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Currently the standard treatment for bilateral cryptorchidism is bilateral surgical orchidopexy. Whether a hormonal treatment should be routinely administered postoperatively to increase fertility is debatable. Low-dose postoperative luteinizing hormone releasing hormone (LHRH) can increase spermatogonial numbers, but the effect of native LHRH (Kryptocur®) on adult fertility is unclear.To determine if low-dose every-second-day postoperative LHRH administration in children with bilateral cryptorchidism improves fertility in adulthood and if Nistal testicular histological grading could guide the decision to administer LHRH.All patients, actually at least 16yr of age, that underwent a bilateral orchidolysis and orchidopexy for bilateral cryptorchidism (surgery between 1997 and 2018) were contacted and offered a clinical exam, hormone levels, sperm analysis, and a scrotal ultrasound. At the original surgery, testicular biopsy was performed (if 60% of the tubuli contain1 spermatogonia, this is normal = Nistal-1, if 30-60% filled = Nistal-2, if30% = Nistal-3 and if Sertoli only = Nistal-4) and if in at least one testis impaired. A low dose native LHRH treatment was offered to the patients, as this treatment is known to increase the number of spermatogonia in a short term. Kryptocur® (LHRH, Gonadorelin, Hoechst®) was prescribed and dosed at 200 μg (one spray in one nostril) every other day for 6-8 months.Forty-two men were eligible for this study. 20/42 accepted the invitation for a clinical and hormonal evaluation. 16/20 men accepted the invitation for an additional sperm analysis. Fourteen of 20 men received low-dose LHRH postoperatively in a nonrandomized manner. Three men had Nistal grade 1, eight grade 2, seven grade 3, and two had grade 4. Inhibin B levels were higher in men with Nistal 1 and 2 compared with Nistal 3 and 4 P ≤ 0.037). Severe oligospermia/azoospermia (1 × 10

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 aim of our study was to determine the risk for azoospermia by histologic criteria in a cohort of unilateral cryptorchid boys undergoing orchidopexy and bilateral testicular biopsy. We performed a retrospective analysis of data available in the library of the Cryptorchidism Research Institute, Liestal, Switzerland. Complete histological evaluations were available for 319 boys operated on for unilateral cryptorchidism with simultaneous biopsy of the contralateral descended testicle. The median age was 39 (5–192) months and 58 patients were

Little is known about the histology of contralateral descended testes in boys with unilaterally absent testis. We investigated whether absence of one testis is associated with abnormal tissue architecture of the solitary contralaterally descended testis.For this retrospective study, we examined the results of biopsies of the contralateral descended testis in 43 boys with monorchidism. Data from 26 control testes from boys of matching ages were selected from results published in 1977 and 2009. During surgery, any nubbins were removed. In each case, the scrotal testis was biopsied, and the testis fixed by subdartos pouch or suture.Of the 43 affected boys, 23 had normal testicular histology in the contralateral descended testis, whereas 20 (46%) had abnormal histology. Eight of the abnormal biopsies matched the criteria for high infertility risk. Samples from three boys in this latter group revealed a Sertoli-cell-only phenotype. Immunohistochemical assays were positive for steroidogenic acute regulatory (STAR) protein in Leydig cells and spermatogonia. STAR expression was stronger in the monorchid group with normal testicular histology.Almost half of the patients with unilateral absent testis were at risk for subfertility or infertility. Our results emphasize the need for testicular biopsy of the solitary testis in boys with monorchidism to appropriately assess infertility risk.Peux d’études ont analysé la structure des tissus testiculaires des testicules descendus controlatéraux chez les garçons avec des testicules unilatéraux absents. Nous avons investigué si l’absence congénitale d’un testicule est. associée à une histologie anormale des testicules descendus controlatéraux solitaires.Cette étude rétrospective a examiné les résultats des biopsies des testicules descendus controlatéraux de 43 garçons monorchides. Les données de 26 testicules témoins ont été appariées surl’âge et sélectionnées à partir des données publiées en 1977 et 2009. Pendant l’opération, les nubins (reliquats) détectés ont été enlevés. Dans chaque cas, les testicules scrotaux ont fait l’objet d’une biopsie et d’une fixation par la technique de la valise ou par suture au subdartos.Parmi 43 garçons, 23 avaient une histologie testiculaire normale dans les testicules descendus controlatéraux, tandis que 20 (46%) avaient une histologie anormale. Huit biopsies anormales correspondaient aux critères de risque élevé d’infertilité. Trois garçons de ce groupe avaient une histologie testiculaire montrant la présence de cellules Sertoli seules. L’analyse immunohistochimique de la protéine STAR a montré un signale dans les cellules de Leydig et dans les spermatogonies. L’expression STAR était plus forte dans le groupe des monorchides avec une histologie testiculaire normale.Près de la moitié des patients ayant des testicules congénitaux unilatéraux absents couraient un risque d’hypofertilité ou d’infertilité. Nos résultats soulignent la nécessité d’une biopsie testiculaire des testicules solitaires chez les garçons monorchides afin d’évaluer le risque d’infertilité de ces patients.

