Predictors of sperm retrieval success in first-time and repeated Micro-TESE for nonobstructive azoospermia

Abstract

Introduction

Microdissection testicular sperm extraction (micro-TESE) is the preferred sperm retrieval method, although success rates vary based on factors such as age and hormone levels. We evaluate sperm retrieval outcomes in first-time and repeated micro-TESE, identifying predictors of success to guide clinical management.

Material and methods

Retrospective study analyzed 152 men with nonobstructive azoospermia (NOA) who underwent micro-TESE between 2020 and 2022. Patients were grouped based on sperm retrieval outcomes, and clinical, demographic and hormonal factors were assessed. Comparisons were made between first-time and repeat micro-TESE cases.

Results

Sperm retrieval rate was higher for first-time TESE cases (64.6%) than repeated TESE cases (28.8%, p < 0.01). Repeated TESE cases were older, had higher smoking rates, and exhibited significantly elevated follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, with lower total testosterone (p < 0.01). In repeated TESE cases, no significant differences in age, smoking status or hormone levels were found between successful and failed attempts.

Conclusion

First-time micro-TESE users had a significantly higher sperm retrieval rate than those undergoing repeated attempts. Repeated TESE was associated with older age, higher smoking rates and adverse hormonal profiles. Larger studies are needed to validate these findings and optimize patient selection for repeated micro-TESE.

ARTICLE HIGHLIGHTS

• This study evaluates sperm retrieval outcomes in first-time and repeated micro-TESE procedures among men with nonobstructive azoospermia (NOA).

• The overall sperm retrieval rate was significantly higher in first-time micro-TESE cases compared to repeated procedures (64.6% vs. 28.8%).

• Repeated micro-TESE cases were associated with older age, higher smoking prevalence, and more adverse hormonal profiles.

• Klinefelter syndrome and undescended testes were more prevalent in first-time attempts but showed lower retrieval rates in repeated procedures.

• Some patients achieved successful sperm retrieval on repeat attempts despite prior failures, potentially due to improved hormonal preparation or surgical factors.

• The study highlights key clinical and hormonal predictors of micro-TESE outcomes and emphasizes the need for individualized patient selection and counseling.

1. Introduction

Infertility is defined as the inability of sexually active non-contraceptive-using couples to have a child in one year or more [1]. Nearly 15% of couples experiencing infertility seek medical consultation. In general, infertility can be attributed to male factor, female factor, both female and male or idiopathic. (1) There are many causes of male infertility; where nearly 90% of cases are due to low sperm number, poor sperm motility and morphology or both. Other causes may include anatomical problems, hormonal problems including diseases of hypothalamus or pituitary, genetic defects in addition to ejaculatory problems such as retrograde ejaculation or absence of ejaculation [2]. As an important cause of male infertility, azoospermia is diagnosed if no sperm is found in two seminal fluid tests, and it is considered the severest form of male infertility; nearly 15% of infertile males could have azoospermia. Any condition that affects sperm production, function or transport may lead to male infertility [3,4]. In general azoospermia classified into two major categories: obstructive and non-obstructive. Obstructive azoospermia occurs due to obstruction of the duct along the way between the testicular tissues and the urethra or in the ejaculatory duct despite the presence of normal spermatogenesis [5]. On the other hand, non-obstructive azoospermia (NOA) is caused by defect in spermatogenesis process where primary testicular tissues develop into mature motile sperms, while the duct that transport sperms is patent [6]. About 60% of azoospermia cases are due to nonobstructive causes. The etiologies of NOA can be divided according to the anatomical position of the etiology: pre-testicular or testicular. Pretesticular NOA results from the presence of normal functioning testes that are not sufficiently stimulated to produce viable sperms, secondary to hormonal insufficiency due to hypothalamic-pituitary disorders, which is also known as secondary hypogonadism. On the other hand, testicular azoospermia can be an outcome of primary testicular failure or intrinsic dysfunction, and it is called a primary hypogonadism, leading to impaired spermatogenesis [7]. Nonobstructive azoospermia (NOA) is usually caused by a problem in spermatogenesis, which could result from primary testicular failure or from secondary causes like hypothalamus or pituitary dysfunction [8]. There are different other causes of non-obstructive azoospermia, including genetic abnormalities, hormonal imbalance, exposure to radiation, toxins and medications [9]. However, the exact etiology is not well known. For patients with NOA, testicular sperm extraction to retrieve viable sperms and intracytoplasmic sperm extraction are the methods of choice for management [10].

