Azoospermia is defined as the complete absence of spermatozoa upon microscopic evaluation of two centrifuged semen samples (1, 2). It occurs in 1% of men and approximately 10%–15% of the infertile male population, and can be either non-obstructive (NOA) or obstructive azoospermia (OA) depending on the etiology (1).

NOA is caused by testicular failure, which can be primary (testicular) or secondary to hypothalamo-pituitary dysfunction (pre-testicular). OA is caused either by a blockage in the genital tract or ejaculatory dysfunction (post-testicular), such as the absence of the seminal vesicles, as occurs in congenital bilateral absence of the vas deferens (CBAVD) (3, 4). Pre-testicular azoospermia arises when the hypothalamus and pituitary are not working properly to stimulate intrinsically normal testes in terms of both gonadal function (testosterone synthesis in the Leydig cells and spermatogenesis within the seminiferous tubules) (5). However, testicular azoospermia can also occur when the hypothalamus and pituitary are functioning normally, but there is a defect in the spermatogenic process resulting in no sperm production. Since the reproductive ductal structures are present and patent, the two mentioned etiologies fall under non-obstructive azoospermia (5).

The management of NOA or OA patients relies on conventional testicular sperm extraction (cTESE) or microscopic testicular sperm extraction (microTESE) followed by an intracytoplasmic sperm injection (ICSI) [artificial reproductive technology (ART)] (6). In NOA patients, sperm recovery is successful in only 50% of cases, and therefore the ability to predict the patients with a high probability of achieving a successful sperm retrieval would be of great value when counseling the patient and his partner (7, 8). However, several studies have tried to suggest predictors of a positive TESE (e.g. FSH concentration, Y chromosome microdeletions), but most concluded that testicular histology (diagnostic testicular biopsy) is best (9–16).

In this study, we investigated and compared cTESE, microTESE, and testicular histology outcomes in order to better understand the different criteria adopted by the two laboratory methodologies (IVF lab and pathological anatomy laboratory).

For many years testicular biopsy was considered the primary diagnostic tool for men with risk factors for testicular malignancy or male infertility in cases of unexplained infertility and azoospermia; however, currently, the guidelines for male infertility have limited the indications for a diagnostic testicular biopsy to the confirmation of obstructive or non-obstructive azoospermia (20). It may be part of the diagnostic process for infertile men, but it only confirms a disruption of spermatogenesis in cases where sperm count is low and follicle-stimulating hormone is high. Testicular biopsy is most useful in diagnosing obstructive azoospermia, where surgical repair can restore the presence of spermatozoa, increasing the chances of pregnancy. However, a standard testicular biopsy typically does not change the therapeutic options (20). Before the advent of the TESE technique, couples in which the male partner was diagnosed with azoospermia had to resort to heterologous fertilization through sperm donation (23–26). This is why it is important to emphasize the value of the combination of the TESE technique and an ICSI due to the possibility for NOA and OA patients to father biologically related children (8, 27, 28). Sperm retrieval rates in OA are approximately 100%, while in NOA they are approximately 60% (7, 8, 27, 28).

At our institute, TESE samples are meant to be cryopreserved. Indeed, no TESE procedures are performed at the time of oocyte pick-up. This is to prevent the partner’s stimulation from failing because it is possible that no sperm (destined for a subsequent ICSI) is found during the TESE, necessarily resulting in oocyte cryopreservation.

In this study, we compared the retrieval of 526 TESE samples with their histological outcomes from between 2018 and 2020. We found a discrepancy of almost 30% (29.3%, 154/526 samples) between the two techniques, as reported in Supplementary Table S2 ( Supplementary Material ). In many cases, we cryopreserved testicular spermatozoa while the histological result reported the absence of germ cells in the seminiferous tubules (e.g., Sertoli cells only). Unfortunately, in 8 cases, we did not find any spermatozoa or did not find enough compared to the histology report, resulting in no sperm cryopreservation. This may be due to the fact that histological staining mainly highlights spermatozoa heads while embryologists need to observe the entire sperm structure to define it as one for the ICSI procedure. It is noteworthy that 61 female partners of TESE patients who were among those with discordant outcomes became pregnant with cryopreserved TESE samples.

As described in the materials and methods section, the histopathology specimen is smaller and above all different from that which is designated for TESE and our evidence highlights the presence of a heterogeneous histological area within the testis, representing a multifocal distribution of spermatogenesis (19, 29, 30). As other authors have reported, the histology of a small piece of tissue may not be representative of the tissue extracted or of the whole testis (31, 32). Furthermore, the tissue should not be squeezed since the testicular specimen may be disrupted, hampering a proper evaluation of the seminiferous tubules (20). Because of these reasons, regarding ART, some authors have concluded that testicular histology is of limited value (20, 33). The lack of clarity in the findings may stem from the incorrect identification of germ cells and confusing biopsy categorization, as the histological method is often inadequately described or omitted (20, 32). As Hessel M. et al. reported, to increase diagnostic accuracy, a larger biopsy should be sent to the histopathology laboratory, but the most representative and clinically relevant option may be to use a TESE cell suspension for a cytological evaluation of spermatogenesis (31).