Undescended (cryptorchid) testes in patients with defective mini-puberty and low testosterone levels contain gonocytes that fail to differentiate normally, which impairs the development of Ad spermatogonia and ultimately leads to adult infertility. Treatment with the gonadotropin-releasing hormone agonist GnRHa increases luteinizing hormone and testosterone and rescues fertility in the majority of pathological cryptorchid testes. Several Y-chromosomal genes in the male-specific Y region (MSY) are essential for spermatogenesis, testis development and function, and are associated with azoospermia, infertility and cryptorchidism. In this study, we analyzed the expression of MSY genes in testes with Ad spermatogonia (low infertility risk patients) as compared to testes lacking Ad spermatogonia (high infertility risk) before and after curative GnRHa treatment, and in correlation to their location on the Y-chromosome.Twenty genes that are up- or down-regulated in the Ad- group are in the X-degenerate or the ampliconic region, respectively. GnRHa treatment increases mRNA levels of 14 genes in the ampliconic region and decreases mRNA levels of 10 genes in the X-degenerate region.Our findings implicate Y-chromosomal genes, includingLa non descente des testicules chez les garçons cryptorchides qui présentent une mini-puberté défectueuse et un taux réduit de testostérone (T) ont des gonocytes incapables de se différencier normalement en spermatogonie Ad. Cette dernière entraîne une infertilité. Le traitement avec l’agoniste du GnRH (GnRHa) augmente les taux de LH et T et permet de sauvegarder la fertilité chez la majorité des testicules cryptorchides pathologiques. Plusieurs gènes du chromosome Y localisés dans la région spécifique du mâle (MSY) sont essentiels pour la spermatogénèse, ainsi que pour le développement et la fonction testiculaires, et sont associés à l’azoospermie, l’infertilité et la cryptorchidie. Dans cette étude, nous avons analysé l’expression des gènes dans la région MSY des testicules avec et sans spermatogonies Ad, avant et après traitement par GnRHa. Les résultats sont corrélés avec la localisation des gènes dans le chromosome Y.Dans le groupe Ad-, vingte gènes dont l’expression est. régulée à la hausse ou à la baisse sont respectivement localisés dans la région dégénérée du X ou dans la région ampliconique. Le traitement par GnRHa augmente les taux de mRNA de 14 gènes dans la région ampliconique et réduit l’expression de 10 gènes dans la région dégénérée du X.Nos résultats impliquent une participation des gènes du chromosome Y, compris

Introduction: GnRHa treatment following surgery to correct cryptorchidism restores mini-puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. A large group of genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed both in brain and testis. However, the expression of these genes in prepubertal gonads has not been systematically studied and little is known about the effect of hormone therapy on their testicular and neuronal expression levels. Here, we interpret histological sections, data on hormone levels and RNA profiling data from adult normal testis in comparison to pre-pubertal low infertility risk (LIR) and high infertility risk (HIR) patients randomized for treatment with surgery in combination with GnRHa or only surgery. Major Results: We organize 31 target genes relevant for hypogonadotropic hypogonadism and cryptorchidism into five classes depending on their expression levels in HIR versus LIR samples and their response to GnRHa treatment. Only the mRNA encoding the DNA binding transcription factor NHLH2 is decreased in HIR as compared to LIR samples, increased after GnRHa treatment, and expressed in both brain and testis. GnRHa treatment of cryptorchidism that restores mini-puberty and rescues adult fertility increases NHLH2 mRNA levels in testis from HIR patients. Conclusion: This phenomenon may reflect a broader effect of hormone treatment on gene expression in both testicular and central nervous tissues, which could explain why the hypothalamus-pituitary-testicular axis is permanently restored by the administration of GnRHa.