Microtesticular sperm extraction was first described by Shlegel [11]. It yields a higher sperm retrieval rate than other methods, such as testicular sperm aspiration (TESA) and conventional testicular sperm extraction (cTESE) [12]. Advanced assisted reproductive techniques such as Micro-TESE have given hope to infertile patients with NOA, especially those who are diagnosed with primary testicular failure [8]. With a success rate of approximately 63% [13], Micro-TESE is considered as a remarkable improvement over traditional methods used for sperm extraction like conventional testicular sperm extraction, providing the chance for higher sperm retrieval rates and reduced tissue damage and fibrosis post operation. Micro-TESE utilizes a high-powered surgical microscope to search for and extract small amounts of testicular tissue, giving a more targeted and successful sperm retrieval. After that, the extracted Spermatozoa are analyzed under a second magnification microscope to choose the most competent sperm in morphology and motility to use it for intracytoplasmic sperm injection. (ICSI) [13]. The sperm retrieval rate after microdissection testicular sperm extraction (micro-TESE) is influenced by many factors, such as age, smoking status, blood levels of follicle-stimulating hormone (FSH), Luteinizing hormone (LH), in addition to blood levels of total and free testosterone, and previous varicocele ligation [14]. Falcone et al. conducted a broader analysis of the Sperm Retrieval Rate (SRR) of micro-TESE and demonstrated great variability in the results, ranging from 43% to 63%. This variability could be attributed to the diverse nature of NOA, which is influenced by testicular volume and histopathology patterns [15].

In cases of obstructive azoospermia, sperm retrieval is successful in nearly all patients [16]. In contrast, among men with non-obstructive azoospermia (NOA), conventional testicular sperm extraction (TESE) yields sperm in approximately 50% of cases [17]. Moreover, even when sperm is retrieved in NOA patients, the pregnancy rate following a single intracytoplasmic sperm injection (ICSI) cycle remains low [18], often necessitating multiple ICSI attempts and repeated testicular biopsies to obtain viable spermatozoa. In the field of NOA, there has been wide debate about the rationale of using hormonal preparation before Micro-TESE and what type of preparation to use. While it has been believed to be ineffective for NOA due to already elevated gonadotropin levels; on the other hand, several studies have stimulated sperm production before Micro-TESE by using different medications like antiestrogens, aromatase inhibitors, and gonadotropins [19]. The success rate for sperm extraction from Micro-TESE is only 40–60%. Thus, hormonal preparation before Micro-TESE could prove to be an effective adjunctive therapy to increase SSR rates [20]. In patients with negative Micro-TESE, repeating the procedure is the only remaining option before the next steps of sperm donation or adoption. Before proceeding with the next procedure, it is important to discuss the adverse effects and success rates of this procedure with patients. Additionally, factors that affect the outcomes of repeated micro-TESE should be evaluated and discussed carefully as part of the decision-making process [21].

While many studies have addressed sperm retrieval in micro-TESE, differences in study populations and surgical approaches make continued investigation important. In this study, we aimed to evaluate the success rates of both initial and repeated Micro-TESE procedures in patients with non-obstructive azoospermia (NOA), and to examine how outcomes relate to various factors including patient age, smoking status, testicular volume, hormonal profile (FSH, LH, and testosterone levels), preoperative hormonal treatment, and testicular histopathology. Ultimately, our goal is to contribute data that may help improve Micro-TESE success rates in the future and support existing findings in the literature.

2. Materials and methods

2.1. Study design

We conducted a retrospective cohort study involving 152 infertile men diagnosed with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction (micro-TESE). The study included adult participants aged 18 years or older and aimed to assess clinical, demographic, and hormonal predictors of sperm retrieval outcomes.

2.2. Study setting and ethical considerations

This study was carried out at a single tertiary hospital between January 2020 and December 2022. Written informed consent was obtained from each participant prior to inclusion. Ethical approval was granted by the Institutional Review Board (IRB) of AL-Balqa Applied University (reference number: 18/1/2024/2025). Data access was approved on September 15, 2024. All data were anonymized, and no personally identifiable information was available to the investigators during or after data collection.

