by an enrichment of rare deletions in cases, all of which are clustered at the 5′ end of the gene within introns 1–3 . After CNV QC and rigorous case/control selection, we obtained a cohort of 63,064 women with both reliable phenotype data and CNV calls . We again observed a significant association between early menopause and rare 5′-deletions of CSMD1  from exome sequencing performed on a subset of the WHI cohort (n = 1526). We performed a single-locus quantitative trait analysis and found a statistically significant association between rare, deleterious CSMD1 SNVs and age at menopause  and Sushi/CCP (complement control protein) domains , but not Sushi (βSUSHI = 0.046) domains 29.We further tested our association using genotypes ascertained by an orthogonal genotyping modality, by analyzing rare MAF < 0.01)  CSMD1 siins Fig. . Thus, iCSMD1 deletions in female reproductive traits, we tested for association with 4 additional reproductive phenotypes available from the UKBB (see Methods section). Of these traits, we identified a significant association with age at last live birth . The maximum age at last birth among our controls is 51 years, versus 40 years for CSMD1 deletion carriers. However, we note that 99.6% of control individuals have completed childbearing by age 36, indicating that traditional measures of fertility (e.g. fecundity) would not necessarily capture ovarian dysfunction related to CSMD1 deletions.The phenotype of idiopathic early menopause indicates problems in ovarian function. Predictable consequences of such ovarian dysfunction are irregular or absent menses and reduced fecundity. To further clarify the role of CSMD1 and gonadal dysfunction (i) across multiple classes of genetic variation; (ii) ascertained by orthogonal genotyping platforms; (iii) occupying multiple points along the allele frequency spectrum; and (iv) in multiple populations and cohorts.In summary, we detected associations between rare variants in CSMD1 encodes for an extremely large  transmembrane protein with a large extracellular portion consisting of alternating CUB and Sushi complement-interacting domains15. The protein encoded by CSMD1 is conserved between human and mouse, with 93% amino acid sequence identity and 100% domain synteny  Csmd1 wildtype, heterozygous, and knockout mice and observed the effect of genotype on fertility, gonadal morphology and function in male and female mice. In males, Csmd1 knockout leads to variable, but statistically significant, defects across multiple dimensions of gonad function and fertility. First, we observed an increase in infertility rate among knockout males (30%) compared to wildtype (4.5%) . We recapitulated the degeneration phenotype in these backcrossed male knockouts  increased germ cell death; (ii) failure of germ cell proliferation/maturation; and (iii) increased phagocytic clearance of germ cells. We did not observe obvious differences in the abundance of apoptotic germ cells apparent by H&E staining. We excluded systemic endocrine defects that would be observed in the case of failure of the hypothalamus or pituitary  and knockout (7.0%) testes.Three non-exclusive scenarios may account for the net loss of germ cells in C3 and Csmd1 mRNA expression are negatively correlated throughout spermatogenesis . C3 is a secreted, circulating protein whose mRNA is expressed by multiple cell types — including the germ cells themselves. Therefore, we also measured C3 mRNA expression in Csmd1 wildtype versus knockout purified male germ cells . A small minority of knockout females required many cycles to achieve pregnancy (>60 days). Circulating gonadotropin levels did not differ between wildtype and knockouts after controlling for estrous stage, suggesting that this reduction in mating success was not secondary to impaired hormonal input , but at significantly accelerated rates and higher proportions in knockouts.The observed rate of infertility in knockout females 3/37, 8.1%) was not significantly higher compared to controls ; x̅ko = 6.9 (95% CI [5.7−8.1]); Table Csmd1 knockout mothers suffered from significantly higher mortality rates during the neonatal period (1–10 days) when compared to wildtype/heterozygous mothers ; % mortalityKO = 50.0% (95% CI [30.0−70.0%]); Poisson regression p-value = 7.93 × 10−7; Fig. Csmd1-deficient mothers. CSMD1 is expressed in the normal mammary gland through the adult life cycle of wildtype females on both lumenal epithelial cells and myoepithelial cells of the mammary ducts, and on numerous stromal cells ; Table lls Fig. . Mammarylls Fig. . Quantiflls Fig. . Finallylls Fig. . As earllls Fig. .Fig. 7CsCsmd1 knockout mice. To test this prediction, we generated colonies of C3−/− single KO mice and C3/−−Csmd1/−− double KO mice. We did not observe any severely deranged C3−/− testes or ovaries , SYCE1 (refs. 47), SPIDR48). We found that multiple types of genetic perturbations of CSMD1 associate with infertility traits in humans, most notably deletions within introns 1–3 of the gene. Intronic deletions can affect gene function by altering splicing, RNA stability, and transcriptional regulation, and recent work has shown that such deletions tend to reduce transcript levels when they perturb expression of their host gene, although upregulation is also possible49. All infertility-associated deletions found in this study fall within 500 kb of 3 independent GWAS peaks for age at menarche. In the GTEx atlas, we found that only two tissues show significant CSMD1 expression-associated quantitative trait loci (eQTL): thyroid and testis50  are not yet available for testis, ovary or mammary gland; instead we used functional summaries derived from 6 human cell lines and identified 4 consensus CTCF sites within the region . Likewise, CSMD1 is massively depleted for rare loss-of-function (LoF) variation in the gnomAD reference database53, with a pLI score of 1.0. This depletion of LoF variation is especially striking given the local increase in CSMD1 de novo SNV mutation rate55, which is also corroborated by the substantial enrichment of rare synonymous variation in CSMD1 (ref. 53). Altogether we favor a LoF effect of CSMD1 mutations in human primary gonadal dysfunction.Furthermore, we found that rare protein-coding SNVs within the CUB domains of CSMD1 are significantly associated with age at menopause. According to the Residual Variation Intolerance Score (RVIS) only 0.169% of genes in the genome are more intolerant to protein coding changes than Csmd1 genetic knockout construct that was not strictly identical to any of the rare patient mutations found in our study. Further complicating our interpretation of the mouse data is our finding that Csmd1 KO gonads display nonspecific and/or residual expression signal; a phenomenon previously observed in Csmd1 KO brain36. Given the exceptional intolerance of CSMD1 to LoF variation in humans, we hypothesize that incomplete Csmd1 loss-of-function is buffered by functional redundancy56 (e.g. from Csmd2 and Csmd3)57, and that complete loss-of-function of Csmd1 would lead to much more severe and/or systemic phenotypes in both humans and mice. A complete tissue-specific and/or inducible loss-of-function knockout of Csmd1 could help to better disentangle these potential effects but would be technically prohibitive due to the size of the gene.How do our mouse studies relate to the human genetic findings and what are their limitations? We characterized a CSMD1 and reproductive function. Our human samples were ascertained based on pathologic loss of germ cells, and/or reduced reproductive lifespan. We observed heterogeneous gonadal defects corresponding to reduced reproductive lifespan and premature germ cell loss in both male and female Csmd1 KO mice, and these phenotypes were unexpectedly exacerbated in Csmd1/−−, C3/−− double KO mice. While we have neither explicitly nor implicitly defined the mechanism of CSMD1 mutations on gonadal function here, we have constructed a framework that implicates overzealous activation of the complement pathway as a potential contributor to the pathophysiology of the disease, due to the known molecular function of CSMD1 as a complement inhibitor15, the spatiotemporal patterns of complement-related proteins and cell types in the gonads, and the excess complement deposition on Csmd1 KO testes.Despite these practical limitations, the mouse mutants provided an invaluable and previously undescribed functional link between 58. Here, we have reported abnormal post-natal development in Csmd1 knockout animals for three additional tissues; we have observed complement and CSMD1 protein expression in all three tissues, and previous work has shown that macrophages are essential for proper development in all three tissues. A parsimonious model to describe the set of defects we observe here is that macrophages  regulate and refine developing cells in testis, ovaries, and mammary by controlled deposition of complement onto their cell surface, accompanied by subsequent phagocytosis.While complement has a long-appreciated role in innate immunity, it also plays a prominent role in the regulation of self cells during normal postnatal development. A classic example of this phenomenon is complement-mediated synaptic pruning in the developing brain, a process that is under genetic influence and can confer risk for disease when dysregulatedCsmd1 knockout taken in context of the known complement-inhibitory function of CSMD1 (ref. 23) suggests that overzealous complement-mediated phagocytosis of developing