Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

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.

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.

In vitro obtainment of stem-like cells from gubernaculum testis biopsies of cryptorchid pediatric patients.

Testicular descent is a crucial event in male sexual development, and alterations in this process during gestation can lead to reduced fertility in adulthood. Cryptorchidism, i.e., failure of one or both testicles to descend into the scrotum, is one of the most common birth defects and represents a principal cause of infertility in adulthood. Therefore, identifying effective approaches for preserving fertility in childhood is of primary importance. In this context, the key role played by the gubernaculum testis during the placement of the testes in the scrotal bursa emerges. Given its close affinity to testicular tissue and its richness in mesenchymal cells, our prime aim is to characterize this para-testicular tissue to explore its potential ability to differentiate into testicular cells for fertility preservation. The first step of our task is represented by the present study that aimed to obtain in vitro stem-like cells starting from gubernaculum testis biopsies of four pediatric patients affected by cryptorchidism, aiming to differentiate them into testicular functioning cells. Our results show that the obtainment of aggregates with stem features is not dependent on the age of the patients and, therefore, not even on the damage suffered by the testis during its stay in the abdomen. This study opens the possibility of extending this approach to older patients, offering a new potential approach to support their fertility potential.

E2F1 regulates testicular descent and controls spermatogenesis by influencing WNT4 signaling.

Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.

Cryptorchidism and testicular cancer in the dog: unresolved questions and challenges in translating insights from human studies†.

Cryptorchidism, the failure of one or both testes to descend into the scrotum, and testicular cancer show a strong correlation in both dogs and humans. Yet, long-standing medical debates persist about whether the location of undescended testes directly causes testicular cancer in humans or if both conditions stem from a common origin. Although testicular cancer is a prevalent disease in dogs, even less is known about its cause and correlation with testicular descent in this species. This review investigates the relation between these two disorders in dogs, drawing insights from human studies, and examines key biomarkers identified thus far. In addition, it explores potential causal links, including the impact of temperature on maturing testicular cells and a potential shared genetic origin. Notably, this literature review reveals significant differences between men and dogs in reproductive development, histological and molecular features of testicular tumors, and the prevalence of specific tumor types, such as Sertoli cell tumors in cryptorchid dogs and germ cell tumors in humans. These disparities caution against using dogs as models for human testicular cancer research and underscore the limitations when drawing comparisons between species. The paper concludes by suggesting specific research initiatives to enhance our understanding of the complex interplay between cryptorchidism and testicular cancer in dogs.

Biallelic RXFP2 variants lead to congenital bilateral cryptorchidism and male infertility, supporting a role of RXFP2 in spermatogenesis.

STUDY QUESTION: Does RXFP2 disruption impair male fertility? SUMMARY ANSWER: We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation. WHAT IS KNOWN ALREADY: Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown. STUDY DESIGN, SIZE, DURATION: A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations. PARTICIPANTS/MATERIALS, SETTING, METHODS: The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay. MAIN RESULTS AND THE ROLE OF CHANCE: An intragenic deletion of exon 1-5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility. LIMITATIONS, REASONS FOR CAUTION: Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B., 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C., FIKO-IV institutional fund). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Anogenital distance in a cohort of 169 infant boys with uni- or bilateral cryptorchidism including 18 boys with vanishing testes.