2.3. Participants

Eligible participants were infertile men with primary infertility (had no previous successful conceptions) due to NOA who underwent micro-TESE during the study period. Patients were excluded if they had secondary infertility (had any previous successful conception), primary infertility due to female factor, infertility due to obstructive azoospermia, incomplete hormonal profile data (FSH, LH, total testosterone or free testosterone), or genetic causes of non- obstructive azoospermia, including AZFa or AZFb microdeletions. At the end, a total of 152 patients met the study inclusion criteria and were included in the analysis. The patients were divided into two groups based on the sperm retrieval outcome of micro-TESE procedure: those with successful sperm retrieval (finding of sperms during Micro-TESE procedure) and those with negative results (no sperms found during Micro-TESE procedure). Sperm retrieval success was defined as the detection of any sperm (motile or immotile) at the time of the micro-TESE procedure.

2.4. Surgical procedure of Micro-TESE

All procedures were conducted under general anesthesia. After patient counseling, Surgical technique started by doing a midline scrotal incision, after that scrotal layers opened started from dartos muscle and fascia, external spermatic fascia, cremastric muscle and fascia, internal spermatic fascia, tunica vaginalis down to the tunica albuginea, which was incised at its mid-portion in a relatively avascular area to expose testicular tissues. An operating microscope (Karl Zeiss, Germany) was then used to visualize the testicular parenchyma, aiding in the dissection and examination of the seminiferous tubules for the presence of viable sperms, with the magnification set at 15–20×. Thick, dark yellow seminiferous tubules were then extracted and fragmented using sterile 1 mL syringes with attached needles, creating a homogeneous suspension in a dish containing G-MOPS-plus medium (Vitrolife, Vastra Frolunda, Sweden). An inverted microscope at 200× magnification was subsequently used to detect viable sperm. Sperm detection was assessed immediately after retrieval. The presence of any sperm, regardless of motility, was considered a successful retrieval, as motility may develop several hours later. If no sperm were found, good homeostasis achieved using mono-polar electro-cautery, the tunica albuginea and other scrotal layers till scrotal skin were closed with a 2-0 Vicryl suture, after that, the contralateral testis was delivered through the same scrotal incision, scrotal layers also incised till the tunica albuginea which was then opened, and the sperm search process under the microscope was repeated. Micro-TESE was performed as a day case procedure under general anesthesia, where all patients discharged on the same day after full recovery from the procedure with no significant complications, except for one patient who developed a scrotal hematoma that was managed conservatively.

2.5. Variables

The primary outcome in this study was sperm retrieval success, defined as the identification of viable spermatozoa during the microdissection testicular sperm extraction (Micro-TESE) procedure. Based on this outcome, patients were categorized into two groups: those with positive sperm retrieval and those with negative sperm retrieval. The analysis also distinguished between patients undergoing Micro-TESE for the first time and those undergoing repeated procedures. A range of clinical and hormonal variables were collected to evaluate their potential role in predicting Micro-TESE outcomes. These included patient age, recorded in years, and smoking status. Hormonal profiles were assessed through preoperative blood tests, which included serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (ng/mL), and free testosterone (pg/mL). Preoperative hormonal preparation was defined as the administration of clomiphene citrate and human chorionic gonadotropin (HCG) for a minimum of three months prior to the procedure and was recorded as a binary variable (yes or no). Additional variables included the presence of Klinefelter syndrome, confirmed by chromosomal analysis. All patients were diagnosed with non-obstructive azoospermia (NOA) based on repeated semen analyses showing complete absence of spermatozoa after centrifugation, according to the World Health Organization (WHO) guidelines.

2.6. Statistical analysis

The statistical analysis was performed using Jamovi (Version 2.5) [22]. Categorical variables, such as smoking status, were analyzed using Pearson’s chi-square test to assess their association with the two groups, with results presented as counts and percentages. Continuous variables were summarized using interquartile ranges (IQR), including the first quartile (Q1), median, and third quartile (Q3), and were analyzed using the Wilcoxon rank-sum test (Mann–Whitney U test). Statistical significance was set at p < 0.05.

