Introduction
In several species, primordial germ cells (PGCs) are the first germline cell population to colonise the developing gonads through active migration (Richardson and Lehmann, Reference Richardson and Lehmann2010; Saitou and Yamaji, Reference Saitou and Yamaji2010). PGCs initiate differentiation in the gonads along a spermatogenic (male) or oogenic (female) pathway. The spermatogenic path is most commonly associated with spermatogonial or male germline stem cells (Spradling et al., Reference Spradling, Fuller, Braun and Yoshida2011). Germ cells are the embryonic progenitor cells that give rise to gametes, which are the reproductive cells that produce offspring. Early in the development of most species, they are separated from the somatic cell lineages (Cinalli et al., Reference Cinalli, Rangan and Lehmann2008). There are two morphologically distinct kinds of spermatogonia: A dark and A pale spermatogonia. A dark has features that reveal it acts as a testicular stem cell. The A pale, on the other hand, has typical progenitor characteristics. Type A pale cells divide and undergo active mitosis to generate Type B cells, which then develop into primary spermatocytes (Niazi Tabar et al., Reference Niazi Tabar, Azizi, Nazm Bojnordi and Ghasemi Hamidabadi2021; Tabar et al., Reference Tabar, Sojoudi, Henduei and Azizi2022). Sertoli and Leydig cells are the two primary somatic cells in the testis that are required for testicular structure and function. Sertoli cells, which act as an anchor for germ cell growth, are found only in the tubular compartment of the testis and contribute to the progression of germ cells to spermatozoa through direct interaction and regulation of the environmental milieu (Griswold, Reference Griswold1998). In contrast, Leydig cells are located in the intertubular or interstitial region and contribute to testosterone production (Krishnamurthy et al., Reference Krishnamurthy, Kats, Danilovich, Javeshghani and Ram Sairam2001; Chen et al., Reference Chen, Ge and Zirkin2009). In the seminiferous epithelial tissue, spermatogenesis plays an essential role in male fertility (Goh et al., Reference Goh, Falciatori, Tam, Burgess, Meikar, Kotaja, Hammell and Hannon2015; Gunes et al., Reference Gunes, Al-Sadaan and Agarwal2015; O’Hara and Smith, Reference O’Hara and Smith2015; Griswold, Reference Griswold2016). Male germ cells, in conjunction with Sertoli cells, are essential contributors to spermatogenesis, the intricate biological process that transforms diploid spermatogonia into spermatozoa (Heller and Clermont, Reference Heller and Clermont1963; Auger, Reference Auger2018). In the last step of spermatogenesis, mature spermatids are discharged into the lumen of the seminiferous tubules from the supporting Sertoli cells (Sharpe, Reference Sharpe1987; Phillips et al., Reference Phillips, Gassei and Orwig2010). Even slight deviations from the natural spermatogenesis pathway may result in infertility in men (Babakhanzadeh et al., Reference Babakhanzadeh, Nazari, Ghasemifar and Khodadadian2020). Infertility is defined as the failure to achieve a clinical pregnancy following 12 months of consistent and unprotected sexual intercourse, which may stem from either female or male factors (Vander Borght and Wyns, Reference Vander Borght and Wyns2018; Babakhanzadeh et al., Reference Babakhanzadeh, Nazari, Ghasemifar and Khodadadian2020). Men’s infertility is mainly diagnosed by semen analysis. Unusual sperm parameters include sperm concentration, morphology and motility (Thonneau et al., Reference Thonneau, Marchand, Tallec, Ferial, Ducot, Lansac, Lopes, Tabaste and Spira1991; De Kretser and Baker, Reference De Kretser and Baker1999). The most likely reasons for male infertility include hormonal deficiencies, physical causes, sexually transmitted diseases, environment and lifestyle and genetic factors (Winters and Walsh, Reference Winters and Walsh2014; Krausz and Riera-Escamilla, Reference Krausz and Riera-Escamilla2018; Lee and Ramasamy, Reference Lee and Ramasamy2018; Ho et al., Reference Ho, Le, Truong, Nguyen and Cao2020). Roughly 1% of all men, 10% to 15% of infertile males are diagnosed with Azoospermia, the absence of sperm in the ejaculate (Cocuzza et al., Reference Cocuzza, Alvarenga and Pagani2013). Obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) represent two classifications of this condition. OA, responsible for 40% of azoospermia cases, results from a physical blockage in the male excurrent ductal system, potentially occurring at any point from the rete testis to the ejaculatory ducts. (Jow et al., Reference Jow, Steckel, Schlegel, Magid and Goldstein1993; Wosnitzer et al., Reference Wosnitzer, Goldstein and Hardy2014). Non-obstructive azoospermia, however, is responsible for approximately 60% of azoospermic cases (Wosnitzer et al., Reference Wosnitzer, Goldstein and Hardy2014). NOA is characterised by the lack of sperm in the ejaculate due to a failure in spermatogenesis (Chiba et al., Reference Chiba, Enatsu and Fujisawa2016). Men with obstructive azoospermia have two alternatives for fathering children: surgical repair of the blockage and collection of sperm straight from the epididymis or testis, followed by in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) (Jarow