Therapeutic Testicular Biopsy Research Articles

Evaluating the Predictive Value of Diagnostic Testicular Biopsy for Sperm Retrieval Outcomes in Men with Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA) presents a challenge in male infertility management. This study aimed to assess the efficacy of diagnostic testicular biopsy (DTB) in predicting sperm retrieval success via therapeutic testicular biopsy (TTB) and to understand the role of systemic inflammation in microdissection testicular sperm extraction (mTESE) outcomes. A retrospective analysis was conducted on 50 NOA males who underwent mTESE at the University of Ioannina's Department of Urology from January 2017 to December 2019. All participants underwent thorough medical evaluations, including semen analyses and endocrinological assessments. DTB did not detect spermatozoa in half of the patients who later showed positive sperm findings in TTB. Preoperative variables, such as age, plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (TT), prolactin (PRL), estradiol (E2), and inflammation biomarkers (neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), monocyte-eosinophil ratio (MER)), were not consistently predictive of sperm retrieval success. Notably, TTB-negative patients had elevated NLR and PLR values, suggesting a possible link between systemic inflammation and reduced sperm retrieval during mTESE. The findings question the necessity of an initial DTB, which might provide misleading results. A negative DTB should not deter further TTB or intracytoplasmic sperm injection (ICSI) attempts. The study emphasizes the need for further research to refine diagnostic approaches and deepen the understanding of factors influencing sperm retrieval in NOA patients, ultimately enhancing their prospects of biological parenthood.

252 Telomerase assay in percutaneously recovered testicular cells predicts the outcome of a subsequent therapeutic testicular biopsy

252 Telomerase assay in percutaneously recovered testicular cells predicts the outcome of a subsequent therapeutic testicular biopsy

Recovery of ejaculated spermatozoa for intracytoplasmic sperm injection after anti-inflammatory treatment of an azoospermic patient with genital tract infection: a case report

In this paper our experiences with anti-inflammatory treatment of an infertile patient with azoospermia and concomitant silent genital infection are reported. The patient was referred to our fertility centre with prediagnosed non-obstructive azoospermia and no spermatozoa were found in the ejaculate on two occasions. The patient showed leukocytospermia and was suspected to be affected by genital infection. Therefore, anti-inflammatory treatment was initiated and 8 weeks later examination of the ejaculate revealed a decreased number of leukocytes and the presence of few but motile spermatozoa. Subsequently, two ICSI cycles were performed with anti-inflammatory therapy in parallel and a sufficient number of spermatozoa could be retrieved for injection. However, in a third cycle without previous treatment, examination of the ejaculate again revealed azoospermia and leukocytospermia. It is concluded that, in cases of azoospermia and chronic genital infection, some patients will benefit from anti-inflammatory treatment prior to and during ICSI treatment. This may allow retrieval of spermatozoa from the ejaculate and thus may avoid the need for a therapeutic testicular biopsy. Using this approach, sufficient spermatozoa in three out of four ICSI cycles could be retrieved and a pregnancy was finally achieved.

Commentary on Incidental testicular lesions found during infertility evaluation are usually benign and may be managed conservatively

Hypoechoic lesions on scrotal ultrasonography are often considered germ cell tumors and radical orchiectomy is recommended. We retrospectively reviewed the findings at 1 center in men with ultrasonographically detected testicular lesions found during evaluation of severe male infertility. A total of 145 men with nonobstructive azoospermia at 1 center underwent ultrasonographic analysis before diagnostic or therapeutic testicular biopsy. Mean age was 34 ± 0.6 years (range 21–63). All men were azoospermic. Mean serum follicle-stimulating hormone was 25 IU/l. Of the men 26% had a history of cryptorchidism and 3 patients had a history of testis tumor. No other risk factors for testis cancer were identified for any patient. All sonographic lesions were followed with serial ultrasound examinations or were biopsied/excised. All men had tumor markers tested and the results were negative. Of 145 men referred for azoospermia who underwent ultrasonographic analysis before biopsy, 49 (34%) showed a focal sonographic abnormality. A hypoechoic lesion was seen in 20 patients (14%), a hyperechoic lesion was seen in 10 (7%) and a heterogeneous appearance to a region of testicular parenchyma was seen in 19 patients (13%). Some lesions classified as hypoechoic demonstrated hyperechoic or heterogeneous interior components. Two of the patients with hypoechoic lesions were lost to follow-up. Of the remaining 18 patients, 11 had lesions less than 5 mm in greatest diameter and all of these were confirmed to be benign. Only 1 patient had a seminoma, and that patient had an inguinal testis with a mass detected on routine ultrasound. All other patients with hyperechoic or heterogeneous areas on ultrasound with subsequent tissue diagnoses were found to have benign lesions. Men with severe infertility who are found to have incidental testicular lesions and negative tumor markers, especially lesions less than 5 mm, may be initially observed with serial scrotal ultrasound examinations. Enlarging lesions or those of greater dimension should be considered for histological examination.