germ cells by macrophages and/or Sertoli cells underlies at least part of the testicular defects. C3 mRNA and protein are much more highly expressed in the interstitial spermatogonial layer, which is physically sequestered from the lumenal aspect of the tubules by the blood–testis barrier;59 Conversely, CSMD1 is largely expressed in the adlumenal compartment  has been shown to clear apoptotic germ cells in the mosquito testis by this very process64Specifically, in the testis, increased C3 deposition in ent Fig. . Spermat67. After ovulation, macrophages invade the ruptured follicle which undergoes apoptosis/phagocytic luteolysis, forming the corpus luteum68. We showed that CSMD1 is also highly expressed on oocytes of the developing follicle, but not in the corpus luteum . The characteristic directionality of mammary branching is conferred by polarized cell proliferation and phagocytosis mediated by macrophage remodeling, especially in anticipation of nursing70. Furthermore, multiple complement and complement-regulatory components are robustly upregulated during periods of apoptosis and phagocytosis in the mammary tissue of multiple species including humans72, though the functional significance of this regulatory program is unknown. Breast milk itself also suppresses complement activation73. Finally, CSMD1 is expressed on the lumenal aspect of mammary ducts and terminal end buds, where much of the pregnancy-associated breast remodeling occurs74  and factor P (CFP) unexpectedly converts mild C3 glomerulonephritis to lethal C3 glomerulonephritis in mice76. Similarly, CFH/C3 DKO unexpectedly unmasks a more severe form of age-related macular degeneration in mice77. Multiple explanations for this phenomenon have been set forth, including a dual role of C3, differences between fluid-phase and local C3 activation, and context-specific C3 gain-of-function. Finally, we cannot exclude the possibility of primary defects outside of the gonads causing pathology in our double knockouts . More extensive mutation constructs including conditional knockouts and allelic series may help to distinguish among these scenarios.The unmasking of a more severe phenotype in CSMD1 across multiple tissue types, and unveil a potential role for the complement system in postnatal developmental processes across multiple tissues in the body. When interpreted in context of existing observations from mammalian brain and other model organisms, we predict that macrophage mediated complement activity on self cells is a normal and highly controlled process in many developmental systems in metazoans. Furthermore, a wealth of literature shows that genetic dysregulation of complement and complement-regulating factors is a core feature of several heritable diseases beyond infertility;87; we therefore consider CSMD1 to also be a reasonable candidate for many of these diseases. Our work highlights the need for deeper investigation into the role of immune system components in reproductive tissues, and the opportunities that such work can have to illuminate and connect common biological processes that produce disease in more complex contexts across the body.Altogether, our human genetic and model organism findings lend support to a role of 14. WHI-SHARe. To create an analogous case-control cohort of female gonadal dysfunction, we turned to the SNP Health Association Resource (SHARe)88. We constructed early idiopathic case and control definitions from the dense reproductive phenotype data collected on each subject. A self-reported age of menopause before 40 years was used as the only case inclusion criterion. Case exclusion criteria were oophorectomy prior to age 40, a diagnosis of lupus or rheumatoid disease, and a “yes” answer to the question “Did a doctor ever say that you had cancer, a malignant growth, or tumor?”. Smoking history, which is a known factor influencing ovarian reserve, was controlled for during the analysis of genetic data.All human genetic studies were reviewed and approved by the Institutional Review Board of Washington University in St. Louis, under protocol numbers #201107177 and #201109261 and all participants provided informed consent. Male idiopathic infertility cases were clinically ascertained and pre-screened for chromosome Y deletions and compared against normospermic controlsUK Biobank. This research has been conducted using the UK Biobank Resource under Application Number ‘17085’. We generated a table of phenotype data for constructing early idiopathic menopause case and control labels using controlled-access data from the UK Biobank. Exclusion criteria for the study were: withdrawn consent, poor heterozygosity or missingness as defined by the UK Biobank; >10 relatives in the UK Biobank cohort; not used in autosome phasing, apparent sex chromosome aneuploidy; mismatch between genetic and self-reported sex; ever smoker; any self-reported non-Caucasian ancestry; oophorectomy prior to age 40; prior diagnosis of rheumatoid arthritis, lupus, or pelvic cancer; mismatch between self-reported ethnicity or age at menopause among three assessments; SNP array call rate <98%. In the case of pairs of 2nd degree relatives or closer, the one individual with the lower SNP-call rate was dropped. The inclusion criteria for POI case status were self-reported age of menopause <40 years old, and all remaining individuals in the cohort (after exclusions mentioned above) were used as controls.Csmd1 knockout mouse (Mus musculus) on a mixed 129SvEvBrd:C57BL/6 background from the UC Davis KOMP Repository (Project ID CSD118901)89. A 1.086 kb deletion encompassing Csmd1 exon 1 and part of intron 1 were replaced with a lacZ/neomycin cassette. Deletion of this segment was confirmed with Southern blot and PCR. Due to the extreme size of Csmd1 (1.6 Mb), we also analyzed RNA seq data across all 70 exons in knockout testes and ovaries. In ovaries, knockout read counts relative to wildtype are suppressed across all 70 exons. In testes, knockout read counts relative to wildtype are broadly suppressed across exons 1-57 and upregulated from exons 58-70. The amino acid coding portion of these upregulated exons range in size from 45 bp to 180 bp. The translational viability of these fragments is unknown. All littermate tissue comparisons in this study (described below) were generated from damheterozygous × sireheterzygous crossings from this original colony. Next, to eliminate variance in phenotype explained by variance in background genotype (if any), we serially backcrossed the Csmd1 mutation onto a constant C57BL/6 background for 5 generations. From this F5 backcross generation we performed a damheterozygous × sireheterozygous cross from this to create wildtype and knockout littermates, and performed analogous histology and immunofluorescence experiments as with the original colony  for data processing and QC. We obtained raw SNP array data from the UK Biobank and performed single sample CNV discovery using PennCNV92. Individuals with >200 CNV calls were dropped. CNV calls with PennCNV quality score >30 were retained, and adjacent CNVs in the same sample were merged. All qPCR validation of CSMD1 CNV calls were performed on Applied Biosystems TaqMan assay # Hs0367742_cn . We used Human RNase P for the reference assay . We used standard Applied Biosystems TaqMan protocols in 10 µL reaction volumes. We generated 4 to 8 technical replicates for each sample in a 384 well plate. PCR conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. TaqMan copy number was estimated using the CopyCaller software and compared to PennCNV copy number estimates.Array data for the Women’s Health Initiative SHARe cohort were downloaded from the NCBI Database of Genotypes and Phenotypes (dbGAP accession number phg0000g1.v2). SHARe samples were genotyped on the Affymetrix 6.0 platform. We created a high-quality set of CNV calls for all cohorts using our own internal pipelines. SHARe samples were processed with Affy6CNV ; all available WHISP BAM files were downloaded from the NCBI Database of Genotypes and Phenotypes (dbGaP accession phs000200.v10.p3.c1 and phs000200.v10.p3.c2)CNVs. Rare CNVs were associated with case-control status using generalized linear models. For the SHARe association analysis, we included the top 10 ancestry eigenvectors  and smoking history as covariates. For the UK BioBank association analyses, and we included as covariates BMI, smoking history  and the top 10 ancestry eigenvectors calculated from the full UK Biobank SNP genotype matrix.In addition to idiopathic early menopause (defined above in Human patient populations), we performed association testing for the following 4 traits for UKBB subjects (Unique Data Identifier codes in parentheses): Length of menstrual cycle (3710), Number of live births (2734), Age at last live birth (2764), all-cause female infertility IC10 code (N97).CSMD1 and age at menopause in the WHI samples using the Sequence Kernel Association Test (SKAT)94, weighting each variant with the Combined Annotation Dependent Depletion (CADD) value95. Five ancestry eigenvectors and smoking history were included as covariates; significance was evaluated using bootstrapping with 5000 samples.SNVs. We tested for an association between rare SNVs in 96 which we describe below. Sexually mature (40 ± 1 days old), male mice were sacrificed, and their testes were decapsulated and homogenized in a 1× MEM solution (Gibco 11430-030) containing 120 U/mL Type I Collagenase  and 1 mg/mL DNAse I (Roche 10104159001), and agitated for 15 min. 