Cryptorchidism is a frequent endocrinopathy in boys that has been associated with an increased risk of developing testicular cancer and infertility. The condition is curable by combined surgery and hormonal treatment during early pre-pubertal stages using gonadotropin releasing hormone agonist (GnRHa). However, whether the treatment also alters the expression of testicular long non-coding RNAs (lncRNAs) is unknown. To gain insight into the effect of GnRHa on testicular lncRNA levels, we re-analyzed an expression dataset generated from testicular biopsies obtained during orchidopexy for bilateral cryptorchidism.We identifiedGiven that lncRNAs can cooperate with chromatin-modifying enzymes to promote epigenetic regulation of genes, GnRHa treatment may act as a surrogate for mini-puberty by triggering the differentiation of Ad spermatogonia via lncRNA-mediated epigenetic effects. Our work provides additional molecular evidence that infertility and azoospermia in cryptorchidism, resulting from defective mini-puberty cannot be cured with successful orchidopexy alone.La cryptorchidie est. une endocrinopathie fréquente chez les garçons. Elle est. associée à un risque élevé de cancer des testicules et d’infertilité. La cryptorchidie peut être soignée par une thérapie incluant une intervention chirurgicale et un traitement hormonal par l’agoniste de l’hormone GnRH. Alors que l’effet de la thérapie sur l’expression des ARNm a été analysé, ses conséquences pour la transcription des longs ARNs non codants (ARNlnc) testiculaires restent inconnues. Afin de mieux comprendre les effets du GnRHa sur les concentrations cellulaires des ARNlnc dans le testicule, nous avons analysé des données d’expression d’ARN par séquençage (ARN-Seq) générées en utilisant des biopsies testiculaires obtenues dans le cadre d’une orchidopexie pour cryptorchidie bilatérale.Nous avons identifié lesÉtant donné que certains ARNlnc coopèrent avec des enzymes ayant un effet sur la structure chromatinienne et la régulation épigénétique des gènes, le traitement par GnRHa pourrait substituer la mini-puberté en déclenchant la différenciation des spermatogonies Ad par un mécanisme épigénétiques qui dépendrait des ARNlnc. Notre travail révèle des nouvelles pistes moléculaires soutenant l’hypothèse que l’infertilité et l’azoospermie associées avec la cryptorchidie sont la conséquence d’une anomalie de la mini-puberté. Cela explique pourquoi une thérapie efficace de cette pathologie ne nécessite pas seulement l’orchidopexie mais aussi un traitement hormonal.

Abnormal germ cell development in cryptorchidism is not a result of a congenital dysgenesis but is preceded by a hormone imbalance and perturbation in germ cell-specific gene expression during abrogated mini-puberty. Adequate treatment with low doses of GnRHa enables 86% of men to achieve a normal sperm count and, most importantly, prevent development of azoospermia. GnRHa treatment induces a significant transcriptional response, including protein coding genes involved in pituitary development, the hypothalamic-pituitary-gonadal axis, and testosterone synthesis. Furthermore, hormonal treatment to achieve epididymo-testicular descent as a first choice of treatment of cryptorchidism has a long tradition in Europe. It eliminates the necessity of subsequent surgery. Moreover, in the cases of non-responders it facilitates orchidopexy and contributes considerably to a reduced incidence of unilateral and the more serious bilateral complete post-surgical testicular atrophy.

