Diagnostic Testicular Biopsy Research Articles

Operating characteristics of follicle-stimulating hormone in azoospermic men

To validate factors predictive of nonobstructive azoospermia (NOA) and to determine the operating characteristics of FSH for predicting NOA. Retrospective cohort study. Tertiary care military treatment facility. One hundred forty azoospermic males undergoing infertility evaluation. Standard evaluation included history and physical, hormonal workup, and genetic evaluation. Diagnostic testicular biopsy was offered to characterize patients as obstructive azoospermia (OA) or NOA. Semen volume, semen fructose, FSH, T, E₂, PRL, testicular atrophy. Seventy-eight of 140 azoospermic patients underwent a biopsy. The ability to predict NOA based on logistic regression was statistically significant for FSH and testicular atrophy. On multivariate analysis, only FSH remained predictive of NOA. The area under the FSH receiver operating characteristic curve was 0.847, which is significant. The cut point of FSH with the highest likelihood ratio of predicting NOA on biopsy was ≥12.3 mIU/mL. FSH remains the best predictor of NOA. With full knowledge of the operating characteristics of FSH in this population, a patient can be properly educated and treatment can be individualized, based on the specific risk associated with that subject's measured FSH.

032 Cryobanking of testicular tissue in men of reproductive age

Cryopreservation of ejaculated or testicular spermatozoa is recommended for fertility preservation in adolescence and adult men. However cryobanking of the properly preserved human testicular tissue in men of reproductive age is an essential source for further clinical application and additional examination for diagnostic purposes. During the last three years 107 infertile patients were referred for diagnostic testicular biopsy to the Andrology Laboratory, Center of Andrology and Sexual Medicine at Karolinska University Hospital, Stockholm, Sweden. All patients had azoospermia with a testis volume of 1–15 mL. An open biopsy was performed in all cases. The primary section of the biopsy was disaggregated for testicular spermatozoa retrieval for cryopreservation and subsequent use for intracytoplasmic sperm injection (ICSI). Two fresh pieces were fixed for histological evaluation and early cancer detection. As a part of our routine clinical protocol, we typically assess spermatogenesis in semi-thin plastic embedded sections and cryopreserve a further piece of each testis for an additional diagnostic examination. The following three protocols – conventional cooling in two different devices and vitrification, were applied and adjusted for a proper cryopreservation of the testicular tissue as a multicellular system: 1. Small 1–2 × 2 × 2 mm pieces were washed in basic solution, equilibrated in cryoprotectant medium with 0.7 M Me 2 SO for 30 min at +4 °C and cryopreserved by slow programmed cooling protocol, established for cryopreservation of prepubertal testicular tissue in 1.8 mL Nunc cryovials according Keros et al., Hum Reprod., 2007. 2. To preserve tissue for possible clinical application and avoid leaking of LN2 when storing in a liquid faze nitrogen, cryovials were replaced by CBS™ High Security tissue 2 mL straw (CryoBioSystem) and cryopreserved in a closed system using the same protocol. This resulted in a visible structural damage of the tissue. To achieve an appropriate survival of all types of testicular cells, the protocol was optimised, 1.1 M Me 2 SO was used as a cryoprotectant (CPA) and the temperature of initiated seeding was changed to −5 °C. 3. It is well established that cryopreservation of the tissue in pieces as a multicellular system is more complicated and conventional cryopreservation results in harmful ice formation in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation we applied the vitrification protocol developed for human ovarian tissue (Keros et al., 2009, Hum Reprod ). For this combination of Me 2 SO, Propanediol, Ethylene glycol, PVP was applied. Ice crystal formation in human testicular tissue at ultra rapid cooling and warming rates was analysed by freeze substitution technique and transmission electron microscopy. On the basis of our experience, we summarize that human testicular tissue can be successfully cryopreserved by programmed slow freezing using cryovials or tissue straws as well as by vitrification. All types of testicular cells, including cells of different stages of spermatogenesis, motile spermatozoa, Leydig cells and Sertoli cells can survive cryopreservation and long-term storage in liquid nitrogen when an optimized cryopreservation protocol is used. Source of funding: The National council for human tissues and cells (Nationella Vävnadsrådet) at The Swedish Association of Local Authorities and Regions (SKL). Conflict of interest: None declared. Victoria.Keros@ki.se

