Fine needle aspiration biopsy of azoospermic testes. Could it replace histologic biopsy?

OBJECTIVE: To evaluate the possibility of replacing histologic biopsy of azoospermic testes by fine needle aspiration (FNA) for diagnostic and management purposes. STUDY DESIGN: Twenty-seven patients were examined under general anesthesia, and 53 testes were biopsied by FNA. Fifty-four testes were biopsied for histologic examination. Histology and cytology of each testis were compared to assess the discrepancy or concordance between the diagnoses. RESULTS: Twenty-seven testes showed complete concordance of cytology and histology, and 13 testes showed a mild degree of discrepancy. A severe degree of discrepancy was seen in 13 testes. In the majority of discrepant cases, FNA biopsy diagnosis was more sensitive in detecting evidence of full or advanced maturation. In one case of discrepancy, the histologic finding was Sertoli cells only, and cytology revealed focal full maturation. In vitro fertilization using sperm from this case was successful. CONCLUSION: FNA biopsy of testes in azoospermia is a fast, reliable and minimally traumatic method. The prospects for utilizing FNA biopsy material in assisted and microassisted fertilization are promising.     

Aging of male germ line stem cells in mice

In the present study, we investigated the effect of aging on spermatogonial stem cells (SSCs) and on the testicular somatic environment in ROSA26 mice. First, we examined testis weights at 2 mo, 6 mo, 1 yr, and 2 yr of age. At 1 and 2 yr, bilateral atrophied testes were observed in 50% and 75% of the mice, respectively; the rest of the mice had testis weights similar to those of young mice. Next, we evaluated the number and the activity of aged SSCs using spermatogonial transplantation. Numbers of SSCs in atrophied testes decreased in an age-dependent manner to as low as 1/60 of those in testes of young mice. Numbers of SSCs in nonregressed testes were similar regardless of age. The colony length, which is indicative of the potential of SSCs to regenerate spermatogenesis, was similar with donor cells from atrophied testes of 1-yr-old mice and those from testes of young mice, suggesting that SSCs remaining in 1-yr atrophied testes were functionally intact. Colonies arising from SSCs derived from 2-yr atrophied testes were significantly shorter, however, indicating that both SSC numbers and activity declined with age. Finally, we transplanted donor cells from young animals into 1- and 2-yr atrophied testes. Although the weight of 2-yr testes did not change after transplantation, that of 1-yr testes increased significantly, indicating that 1-yr, but not 2-yr, atrophied testes are permissive for regeneration of spermatogenesis by SSCs from young mouse testes. These results demonstrate that both SSCs and somatic environment in the testis are involved in the aging process.     

Human testicular cultures. II. Sertoli cells.

Two cell types, one epitheloid and the other fibroblast-like, were found in human testicular cultures derived from testes of patients. Ultrastructural studies indicated that, whereas the epitheloid cells were Sertoli cells, the fibroblast-like cells were fibroblasts. The Sertoli cells could maintain growth for a period of more than 4 months. In cultures derived from normal testes, only fibroblasts were observed.     

The histochemistry of complex carbohydrates in the testes of patients of idiopathic male infertility

Summary In testis tissues from patients with idiopathic male infertility, complex carbohydrates have been studied by means of light microscopic histochemical methods, comparisons being made with the normal testes. In the infertile testes, histochemical reactions for acidic and neutral complex carbohydrates were in general weaker in intensity than in the normal testes. In the seminiferous tubules and interstitium of the infertile testes, a histochemical finding of primary importance was obtained with the demonstration of galactose deficiency in the complex carbohydrates involved. These glycoproteins contained only small numbers of sulfhydryl and disulfide groups. The possible histophysiological significance of these complex carbohydrates is discussed, with special reference to their histochemical properties determined in the present study.     

The histochemistry of complex carbohydrates in the testes of patients of idiopathic male infertility.

In testis tissues from patients with idiopathic male infertility, complex carbohydrates have been studied by means of light microscopic histochemical methods, comparisons being made with the normal testes. In the infertile testes, histochemical reactions for acidic and neutral complex carbohydrates were in general weaker in intensity than in the normal testes. In the seminiferous tubules and interstitium of the infertile testes, a histochemical finding of primary importance was obtained with the demonstration of galactose deficiency in the complex carbohydrates involved. These glycoproteins contained only small numbers of sulfhydryl and disulfide groups. The possible histophysiological significance of these complex carbohydrates is discussed, with special reference to their histochemical properties determined in the present study.     