Reikšminiai žodžiai: kriptorchizmas, prielipas, Ad spermatogonijos, tubulių fertilumo indeksas, nevaisingumas. Darbo tikslas. Nustatyti sėklidės – prielipo santykio ir sėklidės padėties sąsajas su tubulių fertilumo indeksu bei tamsių (Ad) spermatogonijų ir kanalėlių santykiu sergantiems kriptorchizmu pacientams. Tyrimo medžiaga ir metodai. Prospektyvinėje studijoje ištyrėme 57 berniukus, 2013–2015 metais Vilniaus universiteto ligoninės Santariškių klinikų filialo Vaikų ligoninės Urologijos skyriuje operuotus dėl vienpusio kriptorchizmo. Berniukai su abipus nenusileidusiomis sėklidėmis ir įgytu kriptorchizmu nebuvo įtraukti į tyrimą. Orchidopeksijos metu įvertinta nenusileidusios sėklidės – prielipo morfologija ir sėklidės padėtis. Atliktas nenusileidusios sėklidės biopsijos histologinis tyrimas. Bioptatas fiksuotas 2 proc. glutarataldehidu, nudažytas toluidino mėliu ir ištirtas šviesiniu mikroskopu, padidinus 400 kartų. 1 μm storio preparatuose buvo skaičiuojamas tubulių, germinacinių ląstelių ir Ad spermatogonijų skaičius; apskaičiuotas tubulių fertilumo indeksas bei Ad spermatogonijų ir kanalėlių santykis. Statistinė duomenų analizė atlikta SPSS 20.0 statistine programa. Skirtumai laikyti statistiškai reikšmingi, kai p < 0,05. Rezultatai. Pacientų amžiaus vidurkis – 1,42 ± 0,83 metų (nuo 0,58 iki 4,6 m.). Pagal nenusileidusios sėklidės ir jos prielipo santykį sudarytos 4 grupės: (I) normalus sėklidės – prielipo suaugimas 11 (19,3 proc.), (II) suaugimas tik prielipo galva – 22 (38,6 proc.), (III) suaugimas tik prielipo galvos dalimi – 17 (29,8 proc.) ir (IV) visiškas nesuaugimas – 7 (12,3 proc.). Pagal padėtį pacientai pasiskirstė 3 grupėse: (A) nenusileidusi sėklidė pilvo ertmėje 2 (3,5 proc.), (B) kirkšniniame kanale 16 (28,1 proc.) ir (C) ties išoriniu kirkšninio kanalo žiedu 39 (68,4 proc.). Ad spermatogonijų ir kanalėlių santykis buvo didesnis sėklidėse su normaliu komplekso suaugimu ir žemesne nenusileidusios sėklidės padėtimi. Statistiškai reikšmingas šio histologinio rodiklio skirtumas rastas tarp I ir II (p = 0,036), II ir III (p = 0,005), I ir IV (p = 0,027), III ir IV (p = 0,003) bei B ir C (p = 0,02) grupių. Tubulių fertilumo indeksas tarp grupių statistiškai reikšmingai nesiskyrė. Išvados. Ad spermatogonijos, kaip normaliai vykstančio mažojo brendimo rodiklis, turi ryšį su sėklidės – prielipo suaugimo laipsniu. Taip pat germinacinių ląstelių vystymasis, išreikštas Ad spermatogonijų atsiradimu, yra susijęs su nenusileidusios sėklidės padėtimi. Vis dėlto tyrimo rezultatai turėtų būti vertinami atsargiai dėl mažo tiriamųjų skaičiaus ir nenusileidusios sėklidės padėties bei morfologijos aprašymo sudėtingumo.

The whole genome RNA profiling of testicular biopsies by DNA strand-specific RNA sequencing was examined to determine a potential causative role of isolated congenital cryptorchidism in azoospermia and/or infertility in the context of our previously published GeneChip data. Cryptorchid patients, aged 7 months to 5 years and otherwise healthy, were enrolled in this prospective study. During surgery, testicular tissue biopsies were obtained for histological examination and RNA sequencing. Fifteen patients were selected based on the histological results and were divided into 2 groups. Seven were classified as belonging to the high infertility risk (HIR) and 8 to the low infertility risk (LIR) group. Cryptorchid boys in the HIR group lacked transformation of gonocytes into Ad spermatogonia due to impaired mini-puberty. This group of patients will be infertile despite successful surgery. The new important finding was a decreased PROK2, CHD7, FGFR1, and SPRY4 gene expression in the HIR group. Furthermore, identification of multiple differences in gene expression between HIR and LIR groups underscores the importance of an intact hypothalamic-pituitary-gonadal axis for fertility development. Our RNA profiling data strongly support the theory that in the HIR group of cryptorchid boys insufficient PROK2/CHD7/FGFR1/SPRY4 gene expression induces deficient LH secretion, resulting in impaired mini-puberty and infertility. We therefore recommend hormonal treatment for this cohort of cryptorchid boys with defective mini-puberty following a seemingly successful orchidopexy.

There is a correlation between cryptorchidism and an increased risk of testicular cancer and infertility. During orchidopexy, testicular biopsies are performed to confirm the presence of type A dark (Ad) spermatogonia, which are a marker for low infertility risk (LIR). The Ad spermatogonia are absent in high infertility risk (HIR) patients, who are treated with a gonadotropin-releasing hormone agonist (GnRHa) to significantly lower the risk of infertility. Despite its prevalence, little is known about the molecular events involved in cryptorchidism. Previously, we compared the transcriptomes of LIR versus HIR patients treated with and without hormones. Here, we interpreted data regarding members of the positive regulatory domain-containing (PRDM) family, some of which encoded histone methyltransferases that are important for reproduction. We found there were lower levels of PRDM1, PRDM6, PRDM9, PRDM13, and PRDM14 mRNA in the testes of HIR patients compared with LIR patients, and that PRDM7, PRDM9, PRDM12, and PRDM16 were significantly induced after GnRHa treatment. Furthermore, we observed PRDM9 protein staining in the cytoplasm of germ cells in the testes from LIR and HIR patients, indicating that the mRNA and protein levels corresponded. This result indicated that the curative hormonal therapy for cryptorchidism involved conserved chromatin modification enzymes.

Introduction. This prospective study investigated the efficacy of a gonadotropin-releasing hormone agonist (LH-RHa) in restoring defective mini-puberty. Materials and Methods. Boys with isolated bilateral cryptorchidism and defective mini-puberty were randomly divided into two groups. The “surgery only” group underwent a second orchidopexy without hormonal treatment (control). The “LH-RHa” group received LH-RHa therapy followed by a second orchidopexy. The number of Ad spermatogonia and the total germ cell count per tubule (S/T) were analyzed. Results. Five boys were included in each arm. In the LH-RHa group, the median S/T increased from 0.11 to 0.42, p=0.04. In the surgery only group, the median S/T did not change. In the surgery only group, none of the testes had Ad spermatogonia. In contrast, in the LH-RHa group, all testes completed the transition from gonocytes to Ad spermatogonia (p=0.008). Conclusions. Treatment with LH-RHa was effective in rescuing defective mini-puberty in boys with bilateral cryptorchidism.

Follicle stimulating hormone and testosterone stimulate Sertoli cells to support germ cell function and differentiation. During mini-puberty, when gonadotropin (GnRH) stimulates increases in plasma luteinizing hormone (LH) and testosterone levels, gonocytes are transformed into Ad spermatogonia. In cryptorchidism, impaired gonadotropin secretion during mini-puberty results in insufficient LH and testosterone secretion, impaired gonocyte transition to Ad spermatogonia, and perturbed Sertoli cell proliferation. Treatment with a gonadotropin-releasing hormone agonist (GnRHa/Buserelin) induced gonocytes to differentiate into Ad spermatogonia and rescued fertility. The present study evaluated the impact of low LH secretion on Sertoli cell function by comparing differential gene expression data between testes with low LH that lacked Ad spermatogonia (Ad-) and testes that completed mini-puberty (Ad+). Furthermore, we analyzed changes in the transcription of selected Sertoli cell specific genes in response to GnRHa treatment.Ad- testes showed reduced expression of nine out of 40 selected Sertoli cell specific genes compared to Ad+ testes. GnRHa treatment repressed most of the Sertoli cell specific genes, including the inhibins, but it increased the expression of genes that regulate apoptosis (Impaired-minipuberty with decreased LH and testosterone levels affected Ad and Sertoli cell development through positive and negative regulation of morphoregulatory and apoptotic genes. GnRHa treatment had a repressive effect on most Sertoli cell specific genes, which suggested that Sertoli cells underwent a cellular rearrangement. We propose that gonadotropin-dependent increases inL’hormone folliculostimulante et la testostérone stimulent les cellules de Sertoli pour soutenir la fonction et la différentiation des cellules germinales. Pendant la minipuberté, lorsque la gonadotrophine (GnRH) stimule les augmentations des taux plasmatiques d’hormone lutéinisante (LH) et de testostérone, les gonocytes sont transformés en spermatogonies Ad. Dans la cryptorchidie, une sécrétion altérée de gonadotrophine lors de la minipuberté entraine une sécrétion insuffisante de LH et de testostérone, une altération de la transition des gonocytes en spermatogonies Ad, et une perturbation de la prolifération des cellules de Sertoli. Un traitement par agoniste de la gonadolibérine (GnRHa/Buserelin) induit une différenciation des gonocytes en spermatogonies Ad et sauvegarde la fertilité.Cette étude a évalué l’impact d’un taux de LH bas sur la fonction des cellules de Sertoli en comparant les données d’expression différentielle de gènes entre des testicules avec LH basse qui sont dépourvus de spermatogonies Ad (Ad-) et des testicules qui ont fini la minipuberté (Ad+). En outre, la réponse au traitement par GnRHa a été analysée par les modifications de la transcription de gènes sélectionnés pour être spécifiques de la cellule de Sertoli.Les testicules Ad- présentent une expression réduite de 9 des 40 gènes sélectionnés pour être spécifiques de la cellule de Sertoli par comparaison aux testicules Ad+. Le traitement par GnRHa a réprimé l’expression de la plupart des gènes spécifiques de la cellule de Sertoli, y compris les inhibines, mais a augmenté l’expression de gènes qui régulent l’apoptose (FASLG) et la prolifération (GDNF).Une minipuberté altérée par des taux diminués de LH et de testostérone affecte le développement des spermatogonies Ad et des cellules de Sertoli par une régulation positive et négative de gènes morphorégulateurs et apoptotiques. Le traitement par GnRHa a un effet inhibiteur sur la plupart des gènes spécifiques de la cellule de Sertoli, ce qui suggère que les cellules de Sertoli subissent un réarrangement cellulaire. Nous proposons que les augmentations gonadotrophine-dépendantes de l’expression de FASLG et de GDNF dirigent la prolifération des cellules de Sertoli et l’auto-renouvèlement des cellules germinales, et qu’elles sont le support de la transition gonocytes vers spermatogonies Ad, indépendante des inhibines.

Cryptorchidism is the most common cause of non-obstructive azoospermia in man. In contrast to the general belief that temperature-dependent effects on the undescended gonad damage cryptorchid testes before sexual maturation is complete, molecular pathology strongly supports the theory that impaired mini-puberty is responsible for azoospermia and infertility in cryptorchidism. Molecular biological observations favor LH deficiency, with EGR4 as a master regulatory gene in Leydig cell dysgenesis, as the reason for impaired mini-puberty, and recent evidence supports the idea that infertility in cryptorchidism is a consequence of alterations in the Piwi pathway.

Background: Increasing evidence of progressive damage to germ cell development in boys with cryptorchidism suggests recommending surgery until one year of age. However, despite early and successful orchidopexy, cryptorchid boys with impaired mini-puberty will suffer from infertility. We reviewed changes in the timing of surgery during the past decade and the incidence of unilateral cryptorchid boys with defective mini-puberty. Methods: Medical registries were reviewed for all patients who were operated on for cryptorchidism at the main pediatric urological center of the country. The ages of surgery in cases of unilateral cryptorchidism were compared between the years 2000-2001 and 2012-2013. A high risk of infertility was considered when no Ad spermatogonia were found. Two groups were compared: group I - operated on until the age of 1.5 years and group II - older than 1.5 years. Results: The average age at operation decreased from 5.3 to 4.1 years. Forty-six biopsies in boys with unilateral cryptorchidism were made during orchidopexy on undescended testicles. Overall, 44% in group I and 50 % in group II (p > 0.05) had no Ad spermatogonia. Conclusions: The average age of operation for cryptorchidism has decreased, but remains far above the recommended age. The high prevalence of histologically proven risk of infertility underscores the necessity of more education regarding the importance of earlier surgery and the research on hormonal prevention of infertility.

Spermatogonia contain processing bodies that harbor P-element-induced wimpy testis (Piwi) proteins. Piwi proteins are associated specifically with Piwi-interacting RNAs to silence transposable DNA elements. Loss-of-function mutations in the Piwi pathway lead to derepression of transposable elements, resulting in defective spermatogenesis. Furthermore, deletion of gametocyte-specific factor 1 (GTSF1), a factor involved in Piwi-mediated transcriptional repression, causes male-specific sterility and derepression of LINE-1 (L1) retrotransposons. No previous studies have examined GTSF1, L1 and PIWIL4 expression in cryptorchidism. We examined transposon-silencing genes and L1 transposon expression in testicular biopsies with Affymetrix microarrays and immunohistology. Seven members of the Tudor gene family, 3 members of the DEAD-box RNA helicase family, and the GTSF1 gene were found to show significantly lower RNA signals in the high-infertility-risk group. In the immunohistochemical analysis, patients from the low-infertility-risk group showed coherently stronger staining for GTSF1 and PIWIL4 proteins and weaker staining for L1 transposon when compared to the high-infertility-risk samples. These new findings provide first evidence consistent with the idea that infertility in cryptorchidism is a consequence of alterations in the Piwi pathway and transposon derepression induced by the impaired function of mini-puberty.