3. Results

3.1. Demographics of study participants

A total of 152 patients underwent Micro-TESE at our center. The median age of the cohort was 31 years, with an interquartile range (IQR) of 28 to 36 years. Among the entire group, 47 patients (31%) were diagnosed with Klinefelter syndrome, while 69 patients (45%) were identified as current or former smokers. Preoperative hormonal preparation was administered to 78 patients (51%), following the institutional protocol that included daily oral clomiphene citrate (25 mg) and subcutaneous injections of human chorionic gonadotropin (HCG) at a dose of 2000 IU three times per week for a minimum of three months prior to the procedure. Hormonal data were available for all patients except for free testosterone, which was recorded in 120 patients. The median follicle-stimulating hormone (FSH) level was 17 IU/L (IQR: 10–26), while the median luteinizing hormone (LH) level was 14 IU/L (IQR: 9–23). The median total testosterone level was 2.60 ng/mL (IQR: 1.98–4.00). The median free testosterone level among the 120 available cases was 18 pg/mL (IQR: 11–25). In terms of surgical history, 73 patients (48%) had previously undergone at least one testicular sperm extraction (TESE) procedure before presenting to our center. The remaining 79 patients (52%) were undergoing Micro-TESE for the first time. A comprehensive summary of demographic variables, clinical features, and hormonal parameters is presented in Table 1.

Table 1.

Demographics and hormonal parameters.

Characteristics	N	Median (IQR); n (%)
Age (Years)	152	31 (28, 36)
Smoking	152	69 (45%)
Klinefelter Syndrome	152	47 (31%)
Pre-operation Preparation (Yes)	152	78 (51%)
Follicle-stimulating hormone (FSH) (IU/L)	152	17 (10, 26)
Luteinizing hormone (LH) (IU/L)	152	14 (9, 23)
Total testosterone (ng/mL)	152	2.60 (1.98, 4.00)
Free testosterone (pg/mL)	120	18 (11, 25)
Previous TESE attempts	152	73 (48%)

Preoperative preparation consisted of clomiphene citrate (25 mg daily) and subcutaneous HCG injections (2000 IU, three times weekly) for at least 3 months.

3.2. Comparison of clinical, demographic and hormonal parameters based on previous TESE attempts

A comparison of demographic, clinical and hormonal parameters between individuals undergoing first-time Micro-TESE (n = 79) and those undergoing a repeated Micro-TESE (n = 73) revealed significant differences. The positive rate outcome was significantly higher in the first-time group (64.6%) than in the repeat group (28.8%) (p < 0.01). The median age was also significantly lower in the first-time group (28.0 years) than in the repeat group (34.0 years, p < 0.01). Smoking prevalence found to be higher in the repeat group than first time (58.9% vs. 32.9%, p < 0.01). While Klinefelter syndrome was more prevalent in the first-time than repeat group (40.5% vs. 20.5%, p = 0.01). Hormonal analysis revealed significantly higher levels of FSH (p < 0.01) and LH (p < 0.01) in the repeat group, while total testosterone was lower in this group (p = 0.01). Free testosterone levels revealed no significant differences (p = 0.06) (Table 2).

Table 2.

Comparison of clinical and hormonal characteristics between first-time and repeated Micro-TESE patients.

		First-time	Repeated
	N	(n = 79)	(n = 73)	p-value
Micro-TESE result: Positive	152	0.6  51/79	0.3  21/73	p < 0.01a
Age (Years)	152	26.0 28.0 31.0	31.0 34.0 38.0	p < 0.01b
Smoking: Yes	152	0.3  26/79	0.6  43/73	p < 0.01a
Klinefelter: Yes	152	0.4  32/79	0.2  15/73	p = 0.01a
FSH (IU/L)	152	8.2 14.0 23.0	11.0 18.0 32.3	p < 0.01b
LH (IU/L)	152	7.0 12.0 20.0	10.0 19.0 28.0	p < 0.01b
Total testosterone (ng/mL)	152	2.0 3.0 4.0	1.5 2.2 3.4	p = 0.01b
Free testosterone (pg/mL)	120	14.2 20.0 25.0	10.0 15.0 25.0	p = 0.06b

N is the number of non-missing values. ^aPearson’s chi-square test; ^bWilcoxon rank-sum test.

Continuous variables presented as Q1, Median, Q3.