1× MEM was replaced and added with 50 mg/mL Trypsin  and 1 mg/mL DNAse I and agitated for 15 minutes, then mechanically homogenized for 3 min. 50 mg/mL Trypsin and 1 mg/mL DNAse I were added and agitated again for 15 min. We added 0.4 mL heat inactivated Fetal Bovine Serum (Sigma F1051), 5 μL Hoescht 33342 (Life Technologies H3570), and 1 mg/mL DNAse I, and agitated for 15 minutes. Individual cells were dissociated by pipetting sequentially through two 40 μm cell strainers . For each individual mouse, one dissociated testis was used for whole tissue RNA extraction and sequencing, and the other testis was used for germ cell purification, RNA extraction, and sequencing. All dissociation steps were performed at 33 °C. Dissociated testes were sorted on a modified MoFlo cytometer (Beckman Coulter) at the Washington University Siteman Flow Cytometry Core using a krypton-ion laser97. Cells that are stained with Hoechst can be clustered in two wavelengths: (i) blue fluorescence, which is informative of DNA content, and (ii) red fluorescence, which is informative about chromatin state and Hoechst efflux from the cell. Our gating strategy is graphically summarized in Supplementary Fig. We adapted existing methods for dissociation and germ cell purificationGibco 1140-030 conCsmd1 null siblings (summarized in Supplementary Data Mus musculus reference R72 and transcript expression levels were summarized as reads-per-kb of exon per million-mapped reads (RPKM) using the TopHat2 package98. RPKMs were adjusted for batch effects and cryptic covariates using PEER99, quantile normalized, and then the R package poissonSeq was used for differential expression analyses100.Whole testis, whole ovaries, and purified male germ cell subpopulations were obtained from wildtype and °C for 1 h, deparaffinized in Xylenes, and rehydrated into PBS (Corning). Antigen retrieval was done in boiling citrate buffer  for 20 min. Sections were blocked in PBS containing 0.2% Triton X-100 and 5% normal donkey serum (Jackson Laboratories) for 1 h at room temperature and then incubated with primary antibodies diluted in blocking solution over night at 4 °C. After washing with PBS-Tx (PBS containing 0.2% Triton X-100), they were incubated with fluorescent secondary antibodies in blocking solution for 1 h at room temperature, washed with PBS-Tx, and treated with 0.2% Sudan Black in 70% EtOH for 10 min, followed by PBS washes. The sections were then counterstained with Hoechst dye 33342 diluted 1:500 in PBS for 5 min, washed once with PBS-Tx for 2 min and then with PBS, and mounted in ProLong Diamond anti-fade mounting medium (Molecular Probes). Imaging was done on an Olympus LSM700 confocal microscope using Zen software, and images were processed using Photoshop CS5 (Adobe). Antibodies used were gt α-CSMD1 N20 (Santa Cruz Biotechnology sc-68280 1:100), rb α-mouse vasa homolog (MVH) , donkey α-gt CF594 (Biotium #20116 1:300), and donkey α-rb Alexa488 (Life Technologies #A-21206 1:300), rb α-β-gal (Cappel #55976 1:333), rat F4/80 BM8 (Santa Cruz Biotechnology sc-52664 1:50), donkey α-rat Alexa488 (Life Technologies #A-21208 1:300), rb α-C3 , and gt α-rb Alexa568 . Testes were decapsulated in 1x PBS and the tubules were gently teased apart and fixed in 4% paraformaldehyde in 1× PBS for 2 h at 4 °C. After washing with PBS and blocking in PBS containing 0.2% Triton X-100 and 5% normal donkey serum for 1.5 h at room temperature, they were incubated in primary antibodies overnight at 4 °C in blocking solution and processed as the cross-sectional IF experiments were described above33. For immunohistochemistry, 5 μm paraffin sections were treated as above, except the secondary antibody was biotin-coupled horse α-goat , and detection was done using the Vectastain Elite ABC kit  and DAB Peroxidase Substrate kit (Vector Laboratories SK-4100) per the manufacturer’s instructions. Sections were counterstained with hematoxylin, mounted in Cytoseal Xyl (Thermo Scientific), and imaged on a Zeiss Axioplan 2 microscope equipped with an Olympus DP71 camera and DP software. For X-gal staining, whole tissues were fixed in 4% PFA in PBS for 2 h, washed with PBS, and stained in X-gal (GoldBio #X4281C1) overnight101. Tissues were embedded in paraffin, sectioned at 5 μm, and counterstained with hematoxylin.Testes and ovaries were dissected, fixed in 4% paraformaldehyde (Electron Microscopy Sciences), and embedded in paraffin. We baked 5 μm sections at 60 102 or eosin. Stained testes from 65 individual mice of known age and genotype  were provided to a single mouse pathologist in a blinded fashion. All samples received a score of 0 (no damage), 1 (mild damage), or 2 (severe damage)  for 24 h and Bouin’s fixative (Electron Microscopy Sciences 26367-01) for 24 h. Fixed tissues were embedded in paraffin and sectioned at 5 μm. Sectioned tissues were stained with hematoxylin and counter-stained with either Periodic acid-Schiff reagentyi is the TRA98-positive count in image i, and X1 is the genotype (Csmd1 wildtype versus knockout), and X2 is the total cell count. εi is the nuisance variable for image i.We performed immunofluorescence as described above on a pair of 34 day old male littermates , and measured body weights and bilateral gonad weights at necropsy. For males, mean body weight was 37.1 g, mean testes weight was 273 mg, and mean age was 201 days. For females, mean body weight was 31.3 g, mean ovary weight was 32 mg, and mean age was 234 days. In order to estimate the effect of genotype on gonad weight, we fit the following linear model:103. Frozen testes were quickly thawed at room temperature and approximately half was used to estimate DSP. Sample weight was registered for full testis and tissue fragment used for the assay. To burst all cells, except for spermatid heads, the tissue was sonicated in DSP buffer , 0.05% Trition-X-100) using short pulses (3 × 10 s at 30% amplitude). Sperm heads were then collected by centrifugation (10 min at 500×g) and used in 1:1 dilutions with 0.4% Trypan Blue for estimation of cell density per homogenate in a hemacytometer. These values were then used to estimate the total number of spermatids per testis [number spermatids in homogenate divided by sample weight (g) and multiplied by the full testis weight (g)]. In mouse spermiogenesis, steps 14–16 take 4.84 days (developing spermatids), therefore, DSP was calculated by dividing the total number of spermatids in one testis per 4.84 days.Daily sperm production (DSP) was estimated using the Triton-X 100 nuclear solubilization methodcorpora lutea across each section based on histologic appearance104. In order to estimate the effect of genotype on gonad weight, we fit the following model:yi is the number of total oocytes in bilateral ovaries of individual i, and X1i and X2i are genotype and age, respectively. εi is the nuisance variable for individual i.We sacrificed 15 sexually mature female mice, of which 10 were wildtype and 5 were knockout genotypes. Bilateral ovaries were fixed, sectioned to 5 μm, and stained with H&E. We identified primordial follicles, primary follicles, secondary follicles, early antral follicles, antral follicles, preovulatory follicles, atretic follicles, and Csmd1 wildtype, heterozygote, and knockout sire/dam breeding combinations. We calculated the number of days between first sire/dam co-habitation and birth of each litter. Next we subtracted an estimated C57BL/6 gestation time of 19 days105 to estimate time to conception. We also calculated parental ages at conception. All density plots depicted in Fig. yi is the estimated time to conception for mating pair i, X1i is maternal genotype , X2i is maternal age at conception, and X3i is paternal age at conception. εi is the nuisance variable.We compiled comprehensive husbandry information over a period of greater than 1 year corresponding to 151 litters born representing all possible dam × wtsire, wtdam × hetsire … homdam × homsire] were represented multiple times (excepting hetdam × wtsire). We counted deaths in during the neonatal period  and subtracted from the live birth total to obtain the final number of surviving pups . Next, we stratified each litter by maternal and paternal genotype status (Csmd1 wildtype or heterozygous versus knockout) and fit the following model:yi is the number of surviving pups in litter i, and X1i and X2i are the maternal and paternal genotypes, respectively. εi is the nuisance variable for litter i.We bred 44 females  with 41 males  over a period of 10 months to produce 99 litters, totaling 688 live births. All 9 parental genotype permutations [wtConrad Lab website. To perform statistical comparison of duct morphology between genotypes, measurements of mammary gland ducts were derived from images using AngioTool64 v0.6a106. First, a skeleton representation of the branched duct structure is generated from the input image, which is then used to compute a variety of morphological and spatial parameters for branching characterization. Since this software detects the branches by contrast on a black background, the images of whole mount mammary glands of adult mice were transformed into a compatible input using ImageJ 1.51n.Female littermates were collected at four developmental time points: (i) pre-pubescent (<30 days of age); (ii) adult virgins; (iii) mid-pregnancy (estimated 14 days after copulation); (iv) post-weaning (7 days after weaning pups from mother’s nursing). Freshly-dissected whole inguinal mammary glands were fixed overnight in Carnoy’s solution . Fixed tissues