et al., Reference Jarow, Espeland and Lipshultz1989, Reference Jarow, Sigman, Kolettis, Lipshultz, McClure, Nangia, Kim Naughton, Prins, Sandlow and Schlegel2010). For men with non-obstructive azoospermia, current management relies primarily on medical therapy and microsurgical testicular sperm extraction (microTESE) (Kumar, Reference Kumar2013; Birowo et al., Reference Birowo, Tendi, Rasyid, Turek, Sini and Rizal2021). Beyond these approaches, stem cell transplantation has emerged as a promising strategy for treating male infertility rooted in impaired spermatogenesis (Ahn et al., Reference Ahn, Ryu, Jung, Shin, Won, Um, Oh, Kim and Ryu2020). This technique begins with isolating and cryopreserving spermatogonial stem cells (SSCs) obtained through a testicular biopsy, which are later reintroduced into the testis through targeted stem cell transplantation (Abdelaal et al., Reference Abdelaal, Tanga, Abdelgawad, Allam, Fathi, Saadeldin, Bang and Cho2021). Once delivered back into the testicular environment, SSCs have the capacity to regenerate the spermatogonial population and restore spermatogenesis when native cells are damaged or depleted (Kanatsu-Shinohara et al., Reference Kanatsu-Shinohara, Toyokuni, Morimoto, Matsui, Honjo and Shinohara2003; Han et al., Reference Han, Park, Lee, Im Yun, Choi, Lee and Lee2018). Consequently, this approach offers hope not only for cancer survivors exposed to gonadotoxic treatments but also for patients whose fertility is compromised by severe defects in spermatogenesis (Hanna Valli et al., Reference Valli, Phillips, Gassei, Nagano and Orwig2015; Forbes et al., Reference Forbes, Flannigan and Schlegel2018).
Spermatogonial stem cell
Mammalian adult stem cells share at least two main features: they can undergo ‘indefinite’ self-renewal, indicating low mitotic activity throughout the organism’s lifetime without undergoing differentiation. They can also generate differentiating daughter cells (Leblond, Reference Leblond1964; Robey, Reference Robey2000). Germinal stem cells, also known as SSCs, maintain the cell pool while generating sperm through the process of self-renewal. (Aponte, Reference Aponte2015). Antibody-based research on intact (whole-mount) seminiferous tubules has allowed researchers to identify proteins whose expression patterns are exclusive to undifferentiated spermatogonia and, by extension, SSCs. Undifferentiated stem and progenitor spermatogonia, including A single, A paired and A aligned 4–16, have shown the expression of CDH1, GFRA1, LIN28, NANOS2, NANOS3, NEUROG3, ZBTB16 and POU5F1 genes (Azizi et al., Reference Azizi, Niazi Tabar and Mohammadi2020a, Reference Azizi, Tabar, Skutella and Govahi2020c). Moreover, ID4, PAX7 and BMI1 might be considered marker proteins for ultimate SSCs. However, whether these variables are relevant for SSC function and behaviour in the long run is unclear. This is because the consequences of protein shortage are relatively small (Oatley et al., Reference Oatley, Kaucher, Racicot and Oatley2011). Undifferentiated spermatogonia constitute a cohort of early spermatogonia, which includes a subset of SSCs. In rodents, they are composed of spermatogonia that are either single (A s) or paired (A pr) or arranged in chains of four, eight or sixteen cells (A al) (De Rooij, Reference De Rooij2017). The daughter cells formed after the division of undifferentiated spermatogonia in rodents may either move apart to facilitate stem cell renewal or remain together as a pair of so-called A pr spermatogonia, which is the first stage in the differentiation process. Then, the pairings may grow to produce clones of 4, 8 or 16, resulting in aligned spermatogonia (A al) cells interconnected by intercellular cytoplasmic bridges (Oatley and Brinster, Reference Oatley and Brinster2012; De Rooij, Reference De Rooij2017). Following their initial development as A al spermatogonia, the A spermatogonia complete six rounds of division to become spermatocytes and, finally, spermatids. At this point, the A spermatogonia have committed permanently to meiosis (De Rooij, Reference De Rooij2017). There are several spermatogonial stem cell renewal models. According to the A model (traditional model), the whole A single pool represents the SSCs, and the development of an A-paired syncytium indicates irreversible entrance into a progenitor stage destined for differentiation. This SSC renewal approach has been the most popular for many years. However, two different models have been presented in the recent decade (Lord and Oatley, Reference Lord and Oatley2017). All As, Apr and Aal spermatogonia in the newly-emerged ‘fragmentation model’ possess stem cell potential, and stem cell renewal is promoted by pair and chain fragmentation (Nakagawa et al., Reference Nakagawa, Nabeshima and Yoshida2007, Reference Nakagawa, Sharma, Y-i, Braun and Yoshida2010; Klein et al., Reference Klein, Nakagawa, Ichikawa, Yoshida and Simons2010; Hara et al., Reference Hara, Nakagawa, Enomoto, Suzuki, Yamamoto, Simons and Yoshida2014). Another model that has been suggested is known as the ‘hierarchical model.’ According to this model, there is an SSC hierarchy, as certain spermatogonia are capable of long-term self-renewal, while others are not (Oatley et al., Reference Oatley, Kaucher, Racicot and Oatley2011; Aloisio et al., Reference Aloisio, Nakada, Saatcioglu, Peña, Baker, Tarnawa, Mukherjee, Manjunath, Bugde and Sengupta2014; Chan et al., Reference Chan, Oatley, Kaucher, Yang, Bieberich, Shashikant and Oatley2014; Komai et al., Reference Komai, Tanaka, Tokuyama, Yanai, Ohe, Omachi, Atsumi, Yoshida, Kumano and Hisha2014; Sun et al., Reference Sun, Xu, Zhao and Chen2015 Helsel and Oatley, Reference Helsel and Oatley2017; Helsel et al., Reference Helsel, Yang, Oatley, Lord, Sablitzky and Oatley2017). The undifferentiated spermatogonial population in non-human primates is divided into two types: A dark and A pale. A dark spermatogonia is a regenerative reserve with slow cycling, which cannot participate in spermatogenesis pathways and can only convert to A pale cells. The A pale spermatogonia act as progenitors, dividing mitotically to produce Type B and primary spermatocytes (Clermont, Reference Clermont1963; Ehmcke et al., Reference Ehmcke, Wistuba and Schlatt2006; Hermann et al., Reference Hermann, Sukhwani, Hansel and Orwig2010). Before the derivation of spermatocytes, the A pale is followed by just one generation of B spermatogonia. Therefore, technically, only two mitotic stages occur before the generation of spermatocytes from the progenitor cells in the human testis. Only four spermatocytes, leading to the formation of 16 spermatids, are thus derived from each initial division of a germline progenitor (Ehmcke et al., Reference Ehmcke, Wistuba and Schlatt2006). In humans, the A0/A1 model of SSC self-renewal is an alternative to the A model, which is very similar to the A dark and A pale models used in non-human primates to define stem cell potency (Valli et al., Reference Valli, Phillips, Gassei, Nagano and Orwig2015). During all stages, A0 spermatogonia were detected as single cells or pairs of cells, observed in the seminiferous epithelium. Mitotic measures were seldom found in these cells; hence, they were deemed incapable of sustaining spermatogenesis as resource stem cells. Radiation and other potentially hazardous substances are the only factors that can activate these reserve stem cells, which then suppress spermatogenesis. The functional stem cell pool includes A1–A4 spermatogonia. When A4 spermatogonia divide, they always create new A1 spermatogonia (a process called self-renewal) or intermediate spermatogonia (a process called differentiation) (Clermont and Bustos-Obregon, Reference Clermont and Bustos-Obregon1968; Dym and Clermont, Reference Dym and Clermont1970; Clermont and Antar, Reference Clermont and Antar1973). SSCs are essential for maintaining spermatogenesis throughout life; therefore, understanding how they function is crucial for determining the aetiology of specific infertility cases and identifying new avenues for stimulating or inhibiting the spermatogenic process (De Rooij, Reference De Rooij2017).
Male infertility and azoospermia
Infertility – whether in a woman or a man – is defined as the inability to achieve a clinical pregnancy after twelve months of regular, unprotected intercourse (Vander Borght and Wyns, Reference Vander Borght and Wyns2018; Babakhanzadeh et al., Reference Babakhanzadeh, Nazari, Ghasemifar and Khodadadian2020). In men, infertility specifically refers to the inability to impregnate a fertile female partner after at least one year of unprotected sexual activity (Leslie et al., Reference Leslie, Siref, Soon-Sutton and Khan2026). This challenge affects roughly 16% of couples worldwide, with male factors contributing to nearly half of all infertility cases (Tharakan et al., Reference Tharakan, Luo, Jayasena and Minhas2021). A wide range of conditions can underlie male infertility, some reversible and others permanent; factors such as age, medications, past surgeries, environmental exposures, genetic abnormalities and systemic illnesses can influence reproductive potential in either partner (Shih et al., Reference Shih, Shen, Wu and Kang2019). Therefore, evaluating male fertility centres on identifying the causes of infertility, treating those that are reversible, determining suitability for assisted reproductive technologies (ART) and providing counselling when the condition is irreversible (Cocuzza et al., Reference Cocuzza, Alvarenga and Pagani2013) (Figure 1). Among men, about 1% – and 10% to 15% of those who are infertile – are diagnosed with azoospermia, a condition characterised by the complete absence of sperm in the ejaculate (Safran et al., Reference Safran, Reubinoff, Porat-Katz, Schenker and Lewin1998). Azoospermia itself can arise from diverse causes, including obstruction of the genital tract or intrinsic failure of testicular function (Tharakan et al., Reference Tharakan, Luo, Jayasena and Minhas2021). Although azoospermia once implied absolute infertility, advances in testicular sperm extraction and ART now allow many affected men to biologically father children (Jarow et al., Reference Jarow, Espeland and Lipshultz1989). Clinically, azoospermia is classified as either OA or NOA, with OA resulting from blockages within the testicular or ductal systems and NOA caused by impaired spermatogenesis (Roshan et al., Reference Roshan, Azizi, Majelan and Tabar2024).