STUDY QUESTION: Do different boys with different types of cryptorchidism exhibit different anogenital distances (AGDs)? SUMMARY ANSWER: Length of AGD seemed to differ in different groups of patients with cryptorchidism. WHAT IS KNOWN ALREADY: AGD, which is used as an indicator of prenatal androgen action, tends to be shorter in boys with cryptorchidism compared to unaffected boys. Shorter AGDs have also been reported in boys with hypospadias, in men with poor semen quality, and in men with testicular cancer. STUDY DESIGN, SIZE, DURATION: A prospective descriptive cohort study was performed using data from consecutively selected boys with cryptorchidism (n = 169) operated in a single center over a period of 3 years (September 2019 to October 2022). PARTICIPANTS/MATERIALS, SETTING, METHODS: AGD was measured in 169 infant boys, at 3 to 26 months of age, during anesthesia with a vernier caliper measuring the distance from the anus to the base of the scrotum (AGDAS) and from the anus to the anterior base of the penis (AGDAP) in two body positions according to the methods by 'The Infant Development and the Environment Study' (TIDES) and 'Cambridge Baby Growth Study', resulting in four mean values per patient (TIDES AGDAS/AP and Cambridge AGDAS/AP). Normal values for AGD by age were set by our hospital Department of Growth and Reproduction based on a large cohort of healthy infant boys (n = 1940). Testicular biopsies were performed at orchidopexy as a clinical routine. The germ cell number (G/T) and type Ad spermatogonia number (AdS/T) per cross-sectional tubule of at least 100 and 250 tubules, respectively were measured and related to normal samples. Blood samples were obtained by venipuncture for measuring serum LH, FSH, and inhibin B. They were analyzed in our hospital Department of Growth and Reproduction where the normal reference was also established. Correlations between the four mean AGD measurements for each boy were evaluated by Spearman rank correlation analyses. The AGD measurement of every boy was transferred to the multiple of the median (MoM) of the normal AGD for age and named MoM AGD. MAIN RESULTS AND THE ROLE OF CHANCE: There were 104 boysoperated for unilateral, and 47 boys operated for bilateral, undescended testes, whereas 18 boys had vanished testis including one boy with bilateral vanished testes. Only 6% of cases with vanished testes had a MoM AGD higher than the normal median compared to 32% with undescended testes (P < 0.05). MoM AGD increased with the age at surgery for boys with vanished testis (Spearman r = 0.44), but not for boys with undescended testes (Spearman r = 0.14). Boys with bilateral cryptorchidism had longer AGDs and more often had hypogonadotropic hypogonadism than boys with unilateral cryptorchidism (P < 0.005) and (P < 0.000001). LIMITATIONS, REASONS FOR CAUTION: Although being the largest published material of AGD measurements of infant boys with cryptorchidism, one limitation of this study covers the quite small number of patients in the different groups, which may decrease the statistical power. Another limitation involves the sparse normal reference material on G/T and AdS/T. Finally, there are currently no longitudinal studies evaluating AGD from birth to adulthood and evaluating childhood AGD in relation to fertility outcome. Our study is hypothesis generating and therefore the interpretation of the results should be regarded as exploratory rather than reaching definite conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The study findings are in agreement with literature as the total included group of boys with cryptorchidism exhibited shorter than normal AGDs. However, new insights were demonstrated. Boys with vanished testis had shorter AGDs compared to unaffected boys and to boys with undescended testes. This finding challenges the current concept of AGD being determined in 'the masculinization programming window' in Week 8 to 14 of gestation. Furthermore, boys with bilateral cryptorchidism had longer AGDs and more often had hypogonadotropic hypogonadism than boys with unilateral cryptorchidism, suggesting that the lack of fetal androgen in hypogonadotropic hypogonadism is not that significant. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used and no competing interests are declared. TRIAL REGISTRATION NUMBER: The trial was not registered in an ICMJE-recognized trial registry.

Treatment trends for undescended testis and impact of guideline changes a medical health care analysis of orchidopexy and cryptorchidism in Germany between 2006 und 2020.

BACKGROUND: The last decades revealed new scientific knowledge regarding the fertility and potential malignancy of undescended testis AQ2(UDT). Accordingly, many guidelines changed their recommendation concerning timing of therapy, with the goal of an earlier time of surgery. METHODS: We analyzed the number of new diagnosis and performed surgeries in predefined age groups provided by the obligatory annual reports of German hospitals in the reimbursement.INFO"-tool between 2006 and 2020. RESULTS: Overall, 124,741 cases were analyzed. We showed a slight increase in performed surgeries in the first year by 2% per year with a main increase till 2011, a constant number of surgeries between first and 4th year and a decrease of surgeries between 5 and 14th year of living with a main decrease till 2009 by 3% per year. CONCLUSION: Even if our results illustrate an increasing adaption of the guideline's recommendation, there is still a significant number of patients who receive later treatment. More research about the reasons and circumstances for the latter is needed.

Use of Ultrasound in the Evaluation of Cryptorchidism: A Single-Institution Analysis.