were washed and rehydrated in ethanol and water and stained in Carmine alum histological stain  for 48 h. Stained tissues were dehydrated with increasing concentrations of ethanol and stored in xylene to clear lipids for 48 h. Finally, tissues were flattened mechanically and suspended in pure methyl salicylate prior to imaging. Due to the large size of whole mammary tissues, overlapping fields of view were captured and stitched together using the “Photomerge” function in Adobe Photoshop. Gaps in the backdrop of the merged images were filled using the “Content aware fill” function in Adobe Photoshop — if and only if the gaps did not overlap any portion of the tissue proper. All original images are available on the 107. Vaginal swabs were collected, transferred to a glass slide, and inspected under light microscopy. Proestrus, estrus, metestrus, and diestrus staging was then classified based on the relative counts of leukocytes, nucleated epithelial cells, and cornified epithelial cells. Only confirmed proestrous females were selected for blood draws and hormone measurements. All blood was drawn at approximately the same time of day, clotted for 90 min at room temperature, and centrifuged at 2000×g for 15 min. Samples were stored at −20 °C prior to hormone measurements. Male samples were quantified for LH/FSH (EMD Millipore) and testosterone , and female samples were quantified for LH/FSH and estradiol (CALBIOTECH) by the University of Virginia Ligand Assay and Analysis Core.We collected serum from 9 males  and 16 females in the proestrous stage  via submandibular collection. Each wildtype individual was matched with ≥1 knockout littermate. Female estrous cycle was determined first by external inspection of the vaginal opening, then by vaginal cytologyCsmd1 knockout and wild-type mice were decapsulated and washed in 1× PBS before mincing. Minced tissue was subjected to enzymatic dissociation as described above. The crude cell preparation thus obtained was treated with ACK buffer (Life Technologies) for 5 min at room temperature to lyse erythrocytes present if any in the cell preparation. The isolated cells were incubated in α-C3 (B9) primary antibody  diluted in FACS buffer  along with 10% Fc block (to minimize non-specific binding and background fluorescence) for 45 min at room temperature, followed by gt α-mouse Alexa488 (Abcam #A11029 1:250) incubation of 90 mins at RT in the dark with 3 washes of ice cold FACS buffer after each antibody incubation. Flow cytometry was performed with an Accuri C6 cytometer (BD Biosciences).Testes obtained post-dissection from Further information on research design is available in the Supplementary InformationDataset 1Dataset 2Dataset 3Reporting SummaryPeer Review FileDescription of Additional Supplementary FilesSource Data


Numerous bioactive biophenol secoiridoids (BPsecos) are found in the fruit, leaves, and oil of olives. These BPsecos play important roles in both the taste of food and human health. The main BPseco bioactive from green olive fruits, leaves, and table olives is oleuropein, while olive oil is rich in oleuropein downstream pathway molecules. The aim of this study was to probe olive BPseco downstream molecular pathways that are alike in biological and olive processing systems at different pHs and reaction times. The downstream molecular pathway were analyzed by high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI/MS) and typed neglected of different overlap (TNDO) computational methods. Our study showed oleuropein highest occupied molecular orbital (HOMO) and HOMO-1 triggered the free radical processes, while HOMO-2 and lowest unoccupied molecular orbital (LUMO) were polar reactions of glucoside and ester groups. Olive BPsecos were found to be stable under acid and base catalylic experiments. Oleuropein aglycone opened to diales and rearranged to hydroxytyrosil-elenolate under strong reaction conditions. The results suggest that competition among olive BPseco HOMOs could induce glucoside hydrolysis during olive milling due to native olive β-glucosidases. The underlined olive BPsecos downstream molecular mechanism herein could provide new insights into the olive milling process to improve BPseco bioactives in olive oil and table olives, which would enhance both the functional food and the nutraceuticals that are produced from olives. Olive oil and table olives are a major part of the traditional food in the Mediterranean Aliment Culture (MAC) . Olives + isomers 3 as the Lewis catalyst [3. Olive BPseco C7 esters cleaved after 350 days in darkness and lipid medium at 35 °C under simulated storage conditions and were affected by oil acidity and filtration [+ transfers as Bronsted acid or unshared pair/empty orbital interactions were alike in the Lewis base that occurred in olive BPsecos downstream pathways. In fact, olive BPseco hydrolytic downstream mechanisms were inferred under the frontier molecular orbital (FMO) mechanism [Olive milling simulation showed that olive BPseco mixtures at pH = 4.2, 120 min, 25 °C remained intact without any hydrolytic reaction . Simulatrization . This rerization ,47. The rization . Oleuropcatalyst . Apparenltration . Overallltration . H+ tranechanism .3O+ and enzyme catalysis. Olive BPseco LUMOs involve the conjugated seco-ring system on the secoiridoid, similar to Lewis acids, and are energetically the easiest to increase via an increase in electrons through reduction pathways. Polar processes depend on LUMOs for base-catalyzed hydrolysis, which relates to the polar interaction with OH− HOMOs that generate the most probable olive BPseco hydrolytes [− interacting with FMOs, similar to Bronsted behavior. The downstream molecular hydrolytic dynamics of olive BPsecos have been directed at ester functionalities by straight interactions of filled OH− HOMO and LUMO antibonding FMOs at C7=O in competition with C11=O empty orbitals [Oleuropein Lewis acid and basic behavior were evaluated by TNDO calculation. These gave new semiempirical merges of molecular mechanics and semiempirical quantum mechanics for olive BPsecos molecular dynamics. TNDO combines olive BPseco atom typing with a basic quantum mechanical method that shows the difference between atoms with different hybridizations that are suitable for secoiridoid-ring conjugated systems. This rapid semiempirical mapping offers more reliable olive BPseco hydrolytic downstream than AM1 ,17. Olivdrolytes ,17. Figuorbitals . − through the polar interaction of OH− FMOs that filled the electron pair as a Lewis base and revealed the first empty FMO on oleuropein. TNDO calculations exposed oleuropein HOMO = −10.94 eV and HOMO + 1 = −11.75 over hydroxytyrosil moiety, while HOMO-2 = −12.13 eV overlapped the conjugated system –O2–C3=C4–C11=O<―> –O2+=C3–C4=C11–O−  of the OLEC11=O + H+ isomer reveals low kinetic stability and high molecular reactivity. A unimolecular rate determining the transition state of C1–oleuropeinGlu bond cleavage may lead to glucose formation. The alternative glucosyl-carbonium route was generally excluded [3O+ pre-equilibrium  and isomers, such as oleuroside, were obtained from olive fruits rotocols ,51. pHs at 25 °C .2O/0.1% CH3CO2H at pH = 3.3 with selected gradient of 10% (A) and 90% (B), then (A) up to 90% in 60 min [2 nebulizing gas was used at 25–30 L/h source and desolvation at 100 °C and 250 °C, respectively, in negative ion mode with capillary 1.9 kV and cone 28 V; for MS/MS/CAD, Ar gas was used with collision energy of 20 eV [HPLC analysis was performed using a 1100 LC system  with an RP 25 cm × 4.6 mm id, Phenomenex Luna 5 μ C18 column coupled to a UV/VIS detector with a stainless steel loop of 20 µL and a split 1/15 to detector at 280 and 240 nm. Data were processed with both HPChem-Station and online to an ESI/MS interface. Solvents at 0.5 mL/min flow rate were: (A) MeOH, and (B) Hof 20 eV .+, oleuropein GluO + H+, and oleuropein C11=O + H+ isomers were exploited using the TNDO method, a new semiempirical method that merges molecular mechanics and semiempirical quantum mechanics [The semiempirical molecular orbital for the highest occupied molecular orbital (HOMO)/lowest unoccupied molecular orbital (LUMO) evaluation of oleuropein, oleuropein O2 + Hechanics .In our study, we found that olive BPsecos were stable under base catalysis and were hydrolyzed under acid and enzyme catalysis. The oleuropein FMO results suggested that downstream pathways competed with free radical and polar molecular dynamics resulting from HO-aromatics and secoiridoid moieties, respectively. Oleuropein HOMO and HOMO-1 could trigger free radical processes, HOMO-2, and LUMO polar reactions of glucoside and ester groups. Oleuropein aglycone opened to diales and rearranged to hydroxytyrosil-elenolate under acid reaction conditions. The downstream pathway olive BPseco HOMOs activated glucoside hydrolysis under native olive β-glucosidases and general acid catalysis. The probed pH, reaction time, and molecular mechanism—proven using TNDO molecular orbital theory—also shed light on how olive BPsecos changed during fruit maturation, how they were hydrolyzed during both olive milling and the intestinal digestion process, and how they were then transferred to olive oil. The results clearly demonstrate that the use of suitable milling process of olives fruits could be exploited towards the complete release of bioactive BPsecos into extra virgin olive oil, which could contribute to improve functional food and nutraceuticals for the wellbeing and health of consumers.