Sperm Concentration, Testicular Volume and Age Predict Risk of Carcinoma In Situ in Contralateral Testis of Men with Testicular Germ Cell Cancer

We investigated whether semen quality or some easily attainable clinical parameters might be used to estimate the risk of contralateral carcinoma in situ in patients with unilateral testicular germ cell tumors. A total of 264 Danish patients with testicular germ cell tumor with or without contralateral testicular carcinoma in situ were retrospectively investigated. Clinical data included andrological history, physical examination, testis ultrasonography, semen quality and testis histology. Study groups were compared by univariate linear regression analysis and the chi-square test. Associations between contralateral carcinoma in situ and risk factors were modeled in 2 stages: Bayes rule was used to assess the probability of carcinoma in situ; the terms in Bayes rule were estimated using regression models. Significant characteristics of patients with contralateral carcinoma insitu were lower sperm concentration, smaller contralateral testis volume, irregular ultrasonic echo pattern of the contralateral testis and younger age. Cutoff values of sperm concentration and testicular volume were defined. However, according to these only a minority of the noncarcinoma in situ cases could potentially have been spared a diagnostic testicular biopsy. Combining information on age and sperm concentration, secondly age and testis volume resulted in models of the estimated contralateral carcinoma in situ risk, from which patients at particular high risk of carcinoma in situ could be identified. The combined information on sperm concentration, age and contralateral testis volume predict the risk of contralateral carcinoma in situ in patients with unilateral testicular germ cell tumor. The proposed models may facilitate selection of patients with testicular germ cell tumor for contralateral testicular biopsy at the time of orchiectomy if this is not routinely done.

Optical coherence tomography: a novel modality for scrotal imaging

BACKGROUND: For patients with nonobstructive azoospermia, sperm retrieval rates remain modest. We describe the use of optical coherence tomography to improve retrieval rates and to decrease tissue destruction. METHODS: Four patients underwent diagnostic testicular biopsy and imaging with the Niris optical coherence tomography device. We performed a descriptive comparison between optical coherence tomographic images and conventional histology. RESULTS: The measured seminiferous tubule diameter differed by 16 mum between comparative imaging from optical coherence tomography and conventional histology using hematoxylin and eosin staining. CONCLUSION: We illustrate the usefulness of optical coherence tomography in the setting of testicular biopsy and the management of nonobstructive azoospermia.

Testicular biopsy: clinical practice and interpretation

Testicular biopsy was considered the cornerstone of male infertility diagnosis for many years in men with unexplained infertility and azoospermia. Recent guidelines for male infertility have limited the indications for a diagnostic testicular biopsy to the confirmation of obstructive azoospermia in men with normal size testes and normal reproductive hormones. Nowadays, testicular biopsies are mainly performed for sperm harvesting in men with non-obstructive azoospermia, to be used for intracytoplasmic sperm injection. Testicular biopsy is also performed in men with risk factors for testicular malignancy. In a subgroup of infertile men, there is an increased risk for carcinoma in situ of the testis, especially in men with a history of cryptorchidism and testicular malignancy and in men with testicular atrophy. Ultrasonographic abnormalities, such as testicular microlithiasis, inhomogeneous parenchyma and lesions of the testes, further increase the risk of carcinoma in situ (CIS) in these men. For an accurate histological classification, proper tissue handling, fixation, preparation of the specimen and evaluation are needed. A standardized approach to testicular biopsy is recommended. In addition, approaches to the detection of CIS of the testis testicular immunohistochemistry are mandatory. In this mini-review, we describe the current indications for testicular biopsies in the diagnosis and management of male infertility.

In the era of micro‐dissection sperm retrieval (m‐TESE) is an isolated testicular biopsy necessary in the management of men with non‐obstructive azoospermia?

To assess the outcome of sperm retrieval using micro-dissection-TESE (m-TESE) and simultaneous diagnostic biopsy in NOA to determine if the final definitive histology correlated with the outcome of sperm retrieval by m-TESE in men with NOA. To determine if there was a correlation between FSH levels and positive sperm retrieval rates and assessed the success rate of m-TESE as either a primary or a salvage procedure after previous negative sperm retrieval. The EAU guidelines (2010) recommend that in men with non obstructive azoospermia 'a testicular biopsy is the best procedure to define the histological diagnosis and the possibility of finding sperm'. However, these guidelines do not identify which patients should have a diagnostic biopsy and if this biopsy should be performed as an isolated procedure or synchronously with sperm retrieval. It is also suggested that there is a correlation between the histological diagnosis and possibility of finding sperm on testis biopsy. 100 men with NOA underwent a m-TESE sperm retrieval between 2005 and 2010 at a single centre. All patients underwent hormonal analysis (serum FSH, Testosterone and LH levels) and genetic analyses after full counselling including; Y-deletion, CF-gene analysis and karyotype. Thirty five men had previously undergone unsuccessful TESA/TESE or diagnostic biopsy at other centres. All patients underwent synchronous sperm retrieval and biopsy of the testis, which was sent for histopathological examination on the day of an ICSI cycle or as an isolated procedure. Mean age of patients was 37.25 (range 29-56 years). The mean serum FSH levels in the Sertoli cell only, maturation arrest and hypospermatogenesis groups were 21.3 IU/L (2.8-75), 16.18 (1.6-67) and 14.17 IU/L (0.8-42.3) respectively. SR rates in the respective groups were 42.85%, 26.6% and 75.86% (P= 0.023). There were no post-operative complications. In the 35 men who had previously undergone unsuccessful procedures elsewhere, the SR rates were 57.1%. The overall sperm retrieval rate was 50%. There was no correlation between SR and FSH levels (P= 0.28). M-TESE should be considered the gold standard for retrieval of testicular sperm in NOA, even in cases where there has been previously unsuccessful attempts. FSH levels and histology cannot be used to predict the success of sperm retrieval. An isolated diagnostic testicular biopsy is not recommended in men with NOA, as a significant proportion of men undergoing m-TESE will have successful a sperm retrieval irrespective of previous histology or previous unsuccessful surgery.

Diagnostic testicular biopsy before varicocele repair plays a realistic and important role

We believe that diagnostic testicular biopsy before varicocele repair is important, despite the risks.

Histopathologic patterns of testicular biopsies in infertile azoospermic men with varicocele

In azoospermic infertile men with varicocele, testicular biopsy revealed histopathologic patterns that varied from disorganized spermatogenesis with low or moderate sperm scores to early (primary spermatocytes stage) or late (spermatid stage) arrested spermatogenesis or germ cell aplasia and Sertoli cells only. Diagnostic testicular biopsy can be helpful for accurate management of azoospermic infertile men with varicoceles before surgical repair.

Microdissection Testicular Sperm Extraction: Effect of Prior Biopsy on Success of Sperm Retrieval

We determined the effect of prior biopsies with no sperm seen on the chance of sperm retrieval with microdissection testicular sperm extraction in men with nonobstructive azoospermia. A total of 311 men with NOA underwent microdissection testicular sperm extraction. Of these patients 135 underwent no prior biopsies, 159 underwent 1 or 2 diagnostic testicular biopsies per testis and 17 underwent 3 or 4. The outcome measure studied was the success of sperm retrieval with microdissection testicular sperm extraction. Serum follicle-stimulating hormone and histopathological diagnosis were examined as predictive factors for sperm recovery. Spermatozoa were retrieved in 150 men by microdissection testicular sperm extraction (48%). The success of sperm retrieval in patients who underwent 3 to 4 biopsies (23%) was lower than the retrieval rate in patients who underwent no prior biopsies (56%) and 1 to 2 biopsies per testis (51%) (p = 0.04). When histopathology was considered, patients with Sertoli-cell-only diagnosis on prior diagnostic biopsy had lower retrieval rates compared to the group with no biopsies (p = 0.02). Men with maturation arrest and hypospermatogenesis had similar microdissection testicular sperm extraction sperm retrieval rates regardless of the number of prior biopsies. Spermatozoa recovery was independent of serum follicle-stimulating hormone. There is no threshold of prior negative biopsies that precludes the success of sperm retrieval using microdissection testicular sperm extraction. A limited number of testicular biopsies provide limited or no prognostic value for sperm retrieval with microdissection testicular sperm extraction.