Indicates the impact of two hormones, LH and FSH, on Leydig and Sertoli cells. LH induces testosterone production in the testes’ interstitial cells (Leydig cells). FSH stimulates testicular growth and increases the synthesis of an androgen-binding protein by Sertoli cells, a component of the testicular tubule required for sustaining the maturing sperm cell.
Figure 1 Long description
The diagram illustrates the process of spermatogenesis and the impact of hormones LH and FSH on Leydig and Sertoli cells in the testes. It shows the hypothalamus releasing GnRH, which stimulates the pituitary glands to release LH and FSH. LH acts on Leydig cells to produce testosterone, while FSH stimulates Sertoli cells to support spermatogenesis. The diagram also depicts the differentiation of spermatogonia into primary spermatocytes, secondary spermatocytes, and finally sperm. It highlights the roles of Sertoli and Leydig cells in testicular structure and function. Additionally, it shows the distinction between normal sperm count and non-obstructive azoospermia, illustrating the absence of sperm in the latter.
The aetiology of azoospermia is conventionally classified according to the anatomical position of the cause: pre-testicular, testicular and post-testicular (Jarvi et al., Reference Jarvi, Lo, Grober, Mak, Fischer, Grantmyre, Zini, Chan, Patry and Chow2015; Tharakan et al., Reference Tharakan, Luo, Jayasena and Minhas2021). Pre-testicular azoospermia, which accounts for 2% of all azoospermic males, is caused by a hypothalamic or pituitary abnormality. Testicular failure or non-obstructive azoospermia affects between 49% and 93% of males with azoospermia. Spermatogenesis in men with testicular dysfunction may be impaired, halted in its maturation process or completely absent, as shown in Sertoli-cell-only syndrome. Men struggling with post-testicular or obstructive azoospermia (7–51% of azoospermic cases) have normal spermatogenesis but suffer from obstruction in the ductal system or ejaculatory dysfunction (Wang et al., Reference Wang, So, Wong, So and Chan1987; Jarow et al., Reference Jarow, Espeland and Lipshultz1989; Matsumiya et al., Reference Matsumiya, Namiki, Takahara, Kondoh, Takada, Kiyohara and Okuyama1994; Fedder et al., Reference Fedder, Crüger, Oestergaard and Petersen2004; Fogle et al., Reference Fogle, Steiner, Marshall and Sokol2006). Other groups of men with the inability to ejaculate are men with spinal cord injuries, psychogenic failure to ejaculate or neurological damage (Jarvi et al., Reference Jarvi, Lo, Grober, Mak, Fischer, Grantmyre, Zini, Chan, Patry and Chow2015). It’s noteworthy that the absence of sperm should be determined by centrifuging the semen sample; for an appropriate evaluation, at least two semen samples should be assessed (Schlegel, Reference Schlegel2004). If sperm analysis confirms azoospermia, the physician must clarify whether the patient has OA or NOA. A thorough medical history, physical examination, hormonal assessment and genetic testing are used to verify the diagnosis. The diagnosis of spermatogenesis failure is further supported by medical history of variables such as anticancer therapy or an undescended testis. Furthermore, identifying the patient’s medicines is crucial, since some drugs, including steroids, can impede spermatogenesis (Ramasamy et al., Reference Ramasamy, Armstrong and Lipshultz2015). Additional tests, such as karyotyping and genetic analysis, should be conducted if NOA has been diagnosed. It has been shown that 13.7% of azoospermia patients have an aberrant karyotype. Tests for azoospermia factor (AZF) – which includes three sub-regions, AZFa, AZFb and AZFc – are the most prevalent and critical genetic tests for NOA diagnosis. AZF is located on the long arm of the Y-chromosome (Yq). (Van Assche et al., Reference Van Assche, Bonduelle, Tournaye, Joris, Verheyen, Devroey, van sterteighen and Liebaers1996; Ferlin et al., Reference Ferlin, Arredi, Speltra, Cazzadore, Selice, Garolla, Lenzi and Foresta2007).
