A review of testicular histopathology in nonobstructive azoospermia

Abstract

One major challenge in male factor infertility is nonobstructive azoospermia (NOA), which is characterized by spermatozoa-deficient semen without physical duct blockage. This review offers a thorough overview of the histopathology of the testes in NOA cases, clarifying its complex etiology and emphasizing the possible value of histopathology inspection for both diagnosis and treatment. Variable histopathological findings have been linked to NOA, such as tubular hyalinization, Sertoli cell-only syndrome, hypospermatogenesis, and germ cell arrest. Understanding the pathophysiology and forecasting the effectiveness of treatment are further enhanced by both morphometric and ultrastructural analyses. The potential significance of testicular biopsy in forecasting reproductive outcomes is assessed, especially concerning assisted reproductive technologies like intracytoplasmic sperm injection (ICSI). Besides, testicular microlithiasis, serum hormone profiles, and testicular size are investigated concerning NOA histopathology. It is concluded that understanding the histopathological patterns in NOA is crucial for its accurate diagnosis and appropriate management. Further research is still warranted to improve understanding of the complex pathophysiology underlying NOA.

INTRODUCTION

As a multiplex disorder of the reproductive system, infertility affects 40% of men, 40% of women, and 20% of people of both sexes.1 Azoospermia is a medical disorder in which there is lacking spermatozoa in the ejaculate even after centrifugation.2 Azoospermia is divided into two main types on the basis of the underlying causes: nonobstructive azoospermia (NOA) and obstructive azoospermia (OA).3 In this context, among the various causes of male infertility, NOA stands as a significant challenge whose etiology is complex, encompassing genetic, environmental, hormonal, and anatomical factors.4 Even though some men with NOA are currently able to father biological children due to advances in assisted reproductive technologies, a significant portion still have limited success, highlighting the need for a deeper understanding of the underlying NOA pathophysiology.5

In an effort to understand the intricate etiology of NOA, there has been a recent surge in interest in studying the histopathological characteristics of testicular tissue biopsies in these affected individuals.6 These biopsies can provide insights into the testicular structural changes of the underlying pathophysiological mechanisms contributing to spermatogenic impairment and prediction of successful surgical sperm retrieval (SSR).7,8

This review provides a comprehensive study of the testis histopathology findings in NOA to enhance understanding of the pathological mechanisms that impair spermatogenesis and shed a light on its potential prognostic indicators and treatment options.

TESTICULAR HISTOPATHOLOGY

OA

The male reproductive tract is physically blocked in OA, which keeps spermatozoa from getting to the ejaculate. Congenital disorders such as congenital absence of the vas deferens (CAVD) or acquired obstructions from surgeries or infections are known causes.9 Since the obstruction is usually present downstream from the site of sperm production, the histopathological findings in OA typically display normal testicular tissue with complete spermatogenesis throughout the biopsy as well as the presence of normal intertubular tissue. However, histopathological analysis may reveal reduced spermatogenesis and hypospermatogenesis in some chronic OA and vasectomy cases.10

NOA

Impaired sperm production in the testes without actual reproductive tract blockage is the hallmark of nonobstructive azoospermia (NOA). Depending on the underlying causes, which can be environmental, hormonal imbalances, testicular trauma, or genetic, histopathological findings in NOA can differ.11 In NOA, testicular histology may appear as hypospermatogenesis (HS, all spermatogenesis stages are existing but reduced to variable degrees), maturation cell arrest (MA, overall arrest at a particular stage, mostly at the spermatogonial or primary spermatocyte stage), Sertoli cell-only syndrome (SCOS, germ cell aplasia, the tubules lack germ cells whereas Sertoli cells only are lining the seminiferous tubules), and tubular hyalinization (tubules without germ cells or Sertoli cells and are usually accompanied by peritubular fibrosis).10

It is crucial to know that different people have different histopathological results, and histology alone may not always be able to determine the precise cause of azoospermia. It may be necessary to make use of clinical assessment, hormone testing, genetic testing, and other diagnostic techniques to identify the underlying cause and the best sequence of actions in each case.12

Johnsen score

Based on the most mature germ cell in each tubule section, the Johnsen score is a commonly used quantitative histological grading method to evaluate the level of sperm maturation on a scale of 1 to 10 in at least 100 seminiferous tubules. The sum score is divided by the total number of tubules evaluated to get the overall Johnsen score.13 A significant disadvantage of this approach is that the average tubule score might not accurately reflect the state of spermatogenesis. For instance, tubules with a SCOS pattern and tubules with normal spermatogenesis could coexist in the same biopsy, leading to an average score that denotes spermatogenic disruption.14

