Lack of causative mutation in the AMH and AMHR2 genes in a cat (38,XY) with persistent Mullerian duct syndrome (PMDS).

A 1-year-old European shorthair male cat with a normally developed penis was subjected to genetic, endocrinological and histological studies due to unilateral cryptorchidism. The blood testosterone level was typical for males, while the level of anti-Mullerian hormone (AMH) was very low. Surgical removal of internal reproductive organs was followed by a histological study, which revealed inactive testicles with neoplastic changes and derivatives of Mullerian ducts. Cytogenetic analysis showed a normal XY sex chromosome complement and molecular analysis confirmed the presence of Y-linked genes (SRY and ZFY). Although the level of AMH was low, two normal copies of the AMH gene were found using droplet digital PCR (ddPCR). Analysis of the coding sequences of two candidate genes (AMH and AMHR2) for persistent Mullerian duct syndrome (PMDS) in the affected cat and in control male cats (n = 24) was performed using the Sanger sequencing method. In the affected cat, homozygosity was found for three novel missense variants in Exon 1 (one SNP) and Exon 5 (two SNPs) of AMH, but the same homozygous genotypes were also observed in one and two control cats, respectively, whose sex development was not examined. Three known synonymous variants with homozygous status were found in AMHR2. We conclude that the DNA variants identified in AMH and AMHR2 are not responsible for PMDS in the affected cat.

Novel mutation leading to splice donor loss in a conserved site of DMD gene causes Duchenne muscular dystrophy with cryptorchidism.

BACKGROUND: As one of the most common congenital abnormalities in male births, cryptorchidism has been found to have a polygenic aetiology according to previous studies of common variants. However, little is known about genetic predisposition of rare variants for cryptorchidism, since rare variants have larger effective size on diseases than common variants. METHODS: In this study, a cohort of 115 Chinese probands with cryptorchidism was analysed using whole-genome sequencing, alongside 19 parental controls and 2136 unaffected men. Additionally, CRISPR-Cas9 editing of a conserved variant was performed in a mouse model, with MRI screening used to observe the phenotype. RESULTS: In 30 of 115 patients (26.1%), we identified four novel genes (ARSH, DMD, MAGEA4 and SHROOM2) affecting at least five unrelated patients and four known genes (USP9Y, UBA1, BCORL1 and KDM6A) with the candidate rare pathogenic variants affecting at least two cases. Burden tests of rare variants revealed the genome-wide significances for newly identified genes (p<2.5×10-6) under the Bonferroni correction. Surprisingly, novel and known genes were mainly found on X chromosome (seven on X and one on Y) and all rare X-chromosomal segregating variants exhibited a maternal inheritance rather than de novo origin. CRISPR-Cas9 mouse modelling of a splice donor loss variant in DMD (NC_000023.11:g.32454661C>G), which resides in a conserved site across vertebrates, replicated bilateral cryptorchidism phenotypes, confirmed by MRI at 4 and 10 weeks. The movement tests further revealed symptoms of Duchenne muscular dystrophy (DMD) in transgenic mice. CONCLUSION: Our results revealed the role of the DMD gene mutation in causing cryptorchidism. The results also suggest that maternal-X inheritance of pathogenic defects could have a predominant role in the development of cryptorchidism.

Undiagnosed RASopathies in infertile men.

RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10-3). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies.

Achieving an optimal pregnancy outcome through the combined utilization of micro-TESE and ICSI in cryptorchidism associated with a non-canonical splicing variant in RXFP2.

PURPOSE: To identify the genetic cause of a cryptorchidism patient carrying a non-canonical splicing variant highlighted by SPCards platform in RXFP2 and to provide a comprehensive overview of RXFP2 variants with cryptorchidism correlation. METHODS: We identified a homozygous non-canonical splicing variant by whole-exome sequencing and Sanger sequencing in a case with cryptorchidism and non-obstructive azoospermia (NOA). As the pathogenicity of this non-canonical splicing variant remained unclear, we initially utilized the SPCards platform to predict its pathogenicity. Subsequently, we employed a minigene splicing assay to further evaluate the influence of the identified splicing variant. Microdissection testicular sperm extraction (micro-TESE) combined with intracytoplasmic sperm injection (ICSI) was performed. PubMed and Human Genome Variant Database (HGMD) were queried to search for RXFP2 variants. RESULTS: We identified a homozygous non-canonical splicing variant (NM_130806: c.1376-12A > G) in RXFP2, and confirmed this variant caused aberrant splicing of exons 15 and 16 of the RXFP2 gene: 11 bases were added in front of exon 16, leading to an abnormal transcript initiation and a frameshift. Fortunately, the patient successfully obtained his biological offspring through micro-TESE combined with ICSI. Four cryptorchidism-associated variants in RXFP2 from 90 patients with cryptorchidism were identified through a literature search in PubMed and HGMD, with different inheritance patterns. CONCLUSION: This is the first cryptorchidism case carrying a novel causative non-canonical splicing RXFP2 variant. The combined approach of micro-TESE and ICSI contributed to an optimal pregnancy outcome. Our literature review demonstrated that RXFP2 variants caused cryptorchidism in a recessive inheritance pattern, rather than a dominant pattern.