Management of non-obstructive azoospermia
NOA is characterised by a complete lack of sperm in the ejaculate resulting from intrinsic failures in spermatogenesis and is regarded as the most severe type of male infertility (Azizi et al., Reference Azizi, Niazi Tabar and Skutella2021a, Reference Azizi, NiaziTabar, Mohammadi and Skutella2021b, Reference Azizi, NiaziTabar, Mohammadi and Skutella2021c). Patients with NOA were unable to conceive children of their own earlier, and their only alternatives were donor sperm or adoption. The first successful live birth using IVF was reported in 1978, while the first successful live birth with the ICSI method was reported in 1992 (Steptoe and Edwards, Reference Steptoe and Edwards1978; Palermo et al., Reference Palermo, Joris, Devroey and Van Steirteghem1992). After that, pregnancy was documented in NOA patients after testicular sperm extraction (TESE) and ICSI, allowing them to father their children [82]. Recent advancements in assisted reproductive technology (ART) have profoundly transformed the treatment of NOA. Furthermore, recent biotechnology developments have increased the possibility of employing germ cells derived from stem cells in the future (Chiba et al., Reference Chiba, Enatsu and Fujisawa2016). A five-step approach is advised to handle patients with NOA. To confirm or demonstrate that NOA is associated with spermatogenic failure, a differential diagnosis of azoospermia must first be administered. Second, genetic testing is performed to identify men who have NOA due to microdeletions in the long arm of the Y chromosome and to inform affected patients about their chances of sperm retrieval success. Third, it is investigated whether any intervention may stimulate sperm production before a surgical retrieval attempt. Fourth, the most effective and efficient retrieval technique for testicular sperm is adopted. Finally, cutting-edge laboratory methods are used to manage retrieved gametes and cultivate embryos produced by sperm injections (Esteves, Reference Esteves2015). For the majority of patients with NOA, there is yet no treatment that can restore spermatogenesis, except for those who develop testicular failure subsequently. So, for impacted couples to become pregnant without a donor, ICSI using spermatozoa extracted from the testes is their only choice (Donoso et al., Reference Donoso, Tournaye and Devroey2007). Techniques for sperm retrieval have been developed, including percutaneous, incisional and microsurgically aided methods. Testicular sperm aspiration (TESA) is an example of the percutaneous approach, in which testicular tissue is aspirated using small or large-bore needles. The needle is usually attached to a syringe, creating suction as the needle tip is moved around within each testis to sample the seminiferous tubular tissue. Incisional techniques are conventional testicular sperm extraction (cTESE) or microdissection testicular sperm extraction (mTESE). cTESE involves extracting seminiferous tube tissue through one or more testicular incisions (Medicine, 2018). Numerous randomly chosen testicular samples are often collected and analysed, since it is unfeasible to predict the presence of sperm or the location of islets of normal spermatogenesis, assuming they exist, before doing cTESE. A drawback of cTESE is that removing huge portions of testicular tissue may imperil the already reduced androgen production transiently or permanently, resulting in severe hypogonadism (Esteves, Reference Esteves2015). The technique of microdissection in instances of NOA, TESE (micro-TESE), has been suggested as an ideal replacement for TESE. In addition to reducing the amount of testicular tissue removed and increasing the spermatozoa yield per biopsy, this approach also has the added benefit of identifying blood arteries, which helps to limit vascular damage. (Schlegel, Reference Schlegel1999). The technique of microdissection for TESE involves making a large incision in the testis. Then, under the optical magnification of an operating microscope, the largest-diameter seminiferous tubules are selected (Medicine, 2018). One other method to consider is FNA. It was first used for diagnostic reasons and is a less intrusive technique for sperm retrieval compared to TESE. Yet, compared to TESE, FNA has a much lower sperm retrieval rate (SRR), according to the majority of research. (Tournaye et al., Reference Tournaye, Camus, Goossens, Liu, Nagy, Silber, Van Steirteghem and Devroey1995; Hauser et al., Reference Hauser, Botchan, Amit, Ben Yosef, Gamzu, Paz, Lessing, Yogev and Yavetz1998). The timing of sperm extraction is a crucial element in the therapy of NOA. Surgical sperm extraction may be performed concurrently with oocyte retrieval during an IVF cycle, using fresh sperm for ICSI if available. If viable sperm are retrieved before ovarian stimulation, they may be cryopreserved for use in future in vitro fertilisation cycles. Each method has theoretical advantages. Using freshly retrieved sperm keeps the sperm from the stress of cryopreservation. Conversely, cryopreserving the collected sperm for future use decouples the time of in vitro fertilisation from sperm extraction, enabling the female partner to circumvent superfluous ovarian stimulation in the absence of identified sperm. (Medicine, 2018). The next step after SR is to transport the sample of testicular tissue to an in vitro fertilisation facility for analysis and possible sperm retrieval. There has been evidence of poorer fertilisation rates, embryo development retardation and pregnancy rates when using spermatozoa collected from males with NOA in ICSI. This is likely because these gametes are often of lower quality and more delicate compared to ejaculated gametes (Ubaldi et al., Reference Ubaldi, Nagy, Rienzi, Tesarik, Anniballo, Franco, Menchini-Fabris and Greco1999; Verza Jr and Esteves, Reference Verza and Esteves2008; Meseguer et al., Reference Meseguer, Santiso, Garrido, Gil-Salom, Remohí and Fernandez2009; Vozdova et al., Reference Vozdova, Heracek, Sobotka and Rubes2012; Esteves and Agarwal, Reference Esteves and Agarwal2013; Esteves et al., Reference Esteves, Prudencio, Seol, Verza, Knoedler and Agarwal2014). The lower the tissue is extracted, the easier it is to analyse and locate the sperm. Micro-TESE can enhance laboratory process efficiency, reduce testicular damage and increase SR success (Esteves and Varghese, Reference Esteves and Varghese2012; Ashraf et al., Reference Ashraf, Singh, Raj, Ramakrishnan and Esteves2013; Esteves, Reference Esteves2013). Micromanipulation can only take place in an environment that adheres to good laboratory practices, which include maintaining a sterile environment, controlling the temperature and pH of the working solution and ensuring optimal air quality. (Esteves and Verza, Reference Esteves, Verza, Nagy, Varghese and Agarwal2012; Esteves and Bento, Reference Esteves and Bento2013).