The well-researched pro-dopamine regulator KB220 and variants result in increased functional connectivity in both animal and human brains, and prolonged neuroplasticity (brain cell repair) having been observed in rodents. Moreover, in addition to increased functional connectivity, recent studies show that KB220Z increases overall brain connectivity volume, enhances neuronal dopamine firing, and eliminates lucid dreams in humans over a prolonged period. An unprecedented number of clinical studies validating this patented nutrigenomic technology in re-balancing brain chemistry and optimizing dopamine sensitivity and function have been published. On another note, it is sad that unsuspecting consumers could be deceived and endangered by false promises of knock-off marketers with look- and- sound-alike products. Products containing ingredients having potential dangers  threaten the credibility and reputation of validated, authentic, and ethical products. We encourage clinicians and neuroscientists to continue to embrace the concept of “dopamine homeostasis” and search for safe, effective, validated and authentic means to achieve a lifetime of recovery, instead of reverting to anti-dopaminergic agents doomed to fail in the war against the devastating drug epidemic, or promoting powerful D2 agonists that compromise needed balance. Therefore, he appointed an Ohio doctor, Hamilton Wright, to be the nation’s first Opium Commissioner. In the decades after the Civil War, the United States developed a deadly narcotics habit. Suffering veterans were “hooked on morphine,” while genteel “society ladies” dosed up with laudanum—a tincture of alcohol and opium. The wonder drug was used widely as a cough suppressant, and proved very effective in treating diarrhea in children. In fact, in 1911, Wright told the NY Times —Remarkably, more than a century later, America has relapsed . The curThe authors, who have numerous scientific publications on this issue, believe that FDA approved Medication Assisted Treatments (MATS), such as, the acute administration of buprenorphine, are helpful in inducing short-term dopamine release –5. Chronnormal physiologically required function [For more than 50 years, our research has provided a significant dossier of peer-reviewed and published evidence in scientific journals showing that balancing dopamine dynamics in the brain reward circuitry is a far more desirable and useful strategy than blocking its function . Fundamefunction . This fofunction .Mark Gold and associates in their “dopamine depletion hypothesis”  suggesteBlum and associates have been developing, by trial and error and genetic testing, a neuro adaptogen, KB220, and many subsequent improved variants, since 1968 . To dateThe first ever confirmed psychiatric genetic discovery by the Blum and Noble’s group, the association of the Dopamine D2 Receptor (DRD2) gene and severe alcoholism, was published in JAMA in 1990 . The assHave We Hatched the Addiction Egg: Reward Deficiency Syndrome Solution System?” This paper was dedicated to all the people who have lost loved ones to substance abuse and “reward deficiency syndrome”-related tragedies. Why are we failing at reducing the incidence of RDS behaviors? Are we aiming at the wrong treatment targets? At that time, a paradigm shift was proposed; the “Reward Deficiency Solution System” and evidence was provided for its adoption. The “Reward Deficiency Syndrome Solution System” provided evidence for the feasibility of its adoption [To highlight the importance of the RDS concept, in 2013, B. William Downs and Kenneth Blum, published a paper entitled “adoption ,20. Whiladoption  and humaadoption . After 5adoption  and balaUnfortunately, a number of companies have decided to include L-Dopa as an ingredient in their nutraceutical to affect craving behavior. While the product contains some of the same ingredients as found in KB220 variants, the inclusion of the drug L-Dopa, an amino acid precursor of dopamine approved by FDA for Parkinsonism, with well documented side effects, is of concern. Of even more concern is the potential of the tainted product to cause more harmful effects, common to products identified as “dietary supplements”. A possible harmful effect notwithstanding, this ingredient disrupts the neurochemical balance, especially of dopamine, and can induce unwanted hyperdopaminergia and dyskinesia, instead of muchneeded balance . It has Over 100 million people in the United States carry the D2 receptor gene A1 allele , which iKnown side effects of the chronic administration of L-Dopa for Parkinson’s patients include mania, dyskinesia , and abnormal involuntary movements (AIMs). Also, reported among the side effects are psychosis, auditory hallucinations, homicidality, hypersexuality, confusion, delusions, orthostatic hypotension, sleep disruption, age-related mental disturbances/cognitive decline, impaired gait, and kaliuresis  are side effects Based on ignoring many studies, the use of L-Dopa is still considered Generally Recognized as Safe (GRAS), but the FDA has provided limitations on the over-the-counter use of L-Dopa and even the associated plant extracts . CertainThe well-researched, original pro-dopamine regulator, KB220, and variants (i.e. KB220Z/ZBR) utilizing recent technical advancements, show increased functional connectivity, in both animal and human neuroimaging studies. Prolonged neuroplasticity (neurogenesis) has been observed in rodents. Moreover, studies have been published showing that KB220Z increased function and brain connectivity volume, enhanced neuronal dopamine firing, and has eliminated lucid nightmares in humans over a prolonged period. An unprecedented number of clinical studies validating the success of a patented nutrigenomic technology to re-balance brain chemistry and optimize dopamine sensitivity and function have been published. The patented formula is the culmination of decades of meticulous trial and error tests on the effects of ingredients individually and on a plethora of combinations in animals and humans to determine the best behavioral benefits and outcomes. Subsequently, the formula outcomes were honed and verified by evaluating improvements in gene expression. Other ingredients have been included to expand the mechanistic reach to optimize gene expression for improved functionality of the neuro-endocrine-immune axis; optimizing the harmonious connectivity between different brain regions to achieve the symphony of neurochemistry. The improved effectiveness of the formulas has been further validated in clinical research including numerous brain scan studies published in peer-reviewed and cited scientific journals. Adding pro-hormones, plant-based hormonal analogs, or bio-identical hormonal substances to the exhaustively and meticulously researched and developed KB220Z/ZBR formulation will only disrupt the elaborate and sequential flow of genetic communication sequelae and the hamonious interconnectivity between neurotransmitters and their homeostatic, regulatory feedback controls in the Brain Reward Cascade. Due to these feed back regulatory controls, such additions result in homeostatic downregulation of neurotransmitter synthesis and reception to compensate for the artificial presence of such pseudoneurotransmitter substances. It is a similar reactive mechanism, when prednisone is introduced to the system, the body stops making cortisone. The same mechanism applies to insulin, thyroid, and any neurotransmitter, especially, dopamine.The KB220Z/ZBR nutrigenomic formulas (and prior variants) have been formulated to re-balance, harmonize, and optimize neurotransmitter functional relationships in the Brain Reward Cascade-not displace, replace, or disrupt those functional relationships. Moreover, products containing ingredients having potential dangers  threaten the credibility and reputation of validated, authentic, and ethical products and may cause unwanted harm to the unsuspecting subject. We encourage clinicians and neuroscientists to continue to embrace the concept of “dopamine homeostasis” and search for safe, effective, validated and authentic means to achieve a lifetime of recovery, instead of reverting to anti-dopaminergic agents doomed to fail in the war against this devastating drug epidemic.


A second meta-analysis was performed for the striatal dopamine receptor including 8 studies with a total of 72 Tourette patients and 71 controls. This analysis revealed a nonsignificant trend toward lower dopamine 2/3 receptor binding in striatum of Tourette patients. Other analyses regarding study population characteristics in both the DAT and receptor meta-analysis did not show any meaningful results. Our results indicate that dopaminergic alterations in TS are likely and thereby this data would be in line with the current pathophysiological hypotheses of a dysfunction in the dopamine system, e.g., the hypothesis of tonic-phasic dysfunction. However, these analyses suffer from low effect sizes probably due to the heterogeneity of TS and highlight the need for further large-scaled neuroimaging studies.Despite intense research, the underlying mechanisms and the etiology of Tourette’s syndrome (TS) remain unknown. Data from molecular imaging studies targeting the dopamine system in Tourette patients are inconclusive. For a better understanding of the striatal dopamine function in adult dopamine-antagonist-free patients we performed a systematic review in August 2017 identifying 49 PET and SPECT studies on the topic of TS. A total of 8 studies appraised the dopamine transporter (DAT) with 111 Tourette patients and 93 healthy controls, and could be included in a meta-analytic approach. We found a significantly increased striatal DAT binding in Tourette patients (Hedges'  According to the Diagnostic and Statistical Manual of Mental Disorders Fifth Edition’s [DSM-V] the tic onset of TS must be prior to the age of 182. Most Tourette patients have associated neuropsychiatric comorbidities such as obsessive–compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD). Those affected also frequently suffer from mood disorders or rage attacks3.Tourette’s syndrome (TS) is a heritable neuropsychiatric movement disorder, which is clinically characterized by the simultaneous presence of at least one vocal tic and multiple motor tics for at least 12 months4. Since the 1970s several classes of psychotropic drugs, especially dopamine-antagonists, have proven their efficacy in reducing frequency and severity of vocal and motor tics. Nonetheless, response rates to pharmacological standard therapy remain unsatisfactory5. There are only a limited number of randomized controlled trials for the pharmacological treatment of TS. This lack of evidence for safety and efficacy of pharmacotherapy leads to the absence of firm recommendations. Furthermore, the drugs commonly used are often associated with severe side effects, leading to discontinuation of medication6. Although there are promising new pharmacological treatment strategies, such as pregabalin7 or aripiprazole8, there is no causal therapy for TS. Modern pharmacological treatment normally only results in a 50% reduction in tic symptoms5. Although the side effect profile of modern therapeutics like aripiprazole