Rescue of ICSI failure in non-obstructive azoospermia by switching from cryopreserved to fresh testicular sperm: a paired clinical analysis

Abstract

Non-obstructive azoospermia (NOA) is a severe form of male infertility. Although cryopreserved testicular sperm are widely used for intracytoplasmic sperm injection (ICSI), a subset of patients experience poor outcomes with a specific frozen-thawed sample. This study aimed to evaluate whether obtaining fresh testicular sperm via repeat mTESE could serve as a rescue strategy for NOA patients with prior poor or failed ICSI outcomes using cryopreserved sperm. This retrospective paired clinical study included 20 men with NOA who underwent microdissection testicular sperm extraction (mTESE) and two ICSI cycles between December 2019 and February 2024 at a tertiary reproductive center. Each patient first underwent ICSI using cryopreserved-thawed testicular sperm, followed by a second ICSI cycle using fresh testicular sperm. Fertilization rate, 2PN rate, number of available embryos, clinical pregnancy rate, and live birth rate were compared between paired cycles using appropriate statistical tests.As a rescue-focused paired design, this cohort was intentionally enriched for patients with poor first-cycle outcomes and therefore does not represent an unselected NOA population. Switching to fresh testicular sperm significantly improved fertilization rate (86.7% vs. 62.7%, P = 0.0045), number of available embryos (3.50 ± 2.24 vs. 1.05 ± 0.60, P = 0.0005), clinical pregnancy rate (55.0% vs. 15.0%, P = 0.021), and live birth rate (45.0% vs. 5.0%, P = 0.008). The 2PN rate was higher but not statistically significant (79.5% vs. 68.5%, P = 0.094). Progressive motility sperm were more frequent in fresh samples (75% vs. 20%, P = 0.0003). No adverse events occurred. In NOA patients with prior poor or failed ICSI outcomes using cryopreserved testicular sperm, repeating mTESE to obtain fresh sperm was associated with significantly improved fertilization and pregnancy outcomes. These findings support fresh testicular sperm as an individualized rescue option rather than evidence of universal superiority over cryopreserved sperm in the general NOA population.

Introduction

Male infertility is a significant global reproductive health challenge, affecting approximately 10%–15% of couples of reproductive age, with male factors accounting for about 50% of cases¹. Azoospermia, the absence of sperm in the ejaculate, is a major cause of male infertility, observed in about 10%–15% of infertile men², among which non-obstructive azoospermia (NOA) accounts for approximately 60% of all azoospermia cases^3,4. NOA is one of the most challenging forms of male infertility, characterized by severely impaired spermatogenesis in the testes, resulting in the absence of sperm in the semen⁵.

With the widespread adoption of microdissection testicular sperm extraction (mTESE), approximately 40%–63% of patients with non-obstructive azoospermia (NOA) can have small quantities of sperm retrieved from focal areas of active spermatogenesis during surgery^6–9. The combination of mTESE and intracytoplasmic sperm injection (ICSI) has become the primary assisted reproductive strategy for NOA patients^10,11. However, clinical practice shows that some NOA patients still experience ICSI failure even when using cryopreserved-thawed testicular sperm, and currently, no definitive optimization strategy has been established.

There is ongoing controversy regarding the ICSI outcomes using cryopreserved-thawed testicular sperm. Some studies suggest that there are no significant differences between cryopreserved and fresh sperm in terms of fertilization rates, embryo quality, and pregnancy outcomes, supporting the use of cryopreserved sperm as a standardized strategy to streamline treatment and reduce the need for repeated surgeries^5,9,12–16. However, other studies indicate that cryopreservation may impair sperm quality and reduce ICSI success, especially in NOA patients, where sperm are already scarce and of poor quality, and freezing may further compromise their function^7,11,17–20. Therefore, for NOA patients who experience ICSI failure, there is still a lack of direct evidence to support whether switching to fresh testicular sperm can improve reproductive outcomes.

Rather than addressing the broad and still debated question of whether fresh testicular sperm is superior to cryopreserved-thawed sperm in unselected NOA populations, we focused on a more clinically actionable scenario. In real-world practice, a subset of men with NOA experience poor or failed outcomes in an initial ICSI cycle using a specific cryopreserved testicular sperm sample, raising the question of how best to optimize subsequent treatment. We therefore examined whether repeating mTESE to obtain fresh testicular sperm could serve as an effective rescue strategy for these selected patients. To this end, we conducted a retrospective paired analysis of NOA men who failed their first ICSI attempt with frozen-thawed testicular sperm and subsequently underwent a second ICSI cycle with freshly retrieved sperm, comparing key laboratory and clinical outcomes across the two cycles. Our study aims to provide practical evidence to guide individualized decision-making in this challenging rescue setting.