Incidental Testicular Lesions Found During Infertility Evaluation are Usually Benign and May be Managed Conservatively

Hypoechoic lesions on scrotal ultrasonography are often considered germ cell tumors and radical orchiectomy is recommended. We retrospectively reviewed the findings at 1 center in men with ultrasonographically detected testicular lesions found during evaluation of severe male infertility. A total of 145 men with nonobstructive azoospermia at 1 center underwent ultrasonographic analysis before diagnostic or therapeutic testicular biopsy. Mean age was 34 +/- 0.6 years (range 21 to 63). All men were azoospermic. Mean serum follicle-stimulating hormone was 25 IU/l. Of the men 26% had a history of cryptorchidism and 3 patients had a history of testis tumor. No other risk factors for testis cancer were identified for any patient. All sonographic lesions were followed with serial ultrasound examinations or were biopsied/excised. All men had tumor markers tested and the results were negative. Of 145 men referred for azoospermia who underwent ultrasonographic analysis before biopsy 49 (34%) showed a focal sonographic abnormality. A hypoechoic lesion was seen in 20 patients (14%), a hyperechoic lesion was seen in 10 (7%) and a heterogeneous appearance to a region of testicular parenchyma was seen in 19 patients (13%). Some lesions classified as hypoechoic demonstrated hyperechoic or heterogeneous interior components. Two of the patients with hypoechoic lesions were lost to followup. Of the remaining 18 patients 11 had lesions less than 5 mm in greatest diameter and all of these were confirmed to be benign. Only 1 patient had a seminoma, and that patient had an inguinal testis with a mass detected on routine ultrasound. All other patients with hyperechoic or heterogeneous areas on ultrasound with subsequent tissue diagnoses were found to have benign lesions. Men with severe infertility who are found to have incidental testicular lesions and negative tumor markers, especially lesions less than 5 mm, may be initially observed with serial scrotal ultrasound examinations. Enlarging lesions or those of greater dimension should be considered for histological examination.

Role of testicular tissue telomerase assay for the prediction of the presence of testicular spermatozoa in azoospermic men with varicoceles, pre- and post-varicocelectomy.

We evaluated the reproductive potential of frozen/thawed testicular spermatozoa of azoospermic men with left varicocele. The role of testicular tissue telomerase assay (TTA) in the prediction of the presence of testicular spermatozoa pre- and post-varicocelectomy was investigated, as well. Therapeutic testicular biopsy and TTA were performed in 82 nonobstructed azoospermic (NOA) men with varicoceles. Testicular spermatozoa were found in 33 men and processed for cryopreservation. Oocytes were later recovered from the spouses of the latter azoospermic men with varicoceles and injected with frozen/thawed testicular spermatozoa. Among the 49 men who were negative for testicular spermatozoa, 22 men underwent subsequently subinguinal microsurgical varicocelectomy. A total of 198 mature oocytes were successfully injected and 101 were normally fertilized and subsequently cleaved. Transfer of these 101 embryos in 26 women resulted in nine full-term pregnancies. Thirteen healthy babies were delivered. A cut-off value of TTA of 39 TPG U microg(-1) protein had an overall diagnostic accuracy equal to 90.2% to predict the presence of testicular spermatozoa pre-varicocelectomy. Within the group of men who were negative for testicular spermatozoa a cut-off value of TTA equal to 28 TPG U microg(-1) protein (pre-varicocelectomy) had a 84.2 % diagnostic accuracy to recognize the men who would become positive for either ejaculated or testicular spermatozoa post-varicocelectomy. Testicular spermatozoa can be found in 40% of NOA men with left varicocele. Ooplasmic injections with frozen/thawed testicular spermatozoa have a role in the therapeutic management of non-obstructive azoospermia associated with varicocele. Pre-varicocelectomy, a TTA cut-off value equal to 39 TPG U microg(-1) protein has a 90.2% diagnostic accuracy to indicate the men positive/negative for testicular spermatozoa. In addition, pre-varicocelectomy, a cut-off value equal to 28 TPG U microg(-1) protein has a 84.2% diagnostic accuracy to identify those men with varicoceles without testicular spermatozoa, who will become positive/negative for spermatozoa (either ejaculated or testicular) post-varicocelectomy.