seems to be more favorable8, the limited knowledge of TS pathophysiology disables the development of a curative therapeutic approach.Today there are several treatment strategies taking the neurobiological pathophysiology of TS into account10. Many neuroimaging studies using structural and functional magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and combined approaches have given us insight into the pathophysiology of TS. Nonetheless, there are still inconsistencies across those studies. Therefore, a comprehensive understanding of the specific neurobiological mechanisms underlying the origin of tics remains incomplete11.Different neuroimaging techniques, electrophysiological studies, animal models, and postmortem studies support the hypothesis of a dysfunction in cortico-striato-thalamo-cortical networks as a neurobiological substrate of tics12. Especially in the last few years many fMRI studies, estimating brain activity by blood oxygen level dependent signal, were carried out. Two recent meta-analyses attempted to summarize this body of evidence. The first study revealed inhibition control deficits via neuropsychological tasks in TS patients by analyzing the data of 1717 TS patients13. The other study focused on the functional circuitry of TS by evaluating task-based neuroimaging studies. They found differences in motor preparation and prefrontal cortices in TS patients by investigating 651 participants14.The number of new publications on TS is steadily increasing15. These studies were driven by the fact that dopamine receptor blocking agents and dopamine-depleting drugs reduce tics in TS patients1. However, postmortem18 and imaging studies focusing on the pathophysiological hypothesis of striatal dopaminergic hyperinnervation showed inconclusive results19. At this point, a meta-analytic approach is needed to synthesize the existing dopamine radiotracer neuroimaging literature to gain a better picture.Early neuroimaging research on TS focused on the basal ganglia and the role of dopamine20 revealed a widespread reduction of serotonin transporter binding in patients with major depressive disorder (MDD) by investigating 18 PET and SPECT studies. Similar meta-analyses of molecular imaging markers have shed light on the role of dopamine in schizophrenia. Two analyses with large sample sizes showed no alterations in the availability of dopamine transporter (DAT) in schizophrenia22. Yet a small increase in dopamine receptor availability22 and striatal dopamine synthesis capacity could be detected in schizophrenic patients when compared to healthy23. These meta-analytic studies have provided clarity about the role of neurotransmitters in MDD and schizophrenia, as one of the main reasons for false-positive findings are small sample sizes and publication bias24. Here, we are using similar methods to clarify the role of striatal dopamine in TS.Meta-analyses of molecular imaging studies have provided important information for the etiology of other mental disorders. Gryglewski et al.25. PET offers greater sensitivity as well as specificity than SPECT, which tends to have low-specific binding to the target structure26. But neuroimaging studies tend to be confounded by other factors such as age, medical treatment, subject matching, and more. This meta-analysis aims to clarify the role of dopamine in TS.There are several ligand studies utilizing SPECT or PET to examine the function of the striatal dopamine system, yet a number of these studies failed to show differences between TS patients and controls. This might reflect the poor spatial resolution of the current scanning techniqueswww.ncbi.nlm.nih.gov/pubmed) in August 2017 using the following search terms: “Tourette” OR “Tourette’s” AND “neuroimaging” OR “PET” OR “positron emission tomography” OR “single-photon emission computed tomography” OR “SPECT”.A systematic literature search was conducted on PubMed  or severe somatic diseases.Studies included TS patients undergoing current therapy with dopamine-antagonists and the absence of an at least 3 month long washout period.Studies included children.Studies were excluded from the meta-analysis if the:We contacted the corresponding authors of all eligible studies in this meta-analysis to obtain further information about missing data or their results per e-mail. Two authors responded and were able to provide data needed for a sufficient analysis.27 as an instrument for the assessment of tic severity. Only three studies used scores other than the YGTSS, namely the Shapiro Tourette Syndrome Severity Scale28 and the Modified Rush Videotape Rating Scale29. Due to the small number of other instruments used we treated them as equivalent to the YGTSS in our analysis.Beside the PET or SPECT outcome for each brain region, we included demographic variables (age and sex), type of outcome measure and tracer, psychotropic drug history  as well as TS severity in our analyses. Due to the high interrater and interstudy variability in the different Tourette severity scores this approach must be viewed as explorative. Most studies used the Yale Global Tic Severity Scale (YGTSS)2/3).During this systematic search a total of 49 PET and SPECT studies in the field of TS were found. Of those, 19 were excluded because they examined regional brain activity (9 SPECT and 10 PET). Seven studies were excluded because they focused on neurotransmitters other than dopamine . Another 9 studies could not be included because they included children, used special targets, e.g., VMAT, or results could not be sufficiently used in a meta-analytic approach. In the remaining 14 published studies two types of dopaminergic targets in the striatum were investigated. The first neurotransmitter target of interest was the DAT. The other was the dopamine receptor , a dimensionless quantity which is proportional to the binding parameters Bmax/Kd, when dynamic emission sequences are obtained. Other studies report the tracer equilibrium distribution volume , if the arterial input function is available. To use all these different outcome measures in a meta-analytic way, standardized effect size estimates were computed. Therefore, standardized mean difference (Hedges' g) for each study and brain region were calculated32. Hedges' g is the difference between the two means which is divided by their pooled standard deviation33.Principally PET and SPECT studies report a number of different outcome measures. This ranges from the simple uptake ratio in a region of interest compared to a nonbinding reference region to the binding potential  this parameter was not analyzed in a mixed-effects model.Fourteen original PET or SPECT studies reflecting ligands to the striatal dopamine system in adult TS patients and matched healthy controls were included in this meta-analysis. Within these studies, six were targeting the DAT while the other six studies investigated the dopamine receptor. Two studies had examined both the dopamine receptor and the transporter in a TS patient group as well as a group of healthy subjects.Two separate meta-analyses for eight DAT studies and eight dopamine receptor studies were performed. For the analysis of the DAT data from 6 SPECT and 2 PET studies comprising a total of 111 TS patients and 93 healthy controls were included. See Table I2 = 58.89%). The summary effect estimates indicated a scarce significant effect of higher DAT availability in TS patients ). However, when a mixed-effects model was performed correcting for age differences between groups (control groups tended to be older), the effect of age difference became significant and the effect of group differences was not significant anymore. It became half the size ). The funnel plot for striatal DAT binding appears symmetrical, except for two outliers at the bottom right and top left  a trend toward a higher DAT availability in TS patients ) was observed. Leave-one-out analysis indicated omitting the study by Wong et al.35 would result in a summary effect size of increased DAT at 0.58 with p = 0.056. The mixed-effect model analyses were not significant. We performed a separate analysis for right and left caudate of this dataset. A nonsignificant increase of DAT binding in patients’ left ) and right ) caudate remained.Six studies in 86 patients and 68 controls appraised the caudate. In this heterogeneous study sample (I2 = 71.07%) a nonsignificant trend toward a higher DAT availability in TS patients ) could be seen. Similar to the caudate, omitting data from Wong et al.35 resulted in significantly increased DAT binding (p = 0.04). Again a separate analysis for right and left putamen was carried out. Once more a nonsignificant increase of DAT binding in patients’ left ) and right ) putamen was seen.The same 6 studies with 86 patients and 68 controls reported on the putamen. In spite of the heterogeneous study sample  of this set in every analysis, a nonsignificant trend toward a higher DAT binding in TS patients was observed. Additional analyses did not show any meaningful influences.2/3 in the striatum . The summary effect estimates indicated a nonsignificant alteration of D2/3 availability in TS patients ). The funnel plot for striatal D2/3 binding appears symmetrical except for one outlier at the bottom left  summary effect estimates ) showed no significant alterations. None of the other performed analyses found any notable effect. No lateralization analysis was possible.Five studies in 40 patients and 38 controls appraised the caudate. In this homogenous study sample (I2 = 66.88%) and no significant difference in D2/3 availability in TS patients ) was observed. None of the other analyses performed showed a meaningful influence of effect estimates in the putamen. Again, no differentiation between hemispheres could be observed.The same 5 studies with 40 patients and 38 controls reported on the putamen. The study sample was heterogeneous  and differences in imaging technique by pooling the available data. Another limitation for consequent conclusions from these findings is the chicken-and-egg problem44. Bigger DAT density in TS patients may reflect a causal pathophysiological mechanism in tic-generation, potentially by striatal dopaminergic hyper-innervation, or it reflects an adaptive change caused by the disease or pharmacological treatment. At this point, both mechanisms seem to be possible according to the available literature.Our findings of higher DAT binding in adult TS patients can test the hypothesis of hyperinnervation and the presynaptic abnormality hypothesis. The DAT is located in presynaptic dopamine neurons and responsible for the dopamine reuptake from the synaptic cleft, thereby controlling extracellular dopamine levels2/3 receptor binding in adult TS patients can test for the hypothesis of supersensitive dopamine receptors in TS. This hypothesis states a postsynaptic abnormality of dopamine receptors due to changes of affinity and/or density of those receptors44. There are two different G protein-coupled receptor types which mediate dopamine neurotransmission47: the excitatory D1 type and the inhibitory D2 type44. D2-like receptors include the dopamine receptor 2, 3, as well as 4, and D1-like receptors include 1 and 549. Most dopamine receptor-expressing neurons in the human