3.3. Comparison of clinical and hormonal factors between patients with positive and negative outcomes in repeated Micro-TSES procedures

A total of 73 patients underwent repeated Micro-TESE, with outcomes categorized as positive sperm retrieval (G1, n = 21) and negative sperm retrieval (G0, n = 52). Clinical and hormonal parameters were compared between the two groups. The median age was 33 years (Q1–Q3: 30–36.3) in G1 and 35.5 years (Q1–Q3: 32.4–38) in G0 (p = 0.112). The proportion of patients who smoked was 10 out of 21 (48%) in G1 and 33 out of 52 (63%) in G0 (p = 0.211). Klinefelter syndrome was present in 1 out of 21 patients (5%) in G1 and in 14 out of 52 patients (27%) in G0, with a statistically significant difference between the two groups (p = 0.031). Regarding hormonal findings, the median FSH level was 21 IU/L (Q1–Q3: 10–43.3) in G1 and 18 IU/L (Q1–Q3: 12–27.2) in G0 (p = 0.592). The median LH level was 17 IU/L (Q1–Q3: 9.7–31.3) in G1 and 20 IU/L (Q1–Q3: 10.4–25.6) in G0 (p = 0.732). The median total testosterone level was 2.9 ng/mL (Q1–Q3: 1.9–4.2) in G1 and 2.0 ng/mL (Q1–Q3: 1.3–3.2) in G0 (p = 0.192). The median free testosterone level was 15.5 pg/mL (Q1–Q3: 11.2–28.3) in G1 and 14.0 pg/mL (Q1–Q3: 10.0–22.3) in G0, based on 120 available samples (p = 0.142). Preoperative hormonal preparation was documented in 15 out of 21 patients (71%) in G1 and 25 out of 52 patients (48%) in G0 (p = 0.071). A full summary of clinical and hormonal characteristics is presented in Table 3.

Table 3.

Comparison of clinical and hormonal factors between successful (G1) and unsuccessful (G0) sperm retrieval in repeated Micro-TESE cases.

		G1 (positive TESE)	G0 (negative TESE)
	N	(n = 21)	(n = 52)	p-value
Age (Years)	152	30 33 36.3	32.4 35.5 38	p = 0.11b
Smoking status: Yes	152	0.5 10/21	0.6 33/52	p = 0.21a
Klinefelter: Yes	152	0.0 1/21	0.3 14/52	p = 0.03a
FSH (IU/L)	152	10 21 43.3	12.0 18 27.2	p = 0.59b
LH (IU/L)	152	9.7 17 31.3	10.4 20 25.6	p = 0.73b
Total testosterone (ng/mL)	152	1.9 2.9 4.2	1.3 2 3.2	p = 0.19b
Free testosterone (pg/mL)	120	11.2 15.5 28.3	10.0 14 22.3	p = 0.14b
Preparation: Yes	152	0.7 15/21	0.5 25/52	p = 0.07a

N is the number of non-missing values. ^aPearson’s chi-square test; ^bWilcoxon rank-sum test.

Continuous variables presented as Q1, Median, Q3.

4. Discussion

About 10% of men with infertility and 1% of males worldwide suffer from azoospermia, the most severe type of male infertility, which is characterized by the total absence of sperm in the ejaculate. It is classified into two main categories: obstructive azoospermia and nonobstructive azoospermia, with this classification being crucial for management and reproductive outcomes. There are many different factors that negatively affect the process of spermatogenesis, such as obesity, diabetes mellitus, hypertension, smoking, history of varicocele and genetic factors like Klinefelter syndrome [23].

A variety of factors, such as genetic or congenital abnormalities, hormonal imbalances, post-infectious damage, exposure to gonadotoxic chemicals (such as chemotherapy or radiation therapy), and testicular trauma, can lead to NOA. However, in many cases, the underlying cause remains unidentified, and NOA is classified as idiopathic [24].

Microdissection testicular sperm extraction, is considered currently one of the most popular sperm retrieval procedures for men with azoospermia, was first described in 1999. Micro-TESE provides the advantage of allowing the urologist to selectively identify testicular seminiferous tubules most likely to contain spermatozoa depending on the presence of larger and more opaque appearance of those tubules. With micro-TESE, successful sperm retrieval has been reported in men with NOA up to 63% of cases [25].

Several studies formally comparing conventional testicular sperm extraction (TESE) vs. Micro-TESE have seen similar results, with sperm retrieval rates significantly higher in Micro-TESE approach compared to conventional approach [26].