Management of different abnormalities in men with NOA
Treatment of azoospermia depends on the cause, and each cause has a different prognosis. A varicocele is a disorder in which the scrotal venous pampiniform plexus drains blood from each testicle, dilates and enlarges abnormally. Being the most often documented cause of abnormalities in sperm analysis, lower sperm count, poor sperm motility and abnormal morphology, varicoceles are clinically significant (Yetkin and Ozturk, Reference Yetkin and Ozturk2018; Paick and Choi, Reference Paick and Choi2019). Surgical varicocelectomy is a crucial therapy for reestablishing fertility, since varicocele is linked to NOA in 5–10% of individuals. (Chiba et al., Reference Chiba, Enatsu and Fujisawa2016). Recent investigations, however, have revealed that even if spermatogenesis improves, postoperative sperm concentration is still poor and ART such as ICSI will be needed. Consequently, it is essential to evaluate the feasibility of doing varicocelectomy or sperm retrieval without concomitant varicocele repair for these patients (Berookhim and Schlegel, Reference Berookhim and Schlegel2014). Undescended testis is a common congenital condition generally noticed and treated during infancy. There is a risk of testicular cancer and spermatogenesis impairment when the testis is improperly located (e.g., abdominal or inguinal) (Hutson et al., Reference Hutson, Hasthorpe and Heyns1997; Barthold and González, Reference Barthold and González2003; Murphy et al., Reference Murphy, Paran and Puri2007; Virtanen et al., Reference Virtanen, Bjerknes, Cortes, Jørgensen, Rajpert-De Meyts, Thorsson, Thorup and Main2007). The best way to treat undescended testicles (UDT) remains a topic of debate. There are, however, several guidelines for the treatment of individuals with UDT: (1) Hormonal therapy is not suggested due to the short-term and potential long-term spermatogenesis consequences. (2) Surgery is the preferred therapy; it is effective in roughly 95% of UDT cases, with a low complication rate (about 1 percent). (3) Orchiopexy should be performed between the ages of 12 and 18 months or at the time of initial contact if the disease is diagnosed later (Niedzielski et al.,) Many infertile men have had NOA due to a genetic disorder in the last twenty years. These include Y-chromosomal microdeletions, karyotype abnormalities and missense mutations in genes crucial for reproduction (Cerván-Martín et al., Reference Cerván-Martín, Castilla, Palomino-Morales and Carmona2020). The most common chromosomal defect among NOA patients is Klinefelter syndrome. In men, small testes, less muscle, less body hair, poor sex desire and gynaecomastia are all symptoms of Klinefelter syndrome. The diagnosis is usually made during a male infertility assessment. A 47, XXY chromosomal complement is seen in approximately 95 percent of men with Klinefelter syndrome (Oates, Reference Oates2008). Micro-TESE coupled with ICSI is the only treatment option available for NOA patients with Klinefelter syndrome (Ando et al., Reference Ando, Yamaguchi, Chiba, Miyake and Fujisawa2013). An increased incidence of sex and autosomal chromosomal aneuploidies may be seen in spermatozoa from individuals with Klinefelter syndrome (Morel et al., Reference Morel, Bernicot, Herry, Le Bris, Amice and De Braekeleer2003; Staessen et al., Reference Staessen, Tournaye, Van Assche, Michiels, Van Landuyt, Devroey, Liebaers and Van Steirteghem2003). Secondary testicular dysfunction induced by hypothalamic or pituitary disorders is known as hypogonadotropic hypogonadism (HH). The hyposecretion of gonadotropins leads to low testosterone production by the testes and impaired spermatogenesis. There are two types of HH: congenital and acquired. Although HH is a rare condition, medical treatment can improve fertility in patients with this condition. The use of gonadotropins is a conventional medical treatment when fertility is a problem (Chiba et al., Reference Chiba, Enatsu and Fujisawa2016).