brain are GABAergic medium spiny neurons (MSNs), which present 95% of striatal neurons50. In TS patients the cortico-basal ganglio-thalamo-cortical (CBGTC) network is altered52. These alterations include both structural and functional changes of the CBGTC network involving several reentry pathways53. In the CBGTC circuit the striatum acts as an input center for the basal ganglia from the cortex. While the so called hyperdirect pathway bypasses the striatum, striatal efferents are classified as direct and indirect pathways. The indirect pathway expresses D2 receptors, which project to the globus pallidus and the substantia nigra, polysynaptically54. Our above-mentioned results with a trend towards a lower D2/3 receptor affinity and/or density are not in line with the classic supersensitive dopamine receptor hypothesis, where a higher binding of receptors would have been expected. The inconsistencies of previous neuroimaging studies could be overcome with this meta-analytic approach. Several PET and SPECT studies showed no D2/3 receptor differences between TS patients and controls. Accordingly, earlier postmortem findings did not show differences between dopamine receptors in TS brains and those of healthy controls. These findings in the brains of TS patients were limited by a very small sample size18. Whereas two PET studies, which assessed extrastriatal D2/3 receptors in a set of six55 and eight TS patients56 found a significant reduction of dopamine receptor density compared to matched healthy controls. Lower D2/3 receptor availability or affinity (hyposensitive dopamine receptors) may be one of the causal mechanisms of TS. But again, the chicken-and egg dilemma confines this statement44. Also an adaptive mechanism, potentially due to drug treatment, may be the reason for decreased receptor availability or once again both reasons may be accountable for these postsynaptic changes.Our findings of a trend toward lower D57. The recent model of dopamine function states the regulation of dopamine activity in subcortical regions operates via two mechanisms58. First, the tonic dopamine release, where dopamine is released into the extracellular space in low concentrations via different mechanisms continuously and second, the spike-dependent phasic dopamine release. In the phasic state dopamine is released in high concentration into the synaptic cleft and rapidly inactivated59. The classic tonic-phasic TS theory suggests that an overactive DAT would cause reduced tonic dopamine transmission. This in turn reduces autoreceptor availability and increases presynaptic dopamine levels due to higher dopamine reuptake. This would result in an increased phasic dopamine release60. Our findings of higher DAT and lower D2/3 availability fit well with this theory. However, this classic hypothesis of tonic-phasic dysfunction has an obvious problem. Psychostimulants which inhibit the DAT do not reduce vocal or motor tics. Therefore, Maia and Conceição developed a new hypothesis of tonic-phasic dysfunction in TS, which involves an increase in tonic and phasic dopamine57. This theory is grounded on the computational understanding of action selection and learning in striatal dopamine function. So the CBGTC network and striatal dopamine are involved in tic execution and especially phasic mediated tic learning61. In the striatum dopamine inhibits D2 MSNs of the indirect pathway and stimulates the activity of D1 MSNs of the direct pathway62. Maia and Conceição postulate a striatal dopaminergic hyperinnervation in patients with TS63. Our findings are in line with this new approach and can serve as a neuroimaging underpinning of this hypothesis.Another popular pathophysiological hypothesis of TS states a dysfunction of the tonic-phasic dopamine releaseNeuroimaging studies on the topic of TS have reported inconclusive results. To gain a better picture we performed the to our knowledge first meta-analysis on molecular imaging of the dopamine system in TS. A systematic literature search in August 2017 found a total of 49 PET and SPECT studies in the field of TS. For investigating the DAT activity 8 studies with 111 patients and 93 healthy controls could be included in our meta-analysis. Significantly increased striatal DAT binding in TS patients was detected, although this effect did not remain significant after correcting for age. For the dopamine receptor activity analysis of 8 studies with a total of 72 TS patients and 71 controls revealed a nonsignificant trend towards lower striatal D2/3 receptor binding in TS patients. Our data suggests striatal dopaminergic alterations in adult TS patients, which emphasizes the current hypotheses of TS pathophysiology. However, the here observed effects were nonsignificant or did not remain significant after correcting for covariates. This might highlight the heterogeneity and age dependency of TS. Nonetheless, our data clearly demonstrate the need for large-scaled, systematic, longitudinal neuroimaging studies with well-matched control groups to understand the pathophysiology of TS and thereby make the development of a curative therapy possible.


X-AMR. This initiative has been inspired by the recent prioritization of AMR as a global health issue by the World Health Organisation and UN Global Health Assembly [Antimicrobial resistance (AMR) is a cross-disciplinary issue, with ground-breaking studies currently bringing together clinicians and modellers, veterinary and soil scientists, microbiologists and anthropologists. Yet finding a home for the unique publications from this research is often difficult. The Microbiology Society is providing such a home with a new pop-up journal for cross-disciplinary research on antimicrobial resistance: Assembly , collaboAssembly , as wellDifferent disciplines have different working practices, logistical issues, language and scientific norms. Outcomes of research can be interpreted or applied in different ways. However, there is enormous value in working together to overcome these issues to fight the growing impact of AMR on human and animal health. For example, social sciences, mathematical modelling and One Health studies can offer unique and crucial insights into develop interventions to reduce AMR.Antimicrobial resistance is a social problem ; its driMathematical modelling can provide a logical framework to underpin and combine distinct components of cross-disciplinary projects. This is especially key for AMR research where the multiple factors interact in complex ways. For example, mathematical modelling can be used to explore underlying mechanisms that drive basic microbiology as well as trends in drug resistance infection incidence . This caEffective surveillance and management of AMR requires a synthetic approach that incorporates a broad range of reservoirs of resistant bacteria, viruses, parasites and fungi. The ‘One Health’ perspective acknowledges that conventional compartmentalization of clinical, veterinary, agricultural and environmental settings belies the interconnectedness of the microbial ecosystem, and that management practices should factor in potential risks extending beyond the sector in which they are applied. Prudent antibiotic stewardship in agriculture and aquaculture can help mitigate the emergence and spread of AMR genes and resistant microorganisms in clinical settings. Moreover, a greater understanding of how microbial communities in soil or water are perturbed due to anthropogenic exposure to antimicrobials will help inform improved policy guidelines and risk assessments.X-AMR has deliberately been kept broad and we are open to new interpretations or conceptualizations of this emerging interdisciplinary field. We also encourage the submission of well-referenced reviews or opinion pieces addressing major unanswered questions and challenges in the field. In addition to providing a home for cross-disciplinary cutting-edge papers, this pop-up journal aims to build an AMR research community and a platform for education. The new interventions desperately needed for AMR control have to consider a range of factors from the microbiological to the anthropological. By providing a forum for high-quality research that cuts across these topic areas, we intend to support the education of a new generation of AMR researchers who have unique knowledge over this wide range of interplaying themes.There are many other scientific approaches that can also contribute to reducing AMR, including the design and discovery of new antimicrobials. The scope for Microbiology, Journal of General Virology, Microbial Genomics or the Journal of Medical Microbiology. Authors can select the most suitable match for their AMR manuscript to any one of these journals; however, please contact the Microbiology Society’s editorial staff at x-amr@microbiologysociety.org if you are not sure which journal to submit to. Articles will be peer reviewed by the parent journal as usual; cross-disciplinary AMR articles that do not normally fall within the scope of our current journals will be assessed by the X-AMR guest editors. All accepted articles will be published in one of the four participating journals and will be available as a collection dedicated to AMR. This site will curate all articles submitted for this special pop-up journal, as well as other AMR content from all our journals. All articles will be published on PubMed and PubMed Central if authors opt for open access.The Microbiology Society publishes four journals that have traditionally included AMR research. Authors can choose to submit their research to X-AMR’ in your cover letter when submitting your paper, as this will ensure that the paper is assigned to the relevant editor. We welcome papers from any field of AMR and particularly those with a cross-disciplinary or interdisciplinary approach, and aim to provide one platform to publish all accepted AMR content submitted to us. We look forward to receiving your manuscripts.Please mention ‘Submission to