Furthermore, we decided to specifically analyze the category of NOA patients, including retrieval rate, ART cycles, and pregnancy rate. We cryopreserved 65 of the 118 NOA TESE samples (55.1%) which was consistent with the literature (7). Moreover, our analysis revealed that while hormonal profiles and testicular volume may not predict TESE success, the overall pregnancy rate remains promising; indeed, 39/49 TESE patients’ partners who underwent ART became pregnant (79.6%). This suggests that TESE combined with an ICSI remains a viable and effective strategy for couples facing severe male infertility. The limitations of histological evaluation in reflecting the true spermatogenic potential reinforce the necessity of combining TESE with ART for an accurate diagnosis and treatment. Thus, in our analysis, we did not focus on the diagnoses of the female partners due to the main topic of the present retrospective study itself. Notably, a marked increase in impaired reproductive success is observed in women over 35 years of age (34). Furthermore, in our cohort, the age of the partners, expressed as mean(± SD), was 34.6(± 4.6) years. This is because when a man is diagnosed with severe spermatogenic failure (SPGF), such as NOA or OA, the immediate decision is to pursue ART with his partner; in particular, the latter does not wait to grow older. During the freezing and thawing processes, spermatozoa are exposed to physical and chemical stress, such as crystal formation or osmotic shock, which can affect membrane integrity or acrosome status (35, 36). Additional molecular alterations may accompany sperm cryopreservation (37). As a result, sperm cryopreservation leads to a decrease in sperm motility and sperm viability, increasing sperm DNA fragmentation (38, 39). However, Torra-Massana and colleagues found no difference between fresh and cryopreserved sperm in terms of oocyte fertilization, ongoing pregnancy, and live birth rates; moreover, comparable pregnancy rates can be achieved in OA and NOA cases (40, 41). It is important to emphasize that in the absence of sperm motility (in terms of twitching tail or wavy flagellar movement) in testicular sperm, hypo-osmotic swelling test (HOST), or laser-assisted immotile sperm selection (LAISS) are techniques recommended for before an ICSI to ensure a higher rate of success (21, 42). Finally, the pregnancy and delivery rates are strictly dependent on the age of the female partner (41).

However, a non-invasive diagnostic test that could predict sperm retrieval outcomes would greatly benefit the clinical management of NOA cases (7). In the past decade, significant advancements in high-throughput sequencing technologies have been made in biomedical research. Within this scenario, single-cell RNA sequencing (scRNA-seq) has emerged as a groundbreaking tool, uncovering the mechanisms behind testicular dysregulation and disorders (43, 44). A genetic cause has been identified in only 20% of infertile men with SPGF, leaving most cases unexplained and idiopathic (45). Growing evidence suggests that common genetic variants, such as single nucleotide polymorphisms (SNPs), may contribute to male infertility by disrupting the molecular processes that regulate spermatogenesis (46, 47). On this basis, researchers worldwide have moved to genome-wide association studies (GWASs) in the field of SPGF, identifying several risk variants for NOA susceptibility (47, 48).

From this perspective, germ cell maturation molecular patterns have been investigated in the literature. Germ cells undergo precise transitions between multiple cell types and cellular processes, including mitosis, meiosis, and spermiogenesis; the latter is accompanied by spermatid elongation and chromatin repackaging (49). The developmental process from spermatogonia into mature sperm depends on the expression of a range of genes in a precise temporal order (50). It was found that gonadotropin-regulated testicular RNA helicase (GRTH) is essential for the completion of spermatogenesis and male fertility. In particular, in the germ cells of male mice, there are two forms of GRTH: the non-phosphorylated and phosphorylated forms (pGRTH). GRTH knock-in (KI) mice with R242H mutation abolished pGRTH, leading to infertility (51). The same mutation was observed in 5.8% of Japanese men diagnosed with NOA (52).

In conclusion, to the best of our knowledge, our analysis confirmed that the TESE procedure combined with an ICSI remains the only strategy for couples facing severe male infertility. The choice of which testicular sperm to inject, especially when no wavy flagellar movement is observed in the sample, should be performed by an embryologist with vast expertise. However, a testicular biopsy is useful for diagnosing obstructive azoospermia where surgical repair can restore the presence of spermatozoa, increasing chances of pregnancy. Furthermore, novel molecular techniques and GWASs could help in understanding the matter of male infertility, which affects thousands of couples worldwide.