A change in the steroid metabolic pathway in human testes showing deteriorated spermatogenesis

During androgen biosynthesis, the human testes normally produce only small quantities of Δ⁴-C₂₁ steroids as these are products of the Δ⁴-pathway and healthy human testes preferentially use the Δ⁵-pathway. However, the Δ⁴-C₂₁ steroid progesterone accumulates in the thickened lamina propria of the seminiferous tubules in testes with deteriorated spermatogenesis. The objectives of this study were to analyse the pregnenolone metabolites in testes with deteriorated spermatogenesis and to establish whether the androgen biosynthesis pathway changes in this condition. Biopsied or orchiectomised testicular samples were obtained from patients with varicocele, non-obstructive azoospermia, obstructive azoospermia, testicular cancer, and cryptorchidism. The samples were segregated into spermatogenesis related Johnsen’s score groups: Low-JS (< 5.0) and High-JS (> 7.8). Higher levels of progesterone and 17α-hydroxyprogesterone were metabolised under in vitro conversion in the Low-JS testes than the High-JS testes when cell-free homogenates from each group were separately incubated with ¹⁴C-labelled pregnenolone. Nevertheless, the serum hormone levels did not differ between groups. Two novel pregnenolone metabolites 5β-pregnan-3β-ol-20-one and 5α-pregnan-3α, 21diol-20-one were identified from in vitro conversion in Low-JS testes and by recrystallisation. Immunohistochemistry revealed the higher βHSD expression in the Low-JS than the High-JS testes. However, the CYP17A1 expression levels did not differ between groups. Infertile testes increase the relative βHSD levels in their Leydig cells and synthesised testosterone from pregnenolone via the Δ⁴- rather than the Δ⁵-pathway. A new insight into a change of metabolites in Low-JS testes will be relevant to understand the mechanism of the deteriorated spermatogenesis under the normal range of testosterone level.     

Testicular Biopsy in Evaluation of Male Infertility

Testicular biopsy findings in 100 infertile men were correlated with the clinical findings. Mild or moderately severe tubular lesions were seen in 57 cases and severe changes in 43. Clinical examination and semen analysis were no guide to the severity of the testicular lesion. Though patients with normal sized testes more commonly had mild tubular lesions, many were severe. Patients with small testes more often had severe lesions but some had only mild tubular changes. Biopsy findings in both aspermic and oligospermic patients ranged from normal to a complete loss of germinal tissue.Testicular biopsy is advocated in infertile men for the complete assessment of the case and for identifying those which are potentially treatable. Patients with a severe lesion can be spared further investigations. The choice and results of treatment are discussed, particularly the surgical treatment of varicocele or obstruction. Only patients with a mild or moderate testicular tubular lesion should participate in future trials with drugs for male infertility.     

Orchitis: a complication of severe acute respiratory syndrome (SARS)

Severe acute respiratory syndrome (SARS) coronavirus has been known to damage multiple organs; however, little is known about its impact on the reproductive system. In the present study, we analyzed the pathological changes of testes from six patients who died of SARS. Results suggested that SARS caused orchitis. All SARS testes displayed widespread germ cell destruction, few or no spermatozoon in the seminiferous tubule, thickened basement membrane, and leukocyte infiltration. The numbers of CD3+ T lymphocytes and CD68+ macrophages increased significantly in the interstitial tissue compared with the control group (P < 0.05). SARS viral genomic sequences were not detected in the testes by in situ hybridization. Immunohistochemistry demonstrated abundant IgG precipitation in the seminiferous epithelium of SARS testes, indicating possible immune response as the cause for the damage. Our findings indicated that orchitis is a complication of SARS. It further suggests that the reproductive functions should be followed and evaluated in recovered male SARS patients.     