The technique for performing Micro-TESE was first described by Schlegel [27]. The procedure is initially performed using 6–8× magnification to optimize visualization of small blood vessels and allow for performing an incision in the tunica albuginea in an avascular plane. Next, as we did in our cases, the magnification is increased to 15–20× for identification of larger and thicker individual seminiferous tubules that are also more opaque than other surrounding seminiferous tubules. These tubules are then cut into small pieces to search for the presence of spermatozoa from the tubules. Finally, this processed sample is examined for viable spermatozoa to use it for fertilization [28].

Our study evaluated demographic, clinical, and hormonal parameters in individuals undergoing first-time versus repeated micro-TESE. Positive sperm retrievals were significantly more common in the first-time group, which is an expected finding. However, our analysis also identified potential predictors of success in repeated TESE cases. Notably, some patients achieved sperm retrieval on a second attempt despite an initial failure, possibly due to improved hormonal status, surgeon skills or lab factors. The first-time group was younger and had a lower smoking prevalence. Klinefelter syndrome was more common in the first-time group. The relatively high overall rate (31%) may reflect our center’s referral pattern and the inclusion of both first-time and repeated TESE cases. Our cohort consisted mainly of repeated TESE cases, which may be associated with increased testicular fibrosis and subsequently lower sperm retrieval success rates, particularly in patients with Klinefelter syndrome and cryptorchidism. Hormonal analysis revealed significantly higher FSH and LH levels and lower total testosterone in the repeat group, although free testosterone levels were similar. These findings suggest that prior micro-TESE attempts may reduce sperm retrieval success by being associated with older age, higher smoking rates, and adverse hormonal profiles. In addition, in patients undergoing repeated TESE, significant differences were observed between those with positive and negative outcomes. Klinefelter syndrome was more common in those with negative outcomes. Age, smoking status, and hormone levels did not differ significantly between the groups.

In one study to look for predictors of Micro-TESE outcome, AL-zubi et al studied several factors that may affect Micro-TESE outcome like patients age at the time of procedure, follicle-stimulating hormone (FSH), luteinizing hormone (LH), total and free testosterone, testicular volume before the operation, previous TESE procedure, smoking status and medical ill nesses. They found that higher free testosterone levels and fewer previous micro-TESE attempts are significant predictors for effective sperm retrieval in males with NOA [29].

Follicle-stimulating hormone (FSH) is a glycoprotein hormone synthesized by the anterior pituitary in response to gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus. Its biological activity is mediated through binding to receptors on Sertoli cells within the testes, thereby promoting the secretion of hormones such as inhibin and activin, along with essential nutrients for germ cell development [30]. Given these functions, FSH has been proposed as a potential marker for predicting the likelihood of successful sperm retrieval during microdissection testicular sperm extraction (Micro-TESE). Multiple investigations have indicated that elevated serum FSH concentrations are often associated with impaired spermatogenesis and a reduced chance of successful sperm retrieval.

During Micro-TESE, serum FSH levels have been observed to differ significantly between patients with successful sperm retrieval and those with failed retrieval, particularly when conventional TESE had not yielded sperm [31]. Moreover, high FSH levels in men with azoospermia have been linked to decreased sperm retrieval rates in Micro-TESE [32]. Some reports advocate for the administration of exogenous recombinant FSH to improve outcomes in Micro-TESE. Aydos et al. conducted a study including 108 men with non-obstructive azoospermia (NOA) and normal FSH levels, where the intervention group received FSH injections thrice weekly for three months. Micro-TESE performed post-treatment showed a significantly higher sperm retrieval rate in the treatment group compared to controls (64% vs. 33%) [33].

Advanced paternal age has also been explored as a potential factor affecting Micro-TESE success. Although aging may reduce the presence of spermatogenically active regions in the testes, it remains unclear whether older age directly correlates with increased azoospermia prevalence. Studies on male age and fertility are limited, and no definitive age threshold indicating infertility has been established [34]. Nonetheless, increasing paternal age, especially beyond 45–50 years, has been linked to lower pregnancy and live birth rates [35]. Notably, no upper age limit has been defined as contraindicating Micro-TESE.