INTRODUCTION: The American Urological Association guidelines recommend against the performance of ultrasound and other imaging modalities in the evaluation of patients with cryptorchidism before expert consultation. We aimed to examine our institutional experience with cryptorchidism and measure adherence to currently available guidelines. METHODS: An institutional review board-approved retrospective review of ultrasound utilization in the evaluation of patients with cryptorchidism was performed from June 1, 2016, to June 30, 2019, at a single tertiary level pediatric hospital. RESULTS: We identified 1796 patients evaluated in surgical clinics for cryptorchidism. Surgical intervention was performed in 75.2% (n = 1351) of the entire cohort. Ultrasound was performed in 42% (n = 754), most of which were ordered by referring physicians (91% n = 686). Of those who received an ultrasound, surgical intervention was performed in 78% (n = 588). Those 166 patients (22%) who did not undergo surgical intervention were referred with ultrasounds suggesting inguinal testes; however, all had normal physical examinations or mildly retractile testes at the time of consultation and were discharged from the outpatient clinic. There were 597 patients referred without an ultrasound, 81% (n = 483) were confirmed to have cryptorchidism at the time of specialist physical examination and underwent definitive surgical intervention, the remainder (19%, n = 114) were discharged from the outpatient clinics. CONCLUSIONS: Ultrasound evaluation of cryptorchidism continues despite high-quality evidence-based guidelines that recommend otherwise, as they should have little to no bearing on the surgeon's decision to operate or the type of operation. Instead, physical examination findings should guide surgical planning.

Morphological identification and distribution comparison of telocytes in pituitary gland between normal and cryptorchid yaks.

BACKGROUND: Telocytes (TCs) is a novel type of interstitial cells in many mammals organs, which participate in the organizational metabolism, mechanical support, immunomodulation and other aspects. The aim of this study was to explore the organizational chemical characteristics of TCs in pituitary gland and their changes in cryptorchid yaks. METHODS: Transmission electron microscopy (TEM), toluidine blue staining, immunofluorescence, qRT-PCR, and Western blotting may enable us to understand TCs distribution characteristics and biological functions. RESULT: TEM confirmed the presence of TCs in the pituitary gland with small bodies and moniliform telopodes (Tps). The Tps extending out from the cell body to the peri-sinusoidal vessels spaces, the number of Tps is closely related to the morphology of the nucleus. The most obvious changes of TCs in the pituitary gland of cryptorchid yaks is the Tps are relatively shorter and decreased secretory vesicles. H.E. and toluidine blue staining revealed that TCs not only distributed between the sinusoidal blood vessels and the glandular cell clusters, but also present on the surface of vascular endothelial cells. The co-expression of TCs biomarkers, such as Vimentin/CD34, CD117/CD34 and α-SMA/CD34, were evaluated by immunofluorescence to further determine the phenotypic characteristics of TCs. Besides, we analyzed the mRNA and protein expression of these biomarkers to determine the characteristics of TCs changes and possible biological roles. Both the mRNA and protein expression of CD117 were significantly higher in the pituitary gland of cryptorchid yaks than in the normal (p < 0.01), the protein expression of CD34 in the cryptorchid yaks was significantly higher than the normal (p < 0.01). There were no significant difference in mRNA expression of Vimentin and α-SMA (p>0.05), while the protein expression were significantly increased in the normal yaks (p < 0.05). CONCLUSIONS: In summary, this study reports for the first time that the biological characteristics of TCs in yak pituitary gland. Although there is no significant change in the distribution characteristics, the changes in biological features of TCs in cryptorchid yaks are clear, suggesting that TCs participated in alteration in the local microenvironment of the pituitary gland. Therefore, our study provides clues for further investigating the role of TCs in the pituitary gland during the occurrence of cryptorchidism in yaks.

Laparoscopic orchidopexy for the treatment of cryptorchidism in adults: a description of the technique and outcomes.

BACKGROUND: There are few studies on cryptorchidism in adults, and its treatment is still controversial. METHODS: To summarize the surgical strategy and clinical efficacy of laparoscopic orchidopexy for the treatment of cryptorchidism in adults, 37 adult cryptorchidism patients were retrospectively analyzed between September 2017 and February 2022. All 37 patients underwent laparoscopic orchidopexy, of whom 33 underwent inguinal hernia repair without tension. The intraoperative procedures and surgical techniques were recorded in detail. Preoperative examination and regular postoperative review of color Doppler ultrasound, and reproductive hormone, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase levels were performed. RESULTS: All testes descended successfully into the scrotum, including 25 through the inguinal route and 12 through Hesselbach's triangle route. No intraoperative or postoperative complications were observed. The follow-up time was 38.6 (± 19.4) months, and no evidence of testicular malignancy was found during the follow-up period. After analyzing the reproductive hormone levels at 1 year postoperatively in 28 patients with more than 1 year of follow-up, it was found that the patients had a significant increase in testosterone levels and a decrease in follicle-stimulating hormone levels after surgery. None of the patients showed any significant improvement in semen quality after surgery. CONCLUSION: Our study suggests that laparoscopic orchidopexy is a safe and feasible surgical procedure for the treatment of cryptorchidism in adults, especially high cryptorchidism, which is difficult to treat. After comprehensive consideration, preserving the testis should be preferred for treating cryptorchidism in adults to maximize the protection of the patient's reproductive hormone secretion function.