Defective mini-puberty results in insufficient testosterone secretion that impairs the differentiation of gonocytes into dark-type (Ad) spermatogonia. The differentiation of gonocytes into Ad spermatogonia can be induced by administration of the gonadotropin-releasing hormone agonist, GnRHa (Buserelin, INN)). Nothing is known about the mechanism that underlies successful GnRHa treatment in the germ cells. Using RNA-sequencing of testicular biopsies, we recently examined RNA profiles of testes with and without GnRHa treatment. Here, we focused on the expression patterns of known gene markers for gonocytes and spermatogonia, and found that DMRTC2, PAX7, BRACHYURY/T, and TERT were associated with defective mini-puberty and were responsive to GnRHa. These results indicate novel testosterone-dependent genes and provide valuable insight into the transcriptional response to both defective mini-puberty and curative GnRHa treatment, which prevents infertility in man with one or both undescended (cryptorchid) testes.

Infertility is sometimes more a man's problem than a woman's, failure of one or both of the testes to descend (cryptorchidism) being the most frequent genital malformation in boys. Untreated, the undescended testis impairs germ cell development and significantly reduces adult fertility. A-dark spermatogonia are the stem cells for all future spermatozoa, and their depletion can be reliably estimated in resin semithin sections. Additionally, there is an increased risk of testicular preneoplasia in the form of carcinoma in situ (CIS) cells. The authors report how the pathologic biopsy examination of juvenile cryptorchid testes can assess infertility and malignancy risk.

It has been known for many years that boys with unilateral or bilateral undescended testis (cryptorchidism) tend to have a low IQ, and those who belong to the high infertility risk (HIR) group perform less well at school than low infertility risk (LIR) patients. However, the molecular biological processes underlying this phenomenon are not understood. In this study, we report the outcome of testicular RNA profiling for genes involved in long-term memory formation. We analyzed the histology and the transcriptome of testicular biopsies from bilateral HIR cryptorchid boys, comparing those who received GnRHa treatment for 6 months after the first surgery with those who did not receive GnRHa before the second surgery. We found that GnRHa treatment alters the testicular mRNA levels of neuronal genes that are involved in long-term memory and testosterone synthesis. These data highlight a possible molecular link between cryptorchidism, impaired mini-puberty, and diminished cognitive functions. Our results are consistent with the hypothesis that hypogonadotropic hypogonadism in cryptorchid boys with altered mini-puberty may affect neuronal genes important for memory and learning, which could help explaining the negative correlation between cryptorchidism and intellectual abilities.

The gonadotropin-releasing hormone agonist (GnRHa; Buserelin) rescues fertility during adulthood in the majority of high infertility risk cryptorchid boys presenting with defective mini-puberty. However, the molecular events governing this effect are not understood. We report the outcome of an RNA profiling analysis of testicular biopsies from 4 operated patients who were treated with GnRHa for 6 months versus 3 operated controls who were not treated. GnRHa induces a significant transcriptional response, including protein-coding genes involved in pituitary development, the hypothalamic-pituitary-gonadal axis, and testosterone synthesis. Furthermore, we observed an increased abundance of long noncoding RNAs (lncRNAs) participating in epigenetic processes, including AIRN, FENDRR, XIST, and HOTAIR. These data are consistent with the hypothesis that hypogonadotropic hypogonadism in boys with altered mini-puberty is the consequence of a profoundly altered gene expression program involving protein-coding genes and lncRNAs. Our results point to molecular mechanisms that underlie the ability of GnRHa to rescue fertility.