Future perspectives and cellular therapy for management of non-obstructive azoospermia
Exciting new studies have demonstrated the great transformation potential of germline cells in recent years (Hübner et al., Reference Hübner, Fuhrmann, Christenson, Kehler, Reinbold, De La Fuente, Wood, Strauss, Boiani and Schöler2003). Primordial germ cells extracted from the embryonic epiblast or teratocarcinoma can colonise the testis and differentiate into male germ cells (Nayernia et al., Reference Nayernia, Li, Jaroszynski, Khusainov, Wulf, Schwandt, Korabiowska, Michelmann, Meinhardt and Engel2004). Approximately 24 days after fertilisation, they become visible in human embryos within the yolk sac wall, near the allantois. During early embryonic development, SSCs emerge from postnatal testicular gonocytes, which in turn come from long-lived PGCs. (Freeman, Reference Freeman2003). SSCs are seen as a potential option for regenerating deficient or impaired spermatogenesis. SSC transplantation is a prospective approach for treating male infertility and restoring spermatogenesis in patients suffering from degenerative conditions such as cancer, radiation and chemotherapy (Abdelaal et al., Reference Abdelaal, Tanga, Abdelgawad, Allam, Fathi, Saadeldin, Bang and Cho2021). The most delicate premeiotic germ cells are destroyed after cytotoxic treatment; therefore, the remaining spermatogonial stem cells must restart the spermatogenesis process from the beginning (Ehmcke et al., Reference Ehmcke, Wistuba and Schlatt2006). Before beginning cancer treatment, SSCs are isolated and cryopreserved from a testicular biopsy, followed by intra-testicular stem cell transplantation (Abdelaal et al., Reference Abdelaal, Tanga, Abdelgawad, Allam, Fathi, Saadeldin, Bang and Cho2021). Before starting treatment, a sterile biopsy is performed on the testis of children with cancer or adults with azoospermia, whatever the cause may be. Extracting testicular tissue in humans is an alternative way for biopsy. To obtain a single-cell suspension, the testicular tissue or biopsy is digested using enzymes such as trypsin and type I collagenase (Amirinezhadfard et al., Reference Amirinezhadfard, Arjmand, Azizi and Rahimi2025). The process of isolating stem cells from spermatozoa begins with this stage. One effective method that utilises density gradient centrifugation is Percoll isolation (Liu et al., Reference Liu, Tang, Xiong and Tang2011; Oatley, Reference Oatley2017). Propagation of SSCs requires cultivation and growth after isolation or preservation. The main culture of SSCs is established by first making mitomycin-treated STO feeders, followed by washing them with HBSS and then adding the SSCs, whether freshly isolated or cryopreserved (Takahashi and Yamanaka, Reference Takahashi and Yamanaka2006). To improve SCC differentiation into spermatocytes and spermatids, a 3D culture technique was employed in humans that utilised agarose gel (Mohaqiq et al., Reference Mohaqiq, Movahedin, Mazaheri and Amirjannati2019). Human GPR125+ spermatogonia were propagated on hydrogel-coated or gelatin-coated plates with growth factors (GDNG, bFGF, GFRA1-Fc fusion protein, NUDT6, TGFB, EGF and LIF) (Azizi et al., Reference Azizi, Skutella and Shahverdi2017). SSC transplantation involves recipient preparation. The preceding protocols focused on preparing SSCs from donors (Azizi et al., Reference Azizi, Tabar and Skutella2020b). There are two methods for SSC transplantation: the first involves the retrieval of complete testis or testicular tissue, which requires a specific preparation protocol, and the second involves the extraction and culture of cells. In the case of whole testis tissues, the tunica albuginea should be removed. The testes are then immersed in HBSS, and collagenase is added to facilitate the separation of the seminiferous tubules. Subsequently, HBSS with trypsin-EDTA is added for digestion and cell dispersion. Following this, centrifugation, washing and resuspension in SFM are performed. In the second case of culture, a cell suspension is performed directly in SFM. In both cases, the same procedure is followed, which involves microinjecting the cells into the lumen of the seminiferous tubules with the aid of trypan blue for easier gross monitoring during the injection process. After that, the recipient males are kept under observation for an extra eight weeks to ensure strong donor-derived spermatogenesis (Goodyear and Brinster, Reference Goodyear and Brinster2017). SSC transplantation as a treatment for male infertility due to impaired spermatogenesis is promising. However, it has not yet reached its full potential or been well implemented and still faces several challenges. The whole SSC transplantation process requires more evaluation. The cell and tissue cryopreservation efficiency must be pursued with extreme caution, as this stage is crucial, and the efficacy and effectiveness of cryopreserved SSCs will determine the subsequent treatment approach for infertility. Preserving SSCs before they are depleted and degraded by radiation and chemotherapy is crucial for the future fertility of children impacted by cancer before therapies. Another major and urgent problem is the possibility of cancerous contamination of cancer-stricken children’s testes, which might undermine cryopreservation efforts. There are significant concerns about improving protocol efficiency without compromising the testicular niche (Geens et al., Reference Geens, Goossens, De Block, Ning, Van Saen and Tournaye2008). A community’s socio-cultural attitude and viewpoint remain the primary barrier to the widespread implementation of the various SSCs being considered. The reason for this is that stem cell treatment is not an option for adult patients who can preserve sperm obtained from ejaculated sperm or TESE. There is a lack of expertise and information regarding the cryopreservation of testicular tissue from prepubertal patients. Furthermore, only a limited number of institutions have the requisite expertise, infrastructure or regulatory authorisation to treat and maintain testicular tissue (Hanna Valli et al., Reference Valli, Phillips, Gassei, Nagano and Orwig2015). Other stem cell types can also