Jorgen Thorup

What is new in cryptorchidism and hypospadias—a critical review on the testicular dysgenesis hypothesis ☆

It has been hypothesized that poor semen quality, testis cancer, undescended testis, and hypospadias are symptoms of one underlying entity--the testicular dysgenesis syndrome--leading to increasing male fertility impairment. Though testicular cancer has increased in many Western countries during the past 40 years, hypospadias rates have not changed with certainty over the same period. Also, recent studies demonstrate that sperm output may have declined in certain areas of Europe but is probably not declining across the globe as indicated by American studies. However, at the same time, there is increasing recognition of male infertility related to obesity and smoking. There is no certain evidence that the rates of undescended testes have been increasing with time during the last 50 years. In more than 95% of the cases, hypospadias is not associated with cryptorchidism, suggesting major differences in pathogenesis. Placental abnormality may occasionally cause both cryptorchidism and hypospadias, as it is also the case in many other congenital malformations. The findings of early orchidopexy lowering the risk of both infertility and testicular cancer suggest that the abnormal location exposes the cryptorchid testis to infertility and malignant transformation, rather than there being a primary abnormality. Statistically, 5% of testicular cancers only are caused by cryptorchidism. These data point to the complexity of pathogenic and epidemiologic features of each component and the difficulties in ascribing them to a single unifying process, such as testicular dysgenesis syndrome, particularly when so little is known of the actual mechanisms of disease.

The Relation between Adult Dark Spermatogonia and Other Parameters of Fertility Potential in Cryptorchid Testes

PURPOSEnThe fertility potential of boys with cryptorchidism may be related to the number of adult dark spermatogonia per tubular transverse section in testicular biopsies taken at orchiopexy. Placental-like alkaline phosphatase positive gonocytes in testes within year 1 of life indicate preserved ability for germ cell transformation. We related these parameters to the total number of tubular germ cells and other factors associated with fertility potential.nnnMATERIALS AND METHODSnThe study comprised 89 boys 0.7 to 3 years old (median age 1.8) who underwent bilateral testicular biopsy at bilateral orchiopexy and provided blood samples for gonadotropins and inhibin B.nnnRESULTSnOf 76 boys with adult dark spermatogonia 44 (58%) had a normal mean number of spermatogonia per tubular transverse section compared to 2 of 13 (15%) without adult dark spermatogonia (p <0.05). In the 30 boys with good fertility potential, including a normal mean number of tubular germ cells, and normal gonadotropins and inhibin B, the mean number of adult dark tubular germ cells was 0.081 vs 0.031 in the 38 with low fertility potential, including impaired tubular germ cells and/or low inhibin B but no reactive increase in gonadotropins (p <0.05). In the 21 patients with increased gonadotropins the mean number of adult dark spermatogonia per tubular transverse section was 0.063. Of the 20 boys with normal mean adult dark spermatogonia per tubular transverse section 12 (60%) had good fertility potential, including a normal mean number of tubular germ cells, normal gonadotropins and normal inhibin B, compared to only 18 of 69 (26%) with an impaired mean number of adult dark spermatogonia per tubular transverse section (p <0.05). Of 46 boys with a normal mean number of tubular germ cells 26 (57%) had placental-like alkaline phosphatase positive cells compared to 14 of 43 (33%) with a decreased mean number of tubular germ cells (p <0.05).nnnCONCLUSIONSnThe number of placental-like alkaline phosphatase positive gonocytes and adult dark spermatogonia per tubular transverse section are important parameters related to the fertility potential of boys with cryptorchid testes.

Bilateral Undescended Testes Classified According to Preoperative and Postoperative Status of Gonadotropins and Inhibin B in Relation to Testicular Histopathology at Bilateral Orchiopexy in Infant Boys

PURPOSEnIn recent series of boys with cryptorchidism gonadotropin levels have been higher and serum inhibin B levels have been lower than normal. To some extent the serum values of inhibin B reflect the state of germinative epithelium in cryptorchid testes. We evaluated whether blood samples of gonadotropins and inhibin B as well as histopathology could be used to classify undescended testes.nnnMATERIALS AND METHODSnA total of 69 boys (median age 2 years) who underwent surgery for bilateral cryptorchidism had blood samples taken preoperatively and 3 months to 2.1 years postoperatively. Testicular biopsies were performed bilaterally at orchiopexy. The average germ cell number per tubular transverse tubule was measured.nnnRESULTSnGroup 1 included 17 patients with increased follicle-stimulating hormone levels. Serum follicle-stimulating hormone and luteinizing hormone decreased significantly after surgery. In 77% of patients (13 of 17) follicle-stimulating hormone levels were normalized. Of these boys 35% (6 of 17) had a low postoperative serum inhibin B. Group 2 consisted of 27 patients with a decreased germ cell number and/or low preoperative inhibin B, but not increased serum follicle-stimulating hormone or luteinizing hormone. There were no significant postoperative changes in follicle-stimulating hormone and luteinizing hormone. Of these boys 22% (6 of 27) had a low serum inhibin B postoperatively. In group 3 there were 25 patients with a normal germ cell number, normal preoperative serum inhibin B and normal gonadotropins. There were no significant changes in luteinizing hormone and follicle-stimulating hormone postoperatively. Only 1 boy in this group had a low postoperative serum inhibin B.nnnCONCLUSIONSnPatients with increased gonadotropin levels may have testicular dysgenesis and some may benefit from early surgery. Patients with normal gonadotropin levels and a decreased germ cell number have transient hypothalamus-pituitary-gonadal hypofunction and a poor fertility prognosis. These patients may benefit from gonadotropin treatment after orchiopexy. Patients with normal gonadotropins, inhibin B and germ cell number have a good fertility prognosis after surgery.