The role of ultrasonographically guided puncture of the human rete testis in the therapeutic management of nonobstructive azoospermia.

We attempted to characterize the cells collected from the rete testis via ultrasonographically guided puncture. Unilateral puncture of the rete testis was performed in nine men with obstructive azoospermia and 51 men with nonobstructive azoospermia. All the aspirated samples from the rete testis were observed via confocal scanning laser microscope and some of them after fluorescent in situ hybridization techniques. Then therapeutic testicular biopsy was performed in the punctured testis of each man. Spermatozoa were found in all rete testis samples and all biopsy samples from obstructed men. Twenty-two nonobstructed men demonstrated absence of spermatozoa in biopsy samples. Twenty-nine nonobstructed men showed spermatozoa in biopsy material and 24 of these men (82%) had demonstrated spermatozoa in rete testis samples. There were no significant differences in fertilization and cleavage rate between intracytoplasmic sperm injection trials using biopsy spermatozoa and rete testis spermatozoa both in obstructed and nonobstructed men. Considering that puncture of the rete testis does not reduce the volume of testicular parenchyma, is less invasive and apparently causes less detrimental effect on testicular vasculature than biopsy, puncture of rete testis is recommended as first line approach for the treatment of azoospermic men. If puncture is negative for spermatozoa in nonobstructed men, biopsy is indicated.

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.

Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter's syndrome.

Klinefelter's syndrome is the most frequent chromosomal abnormality in infertile men. In this study, the chromosomes of round spermatids and spermatogonia/primary spermatocytes from men with non-mosaic Klinefelter's syndrome were examined, together with the Sertoli cell secretory function and sperm morphometry. Twenty-four men with non-mosaic Klinefelter's syndrome and nine men with obstructive azoospermia underwent therapeutic testicular biopsy. When spermatozoa in the final filtrate were present, they were processed for sperm morphometry or ICSI. Sperm morphometry was evaluated by the maximal length and width of the sperm head, the length of the midpiece and the ratio of the acrosomal region to the total surface area of the head. When round spermatids were present, they were processed for fluorescent in-situ hybridization (FISH). FISH was also applied to fragments of seminiferous tubules. Sertoli cell secretory function was measured by the amount of androgen binding protein (ABP) secreted in vitro. More than 93% of the evaluated round spermatids were normal. The proportions of 24,XY and of 24,XX round spermatids to the total number were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Men with Klinefelter's syndrome who had spermatozoa in their testicular tissue (n = 12) were positive for both 46,XY and 47,XXY spermatogonia in their seminiferous tubules. In contrast, men with Klinefelter's syndrome without spermatozoa in their testicular tissue (n = 12) were positive for 47,XXY spermatogonia but negative for 46,XY spermatogonia in their seminiferous tubules. ABP profiles were significantly smaller in men with Klinefelter's syndrome who were negative for spermatozoa compared with men who were positive. Four pregnancies were achieved and five healthy babies were born. This study suggests that few 46,XXY spermatogonia undergo meiosis with an XX pairing and a Y univalent type of pairing. Hyperhaploid round spermatids (24,XY and 24,XX) may be produced by meiosis of 47,XXY spermatogonia. Men with Klinefelter's syndrome who are negative for testicular spermatozoa have a greater degree of Sertoli cell secretory dysfunction compared with men with Klinefelter's syndrome who are positive for spermatozoa. There are several defects in sperm morphometry with functional significance in men with Klinefelter's syndrome.