Genetic abnormalities are another critical factor contributing to NOA. Y chromosome microdeletions—particularly in the AZFa, AZFb, and AZFc regions—have proven useful in prognosticating Micro-TESE outcomes. According to the American Urological Association, men with NOA or severe oligospermia (<5 million/mL) should undergo karyotype analysis and Y-chromosome microdeletion screening prior to intervention [36]. Deletions in the AZFa and AZFb regions portend a poor prognosis, as sperm retrieval has not been reported in such cases [37]. Isolated AZFa deletions are commonly associated with a Sertoli cell-only histological pattern [38], while AZFb deletions often coincide with maturation arrest [37]. As a result, Micro-TESE is contraindicated in men with complete AZFa or AZFb deletions.

Conversely, AZFc deletions as the most frequently observed may still allow for successful sperm retrieval. Some affected men may even have rare spermatozoa in ejaculate [27]. Stahl et al. analyzed 149 men with NOA and Y chromosome microdeletions, finding a 71.4% sperm retrieval rate among those with AZFc deletions, whereas no sperm was retrieved in men with deletions in AZFa, AZFb, AZFb + c, or complete Yq regions [39]. This highlights the necessity of routine Y chromosome microdeletion testing prior to Micro-TESE.

Klinefelter syndrome (KS) represents the most prevalent genetic disorder associated with male infertility. Most men with the non-mosaic form exhibit azoospermia and require assisted reproductive techniques to achieve pregnancy [40]. Despite this, favorable outcomes have been observed with Micro-TESE in KS patients. Various preoperative predictors, including testicular volume, serum testosterone, and hCG levels, have been evaluated to assess their predictive value for successful sperm retrieval [41], although findings have been inconsistent. Some reports indicate that FSH, LH, and testicular volume do not reliably predict retrieval outcomes in this subgroup [42]. Overall, approximately 68% of KS patients had successful sperm retrieval, an outcome slightly exceeding general Micro-TESE success rates.

Testicular volume is another variable thought to reflect spermatogenic activity. Larger testes are typically associated with normal spermatogenesis, while smaller, atrophic testes are more common in NOA [43] suggested that men with a testicular long axis <4.6 cm and FSH <7.6 mIU/mL are more likely to have NOA. Nonetheless, testicular volume alone is a poor predictor of Micro-TESE outcomes. A positive correlation has been noted between testicular volume and success rates in both conventional TESE and Micro-TESE [31]; however, similar to FSH, testicular size does not indicate the density of viable seminiferous tubules during surgery [44].

Cryptorchidism (undescended testes) also impacts testicular volume and function. Ramasamy et al. reported higher sperm retrieval rates in cryptorchid men (74%) than in other NOA cases (58%), although differences in pregnancy outcomes were not statistically significant [45]. In such cases, both testicular size and age at orchidopexy appear relevant to spermatogenic potential.

In a previous study of 134 men with NOA undergoing 323 micro-TESE procedures, the overall sperm retrieval rate was 73.1% (236 procedures). Success rates increased with subsequent attempts, reaching 73.9%, 86.9%, and 92.5% for the second, third and fourth procedures, respectively. The first micro-TESE yielded sperm in 60.4% of cases. Notably, the duration between the first and second biopsies increased success rates by 1.3-fold per month, although this effect diminished in later attempts. Prior success significantly predicted subsequent success, with probabilities increasing by 10.1-fold, 5.6-fold and 16.5-fold for the second, third and fourth attempts, respectively. These findings underscore the potential benefits of repeated micro-TESE in NOA patients, particularly when prior attempts have been successful [46].

In another study of 125 men undergoing a second micro-TESE after an initial failure, sperm retrieval was successful in 18.4% of cases. Men with successful retrieval had smaller testicular volumes (8.2 ± 5.4 mL) than those with unsuccessful attempts (11.3 ± 5.3 mL). Notably, 50% of patients with Klinefelter’s syndrome achieved sperm recovery during the repeat procedure. Histopathological patterns also influenced outcomes, with higher retrieval rates for those with Leydig cell hyperplasia (54.5%) than for the Sertoli cell-only and maturation arrest groups (18.6%). These results suggest that a second micro-TESE may be beneficial for select patients, particularly those with severe testicular atrophy or specific histopathological features [21].