Comparison of testosterone and insulin-like peptide 3 secretions in response to human chorionic gonadotropin in cultured interstitial cells from scrotal and retained testes in dogs

Levels of testosterone and insulin-like peptide 3 (INSL3) secretions in response to different doses of human chorionic gonadotropin (hCG) in cultured interstitial cells were compared between retained and scrotal testes in dogs. Retained (n=10) and scrotal (n=9) testes were obtained from small-breed dogs. The testicular tissues were dispersed in Dulbecco's Modified Eagle Medium with Ham's nutrient mixture containing 2000 PU/ml dispase II and 10% fetal bovine serum. The cells were plated with differing concentrations (0-10 IU/ml) of hCG for 18 h in multiwell-plates. Testosterone and INSL3 in the same spent medium were measured by enzyme-immunoassays (EIA). A new EIA with a reliable detection range of 0.025-5 ng/ml was developed in order to measure canine INSL3 in culture medium. Dose-dependent stimulation of testosterone by hCG was observed in the cells of both retained and scrotal testes. The incremental rate of testosterone secretion was significantly lower at 0.1, 1 and 10 IU/ml hCG in the cells of retained testes than in scrotal testes, however. INSL3 secretion was significantly stimulated at 10 IU/ml hCG relative to unstimulated controls comprising cells of scrotal testes; no such stimulation was observed in the cells of retained testes. At 10 IU/ml hCG, the incremental rate of INSL3 was significantly lower in the cells of retained testes than scrotal testes. These results suggest that LH-induced secretory testosterone and INSL3 responses are lower in the interstitial cells of retained testes than of scrotal testes. Furthermore, the high concentrations of LH may acutely stimulate INSL3 release in scrotal testes of dogs, but not in retained testes.     

Testes weight, body weight and mating systems in marsupials and monotremes

Relationships between testes weight, body weight and mating systems were examined in 40 marsupial species and in the extant monotremes. Relationships between relative testes weight and mating systems in marsupials resemble those previously described for primates. Thus relative testes weights are greatest in those marsupials where females mate with multiple males during the fertile period, i.e. polyandrous species (e.g. Antechinus ftavipes, Isoodon obesulus, Perameles nasuta, Potorous tridactylus, Macropus eugenii and M. agilis) and smallest in monandrous forms (e.g. Petauroides volans and Petaurus breviceps ) where females usually mate with a single male. These findings are consistent with effects of sperm competition upon the evolution of relative testes sizes in marsupials. Where field studies on marsupial mating systems are lacking, we make predictions based upon examination of their relative testes weights. Tarsipes rostratus, Acrobates pygmaeus, Macropus rufogriseus and Sarcophilus harrisii are predicted to engage in multiple matings and sperm competition. Conversely, Lasiorhinus latifrons, Cercatetus concinnus and Pseudoantechinus macdonnellensis are predicted to be monandrous in their mating behaviour. The monotremes ( Ornithorhynchus anatinus, Tachyglossus aculeatus and Zaglossus bruijnii ) are characterized by possession of very large testes; monotremes are shown to have significantly greater relative testes weights than marsupials, primates or avian species. This taxonomic difference is unlikely to be related lo the occurrence of oviparity or to the abdominal position of the testes in the Monotremata. Their mating systems are not known in detail, but some evidence for multiple matings (and hence for sperm competition) exists for Tachyglossus aculeatus so that their large testes may be adaptive in this context.     

13 Years' experience with the combined hormonal therapy of cryptorchidism.

PURPOSE: We analyze the results of the combined treatment with luteinizing hormone releasing hormone (LH-RH) and human chorionic gonadotropin (HCG) of a large series of patients with cryptorchidism. MATERIALS AND METHODS: Between 1987 and 1999 and after strict differentiation between cryptorchid, retractile and gliding testes, 2,467 boys with 2,962 cryptorchid-gliding testes were treated with the combined hormonal therapy. LH-RH was administrated as a nasal spray at a dosage of 1.2 microg daily for a period of 4 weeks. HCG was injected intramuscularly, 5 times at 2-day intervals at a dosage adjusted according to the age. RESULTS: In the prospective study 2,476 boys with 2,962 cryptorchid testes were hormonally treated. Of the 2,962 evaluated cases 1,200 (40.52%) have been treated surgically after the hormone therapy. In 1,762 cases, 59.48% of cryptorchid testes were in the scrotum after combined hormone treatment. CONCLUSIONS: Treatment with LH-RH and HCG induced the descent of the testes to a normal scrotal position of boys with cryptorchidism in 59.48% of the evaluated cases. The combined treatment was effective for inducing descent of cryptorchid and gliding testes. According to the evaluated intraoperative findings, the failure of the combined therapy in 40.52% of the cases is due to the fact that the free descent is limited by local factors such as anatomical alterations of the inguinal canal, epididymal abnormalities or ectopic distal attachment of the lig. testis.     

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.     

Hormonal therapy for the subfertility of cryptorchidism.