Report on evaluation of the azoospermic male

Report on evaluation of the azoospermic male

Evaluation of Seminal Fluid Parameters, Reproductive Hormones and Testicular Biopsy for non-obstructive Azoospermic Patients with Sertoli cell only Syndrome

The aims of this study were (i) to determine the seminal fluid parameters, levels of serum reproductive hormones, testicular size and diameter of seminiferous tubules depending on multiple testicular biopsies from non-obstructive azoospermic (NOA) patients, and (ii) to assess the effect of smoking on concentrations of serum reproductive hormones. Thirty four NOA patients were involved in the present study. The selection of patients was based on an average of 2 pelleting seminal analysis, reproductive hormones profile and diagnostic testicular biopsy. A detailed questionnaire was used to obtain a thorough history from the patients. The results of this study showed that the mean age of NOA patients with Sertoli cell only syndrome (SCOS) was 33.875 years, and 47.05 % of NOA patients have had 2-4 years duration of infertility. Macroscopic examination of seminal fluid appeared to have normal values as reported by WHO. Non-significant (P>0.05) difference was observed for testicular size and diameter of seminiferous tubules between right and left testis. High and abnormal concentrations of serum FSH were registered for NOA patients with SCOS. However, non-significant (P>0.05) differences were reported for concentrations of serum reproductive hormones between smokers and non smokers. In conclusion, testicular biopsy is the useful and predictable diagnostic tool for NOA patients with SCOS. Further studies are needed to explain the etiology, management and prognosis of NOA patients with SCOS.

Expression of Mucin Genes in the Human Testis and Its Relationship to Spermatogenesis

In this study we investigate the expression pattern of mucin genes in the human testis and evaluate the relationship between the expression of mucin genes and impaired spermatogenesis in the human testis. Thirty human testis tissues were collected from patients undergoing diagnostic testicular biopsy to investigate the cause of infertility. One part of the tissue underwent histological observation, and the other part of the tissue was subjected to semiquantitative RT-PCR of mucin genes, that is, mucin1, 2, 3, 4, and 9. The relative amount of mucin mRNAs was calculated by densitometry using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control. The samples were histologically diagnosed as either obstructive azoospermia with normal spermatogenesis (n = 13) or non-obstructive azoospermia with impaired spermatogenesis (n = 17). In the human testis with normal spermatogenesis, mRNA expression of mucin1, 9, 13 and GAPDH were found, but RT-PCR products of mucin 2, 3 and 4 were not detected. In the testis with impaired spermatogenesis, however, RT-PCR product of mucin1 was not found. There was no difference in the other mucin mRNA expression patterns between the testis with either normal or impaired spermatogenesis. To our knowledge, this study is the first that has detected the mRNA of mucin9 and 13 in human testis. This study also shows that mucin1 expression might be closely related to spermatogenesis. Our findings should be substantiated by more direct evidence, such as mucin protein expression and localization.

Should diagnostic testicular sperm retrieval followed by cryopreservation for later ICSI be the procedure of choice for all patients with non-obstructive azoospermia?

This was a retrospective study to determine if diagnostic testicular biopsy followed by cryopreservation should be the procedure of choice for all patients with testicular failure. The first part of the study analysed 97 ICSI cycles scheduled with frozen-thawed testicular sperm for 69 non-obstructive azoospermia (NOA) patients. The second part focused on a subgroup of 32 patients who underwent 42 ICSI cycles with frozen and 44 cycles with fresh testicular sperm. Sperm characteristics, fertilization, embryo quality, pregnancy and implantation rates were evaluated. Part I: The average time needed to find sperm was 113 min per cycle and 17 min per individual sperm. Fertilization rate, embryo transfer rate, ongoing pregnancy and implantation rates were 58.4%, 83%, 20.8% and 11.3%, respectively. Part II: The search time per sperm was higher (P=0.016) in frozen (18 min) than in fresh suspensions (13 min). A higher embryo transfer rate was observed in fresh cycles than in frozen cycles (93.2% vs 76.2%, P=0.028). Fertilization, ongoing pregnancy and implantation rates were comparable for the two groups. Even in a programme with low-restrictive criteria for patient allocation and for sperm cryopreservation, diagnostic testicular biopsy followed by cryopreservation can be the procedure of choice for patients with testicular failure.