Klinefelter Syndrome (KS) is the most common genetic cause of infertility in men. Degeneration of the testicular tissue starts in utero and accelerates at puberty with hyalinisation of seminiferous tubules, spermatogonia apoptosis and germ cell maturation arrest. Therefore, fertility preservation in young KS boys has been proposed, although this measure is still debated due to insufficient knowledge of the pathophysiology of the disease. To better understand the underlying mechanisms of testicular failure and germ cell loss, we analysed functional and morphological alterations in the somatic compartment of KS testis, i.e., Sertoli cells, including the blood–testis barrier (BTB) and Leydig cells (LC). We compared three populations: 35 KS 47,XXY non-mosaic patients, 28 Sertoli-cell-only (SCO) syndrome patients and 9 patients with normal spermatogenesis. In KS patients the expression of BTB proteins connexin-43 and claudin-11 assessed with a semi-quantitative scoring system appeared significantly reduced with a disorganised pattern. A significant reduction in seminiferous tubules expressing androgen receptors (AR) was observed in KS compared to normal spermatogenesis controls. INSL3 expression, a marker of LC maturation, was also significantly reduced in KS compared to patients with normal spermatogenesis or SCO. Hence, the somatic compartment impairment in KS could be involved in degeneration of seminiferous tubules. Klinefelter Syndrome (KS) is the most frequent sexual chromosomal aneuploidy in men, characterised by the presence of an extra “X” chromosome. It occurs in 1/600 newborns . As testIn KS patients, a number of alterations involving Sertoli cells (SCs), which play a central role in both testicular development and germ cell differentiation, have been reported . Indeed,The blood–testis barrier (BTB) has been poorly studied in KS. This dynamic structure take place during puberty  and is cThe syndrome is further characterised by LCs hyperplasia where LCs are grouped in clusters  and showAs data from the literature are still too scarce to enable understanding of testicular tissue and cell impairment in KS, and as only case report and a small series of KS patients were reported, we further investigated the somatic testicular compartment in adult KS patients. We focused on SC and LC maturation and function, as well as on main BTB proteins, in a large cohort of KS patients compared to: (1) Sertoli-cell-only (SCO) patients with normal karyotype and anatomopathological features similar to KS, and (2) to a group of patients with normal spermatogenesis (NSP) and normal karyotype.Expression patterns of CLD11 and CNX43 are shown in p ≤ 0.02) or in SCO group . On the other hand, a mean of 5% ± 10% STs in KS presented a score 2 corresponding to a well-organised distribution of the protein, which was lower in KS than in NSP  and SCO  (A mean of 22% ± 32% STs for CLD11 scored as 0 (absence of protein) in KS, which was significantly higher than in NSP  a,b. No sp ≤ 0.002) or SCO  groups. On the other hand, a mean of 3% ± 10% STs were scored 2 (well-organised protein distribution) corresponding to a reduction of the protein pattern organisation in KS compared to NSP  and to SCO  (A mean of 42 ± 39% STs were scored 0 (absence of expression) for the gap junction protein CNX43 in KS which was statistically higher than in NSP  c,d. No dNo STs presented a score 0 (absence of the protein staining) for ZO1 in all three groups. A mean of 68% ± 31% STs, 59% ± 28% STs and 74% ± 18% STs were scored as 1  in KS, NSP and SCO patients, respectively, with no differences between groups. No differences were found for score 2  ZO1 STs percentages between groups showing a mean of 32% ± 31% STs in KS, 40% ± 28% STs in NSP and 26% ± 18% STs in SCO patients. A total of 3136 STs with a mean of 25.8 ± 24.8 STs per patient were analysed. No significant difference was found when we compared the AMH expression in the three groups. In KS a mean of 67% ± 37% STs did not present the protein AMH (score 0), and a mean of 33% ± 37% STs presented a score 1. In NSP a mean of 71% ± 35% STs presented a score 0 and a mean of 29% ± 35% STs presented a score 1. In SCO group a mean of 59% ± 34% STs had a score 0 and a mean of 41% ± 34% STs had a score 1. Concerning GDNF expression (score 1), a mean of 99% ± 6% STs, 100% ± 0% STs and 100% ± 0% STs presented the protein expression (score 1) in KS, NSP and SCO, respectively. p ≤ 0.01) and score 1 (p ≤ 0.01) were statistically significant (In KS a mean of 31% ± 36 % STs did not present the AR (score 0) and a mean of 69% ± 36% STs showed the presence of ARs (score 1). In NSP group a mean of 100% ± 0% STs presented a score 1, in the SCO group a mean of 40% ± 34% STs presented a score 0 and a mean of 60% ± 34% STs a score 1. The differences between the KS and the NSP groups for score 0  and to SCO (p ≤ 0.02)  per patient for each protein. A significant reduction of INSL3 expression was observed in KS compared to NSP ( ≤ 0.02) a. A meanStAR was expressed in all groups, showing a mean score of 1.41 ± 0.49 in KS, 2 ± 0 for the NSP and 1.45 ± 0.5 for the SCO group with no significant differences found between the three groups of patients.An important loss of spermatogonia occurs during puberty in KS patients, potentially justifying a prepubertal fertility preservation strategy using cryopreservation of spermatogonial stem cells (SSCs), although this is still highly debated . Indeed,In the present study we therefore investigated, for the first time in a series of 35 adult KS patients, the expression pattern of several BTB proteins  and of other components of the testicular somatic compartment, comparing them to patients with normal karyotype and either histological testicular tissue degeneration similar to KS (SCO) or normal spermatogenesis (NSP).In animal studies, it was shown that the BTB is essential for spermatogenesis as CNX43 knockout mice are infertile with small testis and present spermatogenesis arrest at the stage of spermatogonia and STs containing only Sertoli cells. Moreover, CLD11 selective gene knockout murine testes resulted in spermatogenesis arrest . While iCLD11 and CNX43 are tethered to the cytoskeleton by adaptor proteins such as ZO proteins that control tight junction structure and function . We studBesides the BTB proteins, SCs’ function and maturation also have a central role in spermatogenesis and GCs renewal. Indeed, each SC supports a defined number of germ cells  and throWith regard to markers of SC maturation, we examined the expression of AMH which is normally produced in prepubertal but not adult SCs . While WAnother sign of acquisition of maturity in SCs is the presence of AR. An altered immunoexpression of AR with a reduction of AR nuclear immunostaining was described in prepubertal KS boys compared to age-matched controls . In our Furthermore, a heterogenous profile of SC maturation was observed in a series of seven non-mosaic KS adolescents aged 15–17 y) when Rives et al. calculated a score for SCs maturation including AMH, AR and vimentin immunoexpression. In their study the authors also suggested that SCs’ maturity was correlated to the persistence of germ cells as in KS patients presenting STs with only Sertoli cells, an immature phenotype was preferentially detected [ y when RWe also analysed INSL3, an insulin-like factor secreted by LCs  considerDespite a normal or decreased blood circulating testosterone level in KS , the redIn conclusion, our results highlight a compromised somatic compartment demonstrated by some abnormal SCs features and the LCs’ lack of maturation. These findings are also in agreement with recent transcriptome studies where D’Aurora et al. showed differential expression of several genes encoding for proteins of both the germinal and somatic compartments. In particular they described genes responsible of steroidogenic function, impairment of inflammatory pathways and BTB structure in six KS adult patients compared to patients with obstructive azoospermia ,45. In conclusion, to the best of our knowledge, this study involved the largest series of KS adults, providing analysis of the testicular somatic compartment, and for the first time detecting underlying defects in CNX43 and CLD11, the main components of the BTB. This study was approved by the Ethics Review Board of the Catholic University of Louvain (2017/20OCT/492 on the 20th October 2017). Testicular tissue from three groups of adult patients were selected from the surgery records of the andrology department. Testicular fragments were fixed in 4% paraformaldehyde and were alcohol-dehydrated in xylene and embedded in paraffin. All patients underwent a testicular sperm extraction (TESE) or micro-TESE  procedure for infertility between 2007 and 2017. Thirty-five azoospermic non-mosaic KS patients (mean age 32), nine patients presenting with obstructive azoospermia due to a previous vasectomy after failed PESA  who underwent a testicular biopsy showing normal spermatogenesis at histology and having a normal karyotype (mean age 48), and twenty-eight patients with non-obstructive azoospermia with normal karyotype and an anatomopathological diagnosis of Sertoli cell only syndrome (mean age 32) were included. A total of 128 testicular samples were analysed see . Clinica2O2 for 30 min at room temperature (RT). After washing in deionised water, sections were placed in citrate buffer for 60 min at 98 °C before being washed in 0.05 M Tris-buffered saline (TBS) and 0.05% Triton X-100 and incubated at room temperature (RT) with 10% normal goat serum  and 1% bovine serum albumin  to block non-specific binding sites for 30 min. The primary antibody was added to the sections and incubated overnight at 4 °C in a humidified chamber. Nonspecific antibody binding was blocked by incubation of samples in 10% NGS and 1% BSA for 30 min at RT. The following day, the slides were washed in 0.05 M TBS and 0.05% Triton X-100 three times for 2 min each and the secondary anti-mouse  or anti-rabbit  peroxidase-labeled antibody  was added and incubated for 60 min at RT, followed by washing in 0.05 M TBS and 0.05% Triton X-100 three times for 2 min each. The sections were then incubated for 10 min at RT with the chromogen diaminobenzidine  and nuclei were counterstained with Mayer’s hematoxylin after washing in tap water. For positive controls, human mature testicular tissue with normal spermatogenesis was used. The primary antibody was omitted for negative controls. After dehydrations, sections were mounted and scanned with a Leica SCN400 slide scanner . Immunohistochemistry was performed to study the BTB with CNX43, CLD11 and ZO1 antibodies. To assess SCs viability and maturation we used GDNF, AMH and AR antibodies, and to study the LCs maturation and function we used INSL3 and StAR antibodies. References of antibodies are presented in For ST counts we considered all seminiferous tubules with an intact basement membrane. Double blind analyses of immunostained slides were performed by M.G.G. and M.V. We studied a protein for each of the three junction types of the BTB: CLD11, CNX43 and ZO1. A semi-quantitative method (score 0–1–2) was chosen to evaluate the expression of the BTB proteins: CNX43, CLD11, ZO1. A ratio was calculated between numbers of STs of each score and total number of the STs per slide, expressed as a percentage a. A scorTo evaluate the AMH, AR and GDNF immunostainings, we considered the presence (score 1) or the absence of staining (score 0) a–c. The To evaluate LCs, we considered the interstitium staining for INSL3 and StAR  in threeComparisons between the three groups  for each score were tested using a non-parametric Kruskall–Wallis test, followed by Steel–Dwass post-hoc tests with alpha = 0.05 used as the level of significance. In this study we analysed for the first time together three BTB proteins in a big series of 35 adult KS patients. We compared results with NSP and SCO groups to demonstrate that KS adult patients present a reduction of expression of the BTB proteins CLD11 and CNX43 with a different distribution pattern. This pattern was possibly determined by the presence of a dysfunctional somatic compartment with reduced expression of AR in SCs and reduced expression of INSL3 in LCs, both of which are necessary for good cell function and are markers of cell maturity. It remains to be unraveled if KS SSCs are able to differentiate as isolated cells or if another model should be used to replace a compromised somatic compartment necessary for the differentiation process.