Construction and analysis of a joint diagnostic model of machine learning for cryptorchidism based on single-cell sequencing.

BACKGROUND: Cryptorchidism is a condition in which one or both of a baby's testicles do not fully descend into the bottom of the scrotum. Newborns with cryptorchidism are at increased risk of developing infertility later in life. The aim of this study was to develop a novel diagnostic model for cryptorchidism and to identify new biomarkers associated with cryptorchidism. METHODS: The study data were obtained from RNA sequencing data of cryptorchid patients from Nantong University Hospital and the Gene Expression Omnibus (GEO) database. Differential expression analysis was used to obtain differentially expressed genes (DEGs) between the control and cryptorchid groups. These DEGs were analyzed for their functions by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment using GSEA software. Random Forest algorithm was used to screen central genes based on these DEGs. Neuralnet software package was used to develop artificial neural network models. Based on clinical data, receiver operating characteristic (ROC) was used to validate the models. Single-cell sequencing analysis was used for the pathogenesis of cryptorchidism. RESULTS: We obtained a total of 525 important DEGs related to cryptorchidism, which are mainly associated with biological functions such as supramolecular complexes and microtubule cytoskeleton. Random forest approach screening obtained eight hub genes. A neural network based on the hub genes showed a 100% success rate of the model. Finally, single-cell sequencing analysis validated the hub genes. CONCLUSION: We developed a novel diagnostic model for cryptorchidism using artificial neural networks and validated its utility as an effective diagnostic tool.

A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism.

Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid (n = 122) and control (n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3'UTR (16:23716202G>A) in KATA6, responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism (P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3'UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

Advocating hormonal treatment to prevent adult in-fertility in patients diagnosed with congenital un-descended testes.

In 2007 the Nordic group came to the following unanimous conclusions: In general, hormonal treatment is not recommended, considering the poor immediate results and the possible long-term adverse effects on spermatogenesis. Thus, surgery is to be preferred. However, defective mini puberty inducing insufficient gonadotropin secretion is one of the most common causes of nonobstructive azoospermia in men suffering from congenital isolated unilateral or bilateral cryptorchidism. The extent of alteration in the unilateral undescended testis correlate with the contralateral descended testis, indicating that unilateral cryptorchidism is a bilateral disease. Idiopathic central hypogonadism explains the phenomenon of defective mini puberty in otherwise healthy cryptorchid boys. We therefore recommend hormonal treatment for cryptorchid boys with defective mini puberty. Gonadotropin releasing hormone agonist (GnRHa) treatment following surgery to correct cryptorchidism restores mini puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. Several genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed in both the brain and testis. At the molecular level, there is no convincing evidence that heat shock is responsible for the observed pathological testicular changes. Thus, impaired transformation of gonocytes is not the result of temperature stress but rather a hormonal imbalance. Cryptorchidism should therefore be considered a serious andrological problem that cannot be successfully treated by early orchidopexy alone.

CAG and GGN repeat polymorphisms in the androgen receptor gene of a Chilean pediatric cohort with idiopathic inguinal cryptorchidism.