Materials and methods

Study subjects

A total of 322 patients diagnosed with non-obstructive azoospermia (NOA) underwent microdissection testicular sperm extraction (mTESE) at the Reproductive Medicine Center of the First Affiliated Hospital of Hainan Medical University between December 2019 and February 2024. Among them, 131 patients (40.7%) had successful sperm retrieval. Of these, 20 patients met the inclusion criteria and were retrospectively selected for paired analysis. Inclusion criteria were: (1) confirmed diagnosis of NOA; (2) successful sperm retrieval via mTESE; (3) poor outcome in the first ICSI cycle using cryopreserved-thawed testicular sperm, defined by at least one of the following: fertilization rate or 2PN rate < 50%, Fewer than two transferable embryos in cycles with ≥ 4 retrieved oocytes, No transferable embryos, or total fertilization failure; and (4) subsequent ICSI cycle using fresh testicular sperm. Patients with severe female infertility factors or incomplete clinical data were excluded. All procedures were performed at the same reproductive center, and complete, traceable clinical records were available for all included cases.(Fig. 1) Importantly, the inclusion criteria were designed to capture a clinically specific rescue scenario, namely, men who experienced poor outcomes with a prior cryopreserved-thawed testicular sperm cycle and subsequently pursued a second cycle using fresh sperm. This intentional enrichment introduces selection bias and limits the generalizability of our findings. Therefore, our study should be interpreted as evaluating a rescue strategy rather than providing a population-level comparison of fresh versus frozen testicular sperm in unselected NOA patients.

Fig. 1.

Study flowchart illustrating patient selection and paired comparison of ICSI outcomes using frozen versus fresh testicular sperm in NOA patients.

This retrospective cohort study was conducted in accordance with the principles of the Helsinki Declaration and approved by the Institutional Review Board of the First Affiliated Hospital of Hainan Medical University (approval code: 2023-KYL-264, dated December 29, 2023).

Microdissection testicular sperm extraction (mTESE)

The mTESE procedure was performed according to the method described by Schlegel and Li [28], with minor modifications. The surgery was conducted under an operating microscope. A longitudinal incision was made along the median raphe, and the testis was delivered from the tunica vaginalis to allow thorough inspection of the entire testis and epididymis. After full exposure of the testis, the tunica albuginea was incised under 15× to 20× magnification, and microdissection of the seminiferous tubules was performed. Careful hemostasis was achieved using microbipolar forceps. Throughout the procedure, copious irrigation with physiological saline was applied.Only seminiferous tubules suspected to contain active spermatogenesis were harvested. The retrieved tubules were immediately placed into G-IVF Plus culture medium (Lot No. 506873, Vitrolife). Subsequently, an experienced embryologist gently minced the tubules in a culture dish using a 5 mL flow tube and examined the suspension under 400× magnification using an inverted microscope (Olympus CKX41) to detect the presence of sperm. Regardless of sperm retrieval success, a small portion of testicular tissue was randomly excised for pathological analysis to confirm the surgical outcome.

Sperm motility assessment

In this study, all testicular sperm used for ICSI were assessed for motility by experienced embryologists under 400× magnification using an inverted microscope prior to injection. Sperm motility was classified according to the WHO 5th edition guidelines into three categories: progressive motility (PR), non-progressive motility (NP), and immotility (IM)²¹.For sperm initially observed as immotile, pentoxifylline was systematically applied according to predefined criteria, such as when no clearly motile sperm could be identified or when motile sperm were extremely scarce. After a fixed incubation period, any sperm exhibiting induced tail movement or minimal progression were considered viable and could be selected for ICSI when necessary. The same protocol for sperm activation was applied consistently to both cryopreserved-thawed and fresh testicular sperm samples to ensure comparability. The motility status (PR, NP, or IM) of each injected sperm was recorded at the time of injection after any activation attempts and used for subsequent motility distribution analysis.

Cryopreservation and thawing of testicular sperm

During the microdissection testicular sperm extraction (mTESE) procedure, the retrieved seminiferous tubule tissue was first placed in a 35 mm culture dish containing 2 mL of pre-equilibrated Sperm Rinse medium. Blood clots were removed using a 1 mL syringe, and the tubules were then minced in a 5 mL round-bottom tube to release sperm. The tubule fragments were subsequently transferred to a new 35 mm culture dish containing 1 mL of Sperm Rinse medium to further facilitate sperm release. After microscopic examination and motility assessment, the sperm suspension was transferred into a 15 mL conical centrifuge tube. An additional 2 mL of Sperm Rinse was added, the mixture was gently mixed, and centrifuged at 300 × g for 5 min.After centrifugation, the supernatant was discarded, leaving only 0.1–0.2 mL of concentrated sperm suspension. This suspension was aspirated using a 1 mL syringe. Cryoprotectant (Vitrolife) was then added dropwise, gently mixed, and the 15 mL tube was swirled softly to ensure uniform mixing. After equilibration at room temperature for 3 min, the mixture wasloaded into micro-volume plastic straws (micro-capillary “micro-straws”) in small aliquots separated by short air columns, and both ends of each micro-straw were sealed using a flame. This micro-volume straw-based technique was adopted to optimize cryopreservation for testicular sperm samples with very limited sperm numbers. The sealed straws were pre-cooled at 4 °C for 30–90 min and then stored in liquid nitrogen at − 196 °C for long-term preservation.For thawing, a water bath was preheated to 37 °C. The frozen straw was removed from liquid nitrogen and immediately submerged in the 37 °C water bath. It was gently swirled until the ice column was completely melted. The straw was then dried with sterile gauze, one end was cut, and the contents were transferred into a 15 mL conical centrifuge tube with 2 mL of Sperm Rinse medium. The other end of the straw was also cut, and a Pasteur pipette was used to flush the remaining contents into the tube to ensure complete recovery of sperm. The sample was centrifuged again at 300 × g for 5 min. After discarding the supernatant, 0.1–0.2 mL of the sperm suspension was retained and incubated in a culture incubator for subsequent ICSI or further processing.