THE ROLE OF TESTICULAR BIOPSY IN THE MODERN MANAGEMENT OF MALE INFERTILITY

We evaluate the traditional role of isolated testicular biopsy as a diagnostic tool, as opposed to the value as a therapeutic procedure for azoospermic men. The medical records of azoospermic patients who were evaluated, and treated between 1995 and 2000 were retrospectively analyzed for history, physical examination findings, endocrine profiles, testicular histology and sperm retrieval rates. Based on these parameters, cases were placed into diagnostic categories that included obstructive or nonobstructive azoospermia. Diagnostic parameters used to distinguish obstructive from nonobstructive azoospermia were subjected to statistical analysis with the t-test, analysis of variance and receiver operating characteristics curve. A total of 153 azoospermic men were included in our analysis. Of men with obstructive azoospermia 96% had follicle-stimulating hormone (FSH) 7.6 mIU/ml. or less, or testicular long axis greater than 4.6 cm. Conversely, 89% of men with nonobstructive azoospermia had FSH greater than 7.6 mIU/ml., or testicular long axis 4.6 cm. or less. Receiver operating characteristics analysis revealed that FSH, testicular long axis, and luteinizing hormone were the best individual diagnostic predictors, with areas 0.87, 0.83 and 0.79, respectively. In the vast majority of patients obstructive azoospermia may be distinguished clinically from nonobstructive azoospermia with a thorough analysis of diagnostic parameters. Based on this result, we believe that the isolated diagnostic testicular biopsy is rarely if ever indicated. Men with FSH 7.6 mIU/ml. or greater, or testicular long axis 4.6 cm. or less may be considered to have nonobstructive azoospermia and counseled accordingly. These men are best treated with therapeutic testicular biopsy and sperm extraction, with processing and cryopreservation for usage in in vitro fertilization and intracytoplasmic sperm injection if they accept advanced reproductive treatment. Diagnostic biopsy is of no other value in this group. Men with FSH 7.6 mIU/ml. or less, or testicular long axis greater than 4.6 cm. may elect to undergo reconstructive surgery with or without testicular biopsy and sperm extraction, or testicular biopsy and sperm extraction alone depending on their reproductive goals.

Criteria predicting the absence of spermatozoa in the Sertoli cell-only syndrome can be used to improve success rates of sperm retrieval

In patients with non-obstructive azoospermia, testicular sperm extraction (TESE) is a method of choice to recover spermatozoa as a male therapeutic approach in intracytoplasmic sperm injection (ICSI) programmes. However, the efficacy of TESE in this indication is burdened by a frequent failure of sperm recovery, which renders useless both the invasive testicular intervention and ovarian stimulation of the patient's spouse. One of the most frequent pathological pictures characterizing complete absence of spermatozoa is germinal aplasia (Sertoli cell- only syndrome or SCOS). Two different histological patterns of SCOS have been already described during the past five decades. These two patterns can be characterized as the congenital (pure) and the secondary (mixed) forms. Both patterns, with different prognosis to retrieve spermatozoa by therapeutic testicular biopsy, are frequently confused when TESE is performed during ICSI programmes. Useful criteria to predict the absence of spermatozoa can be obtained by a definite recognition of the two typical histological patterns during the diagnostic testicular biopsy. The diagnosis of congenital or acquired SCOS can be refined by endocrine, chemical, immunohistochemical and molecular biology aids. Reduction of both sperm retrieval failure and unnecessary ovarian stimulation can be achieved by combination of these methods.

Effects of Hormones on Spermatogenesis in Men with Histological Diagnosis of Spermatogenic Arrest at the Primary Spermatocyte (PS) Stage or Sertoli Cell-Only Syndrome (SCOS)

Objectives: Men with absence of spermatozoa in the therapeutic testicular biopsy (TTB) material represent the most difficult cases of non-obstructive azoospermia (NOA). Even when alive round spermatids (RSs) are present in the TTB samples of the latter men, the outcome of ooplasmic injections of RSs rarely alleviates the couple’s infertility. We evaluated the effects of a combined administration of Profasi (Serono Co.) and Gonal-F (Serono Co.) on testicular spermatogenic and steroidogenic function. Design: Diagnostic testicular biopsy (DTB; hematoxylin-eosin stain) outcome and TTB (mincing testicular tissue and processing it for assisted reproduction trial) outcome were compared before and after hormonal treatment in three groups of NOA-men. Materials and Methods: All men had peripheral serum LH and FSH values less than twice the upper normal level of each respective hormone. TTB was performed at least four months after the performance of DTB. At the time of DTB, intratesticular testosterone (ITT) was measured, whereas, at the time of TTB, fractions of dispersed cells were processed for fluorescent in situ hybridization (FISH) techniques or observation via transmission electron microscope (TEM). Gonal-F (50 IU three times a week) and Profasi (2,500 IU twice a week) were administered for 12–24 months in a) 21 men whose DTB showed arrest at the PS stage and the TTB revealed absence of RSs and spermatozoa (group A), b) 7 men whose DTB demonstrated arrest at the PS stage and the TTB was positive for RSs but negative for spermatoa/elongating/elongated spermatids (group B), and c) 5 men whose DTB showed SCOS and the TTB demonstrated few PSs but not RSs (group C). New DTB, TTB, FISH, and TEM techniques were performed at the end of the treatment. Results: At the end of the homonal treatement, DTB images showed the same level of spermatogenic arrest as prior to treatment in all men of groups A, B, and C. Peripheral serum testosterone increased in all the participants. Observations of TTB samples post-treatment demonstrated few RSs in 8 men of group A. One of the latter 8 men showed spermatozoa, as well. No RSs were observed in TTB-samples of group C post-treatment. TEM showed elongating or elongated spermatids having tail length 0.004–0.047 mm in the TTB materials of 5 men of group B post-treatment. However, testicular spermatozoa were found in only two of the latter men. When the groups A and B were analyzed together it was found that the 13 men who responded to the treatment (development of RSs in the second TTB for group A; spermatid elongation in the second TTB for group B) had (prior to the hormonal treatment) significantly lower (P<0.05; Wilcoxon’s test) ITT profiles than the 15 remaining men who did not respond to the treatment. There was a large variation in ITT profiles prior to the treatment in groups A and B (taken together the groups A and B: 76–2675 ng/g testis). Four out the five men of group B who responded to the treatment had relatively low ITT profiles (less than 300 ng/g testis) pre-treatment. Conclusions: Hormonal treatment in men with early maturation arrest stimulates spermatogenesis in a clinically significant percentage (46%). Prior to the hormonal administration, the presence of RSs in the TTB of men with histological diagnosis of early maturation arrest or small ITT levels indicates good prognosis for response to the treatment. Hormonal treatment in SCOS-men without foci of RSs/spermatozoa is not recommended. This research was supported by a grant from the JG Fertility Institute.

Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy in men with Sertoli cell-only syndrome.

The role of a telomerase assay in the recognition of Sertoli cell-only syndrome with testicular foci of haploid cells was evaluated. Men with Sertoli cell-only syndrome (n = 23) were given a new diagnostic testicular biopsy. Part of the biopsy was stained and the remainder was processed for the quantitative telomerase assay. After 3-13 months, a therapeutic testicular biopsy was performed. This material was minced and then examined using confocal laser scanning microscopy and fluorescent in-situ hybridization. Histology of diagnostic testicular biopsy material confirmed the diagnosis of Sertoli cell-only syndrome in all the participants. All seven men with a telomerase assay value in their diagnostic testicular biopsy of >42 total product generated (TPG) U/microg protein had haploid cells (i.e. spermatozoa and/or spermatids) in their therapeutic testicular biopsy. Among participants with telomerase assay values <42 TPG U/microg protein, only one man had haploid cells in his therapeutic testicular biopsy. Thus, telomerase assay values >42 TPG U/microg protein in the diagnostic biopsy identified 87.5% of the Sertoli cell-only syndrome men with haploid cells in their therapeutic testicular biopsy. Significantly higher values of the telomerase assay were found in men with testicular foci of haploid cells than in men without these foci. The use of a quantitative telomerase assay biopsy appears to be important for identifying those men with Sertoli cell-only syndrome who have foci of haploid cells and can be candidates for assisted reproduction techniques.

P-107. Testicular testosterone and telomerase activity of diagnostic testicular biopsy have a role in the prediction of the outcome of the therapeutic testicular biopsy

P-107. Testicular testosterone and telomerase activity of diagnostic testicular biopsy have a role in the prediction of the outcome of the therapeutic testicular biopsy

R-016. Experience with therapeutic testicular biopsy for testicular sperm extraction

R-016. Experience with therapeutic testicular biopsy for testicular sperm extraction