Talas et al. investigated the outcomes of repeat microdissection testicular sperm extraction (Micro-TESE) in a cohort of 68 men diagnosed with non-obstructive azoospermia (NOA). Initial Micro-TESE procedures yielded mature spermatozoa suitable for fertilization in 44 patients (64%), while retrieval was unsuccessful in the remaining 24 cases (36%). Among the latter group, all 24 elected to undergo a second Micro-TESE attempt. Spermatozoa were successfully retrieved in 19 of these patients, whereas 5 remained negative. Of those undergoing a second attempt, three of the five previously sperm-negative patients and 16 of the 19 previously sperm-positive individuals had sperm retrieved again. In subsequent procedures, spermatozoa were identified in all four patients during a third Micro-TESE and in one patient who underwent a fourth attempt. Histopathological evaluation revealed Sertoli cell-only syndrome in 16%, maturation arrest in 22%, hypospermatogenesis in 21%, and focal spermatogenesis in 41% of cases. Overall, repeated Micro-TESE resulted in successful sperm retrieval in 24 out of 29 patients (82%) [47]. These findings, consistent with larger series in the literature, suggest that Micro-TESE can be safely repeated in men with NOA to improve the cumulative sperm retrieval rate and facilitate the availability of fresh spermatozoa for use in intracytoplasmic sperm injection (ICSI).

4.1. Limitations of the study

Our study has several limitations. First, histopathological findings were not available for all patients who underwent Micro-TESE and were therefore not included in the analysis, although such data may have provided further insight into factors influencing sperm retrieval outcomes. Second, the retrospective design of the study introduces the potential for selection and information bias and limits the ability to draw causal inferences. Third, the absence of long-term follow-up data precludes assessment of downstream reproductive outcomes such as pregnancy and live birth rates. Fourth, the study was conducted at a single tertiary care center, which may limit the generalizability of the findings to other populations and clinical settings. In addition, although a range of clinical and hormonal variables was assessed, the sample size—particularly in the subgroup of patients undergoing repeated Micro-TESE—may have limited the statistical power to detect modest associations or to support more extensive multivariable analyses. Finally, relevant variables such as genetic subtypes beyond Klinefelter syndrome and testicular histology were not uniformly documented across the cohort, which may have constrained the scope of the analysis. Future studies with larger cohorts, prospective designs, and comprehensive follow-up are needed to validate these findings and to further explore strategies to optimize outcomes in repeated Micro-TESE procedures.

5. Conclusion

In conclusion, our findings demonstrate that the success rate of the Micro-TESE procedure was significantly higher in patients undergoing the procedure for the first time compared to those with prior attempts. This suggests that earlier surgical intervention may be associated with improved sperm retrieval outcomes in men with non-obstructive azoospermia (NOA). Factors such as younger age, lower smoking prevalence, and a more favorable hormonal profile including lower FSH and LH levels and higher total and free testosterone concentrations were associated with higher success rates. These observations highlight the potential importance of timely evaluation and intervention before advancing age or repeated procedures potentially compromise spermatogenic potential. Additionally, the influence of modifiable lifestyle factors such as smoking suggests that preoperative counseling and behavioral modifications may enhance outcomes. The integration of individualized patient assessment, including hormonal evaluation and genetic screening, may guide clinical decision-making and optimize treatment planning. Overall, these findings support a personalized approach to the management of male infertility and reinforce the need for early referral and intervention in patients diagnosed with NOA.

Funding Statement

This paper was not funded.

Authors’ contributions

S.A.: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing–original draft, Writing–review and editing. M.G.D.: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing—original draft, Writing—review and editing H.F.: Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—review and editing J.K.A.: Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—review and editing B.A.B.I.: Investigation, Data curation, Resources, Project administration, Writing—review and editing. H.A.R.: Investigation, Data curation, Resources, Project administration, Writing—review and editing. A.K.A.: Investigation, Resources, Validation, Supervision, Writing—review and editing. M.A.M.: Investigation, Resources, Validation, Supervision, Writing—review and editing. A.F.A.: Investigation, Resources, Validation, Supervision, Writing—review and editing, W.A.B.A.: Investigation, Resources, Validation, Supervision, Writing—review and editing M.A.: investigation, Software, Supervision, Validation, Visualization, Writing—review and editing.

Disclosure statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Ethical consideration

The ethical approval was obtained from the Research Ethics Committee at the Faculty of Medicine and the Institutional Review Board (IRB) at Al-Balqa’ Applied University (18/1/2024/2025).

Availability of data

The data that support the findings of this study are available on request from the corresponding author. The data is not publicly available due to privacy or ethical restrictions.