BACKGROUND: Cryptorchidism is one of the most common congenital disorders in boys and it is associated with a higher risk of sub-fertility and testicular cancer. Testicular descent occurs during embryo-fetal development in two phases, transabdominal and inguino-scrotal. In the latter process, androgens play a leading role. The androgen receptor has in its N-terminal domain, two aminoacidic repeats encoded by polymorphic nucleotide repetitions: (CAG)nCAA and GGN. The number of repetitions of these trinucleotides has been associated with different transactivation capacities and sensitivities of the androgen receptor response. OBJECTIVE: To determine whether pediatric Chilean individuals with idiopathic inguinal cryptorchidism have a different number of CAG and/or GGN repeats polymorphisms compared with controls. MATERIALS AND METHODS: A total of 109 cases with idiopathic inguinal cryptorchidism (26 bilateral and 83 unilateral) were studied by polymerase chain reaction amplification from DNA extracted from peripheral blood, followed by fragment size analysis by capillary electrophoresis, which were compared with 140 controls. RESULTS: The CAG26 repeats allele was increased in the total cases (8.3% vs. 1.4%; p = 0.012; odds ratio = 6.21, 95% confidence interval 1.31-29.4), and in bilateral cases compared to controls (11.5% vs. 1.4%; p = 0.028; odds ratio = 9 CI 95% 1.43-56.8). Similarly, CAG > 22 alleles were increased in the total cases (62.4% vs. 49.3%, p = 0.041), and more significantly in bilateral cases (73.1% vs. 49.3%; p = 0.032; odds ratio = 2.79, 95% confidence interval 1.1-7.1). In addition, CAG < 18 alleles were not observed among cases, but were present in 5.7% of controls (p = 0.01). Regarding the GGN repeats, no differences were observed between cases and controls either when analyzing separately unilateral and bilateral cryptorchidism. The joint analysis of the distribution of CAG and GGN alleles showed that the CAG26 allele was present with GGN23, hence the combination CAG26/GGN23 alleles was equally increased in bilateral cases compared with controls (11.5% vs. 1.4%). In contrast, CAG < 18 was preferably observed in the combination CAG < 18/GGN≠23 and was absent in the total cases (4.3% vs. 0%; p = 0.037). DISCUSSION: These results suggest that greater lengths of CAG alleles may contribute to a diminished androgen receptor function. The CAG26 allele alone or in combination with GGN23 was associated with a higher risk of bilateral cryptorchidism. On the other hand, CAG < 18 and the CAG < 18/GGN≠23 allele combination may reduce the probability of cryptorchidism.

RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism.

Introduction: The fertility of cryptorchidism patients who didn't perform corrective surgery will decrease with age. Herein, we elucidate the histological alterations and underlying molecular mechanism in patients with an increase in the disease duration from 20 to 40 years. Methods: Testicular tissues were obtained from three patients with cryptorchidism, ranging in age from 22 to 44 years. Three benign paracancerous testicular samples of matched ages were used as controls. The normal and undescended testicular tissues were stained with hematoxylin and eosin (HE) and immunofluorescence and all six testicular samples were subjected to RNA sequencing. RNA sequencing data were subjected to gene set enrichment analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) network analysis, and Gene Ontology (GO) searches. Real-time reverse transcriptase polymerase chain reaction was used to confirm the DEGs. Results: The seminiferous tubules' basement membrane thickens with age in healthy testes. As the period of cryptorchidism in the cryptorchid testis extended, the seminiferous tubules significantly atrophy, the number of spermatogenic cells declines, and the amount of interstitial fibrous tissue increases in comparison to normal tissues. The number of germ cells per cross-section of seminiferous tubules was significantly lower in cryptorchidism than in normal testicular tissues, according to immunofluorescence staining, but the number of Sertoli cells remained stable. RNA sequencing analysis identified 1150 differentially expressed genes (DEGs) between cryptorchidism and normal testicular tissues (fold change >2 and p<0.05), of which 61 genes were noticeably upregulated and 1089 were significantly downregulated. These genes were predominantly linked to sperm development and differentiation, and fertilization, according to GO analysis. Meiosis pathways were significantly downregulated in cryptorchidism, according to KEGG pathway analysis and GSEA (P<0.001). PPI analysis was used to identify the top seven downregulated hub genes (PLCZ1, AKAP4, IZUMO1, SPAG6, CAPZA3, and ROPN1L), which were then further verified by qPCR. Discussion: By describing the histological changes and differential gene expression patterns in adult cryptorchid patients of different age groups, we discovered the progression mechanisms of undescended testes in adults with aging and identified seven significantly downregulated hub genes (PLCZ1, AKAP4, IZUMO1, SPAG6, CAPZA3, and ROPN1L) in cryptorchid testis compared to normal testicular tissues. These genes played a role in the process of spermgenesis and are directly linked to the steady decline in fertility caused by cryptorchidism. Our study provided a better understanding of the molecular mechanisms underlying the loss of spermatogenesis in adult cryptorchidism, and give support for the development of adult cryptorchidism treatments.