Etiology of azoospermia in 100 consecutive nonvasectomized men

History was taken systematically for 100 azoospermic, nonvasectomized men referred consecutively to a Danish fertility clinic. The men were examined by ultrasound, and their blood samples were analyzed for karyotype, Y microdeletions, and cystic fibrosis transmembrane conductance regulator gene mutations. In 29% of patients, the condition could be explained by genetic abnormalities; in 22%, by diseases or external influence; and in 27%, by former cryptorchidism. The azoospermic condition remained unexplained in only 22%.

La proacrosine, un marqueur acrosomal pour la détection des cellules spermatiques dans le sperme éjaculé de patients azoospermes

Between January 2003 and April 2004, a prospective study was performed on ejaculates from non-obstructive azoospermic men ( n = 95), for the identification of spermatogenic cells using an immunohistochemical labeling for proacrosin. 48.4% of ejaculates (46/95) displayed labeled spermatogenic cells. A 38/95 (40%) of men had testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were extracted from 21 men (52.5%). The sensitivity of detecting spermatogenic cells is 66.7% in predicting the presence of testicular spermatozoa, and its specificity is 76.5%. Compared to histopathological diagnostic testicular biopsy, the detection of spermatogenic cells using proacrosin immunohistochemical method offers a predictive parameter for successful TESE. The immunohistochemical method for proacrosin has the advantages of simplicity and low cost. It could be used to predict spermatogenesis from non-obstructive azoospermic men. Further evaluation is required with extensive results to improve sensitivity and specificity.

Causes of azoospermia and their management.

Azoospermia may occur because of reproductive tract obstruction (obstructive azoospermia) or inadequate production of spermatozoa, such that spermatozoa do not appear in the ejaculate (non-obstructive azoospermia). Azoospermia is diagnosed based on the absence of spermatozoa after centrifugation of complete semen specimens using microscopic analysis. History and physical examination and hormonal analysis (FSH, testosterone) are undertaken to define the cause of azoospermia. Together, these factors provide a >90% prediction of the type of azoospermia (obstructive v. non-obstructive). Full definition of the type of azoospermia is provided based on diagnostic testicular biopsy. Obstructive azoospermia may be congenital (congenital absence of the vas deferens, idiopathic epididymal obstruction) or acquired (from infections, vasectomy, or other iatrogenic injuries to the male reproductive tract). Couples in whom the man has congenital reproductive tract obstruction should have cystic fibrosis (CF) gene mutation analysis for the female partner because of the high risk of the male being a CF carrier. Patients with acquired obstruction of the male reproductive tract may be treated using microsurgical reconstruction or transurethral resection of the ejaculatory ducts, depending on the level of obstruction. Alternatively, sperm retrieval with assisted reproduction may be used to effect pregnancies, with success rates of 25-65% reported by different centres. Non-obstructive azoospermia may be treated by defining the cause of low sperm production and initiating treatment. Genetic evaluation with Y-chromosome microdeletion analysis and karyotype testing provides prognostic information in these men. For men who have had any factors potentially affecting sperm production treated and remain azoospermic, sperm retrieval from the testis may be effective in 30-70% of cases. Once sperm are found, pregnancy rates of 20-50% may be obtained at different centres with in vitro fertilisation and intracytoplasmic sperm injection.