BACKGROUND: The subfertility of cryptorchidism correlates with severely reduced total germ cell counts in prepubertal testicular biopsies of undescended testes. Reduced total germ cell counts are associated with defects in the two prepubertal steps in maturation and proliferation in germ cells: first, the transformation of the fetal stem cell pool (gonocytes) into the adult stem cell pool (adult dark spermatogonia) at two to three months of age and, second, the transformation of adult dark spermatogonia into primary spermatocytes (meiosis) at 4-5 years. The defects in maturation are associated with blunting of the normal surges in gonadotropins and testosterone. Prepubertal treatment with gonadotropin-releasing hormones would theoretically trigger normal germ cell maturation and proliferation and thereby improve total germ cell counts and improve fertility. Prepubertal treatment of cryptorchidism with the GnRH analogue Buserelin has resulted in improved total germ cell counts and improved spermiograms. The purpose of this report is to describe the results of treatment of cryptorchidism with the GnRH analogue Naferelin. PATIENTS: Twelve boys with cryptorchidism, 6 unilateral and 6 bilateral, and severely reduced germ cell counts in testicular biopsies were treated with Naferelin following successful orchidopexy and bilateral testicular biopsies. Response of the total germ cell counts was assessed in follow-up bilateral biopsies within 5 months of completing the hormonal therapy. RESULTS: Eight of the 12 boys (5 of the 6 with unilateral and 3 of the 6 with bilateral cryptorchidism) showed improvement in the total germ cell counts in one or both testes. All 8 had a poor prognosis for fertility pretreatment and a good prognosis for fertility posttreatment. Of the 5 with unilateral cryptorchidism who improved, 2 showed improvement in both testes; and 3, only in the contralateral descended testes. All 3 of the boys with bilateral cryptorchidism who improved showed improvement in both testes. Testes with absence of germ cells and older patients tended to show no improvement. Of the 6 contralateral descended, 5 (83%) improved, and of the 18 undescended testes, 8 (44%) improved. CONCLUSIONS: In this preliminary study, Naferelin therapy appears to induce improvement in the total germ cell counts and the prognosis for future fertility in 75% of patients.     

Ecdysteroid production by testes of the tobacco budworm,Heliothis virescens,from last larval instar to adult

Testes of Heliothis virescens were examined in vitro for the ability to produce ecdysteroids. Production was detected in testes removed from larvae at mid and late periods in the last instar, in testes from pupae after the third day of purpal development, and in testes taken from young adult males. The testes in the isolated abdomens do not fuse during later stages of spermatogenesis as they do in intact insects. Spermatocysts in testes of larval abdomens isolated from the upper body early in the last instar developed to mature sperm despite low haemolyph and testis ecdysteroid titres. Gonadectomized animals developed and moulted normally, indicating that ecdysteroids from the testes are not essential to general development.     

Abnormal germ cell development in cryptorchidism.

BACKGROUND: Previous studies suggest that two fundamental, probably androgen-dependent, steps in maturation of germ cells normally occur in the prepubertal testis: the disappearance of gonocytes (the fetal stem cell pool) and the appearance of adult dark spermatogonia (the adult stem cell pool) at 2-3 months of age and the appearance of primary spermatocytes (the onset of meiosis) at 4-5 years. Previous studies of small series of cryptorchid boys suggest that both steps are defective in undescended testes and to a lesser degree in descended testes contralateral to unilaterally undescended testes. The purpose of this study is to confirm the previous findings of defective germ cell maturation in a large series of boys with unilateral undescended testes. PATIENTS: Seven hundred and sixty-seven boys with unilateral cryptorchidism who had orchidopexy and bilateral testicular biopsies between birth and 9 years of age were studied. MATERIALS AND METHODS: Total and differential germ cell counts were performed on semithin histologic sections of the biopsies. The results from the undescended and contralateral descended testes were compared using the Wilcoxon signed-rank test and the Wilcoxon-Whitney-Mann U test. RESULTS: Gonocytes failed to disappear and adult dark spermatogonia failed to appear in undescended testes under 1 year of age indicating a defect in the first step in maturation at 2-3 months resulting in failure to establish an adequate adult stem cell pool. Primary spermatocytes failed to appear in undescended testes and appeared in only 19% of contralateral descended testes at 4-5 years of age indicating a defect in the onset of meiosis. CONCLUSION: Unilaterally undescended testes fail to establish an adequate adult stem cell pool which normally occurs at 2-3 months of age and fail to establish adequate meiosis which normally occurs at 4-5 years of age. Similar but less severe changes are seen in the contralateral descended testes. Defects in the two pubertal steps in germ cell maturation are associated with reduced total germ cell counts.     