Clinical characteristics and outcome of children with acute cryptorchid testicular torsion: A single-center, retrospective case series study.

BACKGROUND: Cryptorchidism and testicular torsion (TT) are relatively common conditions in clinical practice; however, sparse information about cryptorchid TT is available in the current literature. METHODS: We retrospectively reviewed the clinical characteristics, treatment modalities, and long-term outcomes of pediatric patients treated for acute cryptorchid TT. RESULTS: We found eight patients with unilateral acute cryptorchid TT with a prevalence of 8.9% (8/90) among all TT cases. The left testis was affected in six patients. The median age of patients at the time of the surgery was 65 months (interquartile range (IQR) 4-136 months). The median duration of symptoms was 16 h (IQR 9-25 h), while the median time to treatment was 60 min (IQR 59-63 min). The most common symptoms were pain (abdominal and inguinal) and inguinal mass with no palpable testis in the ipsilateral hemiscrotum. Preoperative color Doppler ultrasonography revealed absent or decreased testicular blood flow in the affected testes in 7/8 of patients. Various degrees of testicular torsion (median 540°, min 360°, max 1260°) were found during surgery. A necrotic testis that led to orchidectomy was found in 4/8 of patients. The median follow-up period was 42.6 months (IQR 12.5-71.2 months), revealing only one patient with testicular atrophy. The final testicular salvage rate was 35%. CONCLUSIONS: Greater awareness among caregivers and primary care physicians about acute cryptorchid TT is required to improve their timely diagnosis and treatment. A physical examination of the external genitalia and inguinal regions should be mandatory to attain a proper diagnosis and treatment without delay.

Testicular volume at puberty in boys with congenital cryptorchidism randomised to treatment at different ages.

AIM: To assess testicular volume at puberty for boys who underwent orchidopexy at 9 or at 36 months compared to boys with spontaneous postnatal descent. METHODS: At age 6 months, boys with congenital unilateral cryptorchidism were randomised to surgery at 9 or 39 months of age and followed to 16 years in parallel with boys with spontaneous postnatal descent. Ultrasound was done at 11 and 16 years to determine testicular volume. The ratio of the initially undescended testis to its scrotal counterpart was used to assess testicular growth. RESULTS: At age 16, the ratio was lower (p < 0.00) in the late group compared to the early group. At 16 years, the spontaneously descended testes were significantly smaller than their scrotal counterparts but larger than the operated groups (early p < 0.01 and late p < 0.00). CONCLUSION: Our data at 16 years show that orchidopexy at 9 months results in better testicular growth compared to 3 years but did not reach the corresponding volumes of their scrotal counterparts. This indicates that earlier surgery is beneficial to testicular growth. At age 16, the postnatally descended testes were not only larger than the surgically treated testes but also exhibited impaired testicular growth.

Pre-referral ultrasound for cryptorchidism: Still common, still not necessary.

AIM: Evidence-based guidelines do not recommend imaging in cryptorchidism, but anecdotally most referrals include an ultrasound report. We aimed to assess the frequency, utility and burden of imaging in children referred with presumptive disorders of testicular descent, and to assess trends over a 7-year period before and after local and international guidelines have been introduced. METHODS: This was a prospective cohort study of children referred to the Queensland Children's Hospital for anomalies of testicular descent between 2015-2017 and 2023-2024. Data were collected regarding demographics, referral details, imaging performed and surgical diagnosis. RESULTS: A total of 268 children were recruited. Ultrasound frequency has not significantly changed over time (72.8%, 2015-2017; 63.6% 2023-2024; P = 0.11). Currently, 17.6% of families are charged, and 31.9% need to take time off work, for the ultrasound. This is a significant increase from the 2015-2017 cohort. Parents report concern and anxiety, and find it traumatic for their child. Following review, the majority (65.7%) had physiologically normal testes, while 4.9% and 22.4% were diagnosed with bilateral or unilateral cryptorchidism, respectively, and 7.0% had other diagnoses. Ultrasound was concordant with the surgical diagnosis in 25.0% (2015-2017) and 30.7% (2023-2024). Ultrasound did not contribute to diagnosis nor management in any patient. CONCLUSIONS: Despite international and local guidelines, cryptorchid children continue to undergo ultrasound prior to referral. Such ultrasounds do not provide utility, or value for cost. We recommend avoiding their routine use for suspicion of testicular maldescent and working together to improve strategies for translating evidence-based guidelines into clinical practice.