MORPHOMETRIC AND ULTRASTRUCTURAL HISTOLOGICAL ANALYSIS OF NOA

The measurement of the testicular tissue’s structural characteristics, such as tubular diameter, basement membrane thickness, epithelial height, and tubular/tissue ratio, is carried out by morphometric analysis to clarify the cause of NOA. Spermatogenic disorders are frequently indicated by decreased seminiferous tubule diameter and increased tubular wall thickness. Besides, a rise in Sertoli cells, germ cells, or differences in their nuclear size may indicate a maturation arrest.15,16

In ultrastructural analysis, transmission electron microscopy (TEM) is crucial because it allows for the visualization of cell and subcellular organization that is invisible with the light microscopy. In SCOS, ultrastructural analysis may identify a small number of germ cells that are not visible otherwise. In addition, TEM sheds light on components of Leydig cell function, including the endoplasmic reticulum, mitochondria, and intracellular lipid droplets, which offer useful hints regarding the synthesis of testosterone.17

In order to provide an opportunity for early intervention, comprehensive morphometric and ultrastructural analysis helps in the early detection of subtle pathophysiological changes preceding full-blown histological alteration. Identifying these patterns may help forecast the outcome of treatments such as SSR or hormone replacement therapy and the potential need for assisted reproductive techniques.18

TESTICULAR SIZE AND HISTOPATHOLOGICAL CHANGES IN NOA

Given that testicular size is a good indicator of the seminiferous tubule volume, which represents the testes’ capacity for spermatogenetic activity, it is a useful physical examination parameter in male infertility. Each testis typically has dimensions of 3 cm in diameter and 4–5 cm in length.19

In certain conditions, such as testicular atrophy, where the testicles shrink in size due to decreased functioning or loss of testicular tissue, there may be a correlation between testicular size and histopathological findings. Changes in the seminiferous tubules, such as degeneration, fibrosis, or loss of germ cells, may be seen by the histopathological examination.20 In NOA cases, testicular size is often reduced as a result of poor or absent spermatogenesis. As a result, a reduction in the testicular volume typically indicates an underlying histopathological defect.21 Nevertheless, a decrease in testicular size is not exclusively associated with NOA and may also point toward other conditions, such as undescended testis or an associated varicocele.22,23

Prior to SSR, testicular size can be used as a noninvasive prognostic indicator. Smaller testes have a lower chance of successfully retrieving spermatozoa due to the probable severe undersupply of germ cells, whereas larger testes typically show a higher likelihood. Although useful, testicular size alone cannot accurately diagnose or predict SSR success in NOA.24 There is a nonlinear relationship between testicular size and spermatogenetic capacity because smaller testes may still have some degrees of functional spermatogenesis, while larger testes may show marked spermatogenetic impairment. Consequently, testicular size ought to be taken into account as a component of the thorough clinical assessment rather than as a stand-alone diagnostic criterion.25

SERUM HORMONE PROFILES AND TESTICULAR HISTOPATHOLOGY PATTERNS IN NOA

Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels are among the reproductive hormones that are commonly measured as part of the serum hormonal evaluation in NOA. Elevated levels of serum FSH may suggest that the testes are not reacting correctly to the pituitary gland’s regulatory cues.21 In NOA, serum T levels can range from low to normal. Impaired testicular function can lead to low T levels. In certain situations, there may also be an increase in serum LH levels, which is a result of the testes being stimulated by hormones to produce more T.26

Although more severe cases of NOA are frequently linked to elevated serum FSH levels and abnormal testicular histopathology, hormonal levels by themselves may not always be predictive of the histopathological findings in NOA cases; in some individuals, severe testicular histopathology may coexist with normal or nearly normal hormone levels.27

NOA HISTOPATHOLOGY AND TESTICULAR MICROLITHS (TM)

Ultrasonography-detected TM is the presence of five or more hyperechogenic spots.28 A condition known as limited TM is characterized by fewer than five hyperechogenic spots per sonogram, whereas cases classified as classical TM had five hyperechogenic spots per sonogram.29 Along with their study, Barda et al.30 observed that in 16 out of 110 men with NOA (14.5%), the prevalence of TM was correlated with histopathology. In that subgroup, the SSR rate was only 6.2% (1/16), while in men with NOA who did not exhibit TM symptoms, it was 39.4% (37/94; P = 0.009). The only independent predictor of failed SSR was the existence of TM (odds ratio: 7.4, 95% confidence interval: 2.3–12.2; P = 0.01). Histopathology determined that 15 patients with TM who did not have a successful SSR had SCOS. Otherwise, Rassam et al.31 noted that TM in NOA patients was not significantly correlated with SSR rate in microtesticular sperm extraction (micro-TESE) procedures or with other reproductive parameters in their study.