Maldescent of the epididymo-testicular unit can occur as an isolated event or as a component of various syndromes. When part of a syndrome, crypto-epididymis is usually accompanied by other genital and/or extragenital features. Epididymis development is primarily regulated by androgens, and successful epididymo-testicular unit development and descent requires an intact hypothalamic-pituitary-gonadal axis. The developing gonadotropin-releasing hormone system is essential for epididymo-testicular descent and is highly sensitive to reduced fibroblast growth factor (FGF) signaling. Our understanding of the impact of FGFR1 in the process of epididymo-testicular descent has recently improved. At later stages of embryonic development, the undifferentiated epididymal mesenchyme is a specific domain for FGFR1 expression. The majority of individuals with syndromic crypto-epididymis, as well as individuals with isolated maldescent of the epididymo-testicular unit, exhibit some disturbance of FGF, FGFR1 and/or genes involved in hypothalamic-pituitary-gonadal axis regulation. However, the mechanisms underlying FGF dysregulation may differ between various syndromes.

Mini-puberty is the period between 30 and 80 days after birth when testosterone and gonadotropin surges occur in male infants to induce the transformation of gonocytes into adult/dark spermatogonia. Cryptorchid boys with impaired mini-puberty develop infertility despite timely and successful surgical treatment. The decreased germ cell count found in this group of boys could be the result of uncontrolled transposon activity inducing genomic instability and germ cell death. A genome-wide analysis of 18 cryptorchid and 4 control testes was performed with Affymetrix chips. We found that 5 of 8 genes that are important for transposon silencing were not expressed in the high azoospermia risk group of cryptorchid boys but were expressed in the low azoospermia risk and control groups. Two genes, CBX3 and DNMT1, were equally expressed in all 3 groups. Impaired expression of the DDX4, MAEL,MOV10L1, PIWIL2, PIWIL4, and TDRD9 genes in the group of cryptorchid boys at high risk of infertility indicates that gene instability induced by impaired expression of transposon silencing genes contribute to the development of azoospermia. Intact mini-puberty appears to be essential for the development of the endogenous defense system mediated by transposon silencing.

Despite timely and successful surgery, 32% of patients with bilateral and 10% with unilateral cryptorchidism will develop azoospermia. Cryptorchid boys at risk of azoospermia display a typical testicular histology of impaired mini-puberty at the time of the orchidopexy. During mini-puberty increased gonadotropin and testosterone secretion stimulate transformation of gonocytes into Ad spermatogonia. In the azoospermia risk group this transformation is to a great extent impaired. This study aimed to analyze data on whole genome expression signatures of undescended testes at risk of developing azoospermia. Twenty-three testicular biopsies from 22 boys were analyzed (19 testes from 18 boys with cryptorchidism and 4 contralateral descended testes from patients with testicular agenesis). Expression profiling identified 483 genes not or under-expressed in the azoospermia risk group compared with the control and low risk for azoospermia (LAZR) groups. Annotated loci were associated with spermatogenesis. Other significant genes were cellular defense response genes and hormone-controlled loci involved in spermatogenesis. Some genes transcribed in normal adult meiotic and post-meiotic germ cells are activated in healthy juvenile Ad spermatogonia. Thus, molecular events initiating the testicular expression program at the onset of puberty and maintaining it during adulthood occur very early in prepubertal testes. This molecular event is to a great extent impaired in the high risk for azoospermia (HAZR) group lacking Ad spermatogonia (stem cells for spermatozoa) indicating impaired mini-puberty.

Background: In recent years, several genes were found to be involved in the process of epididymo-testicular descent, the most frequently cited ones include INSL3, HOXA10, GNRHR, and KAL1. In this study, we analyzed the differences in gene expression profiles between cryptorchid and descended testes. In particular, we analyzed expression of all recently published genes known to be associated with undescended testis. Patients and Methods: Twenty-two testicular biopsies from 18 boys were analyzed. We analyzed gene expression in 16 cryptorchid and 6 descended testes using Affymetrix Human Genome U133 Plus 2.0 GeneChips, and validated the results with qPCR. Results: 3,688 transcripts were differentially expressed with an adjusted p value of (FGFR1) and its mediators SOS1 and RAF1 were expressed less in undescended testes. Conclusion: Based on our results, it is possible that a subtle dysfunction (expression) of the FGFR1, SOS1 and RAF1 genes is involved in the development of the most common male reproductive tract disorder – unilateral or bilateral cryptorchidism.