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.

Assessment of diagnostic accuracy for cryptorchidism and risk factors for delayed orchidopexy.

OBJECTIVES: Cryptorchidism (CO) diagnosis by palpation is challenging. Patients with suspected CO are primarily referred to pediatric urologists by general pediatricians and urologists. Currently, surgical treatment for CO is recommended earlier than in previous guidelines. In this study, we evaluated factors that lead to diagnosis discordance and delayed orchidopexy in patients referred with suspected CO in addition to timing of initial screening. METHODS: In total, 731 patients (1052 testes) with suspected CO were included. Risk factors for diagnostic discrepancy in CO diagnosis by pediatric urologists and risk of delayed orchiopexy were evaluated. RESULTS: Herein, 659 (90%) patients were diagnosed during routine public health checkups for infants and young children, and 419 (57%) patients were referred by pediatric practitioners. Of 1052 testes, 374 (36%) were diagnosed with CO by pediatric urologists. In multivariate analysis, risk factors of diagnostic discrepancy for CO diagnosis by pediatric urologists were bilateral testis (odds ratio [OR] = 9.17, p < 0.0001), >6 months old at initial diagnosis (OR = 1.036, p < 0.0001), and pediatric referral (OR = 4.60, p < 0.0001). In total, 296 patients underwent orchiopexy for CO. In multivariate analysis, risk factors for delayed orchiopexy were presence of comorbidities (OR = 3.43, p = 0.003) and >10 months old at referral (OR = 12.62, p < 0.0001). CONCLUSIONS: Pediatric referral is a risk factor for discordant CO diagnostics, and late age at referral brings a risk of delayed orchiopexy. It is necessary to enlighten pediatricians, who are mainly responsible for routine health checkups, in teaching CO diagnostic techniques to ensure early referral.

Histological study of Bactrian camel cryptorchidism and expression of immunoglobulin λ light chain in the testicular and epididymis of cryptorchid Bactrian camel.

Lambda light chains (λ-LCs) are frequently responsible for triggering the activation of inflammatory factors in autoimmune disorders, and an increase in their levels will cause various pathological changes in serum. The aim of this study was to determine the histological differences between the epididymis and testis of normal and cryptorchid Bactrian camels and the differences in λ-LC expression in the epididymis and testis of normal and cryptorchid Bactrian camels. Haematoxylin and eosin (H&E) staining was used to examine the pathological changes in cryptorchidism. The gene and protein levels of λ-LC were determined using RT-qPCR and western blot. The distribution of λ-LCs was assessed by immunohistochemistry and immunofluorescence. Compared with that in normal Bactrian camels, the diameter of the epididymal lumen and the thickness of the epithelium were decreased in the epididymis of cryptorchidic animals. Additionally, no sperm was detected in the cavity of the cryptorchidic epididymis. Meanwhile, the expression of λ-LC was significantly increased in the cryptorchidic epididymis at both the mRNA and protein levels (p < .05). The highest protein expression of λ-LC was found in epididymal epithelial halo cells and testicular Sertoli cells. These findings suggested that the structural changes observed in the epididymal epithelium of cryptorchidic camels affect its secretory and absorptive functions. Additionally, the high level of λ-LC expression recorded in halo cells suggested that these cells play an important role in epithelial immunity in cryptorchidic Bactrian camels. Furthermore, the high λ-LC expression levels detected in normal testicular Sertoli cells indicated that λ-LCs may be involved in spermatogenesis. The results of this study provide clues for an in-depth study of immunological sterility in cryptorchidic Bactrian camels.

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.

Expression pattern of germ cell markers in cryptorchid stallion testes.

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions.