Anatomical considerations, testicular, and scrotal anatomy of nonobstructive azoospermia patients

Abstract

Infertility, defined as the inability to conceive after 1 year of regular unprotected intercourse, impacts 10%–20% of couples globally. Both male and female factors contribute equally to this condition. Azoospermia, particularly nonobstructive azoospermia (NOA), which affects 10%–15% of infertile men, represents a significant challenge in male infertility. The advent of assisted reproductive technology (ART), specifically microdissection testicular sperm extraction (micro-TESE) followed by intracytoplasmic sperm injection (ICSI), offers a possibility for men with NOA to father biological children. Recent studies have focused on the predictors of sperm retrieval in NOA patients, such as age, testicular volume, and follicle-stimulating hormone (FSH) level. This review aims to explore the limited data on the anatomical characteristics of NOA patients and provide surgical considerations for micro-TESE, thereby enhancing understanding and improving outcomes for this challenging condition.

INTRODUCTION

Infertility is defined as the inability to conceive after 1 year of regular and unprotected intercourse. It affects 10%–20% of couples globally, with male and female factors each contributing equally.1 Azoospermia is the complete absence of spermatozoa in the centrifuged semen samples at least twice, which accounts for 10% of male infertility.2 It is further divided into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). NOA is more prevalent than OA in azoospermia cases, which affects 10%–15% of infertile men. With the development of assisted reproductive technology (ART), patients diagnosed with NOA could have their own children through microdissection testicular sperm extraction (micro-TESE) combined with subsequent intracytoplasmic sperm injection (ICSI). Micro-TESE, first reported by Schlegel3 in 1999, is considered a standard procedure for NOA patients due to its high sperm retrieval rate (SRR) and less tissue loss.

Although the etiology of NOA can be complicated, including congenital, acquired, and idiopathic causes, SRRs under micro-TESE of different causes of NOA patients vary from 40% to 60%.4

Many studies on micro-TESE have emerged in recent years. Some studies focused on the predictors of testicular sperm retrieval of NOA patients, including age, testicular volume (TV), and follicle‐stimulating hormone (FSH) level,5,6 and others compared the SRRs of NOA patients with different causes.4 However, there are limited data on the anatomical characteristics of different NOA patients. Therefore, our primary focus in this review is to understand further the anatomical alterations of NOA patients and surgical considerations for micro-TESE.

ANATOMY OF THE SCROTUM

The scrotal wall is an extension of the abdominal wall, characterized by its intricate anatomical layers. From the exterior to the interior, these layers include the scrotal skin, dartos muscle, external spermatic fascia, cremaster muscle and its fascia, internal spermatic fascia, parietal layer of the tunica vaginalis, and the visceral layer of the tunica vaginalis. The scrotum’s subcutaneous tissue is a unique layer filled with smooth muscle fibers devoid of adipose tissue. This layer, along with the spermatic cord fascia, extends posteriorly to form the scrotal septum, dividing the scrotum into left and right chambers, each housing a testis. The scrotal skin is thin and corrugated, enriched with sebaceous glands, aiding in perspiration and thermoregulation.7

TV

The testes are bilateral organs situated within the scrotum. In healthy adult males, each testis typically measures 3–5 cm in length, 2–3 cm in width, and 2–3 cm in thickness and typically has a volume of 15–25 ml.8 A study enrolled 1139 average Asian young men concluded that the cut-off TV is approximately 18 ml, which is lower than that of 20 ml for Caucasian and African–American men.1,9 The TV difference among average men in different ethnic groups indicated that TV might be an ethnicity-based variable for NOA patients. In general, the decreased testicular size is associated with impaired spermatogenesis. Previous studies have shown that TVs in patients with NOA are significantly smaller than the average value.4,10,11 Zhang et al.4 reported TVs in five groups of NOA patients, with values (mean ± standard deviation [s.d.]) in Group A (Klinefelter syndrome [KS]), Group B (azoospermia Y chromosome factor C microdeletion [YCMD]), Group C (cryptorchidism), Group D (previous mumps and bilateral orchitis), and Group E (idiopathic nonobstructive azoospermia [iNOA]) of 1.92 ± 0.82 ml, 8.81 ± 3.27 ml, 8.49 ± 3.43 ml, 6.24 ± 3.24 ml, and 8.13 ± 3.93 ml, respectively. Another study conducted by Chen et al.10 demonstrated that the TVs in different groups were as follows: for iNOA, KS, YCMD, cryptorchidism, mumps orchitis, and chemotherapy groups, the right-side TVs (mean ± s.d.) were 8.11 ± 3.28 ml, 2.24 ± 0.91 ml, 9.67 ± 3.32 ml, 8.25 ± 2.49 ml, 7.35 ± 4.22 ml, and 5.16 ± 2.67 ml, respectively; the left-side TVs (mean ± s.d.) were 7.98 ± 3.16 ml, 2.53 ± 0.94 ml, 9.27 ± 3.15 ml, 8.42 ± 2.61 ml, 7.18 ± 3.79 ml, and 5.23 ± 2.54 ml, respectively. While Kizilkan et al.11 reported a positive correlation between TVs and SRRs, identifying a TV above 11 ml as a significant cut-off value associated with successful sperm retrieval. Most studies demonstrated that the correlation between TVs and SRRs is poor.5,12 It is currently accepted that micro-TESE can be considered a therapeutic option for patients with azoospermia, regardless of their TVs.

BLOOD FLOW OF TESTES

Three arteries supply blood to the testes: the testicular artery (internal spermatic artery), the deferential artery, and the cremasteric artery (external spermatic artery). The testicular artery is the primary source of blood for the testicles.13 Upon reaching the testis, it enters the mediastinum and supplies blood to the anterior superior and anterior inferior poles as well as the medial and lateral aspects of the testis. The vas deferens artery branches from either the internal iliac or superior vesical artery. The cremasteric artery, originating from the inferior epigastric artery, primarily supplies the tunica vaginalis but has branches extending into the testis.14,15 Studies consistently show that men with NOA often exhibit reduced testicular blood flow compared to fertile men or those with OA.16,17,18,19,20 This reduction is thought to affect spermatogenesis negatively.16,17 Doppler ultrasonography (US) assessments have measured testicular blood flow in NOA patients. Foresta et al.18 indicated that blood flow might suggest potential spermatogenic activity, especially in the peripheral regions. They conducted fine-needle aspirations (FNAs) in the testicular areas where these vessels were detected. Notably, spermatozoa were found in 12 out of 16 NOA patients.18 Another study demonstrated that testicular perfusion assessment using Doppler US can effectively localize viable sperm areas, thereby guiding biopsies to specific sites.19 Altinkilic et al.17 found no significant blood flow and US differences between micro-TESE positive and negative group. In the study of Westlander et al.,20 it was reported that no differences in testicular echogenicity or intratesticular blood flow resistance were found between 47,XXY men whose sperm recovery was successful and those whose sperm recovery failed. It remains to be further studied whether a preoperative US test to determine testicular blood flow could benefit NOA patients accepting micro-TESE.

MICROANATOMIC ARCHITECTURE OF TESTES

Normal microanatomy and function of testes

The testes are encased in a robust white fibrous layer known as the tunica albuginea. This layer thickens at the posterior border, forming the mediastinum testis, from which septa extend, dividing the testicular tissue into lobules. Each lobule houses numerous convoluted seminiferous tubules, where sperm production takes place in the epithelial lining. These tubules converge at the mediastinum to form the rete testis, which gives rise to 12–15 efferent ductules that connect to the epididymis, providing a pathway for sperm transport. Remarkably, each testis contains 600–900 seminiferous tubules, spanning a combined length of 450–600 m.21

Spermatogenesis is a complex process, in which spermatogonial stem cells (SSCs) undergo self-renewal and differentiation into spermatozoa. The testicular microenvironment (niche) consists of Sertoli cells (SCs), Leydig cells (LCs), and peritubular myoid cells (PTMs).22 SCs are the most pivotal components in a niche that offer physical and nutritional support to germ cells within the seminiferous tubules.

Additionally, SCs form the blood–testis barrier to create an immune-privileged environment for the germ cells.23 Using single-cell analysis, Zhao et al.22 identified three groups of SCs named stage a/b/c cells. Stage a/b cells are the immature SCs, which gradually decrease with age. Stage c cells do not appear until 11 years old and have become predominant since then.22 LCs, located in the interstitial space between seminiferous tubules, produce testosterone, which is vital for spermatogenesis and maintenance of sexual function.24 Additionally, various studies have highlighted that LCs are crucial in regulating spermatogenesis through their impact on growth factor and steroidogenesis; examples include interleukin 1α, transforming growth factor β, inhibin, insulin-like growth factors 1, insulin-like peptide 3, estrogens, and thyroid hormones.25,26 Mahyari et al.27 also proved that progenitor LCs (PLCs), immature LCs (ILCs), and mature LCs (MLCs) coexisted in adult testis. Pseudotime analysis has confirmed that the maturation of LCs begins with PLCs, progresses through ILCs, and culminates in MLCs.27

Moreover, Guo et al.28 reported that prepuberty LCs might have the potential to develop into both mature LCs and PTMs. PTMs are specialized smooth muscle-like cells that reside on the peripheral surface of the basal lamina of the seminiferous tubules combined with SCs in the testis. They are thought to be involved in the tubular contraction associated with the transport of immotile spermatozoa and play an important role in spermatogenesis in collaboration with other somatic cells.29 The blood–testis barrier, a unique structure, exists between the seminiferous tubules and capillaries. It comprises capillary endothelium, basement membranes, and tight junctions between SCs. This barrier protects the spermatogenic epithelium, prevents harmful substances from entering, averts autoimmune responses triggered by sperm antigens, and creates a conducive microenvironment for spermatogenesis.30

Variations of testes in NOA patients

The common causes of NOA include KS, iNOA, YCMD, and cryptorchidism. KS, also known as 47,XXY syndrome, is a congenital chromosomal disorder primarily resulting from meiotic nondisjunction, where X chromosomes fail to separate during oocyte maturation and division, resulting in an oocyte containing two X chromosomes.31 However, the supernumerary X chromosome in KS may originate from several different mechanisms. Nondisjunction of the sex chromosomes can occur during paternal meiosis I, maternal meiosis I or II, or postzygotic mitotic divisions.32 KS is the most prevalent genetic cause of male infertility, affecting 0.1%–0.2% of the general population and up to 3.1% of infertile men.33 Spermatogenesis and hormone production of KS patients are disrupted due to dysfunctions in LCs, SCs, and the hypothalamic–pituitary–gonadal (HPG) axis.

Additionally, changes in the pituitary feedback inhibition threshold have been observed. The testicular abnormalities in KS include small testicular size, hyalinization and sclerosis of seminiferous tubules, decreasing diameter of seminiferous tubules, fibrosis, and reduced elasticity of testicular tissue due to increasing connective tissue within the testes.34 Individuals with KS may present with hypogonadal symptoms such as reduced muscle mass, decreased body and facial hair, and development of breast tissue (gynecomastia).35 Micro-TESE has proven to be an effective procedure for sperm retrieval in KS patients, with reported high SRRs ranging from 47% to 69%.34

iNOA is a condition characterized by the absence of sperm in the ejaculate, normal chromosomal karyotyping, and the absence of Y-chromosome azoospermia factors (AZF) microdeletions due to impaired spermatogenesis without any identifiable underlying cause.36 Nearly 70% of NOA patients are classified as iNOA due to unknown causes. The TV of iNOA patients can vary significantly. In many instances, these individuals may exhibit average testicular size despite impaired spermatogenesis, especially in cases of early maturation arrest. Zheng et al.37 reported a total TV (mean ± s.d.) of 13.4 ± 6.6 ml in NOA patients who underwent micro-TESE, while another study highlighted a subset of patients with early maturation arrest who presented with normal-sized testes (mean ± s.d.: 17 ± 4.4 ml) and FSH levels (mean ± s.d.: 4.1 ± 1.7 IU ml⁻¹).38 This indicates that although the testes may appear anatomically normal, the underlying sperm production process is compromised, making diagnosis challenging when relying solely on external examination or hormone levels. Being the largest number of the NOA population, iNOA patients have the lowest SRRs of micro-TESE with fewer opportunities to father their children. The SRRs could range from 26.2% to 52% after micro-TESE.4,39

The Y chromosome, exclusive to males, plays a pivotal role in typical male development. The Y chromosome harbors spermatogenesis-regulating genes, known as AZF, located in Yq11 and further divided into three regions: AZFa, AZFb, and AZFc,40 of which AZFc is the most commonly deleted locus, with a frequency of 57%. AZFa microdeletions lead to the absence of spermatogonial cells, often resulting in small, soft testes with a volume typically <2 ml. AZFb microdeletions cause spermatogenic arrest at the spermatogonial stage, with TV ranging from nearly normal to smaller than average. AZFc microdeletions have less impact on TV, with mature supportive cells, but semen parameters vary greatly among ages.41,42 Seminal indices can include azoospermia, oligozoospermia, or normazoospermia, with a progressive decline in performance with age. Generally, these patients tend to exhibit smaller TVs and less functional testes than those without genetic deletions. However, the extent of this can depend on the specific region of the Y chromosome affected. This physical manifestation is primarily due to impaired development of the germinal epithelium, the tissue within the testes where sperm is produced.43 In some tubules, spermatogenesis may be arrested early, leading to tubules filled only with Sertoli cells or early-stage spermatocytes, which may cause variable tubular diameters. In contrast, AZFc deletions are more common and associated with a wider range of testicular sizes. Some patients may have average TV. However, their spermatogenesis is often compromised. It was demonstrated that SRRs range from 59% to 63% in AZFc patients undergoing micro-TESE.44,45

Cryptorchidism, commonly referred to as undescended testicle, is a condition, in which one or both of the testicles fail to descend into the scrotum before birth.46 The normal development of testicles involves their descent from the abdomen to the scrotum, which is crucial for the proper temperature regulation necessary for sperm production. When testicles remain undescended, they are subjected to higher internal body temperatures, which might impair their growth and function. This condition often results in smaller-than-normal testicles if left untreated.47 Histopathologically, cryptorchidism is associated with a significant reduction in spermatogenic cells, the absence of supporting cells or mixed pathological alterations, and, in severe cases, the complete disappearance of spermatogenic cells. One of the most common findings in cryptorchid testes is tubular atrophy, with tubules often showing reduced diameter, indicating decreased spermatogenic activity. The tubules may contain only SCs in many cases, with no germ cells present.47,48 Moreover, the longer cryptorchidism persists, the more severe these abnormalities become, indicating the importance of early intervention to mitigate long-term reproductive complications.

The surgical approach during micro-TESE must be meticulous to minimize additional damage while thoroughly exploring the testicular tissue for areas of residual spermatogenesis. Surgical complexity increases in cases where orchiopexy has already been performed due to adhesions and altered testicular positioning. Additionally, the success of micro-TESE in cryptorchidism cases is influenced by the location of the undescended testis. Chen et al.10 found that the SRRs varied significantly by location, with rates of 27.3%, 44.8%, and 66.7% in intra-abdominal, inguinal, and supra-scrotal cryptorchidism, respectively. Multivariate analysis revealed a negative correlation between the testis location and success rate of micro-TESE (odds ratio [OR]: 0.479, 95% confidence interval [CI]: 0.245–0.936, P < 0.032).49 Therefore, it is recommended that patients undergo orchiopexy between 1 year and 2 years of age to reduce the risk of malignant transformation and enhance reproductive potential.50 Interestingly, KS is the most common genetic alteration in patients suffering from cryptorchidism, highlighting the need for karyotype analysis in these patients. Similar to KS, cryptorchidism patients undergoing micro-TESE show SRRs ranging from 47% to 69%.51,52

Given the critical roles of SCs, LCs, and PTMs in the testicular microenvironment, alterations in the niche of NOA patients are likely to manifest in these cells. Due to the SRRs of patients with mumps orchitis and cryptorchidism,4 most studies related to microanatomical variations focused on SCs, LCs, and PTMs. Zhao et al.22 revealed that stage a/b SCs are proven to be dominant in iNOA patients, which indicated that the different gene expressions of immature SCs might influence the spermatogenesis of iNOA patients. They also revealed that the inhibition of the wingless/integrated (WNT) signaling pathway in vitro blocks the SCs proliferation.22 Another study conducted by Alfano et al.53 confirmed the immaturity of SCs and an increasing level of estradiol preproduction, consistent with previous studies.

Furthermore, two separate research groups have found that SCs in KS exhibit increased expression of genes related to immune responses. This suggests that abnormal activation of immune cells may be a standard pathological process stemming from sex chromosomal abnormalities.22,27 The alterations in LCs in NOA patients have been the subject of extensive research over many years. While some studies have noted cell hyperplasia,54,55,56 others have not found evidence of it.57,58 Further studies are still required to confirm the debate. Compared to biopsy samples from normal adult testes, the Leydig cell populations in testes exhibiting dysgenic features show a higher number of undifferentiated cells. This is evidenced by increased expression of delta-like homolog 1 (DLK1) and decreased expression of insulin-like peptide 3 (INSL3).59 Alfano et al.53 reported a decrease in the expression of MLC markers and an increase in ILC markers, suggesting that LC maturation is either delayed or arrested in these patients. PTMs secrete factors essential for spermatogenesis and provide contractile function to the seminiferous tubules.60 A study identified LCs, PTMs, and SCs as the primary somatic contributors to the altered deposition of collagen type I and IV and the disorganization of the extracellular matrix (ECM) associated with idiopathic germ cell aplasia. The uneven distribution of collagen types I and IV in the basal membrane of the seminiferous tubules was closely linked to the absence of germ cells.53