NOA histopathology and reproductive outcome

Although the results can vary, ICSI in conjunction with effective SSR techniques is still a viable option for patients with NOA to achieve fertility. Determining the likelihood of a successful SSR for TESE or micro-TESE-assisted reproductive techniques can be aided by specific histopathological patterns seen in a patient with NOA.32 Except for Y chromosome microdeletions in regions of azoospermic factor a (AZFa)/AZFb that predict a poor outcome, none of the predictors, individually or in combination, could definitively indicate the likelihood of harvesting spermatozoa from men with NOA.33 Klinefelter syndrome and SCO histopathology patterns were linked to decreased SSR success. There is a significant decrease in SSR with the decline in NOA testicular histopathology from HS to MA, and SCOS, using various techniques, including conventional TESE (cTESE), micro-TESE, and fine needle aspiration cytology (FNAC).

According to an earlier research by Seo and Ko,34 spermatozoa were recovered in 16.3% of cases with SCOS, 62.5% of cases with MA, and 89.2% of cases with severe HS. Along with their study, Colpi et al.35 pointed out that the odds of SSR were 8.4 times higher in MA and 44.3 times higher in severe HS when compared to SCOS. Twenty-one in 22 testicles with HS (micro-TESE: 11/11; cTESE: 10/11), 12/14 with MA (micro-TESE: 6/7; cTESE: 6/7), 16/22 with incomplete SCOS (micro-TESE: 8/11; cTESE: 8/11), and 16/80 with complete SCOS (micro-TESE: 11/40; cTESE: 5/40) all had successful SSR results. Kalsi et al.36 demonstrated that the results of the SSR from micro-TESE in 100 men with NOA were 42.9%, 26.6%, and 75.9% for the SCOS, MA, and HS groups, respectively. According to testicular histology, the micro-TESE SSR yields in the SCOS, MA, and HS groups were 36%, 48.6%, and 95.5%, respectively, as demonstrated by Cetinkaya et al.37

In their study, Caroppo et al.38 showed that patients with HS had a significantly higher diagnostic accuracy for predicting SSR in 356 NOA patients undergoing cTESE retrospectively than patients with SCOS or MA (88.2% vs 30.5% and 30.9%, respectively). Toksoz and Kizilkan6 showed that SSR success rate from micro-TESE was significantly higher in HS and incomplete MA groups (93.2% and 72.5%, respectively) than those with complete MA, SCOS, and fibrotic/atrophic patterns (32.3%, 27.5%, and 20.0%, respectively). In line, Falcone et al.39 associated significantly testicular histopathology and successful SSR (HS: 100%; MA: 32%; and SCOS: 22%). Lantsberg et al.40 also observed that the highest SSR rate was observed in patients with HS (92.9%) and the SSR rate was low in patients with SCOS (46.3%). Ghalayini et al.41 showed that the feasibility of SSR in men with NOA was the highest in HS patients (95.6%), followed by MA (58.5%) and SCOS (56.0%). It was demonstrated that the success of the previous trial significantly increased the probability of success by 10.1-fold in the second trial, 5.6-fold in the third trial, and 16.5-fold in the fourth trial. An increased duration between the first and second biopsies significantly increases the success rate by a factor of 1.3-fold per month; however, afterward, the duration did not play a role in the success of micro-TESE.

Ezeh et al.42 concluded that an open testicular specimen is more effective than FNAC for retrieving testicular spermatozoa from patients with NOA (63% vs 14%) according to the amount of testicular tissue retrieved and the status of testicular histology. In their study, Bettella et al.43 reported that spermatozoa were detected by FNAC in 24/42 men with severe HS and 9/13 men with MA, while they were retrieved by TESE in 29/70 men with SCOS, 35/42 men with severe HS, and 10/13 men with MA. Along with their study, El-Haggar et al.44 concluded that micro-TESE is superior to FNAC as regards sperm retrieval rate and lower incidence of complications in NOA patients. A meta-analysis carried out by Yang et al.,45 covering 25 articles, showed that SSR rates of the FNAC, cTESE, and micro-TESE were 23%, 52.2%, and 54.6%, respectively, being closely correlated with the testicular pathological category of the NOA patients in HS, MA, and SCOS (76.7%, 6.2%, and 32.8%, respectively).

Collectively, Esteves et al.46 pointed out that SSR success depends on many different factors, including patient selection criteria, the surgeon’s experience, the embryologist’s experience, and laboratory technique used to process the retrieved specimens.