Causes of azoospermia and their management

Azoospermia may occur because of reproductive tract obstruction (obstructive azoospermia) or inadequate production of spermatozoa, such that spermatozoa do not appear in the ejaculate (non-obstructive azoospermia). Azoospermia is diagnosed based on the absence of spermatozoa after centrifugation of complete semen specimens using microscopic analysis. History and physical examination and hormonal analysis (FSH, testosterone) are undertaken to define the cause of azoospermia. Together, these factors provide a >90% prediction of the type of azoospermia (obstructive v. non-obstructive). Full definition of the type of azoospermia is provided based on diagnostic testicular biopsy. Obstructive azoospermia may be congenital (congenital absence of the vas deferens, idiopathic epididymal obstruction) or acquired (from infections, vasectomy, or other iatrogenic injuries to the male reproductive tract). Couples in whom the man has congenital reproductive tract obstruction should have cystic fibrosis (CF) gene mutation analysis for the female partner because of the high risk of the male being a CF carrier. Patients with acquired obstruction of the male reproductive tract may be treated using microsurgical reconstruction or transurethral resection of the ejaculatory ducts, depending on the level of obstruction. Alternatively, sperm retrieval with assisted reproduction may be used to effect pregnancies, with success rates of 25–65% reported by different centres. Non-obstructive azoospermia may be treated by defining the cause of low sperm production and initiating treatment. Genetic evaluation with Y-chromosome microdeletion analysis and karyotype testing provides prognostic information in these men. For men who have had any factors potentially affecting sperm production treated and remain azoospermic, sperm retrieval from the testis may be effective in 30–70% of cases. Once sperm are found, pregnancy rates of 20–50% may be obtained at different centres with in vitro fertilisation and intracytoplasmic sperm injection.

In-vitro maturation (IVM) of fresh and frozen-thawed testicular sperm in non-obstructive azoospermia (NOA) improves ICSI outcomes

In-vitro maturation (IVM) of fresh and frozen-thawed testicular sperm in non-obstructive azoospermia (NOA) improves ICSI outcomes

Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men.

We evaluated the role of the sensitive quantitative telomerase assay (SQTA) in the management of men with non-mosaic Klinefelter's syndrome (KS). Diagnostic testicular biopsy (DTB) was performed in 24 men with KS. A part of the DTB was stained and the remaining fragment was processed for the SQTA. After 3-18 months, a therapeutic testicular biopsy (TTB) was performed in the same testicle and the recovered specimens were processed to identify spermatozoa. Men with a SQTA outcome equal to 0.00 Units microg-1 protein (n = 7) demonstrated therapeutic testicular biopsy material that was negative for spermatogenic cells. In five men with a SQTA outcome of 8.11-38.03 Units microg-1, the most advanced germ cell was the spermatogonium/primary spermatocyte. In the remaining 12 men, the most advanced spermatogenic cell in the TTB was the spermatozoon. In these men, the SQTA outcome was equal to 25.76-92.68 Units microg-1 protein. Using 39.00 Units microg-1 protein as a cut-off value, the accuracy of the SQTA in identifying men positive for spermatozoa was 91.6%. It appears that the SQTA has a role for identifying non-mosaic KS men who have testicular spermatozoa.

Number, distribution pattern, and identification of macrophages in the testes of infertile men

Objective: To investigate the number, location, and secretory products of macrophages in human testes showing normal and abnormal spermatogenesis. Design: Evaluation of testicular biopsies with the use of immunohistochemistry, laser capture microdissection, and reverse transcriptase polymerase chain reaction. Setting: University research and clinical institutes. Patient(s): Infertile men with germ cell arrest (n = 10), Sertoli cell only (n = 8), or mixed atrophy (n = 7) syndromes, and with cases of idiopathic infertility showing normal spermatogenesis (n = 8). Intervention(s): Diagnostic testicular biopsy was performed on participants. Main Outcome Measure(s): We recorded the location, number, distribution, and cytokine expression of human testicular macrophages. Result(s): CD68-positive macrophages were found in the testes of all groups analyzed. These macrophages expressed the genes for interleukin 1 and tumor necrosis factor-α, and were located in the interstitium, tubular wall, and tubular lumen. In Sertoli cell only and germ cell arrest syndromes, the overall macrophage number was increased over twofold. In all pathologic states, there was a significant shift of these cells from the interstitium to the tubules. Conclusion(s): Our study suggests that increased numbers of CD68-positive macrophages directly (via phagocytosis) or indirectly (via paracrine actions exerted through their secretory products) are involved in the regulation of steroidogenesis, Sertoli cell activity, germ cell survival, and, in consequence, in the pathogenesis or maintenance of infertility states in the human testes.