HORMONES IN NOA PATIENTS

Hormonal evaluation helps make a diagnosis of NOA. Although NOA cannot permanently be excluded when gonadotropins are within the normal range (especially in patients with germ cell maturational arrest), high serum gonadotropin levels typically indicate primary testicular failure. It has been reported that more than 90% of patients with azoospermia could be accurately diagnosed as NOA or OA by combined measurement of FSH, LH, and TV.61

The FSH and LH levels were higher in micro-TESE-negative men, with the testosterone levels lower in these men. Though levels of FSH generally correlate with the predominant pattern of spermatogenesis, they may not predict isolated areas of spermatogenesis within the testis according to the study of Eken and Gulec.62 Ramasamy et al.50 demonstrated that the relationship between FSH and the presence of spermatogenesis is not straightforward in men with NOA, including men with KS. They concluded that serum FSH level has a poor predictive value for successful micro-TESE.

ANATOMICAL CONSIDERATIONS OF MICRO-TESE

TESE is one of the most helpful treatment strategies for NOA. It is divided into simple TESE, in which a small incision is made in the tunica albuginea and the seminiferous tubules are extracted blindly, and micro-TESE, in which a wide incision is made in the tunica and the dilated tubules are examined in detail under a surgical microscope. It was reported by a systematic review and meta-analysis that micro-TESE had a higher SRR than simple TESE (micro-TESE vs simple TESE: 52% vs 35%).63 Micro-TESE is a procedure that takes sperm directly from the testicular tissue of a man’s reproductive system. It was developed from the observation that seminiferous tubules with active spermatogenesis appear larger and more opaque under optical magnification than those without active procedures. With the guidance of an operating microscope during testicular exploration, the testicular blood supply is visualized and preserved; the seminiferous tubules that are most likely to contain spermatozoa are identified and targeted explicitly for extraction and sperm retrieval.64

The procedure was introduced in our previous study,4 and it was performed under general anesthesia. A midline scrotal incision was made on the median raphe of the scrotum. After fully exposing the testicle, a large equatorial or longitudinal incision is made in the tunica albuginea. Then, the testis parenchyma is fully extruded, and opaque seminiferous tubules are biopsied preferentially. Testicular parenchyma was exposed and directly examined for dilated tubule areas under an operating microscope (12× to 24× magnification), and larger tubules were selectively biopsied until enough sperm was collected. In micro-TESE, careful anatomical consideration is crucial to maximize sperm retrieval while minimizing damage to the testicular structure and function.22 The procedure should be designed to preserve the testicular blood supply, which typically runs in a centripetal direction towards the mediastinum testis. Dissections are generally done carefully under an operating microscope to visualize and preserve these blood vessels.3,4,65

The decision between an equatorial or longitudinal incision is based on testicular anatomy and the potential for accessing areas likely to yield sperm. An equatorial incision is often preferred as it provides a larger cross-sectional area of the testicle without compromising blood supply from the poles.66,67 Ichioka et al.68 explored mathematical three-dimensional (3D) simulation models of micro-TESE, revealing that the likelihood of identifying suitable tubules during the procedure depends on the total cut surface area of the parenchyma. Their findings suggest that maximizing this cut surface area could achieve the highest SRRs.68 In contrast, a longitudinal incision may be preferred if specific tubules need to be targeted or if the anatomy of the testis suggests a better approach from the upper or lower pole.69 To prevent testicular damage and minimize bleeding from the sub-tunica venous plexus, dissection should carefully separate the seminiferous tubules, taking care to avoid excessive traction that may damage the delicate tubular structure. Preventing the extrusion of tubules and avoiding their separation from the tunica albuginea requires gentle manipulation and microsurgical techniques to isolate the tubules most likely to contain spermatozoa. It is also essential to avoid unnecessary pressure or trauma to the testicular parenchyma, as this may impact the remaining spermatogenesis and the integrity of the testicular tissue. This refined approach helps preserve testicular function and structure, potentially enabling future sperm production and hormonal function. Our review introduces the alterations of anatomy and microanatomy in testicular tissue in NOA patients and their potential clinical significance. It also discusses surgical considerations during the micro-TESE surgical procedure.

CONCLUSION

Male infertility, particularly NOA, remains a considerable challenge for couples attempting to conceive. A deeper understanding of NOA patients’ anatomical and clinical features, combined with the refinement of surgical procedures, can lead to enhanced reproductive outcomes. Continued research in this field is essential for optimizing treatment efficacy and providing hope to many couples affected by infertility.

AUTHOR CONTRIBUTIONS

KH and HCL conceived and reviewed the manuscript. HCL, YC, and YYF contributed to the literature review and writing of the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.