Role of testicular biopsy

Testicular biopsy was always considered the keystone of male infertility diagnosis for many years in NOA men. Currently, testicular biopsies are mainly performed in NOA men for retrieving spermatozoa for subsequent ICSI.13 Abdel Raheem et al.7 showed that the strongest predictor of the successful TESE is when tubules with mature spermatozoa (Johnsen score ≥8) are found in the histopathology. Diagnostic biopsy is best joined with a preliminary TESE before starting ICSI cycle. Based on the histopathology findings, the patients may be offered a repeated TESE if more spermatozoa are needed on the day of ovum pick-up and ICSI. If the preliminary TESE was negative, the biopsy result might help in deciding whether to repeat TESE. This schedule can be more cost-effective because the ICSI cycle will be ongoing only if satisfactory sperm numbers are retrieved. In a retrospective study, Amer et al.47 evaluated testicular histopathology in determining the successful SSR in 1395 NOA patients who underwent first-time micro-TESE. That study revealed that a preoperative testicular biopsy was unnecessary to predict SSR in NOA patients.

Cell suspension examination with histopathology

Tang et al.48 investigated 1112 NOA patients who underwent TESE and whose testicular tissue was subjected to cell suspension examination and histopathology. The consistency rate of the two methods was 92.63%, with SSR of 41.82% and a nonsperm detection rate of 50.81%. Spermatozoa were retrieved from 96.0% (24/25) of the patients on the day of oval retrieval, in whom spermatozoa were found in the cell suspension examination but not in histopathology. These authors concluded that cell suspension examination combined with histopathology for detecting spermatozoa in the testicular tissue of NOA patients gives instant, accurate, reliable, and consistent results ensuring SSR for NOA patients.

NOA histopathology and ICSI outcome

Kuai et al.49 investigated whether testicular histology influences the clinical outcomes of ICSI in NOA cases (HS: n = 72; MA: n = 21; and SCOS: n = 12). The rates of fertilization, failed fertilization, transferrable embryos, and high-quality embryos and the average number of transferred embryos were 67.0% (553/825), 9.5% (10/105), 85.7% (472/551), 35.0% (193/551), and 2.10, respectively, resulting in 44 (55.7%) pregnancies and 42 (53.2%) live births, without birth defects. No significant differences were observed among different groups (HS vs MA vs SCOS) in the mean age of either men or women, infertility duration, rate of fertilization (68.5% vs 64.4% vs 61.5%), transferrable embryos (85.1% vs 90.5% vs 83.1%), or high-quality embryos (33.1% vs 41.7% vs 39%). The rates of clinical pregnancy and embryo implantation were higher in the HS (60.0% and 37.6%) and SCOS (62.5% and 50.0%) than those in the MA group (37.5% and 21.2%), but without significant differences (all P > 0.05). These authors concluded that once testicular spermatozoa are retrieved, desirable clinical outcomes can be achieved in ICSI for NOA patients, who are not affected by abnormal testicular histopathology.

NOA and germ cell neoplasia in situ (GCNIS)

Testicular cancer, with 74 500 new cases worldwide in 2020, ranks as the 20^th most common cancer globally and the leading cancer among men aged 15–44 years in Europe.50 GCNIS is the precursor of seminomatous and nonseminomatous germ cell tumors consisting of expanded tubules that have intratubular seminoma or embryonal carcinoma cells.51 Zorn et al.52 estimated the incidence of GCNIS in testicular biopsies among infertile men to range from 0 to 3.5% during the 1970s and 1990s, while Mancini et al.53 estimated its incidence after the 1990s to range from 0.54% to 6.3%. This heterogeneity might suggest that infertility itself may not be the true risk factor but rather the conditions associated with infertility.54

Pang et al.55 analyzed 8 nonrandomized studies involving 469 men with testicular tumor (TT; azoospermia: n = 57; no azoospermia: n = 412). Results from azoospermic vs nonazoospermic cases demonstrated that bilateral TT (12.3% vs 2.9%), nonseminoma germ cell tumors (6.4% vs 1.9%), GCNIS (11.1% vs 1.2%), stage 2–3 disease (22.2% vs 0%), SCOS on biopsy (60% vs 37.5%) and a history of undescended testis (66.7% vs 50%) were more common in azoospermic men. Leydig cell tumors and hyperplasia were only detected in men with azoospermia. Ultrasonographic findings from NOA men with TT had a higher percentage of MT compared with those without TT (50% vs 15.2%).

CONCLUSIONS

This review highlights that good understanding of the histopathological patterns in NOA is decisive for its accurate diagnosis, prognosis, and appropriate management. Further research is still warranted to improve understanding of the complex pathophysiology underlying NOA and to find more accurate predictors of SSR.

AUTHOR CONTRIBUTIONS

TM, KB, and VM contributed to data search, drafting the manuscript, reviewing the manuscript, critically revising the manuscript, and read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.