The Sertoli cell hormones inhibin-B and anti Müllerian hormone have different patterns of secretion in prepubertal cryptorchid boys

OBJECTIVES AND HYPOTHESESnThe Sertoli-cells produce inhibin-B and Anti-Müllerian-Hormone (AMH). Much is still unknown about these hormones in prepubertal cryptorchids. The Sertoli-cells are mandatory for germ cell development. The aim of the study was to investigate if there are differences in secretion pattern of Sertoli-cell hormones and their gonadotropin feed-back mechanisms.nnnMETHODSnIncluded were 94 prepubertal cryptorchid boys 0.5-13.1years with measurements of serum-inhibin-B, Anti-Müllerian-Hormone (AMH), Luteinizing Hormone (LH) and Follicle Stimulation Hormone (FSH). The serum values were measured using commercially available kits. The hormonal values were related to age-matched normal values. Testicular biopsy was taken at orchiopexy.nnnRESULTSnInhibin-B positively correlated to AMH for 1-13year-old patients (p<0.0001), but not for 0.5-1year-old patients (p=0.439). For 0.5-1year-old patients inhibin-B-values tended to decrease (p=0.055), in contrast to AMH-values (p=0.852). LH was elevated more often than FSH (p=0.014). FSH and LH were positively associated in patients both 0.5-1year (p=0.042) and 1-13years of age (p<0.0001). LH correlated positively to inhibin- B (p=0.001). In contrast, FSH did not correlate to inhibin-B or AMH (p=0.755 and p=0.528). The number of A-dark spermatogonia per tubular transverse section was positively correlated to inhibin-B serum level.nnnCONCLUSIONnOur new finding of an association between LH and inhibin-B in infancy of cryptorchid boys may be essential for the transformation of gonocytes to A-dark spermatogonia. Previously, LH associated to inhibin-B was described in early puberty only. During the first year of life inhibin-B values decreased faster than AMH. The AMH-levels may just reflect the increased Sertoli cell number that occurs during the first 3months of life.

Immunofluorescent analysis of testicular biopsies with germ cell and Sertoli cell markers shows significant MVH negative germ cell depletion with older age at orchiopexy.

PURPOSEnUndescended testis is the most common defect in male newborns. This condition is associated with increased risks of infertility and testicular malignancy due to abnormal germ cell development in the testes. Early surgery may limit such risks. We analyzed germ cell development vs age at orchiopexy using a germ cell marker and a Sertoli cell marker on testicular biopsies.nnnMATERIALS AND METHODSnA total of 22 testicular biopsies at orchiopexy in 20 patients 5 to 24.5 months old were fixed and embedded in paraffin. Sections were processed and labeled with AMH antibody for Sertoli cells and MVH antibody for germ cells for immunofluorescent histochemical analysis. Confocal images were counted using ImageJ (National Institutes of Health, Bethesda, Maryland) for germ cells and testicular tubules. The data were analyzed using linear regression.nnnRESULTSnSertoli cells were clearly distinguished from MVH positive and negative germ cells located centrally or on basement membranes of tubules. Percentage of tubules with MVH negative germ cells significantly decreased with increasing age at orchiopexy (β = -0.03, p = 0.03). Total tubular numbers and empty tubules without germ cells significantly increased with age at orchiopexy (β = 1.15, p = 0.02 and β = 0.44, p = 0.04, respectively).nnnCONCLUSIONSnAMH antibody distinguished Sertoli cells from germ cells, and MVH antibody distinguished 2 types of germ cells at different developmental stages. Biopsy at orchiopexy in older patients showed significant germ cell depletion. These results lend support to early surgery to optimize germ cell number.

Serum inhibin B values in boys with unilateral vanished testis or unilateral cryptorchidism.

PURPOSEnBoys with cryptorchidism have overall increased gonadotropin and decreased serum inhibin B levels compared to normal. Serum inhibin B levels, produced by Sertoli cells, may reflect the state of germinative epithelium in cryptorchid testes. We evaluated whether serum inhibin B levels differed between boys with unilateral vanished testis and those with unilateral cryptorchidism.nnnMATERIALS AND METHODSnBlood samples from 297 boys 1.5 to 5 years old were included, of whom 222 had unilateral cryptorchidism, 29 had unilateral vanished testis and 46 had undergone unilateral orchiopexy 1 year previously. Serum inhibin B levels were measured using a commercially available ELISA kit and were compared to normal range.nnnRESULTSnSerum inhibin B levels in boys with unilateral vanished testis were not different from those with unilateral cryptorchidism. Serum inhibin B values were above the normal median in 43% of boys previously operated on for unilateral cryptorchidism, compared to 17% at surgery (p = 0.0003). The percentage of patients with inhibin B levels below normal range was 14% in those with unilateral vanished testis, 23% in those with unilateral cryptorchidism and 11% in those who had undergone orchiopexy 1 year previously for unilateral cryptorchidism. The percentage of boys with inhibin B levels above normal median was 24% in those with unilateral vanished testis, 17% in those with unilateral cryptorchidism and 43% in those who had undergone orchiopexy. However, in boys with a vanished testis the frequency of serum inhibin B above normal median was only 5% before age 1.5 years, after which the rate was 67% (p = 0.0022).nnnCONCLUSIONSnOur findings may reflect the development of contralateral testicular hypertrophy in boys with unilateral vanished testis. The initial low inhibin B values may be explained by impaired total number of Sertoli cells. Serum inhibin B values also indicated that in 6-month to 5-year-old boys with cryptorchidism orchiopexy was beneficial for the germinative epithelium.

Bilateral vanished testes diagnosed with a single blood sample showing very high gonadotropins (follicle-stimulating hormone and luteinizing hormone) and very low inhibin B

Abstract Objective. In boys with cryptorchidism median serum values of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are higher and median serum values of inhibin B lower than in normal controls. Serum values of inhibin B reflect the state of germinative epithelium in cryptorchid testes. The aim of the study was to evaluate whether a simple blood sample of gonadotropins and inhibin B could diagnose bilateral vanished testes. Material and methods. Group I included five boys (4 months to 6 years and 3 months old) with bilateral vanished testes at laparoscopy. Group II included 82 boys with bilateral cryptorchidism younger than 7 years of age at surgery for bilateral cryptorchidism (median age 1 year and 9 months). Results. The serum levels of hormones for the patients with vanished testes were: inhibin B 5–18 pg/ml, FSH 41–191 IU/l and LH 3.9–56 IU/l. The patients all had karyotype 46,xy. The serum levels of hormones from group II were: inhibin B median 122 (range 20–404) pg/ml, FSH median 0.8 (range 0.2–3.5) IU/l and LH median 0.2 (range 0.1–3.2) IU/l. The serum levels of inhibin B, FSH and LH from the boys with vanished testes were significantly different from the serum levels of the boys with bilateral cryptorchidism (p = 0.0026, p < 0.0001 and p < 0.0001, respectively). Conclusions. The serum values of gonadotropins and inhibin B from boys with bilateral vanished testes were significantly different from those of bilateral cryptorchid boys, indicating no germinative epithelium, no Sertoli cells and compensatory high gonadotropins. If such abnormal serum values are obtained from boys with bilateral non-palpable testes, tubular tissue is not present and surgery can be avoided.

Classification and Causes of Undescended Testes in Humans

To understand the heterogeneous aetiology of cryptorchidism, a classification system is required. The classification is becoming clearer now that undescended testes (UDT) may be separated into congenital and ‘acquired’ types, and the recognition that ‘retractile’ testes form a grey zone between normally descended testes and those that may be developing to ascending, acquired UDT. Congenital UDT can be classified into intraabdominal UDT, canalicular, superficial inguinal pouch and prescrotal variants, aside from the rare ectopic testes. Acquired, ascending testes present later in childhood rather than at birth, and are thought to be caused by failure of the spermatic cord to elongate normally postnatally, so that the testis remains close to the inguinal canal as the scrotum grows away from the groin.The incidence of UDT was thought to be increasing but recent studies suggest this was just related to the use of different definitions of UDT. The aetiology remains mostly unknown, but is thought to be multifactorial. Genetic screening so far implicates muscle development and connective tissue remodelling in the aetiology.

Conclusions and Future Developments

In this chapter we summarise the main conclusions of this book. Testicular descent has evolved only in mammals and in modern mammals and humans it occurs in 2 separate steps, with different anatomy and hormonal control. The first step occurs when the developing testis produces insulin-like hormone 3 (INSL3) to make the gubernaculum swell. This holds the testis near the groin as the fetus grows. In the second step androgens masculinise the genitofemoral nerve, which controls gubernacular migration to the scrotum. Undescended testes (UDT) can be classified into congenital and ‘acquired’ with ‘retractile’ testes forming a grey zone between descended testes and those developing into ascending, acquired UDT. The effects of UDT are secondary to the high temperature of the maldescended testis, although some have a primary hormonal anomaly. Because of key germ cell development in the first year, orchidopexy should be done between 6 and 12 months of age, while surgical treatment of acquired UDT remains controversial. Standard orchidopexy is required for a palpable UDT and laparoscopy for intraabdominal, impalpable UDT. Hormone treatment remains controversial. It has little role to cause descent, but may have a role as an adjunct to surgery to improve germ cell function. The long-term prognosis remains uncertain, but is predicted to improve with early surgery compared with the past.