[Hormonal and genetic causes of cryptorchidism].

Cryptorchidism is the most frequent congenital disorders of the reproductive system, is present in 2-3% of term newborn boys. Genes involved in embryonic testicular migration are known but their role in cryptorchidism development are not investigated enough. Genetical causes of cryptorchidism are identified in 5-7% of patients. The article contains data on the role of insulin-like peptide 3 and its receptor, anti-Müllerian hormone, gonadotropins, androgens in embryonic testicular migration. INSL3 and AMH are presented as markers of testicular dysfunction associated with cryptorchidism. Hypogonadotropic hypogonadism is also associated with cryptorchidism and can be diagnosed based on it. Results of modern investigations determine the necessary of hormonal and genetical examination of patients with isolated cryptorchidism to detect causes of cryptorchidism and manage of patients.

A novel mutation in the AMHR2 gene, resulting in persistent Müllerian duct syndrome presenting with bilateral cryptorchidism and obstructed inguinal hernia.

OBJECTIVES: To highlight important clinical aspects of Persistent Müllerian duct syndrome (PMDS). PMDS belongs to the group of differences of sex development. It is attributed to mutations in genes encoding for the anti-Müllerian hormone or its type II receptor (AMHR2) and inherited via an autosomal recessive transmission. CASE PRESENTATION: An 18-day-old male infant with known bilateral cryptorchidism, presented with left-sided obstructed inguinal hernia. The diagnosis of PMDS was considered during inguinal exploration as both testes together with uterus and fallopian tubes were recognized in the hernial sac. Histology confirmed the presence of Müllerian-derived tissues. Genetic testing revealed two different mutations of the AMHR2 gene, both with autosomal recessive transmission: a frequently encountered deletion of 27 pairs bases on exon 10 of this 11 exon gene and a novel deletion of 2 pairs bases on exon 6. CONCLUSIONS: This case is notable being the rarest type of PMDS, that of transverse testicular ectopia and associated with a novel AMHR2 gene mutation.

MicroRNA as epigenetic regulators of canine cryptorchidism.

Cryptorchidism, the failed descent of one or both testes into the scrotum, is a common developmental disorder in male dogs. Cryptorchidism may affect canine fertility, reducing the quality of the semen, and may promote spermatic cord torsion and onset of neoplasia. MicroRNAs (miRNAs) are epigenetic regulators of gene expression and their dysregulation is associated with disorders of spermatogenesis and testis neoplasia. The present study aimed at investigating the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) canine retained testes and testes affected by seminoma, and at integrating miRNAs to their target genes. Forty testicular FFPE specimens from 30 dogs were included - 10 scrotal and 10 contralateral retained from 10 unilateral cryptorchid dogs; 10 tumoral testes affected by seminoma from non-cryptorchid dogs; 10 scrotal normal testes from non-cryptorchid dogs included as the control. The expression level of three miRNAs, namely miR-302c-3p, miR-302a-3p, and miR-371-3p, associated with testicular disorders, were quantified using RT-qPCR. The comparative analysis demonstrated that the level of miR-302a-3p and miR-371a-3p were quantifiable exclusively in control testes. The expression level of miR-302c-3p was higher in the control than in the other groups; its expression decreased in retained testes compared to scrotal testes and testes with seminoma. Gene Ontology analysis pointed out that these miRNAs may be involved in the modulation of estrogen and thyroid hormone signaling pathways. In conclusion, this study demonstrated that miRNAs are dysregulated in canine cryptorchid and seminoma-affected testes compared to control tissues, confirming the pivotal role of miRNAs in cryptorchidism.

Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptorchidism and male infertility.

STUDY QUESTION: What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility? SUMMARY ANSWER: Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected. WHAT IS KNOWN ALREADY: The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear. STUDY DESIGN, SIZE, DURATION: Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2. PARTICIPANTS/MATERIALS, SETTING, METHODS: For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein's cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined. MAIN RESULTS AND THE ROLE OF CHANCE: This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients' testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation. LIMITATIONS, REASONS FOR CAUTION: Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism. WIDER IMPLICATIONS OF THE FINDINGS: In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG ('Emmy Noether Programme' project number 464240267). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Microdeletion at ESR1 Intron 6 (DEL_6_75504) Is a Susceptibility Factor for Cryptorchidism and Hypospadias.

CONTEXT: We have previously reported that a specific "AGATC" haplotype in a >34 kb tight linkage disequilibrium (LD) block within ESR1 is strongly associated with cryptorchidism and hypospadias in Japanese boys. OBJECTIVE: We aimed to determine the true susceptibility factor for cryptorchidism and hypospadias linked to the "AGATC" haplotype. METHODS: We performed various molecular studies in hitherto unreported 230 Italian boys (80 with cryptorchidism and 150 with normal genitalia) and previously reported and newly recruited 415 Japanese boys (149 with cryptorchidism, 141 with hypospadias, and 125 with normal genitalia). We also performed ESR1 expression analyses using breast cancer-derived MCF-7 cells. RESULTS: Haplotype analysis revealed the LD block and positive association of the "AGATC" haplotype with cryptorchidism in Italian boys. Whole genome sequencing identified an identical 2249-bp microdeletion (ΔESR1) generated by a microhomology-mediated replication error in both Japanese and Italian boys with the specific haplotype. ΔESR1 was found to be strongly associated with cryptorchidism and hypospadias by Cochran-Armitage trend test and was revealed to show nearly absolute LD with the "AGATC" haplotype. ESR1 expression was upregulated in MCF-7 cells with a homozygous deletion encompassing ΔESR1 and those with a homozygous deletion involving a CTCF-binding site within ΔESR1. CONCLUSION: The results reveal that ΔESR1, which has been registered as "DEL_6_75504" in gnomAD SVs v2.1, is the true susceptibility factor for cryptorchidism and hypospadias. It appears that ΔESR1 was produced in a single ancestral founder of modern humans and has been maintained within the genome of multiple ethnic groups by selection.

Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs.

INTRODUCTION: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. METHODS: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. RESULTS: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. CONCLUSION: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.

Elevated plasma miR-210 expression is associated with atypical genitalia in patients with 46,XY differences in sex development.

BACKGROUND: Differences of sex development (DSD) is a term used for conditions in which the chromosomal, gonadal or phenotypical sex is atypical. 46,XY DSD patients frequently present undervirilized external genitalia. The expression of different miRNAs in many organs of the male genital system has been reported, and these miRNAs have been associated with testicular function and its disorders, but no description has been related to DSD conditions. This study aimed to evaluate the plasma expression of miR-210 in 46,XY DSD patients who presented atypical genitalia at birth. METHODS: Eighteen 46,XY DSD patients who presented atypical genitalia (undescended testis and/or hypospadias, bifid scrotum or micropenis) at birth and 36 male control individuals were selected. Plasma levels of miR-210 and reference miR-23a were measured using RT-qPCR and the data were analysed by the 2-ΔCt method. RESULTS: MiR-210 plasma levels were significantly higher in 46,XY DSD patients with atypical genitalia than in male control subjects (p = 0.0024). A positive association between miR-210 levels and the presence of cryptorchidism and hypospadias (p = 0.0146 and p = 0.0223) was found in these patients. Significantly higher levels of miR-210 were observed in patients with 46,XY DSD and cryptorchidism than in control subjects (p = 0.0118). These results are in agreement with previous literature reports, in which increased levels of miR-210 expression were observed in human testicular tissue from adult males with undescended testes in comparison with samples of descended testes. CONCLUSION: Our study showed a positive association between the presence of atypical genitalia and plasma levels of miR-210 expression in the group of patients with 46,XY DSD of unknown aetiology studied. These findings contribute to reveal a new perspective on the role of miRNAs in the development of male external genitalia and the broad spectrum of phenotypes presented by patients with 46,XY DSD.

Loss of WNT4 in the gubernaculum causes unilateral cryptorchidism and fertility defects.

Undescended testis (UDT) affects 6% of male births. Despite surgical correction, some men with unilateral UDT may experience infertility with the contralateral descended testis (CDT) showing no A-dark spermatogonia. To improve our understanding of the etiology of infertility in UDT, we generated a novel murine model of left unilateral UDT. Gubernaculum-specific Wnt4 knockout (KO) mice (Wnt4-cKO) were generated using retinoic acid receptor ß2-cre mice and were found to have a smaller left-unilateral UDT. Wnt4-cKO mice with abdominal UDT had an increase in serum follicle-stimulating hormone and luteinizing hormone and an absence of germ cells in the undescended testicle. Wnt4-cKO mice with inguinal UDT had normal hormonal profiles, and 50% of these mice had no sperm in the left epididymis. Wnt4-cKO mice had fertility defects and produced 52% fewer litters and 78% fewer pups than control mice. Wnt4-cKO testes demonstrated increased expression of estrogen receptor α and SOX9, upregulation of female gonadal genes, and a decrease in male gonadal genes in both CDT and UDT. Several WNT4 variants were identified in boys with UDT. The presence of UDT and fertility defects in Wnt4-cKO mice highlights the crucial role of WNT4 in testicular development.

Gene dosage changes in KCTD13 result in penile and testicular anomalies via diminished androgen receptor function.

Despite the high prevalence of hypospadias and cryptorchidism, the genetic basis for these conditions is only beginning to be understood. Using array-comparative-genomic-hybridization (aCGH), potassium-channel-tetramerization-domain-containing-13 (KCTD13) encoded at 16p11.2 was identified as a candidate gene involved in hypospadias, cryptorchidism and other genitourinary (GU) tract anomalies. Copy number variants (CNVs) at 16p11.2 are among the most common syndromic genomic variants identified to date. Many patients with CNVs at this locus exhibit GU and/or neurodevelopmental phenotypes. KCTD13 encodes a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (B-cell receptor (BCR) [BTB (the BTB domain is a conserved motif involved in protein-protein interactions) Cullin3 complex RING protein Rbx1] E3-ubiqutin-protein-ligase complex), which has essential roles in the regulation of cellular cytoskeleton, migration, proliferation, and neurodevelopment; yet its role in GU development is unknown. The prevalence of KCTD13 CNVs in patients with GU anomalies (2.58%) is significantly elevated when compared with patients without GU anomalies or in the general population (0.10%). KCTD13 is robustly expressed in the developing GU tract. Loss of KCTD13 in cell lines results in significantly decreased levels of nuclear androgen receptor (AR), suggesting that loss of KCTD13 affects AR sub-cellular localization. Kctd13 haploinsufficiency and homozygous deletion in mice cause a significant increase in the incidence of cryptorchidism and micropenis. KCTD13-deficient mice exhibit testicular and penile abnormalities together with significantly reduced levels of nuclear AR and SOX9. In conclusion, gene-dosage changes of murine Kctd13 diminish nuclear AR sub-cellular localization, as well as decrease SOX9 expression levels which likely contribute in part to the abnormal GU tract development in Kctd13 mouse models and in patients with CNVs in KCTD13.

Identification of endocrine-disrupting chemicals targeting the genes and pathways of genital anomalies in males.

Hypospadias and cryptorchidism are the most common congenital malformations in male neonates, both of which are also the important clinical manifestations of testicular dysgenesis syndrome and share a same origin. Many studies have suggested that prenatal exposure to endocrine-disrupting chemicals (EDCs) is associated with hypospadias and cryptorchidism development. However, the consistent mechanisms remain unclear. To identify the key EDCs, genes and biological networks related to the development of hypospadias and cryptorchidism respectively and commonly, we conduct the present study and found a new method for predicting the correlation between the interactive genes of hypospadias/cryptorchidism and chemicals. Transcriptome profiles were obtained from the Comparative Toxicogenomics Database (CTD). Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses and protein-protein interaction (PPI) network were applied for integrative analyses. The rat model and molecular docking were applied to furtherly verifying the findings of the integrative analyses. Besides the highly related genes, most enriched pathways and chemicals for hypospadias and cryptorchidism respectively, we found hypospadias and cryptorchidism share many same highly associated EDCs (e.g., dibutyl phthalate) and genes (e.g., androgen receptor and estrogen receptor 1) through comparing highly related chemicals or genes of hypospadias and cryptorchidism respectively. GO and KEGG analysis showed that these same interactive genes were mainly enriched in steroidogenesis, response to steroid hormone and nuclear receptor activity. PPI network analysis identified 15 biological hub genes. Furtherly, hypospadias and cryptorchidism were induced by prenatal dibutyl phthalate exposure. Decreased serum testosterone level, downregulation of nuclear androgen-dependent and upregulation of cytoplasmic estrogen-dependent pathways may lead to hypospadias and cryptorchidism. This study proposed a new method for predicting the correlation between the interactive genes of hypospadias/cryptorchidism and chemicals and found that hypospadias and cryptorchidism share many same highly associated EDCs and genes.