High activity of 17 alpha-oxidoreductase in neonatally grafted mouse testes in adult female mice

Male and female (WB-C57BL/6)F1 hybrid mice were used. Two testes from neonatal mice were grafted into the spleen of adult male and female mice, and the grafted testes were removed 30 and 60 days after grafting. Normal testes from 30- and 60-day old mice were also used. Testicular homogenates were incubated with [14C]4-androstene-3,17-dione or [3H]progesterone, and enzyme activities per g wet tissue and progesterone metabolism were examined. Activity of 17 alpha-oxidoreductase in the grafted testes in females (20 nmol/g/h) was approx. 10 times the activity in the grafted testes in males or in the normal testes, whereas 17 beta-oxidoreductase activity in the grafted testes in females was the lowest among these testes. The bilateral ovariectomy performed 1 month before the grafting of neonatal testes, artificial cryptorchidism performed at 20 days of age, and estrogen treatment for 10 days by diethylstilbestrol pellets resulted in no significant changes in 17 alpha-oxidoreductase activities in 30- and 60-day old grafted, cryptorchid or normal testes. The major 17-hydroxy-C19-steroids formed in vitro from progesterone by the grafted testes in female mice were testosterone and 17 alpha-hydroxy-4-androsten-3-one (epitestosterone), but the formation of epitestosterone was insignificant in the normal testes. The present results demonstrate for the first time that epitestosterone is formed as one of major C19-steroids in neonatally grafted mouse testes in females but not in those in males or in normal mouse testes. However, the mechanisms remain unexplained.     

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.     

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.     

Effect of surgical restriction of growth of the testicular artery on testis size and histology in bulls.

The growth of the testicular artery was restricted on one side in young bulls and subsequent testicular development was monitored. After the animals had been killed, the testes were studied histologically and compared with testes from hypoplastic bulls. The growth rate of testes from the experimental side was significantly lower than that of testes from the sham-operated side over a period of 578 days. At death, the experimental testes had a mean (+/- SD) mass of 76 (+/- 41) g compared with 220 (+/- 31) g for the control testes. The sham-operated testes accounted for 0.071 (+/- 0.008)% of live body mass compared with 0.025 (+/- 0.014)% for the experimental testes. The seminiferous tubules in the sham-operated testes had a mean diameter (+/- SD) of 211 (+/- 25) microns, whereas those of the artery-restricted testes and hypoplastic testes were significantly smaller (152 (+/- 37) and 145 (+/- 45) microns, respectively). In the artery-restricted and hypoplastic testes, the interstitial tissue accounted for a significantly greater proportion of the testicular tissue than in the sham-operated testes and spermatogenesis was either totally absent or present in only a small proportion of tubules. It is suggested that the artery-restricted testes could be used as a model for testicular hypoplasia.     

Steroid metabolism in testes of patients with incomplete masculinization due to androgen insensitivity or 17 beta-hydroxysteroid dehydrogenase deficiency and normally differentiated males

For purposes of establishing suitable controls in studies of patients with a suspected enzyme deficiency, activities of enzymes involved in the biosynthesis of testosterone were compared in testes of patients with androgen insensitivity syndrome (AIS) and normally differentiated males with carcinoma of the prostate (Ca prostate) or testis (Ca testis). Activities of 17,20-desmolase and of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were higher in the testes of pre-, peri- or postpubertal patients with AIS than in elderly men (58-80 yr) with Ca prostate. Activities of 17 beta-HSD (reductive direction) and 3 beta-HSD tended to be higher in peri- or postpubertal than in prepubertal patients with AIS. Activity of 3 beta-HSD was low in the patient with Ca testis. In a peripubertal (12 yr) patient with incomplete masculinization due to a severe deficiency of 17 beta-HSD, reductive activity of 17 beta-HSD was very low compared with that of patients with Ca prostate, Ca testis or AIS. In contrast, in testes from the younger sibling (4 yr), in whom the deficiency of 17 beta-HSD was less severe, 17 beta-HSD reduction of dehydroepiandrosterone was as high as that of men with Ca prostate, yet deficient in comparison with that of more closely age-matched patients with AIS. This emphasizes the desirability of using age-matched tissue for control purposes in enzyme studies.