be used in this process, particularly in patients who lack SSCs. As a result, these stem cells give individuals the opportunity to have children in the future. ESCs, pluripotent cells that can differentiate into various cell lineages, are isolated from the inner blastocyst cell mass. They can produce germ cell lines in vitro; however, using embryonic stem cells in humans is fraught with ethical issues. Furthermore, ESCs lose their plasticity during differentiation (Pourmoghadam et al., Reference Pourmoghadam, Aghebati-Maleki, Motalebnezhad, Yousefi and Yousefi2018). In vitro, ESCs differentiate into male germ-like cells; however, this approach has the drawback of being genetically unrelated to patients (Devolder, Reference Devolder2010). VSELs are another type of stem cell that can be utilised in this process. These cells are pluripotent in humans and mice. They possess a self-renewal ability and can regenerate. Since VSELs are not damaged by chemotherapy, they can contribute to the development of germ cells in men who underwent chemotherapy as infants (Kurkure et al., Reference Kurkure, Prasad, Dhamankar and Bakshi2015; Wyns et al., Reference Wyns, Kanbar, Giudice and Poels2021). iPSCs, which can be reprogrammed, can be utilised to replace ESCs, avoiding ethical controversy. The benefits of iPSCs include a straightforward sample approach, in which somatic cells, such as skin cells, can be acquired and reprogrammed to form germ cells (Takahashi and Yamanaka, Reference Takahashi and Yamanaka2006; Forbes et al., Reference Forbes, Flannigan and Schlegel2018; Pourmoghadam et al., Reference Pourmoghadam, Aghebati-Maleki, Motalebnezhad, Yousefi and Yousefi2018). Due to their versatility, immunomodulatory capabilities, excellent safety profile and ease of isolation and culture, mesenchymal stem cells (MSCs) have found applications in a wide range of fields and medical conditions. After injecting MSCs into a busulfan-induced azoospermic rat, researchers found that the animals resumed normal spermatogenesis, indicating that MSCs may be a promising treatment for male infertility (Cakici et al., Reference Cakici, Buyrukcu, Duruksu, Haliloglu, Aksoy, Isık, Uludag, Ustun, Subası and Karaoz2013; Zhang et al., Reference Zhang, Liu, Peng, He, Lin, Zhu, Li, Zhang and Wei2014). Remarkably, even after transplanting half as many SSCs, the reproductive efficiency of SSC transplantation was improved when co-transplanted with TGF1-treated MSCs (Kadam et al., Reference Kadam, Ntemou, Onofre, Van Saen and Goossens2019) (Figure 2).
Types of methods of maintaining fertility in in vitro and in vivo conditions. Obtaining testicular tissue samples from azoospermic individuals or individuals exposed to chemotherapy.
Figure 2 Long description
The diagram depicts the process of maintaining fertility through various methods. It begins with a testis biopsy to obtain testicular tissue samples from azoospermic individuals or those exposed to chemotherapy. The isolated spermatogonial stem cells (SSCs) undergo expansion and can be cryopreserved. The cryopreserved SSCs are later thawed and cultured in 2D or 3D organ cultures to produce spermatozoa. These spermatozoa are then used in assisted reproductive technology (ART) to produce offspring. Alternatively, the isolated SSCs or tissues can be directly transplanted to restore fertility. The diagram shows the flow from the initial biopsy to the final outcome of producing offspring, highlighting key steps such as SSCs isolation, cryopreservation, and transplantation.
Conclusion
NOA represents the most severe form of male infertility, and current clinical management remains limited. Although micro-TESE enables identification and use of rare spermatozoa in nearly half of affected men, it does not restore endogenous spermatogenesis, and it offers no reproductive options for patients in whom sperm cannot be retrieved. The high prevalence of chromosomal and genetic aberrations in NOA underscores the need for comprehensive diagnostic testing and careful counselling; however, these measures do not alter the fundamental therapeutic gap. In this context, spermatogonial stem cell (SSC)–based interventions emerge as a critical frontier. SSC isolation, expansion and transplantation hold unique potential to reestablish sustained spermatogenesis and thereby offer a biological cure – rather than a procedural workaround – for NOA. Yet, despite decades of progress in experimental models, several translational challenges remain unresolved, including optimisation of SSC preservation, validation of transplantation protocols and assurance of genetic and epigenetic safety. Addressing these challenges is particularly urgent for prepubertal boys undergoing gonadotoxic treatments, whose only opportunity to preserve future fertility may lie in banking and later autologous transplantation of SSCs. Ensuring the viability and functionality of cryopreserved SSCs is therefore not merely a technical consideration but a decisive step that will determine the feasibility of SSC-based therapies as the next generation of fertility restoration. Ultimately, the successful clinical deployment of SSC therapy could transform the landscape of NOA management by restoring natural spermatogenesis and expanding reproductive options for patients who currently have none. Continued foundational research, rigorous translational testing and the development of standardised clinical frameworks will be essential to realise this therapeutic promise.
Data availability statement
None.
Acknowledgments
The research protocol was approved & Supported by Student Research Committee, Tabriz University of Medical Sciences (grant number: 74848).
Author contributions
Ψ Mehdi Mehdinezhad Roshan and Maryam Salehi Novin contributed equally to write the manuscript and edited the final document; Hamed Hajipour designed the study; Omid Abouie Mehrizi and Keyvan Kiani designed figures and edited scientific and handwritten writing; the authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Ethical standards
The Ethics Committee of Tabriz University of Medical Sciences gave its approval to the current research (IR.TBZMED.VCR.REC.1403.285).
Funding
None.
Consent for publication
Not applicable.