Ovarian stimulation, ICSI, and pregnancy follow-up

In this study, all 20 NOA patients underwent two controlled ovarian hyperstimulation cycles—one for the ICSI cycle using thawed sperm and one for the ICSI cycle using fresh sperm. The daily dose of FSH was adjusted based on the female partner’s oocyte yield, ovarian reserve, and individual response to stimulation. When at least one or two follicles reached a diameter of 18 mm, or more than three follicles reached 17 mm, ovulation was triggered. Oocyte retrieval was performed 36 h later. Under 400× magnification, morphologically normal sperm were selected for ICSI.

Embryos were graded according to the 2011 Istanbul consensus on cleavage-stage embryo assessment²². Embryos that met the morphological criteria for transfer or cryopreservation according to this consensus were defined as “available embryos”. Available embryos were selected for single embryo transfer on day 3, while surplus available embryos were cryopreserved. Serum β-hCG was measured 13 days after embryo transfer. When β-hCG ≥ 50 U/L, luteal phase support was continued. Clinical pregnancy was confirmed by transvaginal ultrasound showing a gestational sac 4 weeks after embryo transfer.Pregnancy loss was defined as the loss of a clinical pregnancy before 28 weeks of gestation. Live birth was defined as the delivery of at least one living newborn, regardless of gestational age. The outcomes of ICSI-ET (intracytoplasmic sperm injection–embryo transfer) included MII oocyte rate, fertilization rate, 2PN rate, number of available embryos, clinical pregnancy rate, and live birth rate. All pregnancy outcomes were based on the first embryo transfer per ICSI cycle.

Statistical analysis

All statistical analyses were conducted using SPSS version 26.0 (IBM Corp., Armonk, NY). This study employed a paired design to compare clinical outcomes between ICSI cycles using cryopreserved and fresh testicular sperm in the same cohort of patients with non-obstructive azoospermia (NOA). Continuous variables were tested for normality; those with a normal distribution were analyzed using the paired t-test, while those not normally distributed were analyzed using the Wilcoxon signed-rank test. Categorical variables, such as pregnancy and live birth rates, were compared using McNemar’s test or Fisher’s exact test, as appropriate. Sperm motility was classified into three categories based on microscopic observation prior to injection: progressive motility (PR), non-progressive motility (NP), and immotility (IM). Differences in the distribution of sperm motility categories between the cryopreserved and fresh sperm groups were assessed using the chi-square test or Fisher’s exact test when expected cell counts were less than 5. Continuous data are expressed as mean ± standard deviation or median (interquartile range), and categorical data as frequencies (n) and percentages (%). All statistical tests were two-tailed, and a P-value < 0.05 was considered statistically significant.

Results

Baseline characteristics

Among the 20 patients who underwent a second ICSI cycle using fresh testicular sperm after initial failure with cryopreserved sperm, the baseline characteristics were as follows: the mean age of the male partners was 33.20 ± 4.57 years, and that of the female partners was 31.55 ± 4.03 years. The etiology of non-obstructive azoospermia (NOA) was primarily idiopathic (13 cases), followed by Klinefelter syndrome (4 cases), a history of cryptorchidism (2 cases), and AZFc deletion (1 case). The average interval between the two mTESE procedures was 8.85 ± 3.36 months, ranging from 3 to 18 months. The mean body mass index (BMI) was 25.67 ± 3.36 kg/m² for males and 22.42 ± 3.49 kg/m² for females. The mean right and left testicular volumes were 5.95 ± 3.85 mL and 6.05 ± 3.73 mL, respectively. Male baseline hormone levels were as follows: FSH, 23.44 ± 16.47 IU/L; LH, 10.30 ± 8.08 IU/L; testosterone, 11.26 ± 4.43 nmol/L; AMH, 2.18 ± 0.97 ng/mL; and inhibin B, 41.70 ± 18.89 pg/mL. In females, the mean levels of FSH, estradiol (E2), and AMH were 6.42 ± 2.62 IU/L, 142.67 ± 59.50 pg/mL, and 4.59 ± 2.93 ng/mL, respectively(Table 1).

Table 1.

Baseline characteristics of 20 patients undergoing second ICSI cycle using fresh testicular sperm following initial failure with cryopreserved sperm.

Baseline Variable		Second ICSI (Fresh Sperm, n = 20)
Male age (years)		33.20 ± 4.57
Etiology of NOA(n)	Idiopathic	13
	Klinefelter syndrome	4
	AZFc deletion	1
	Cryptorchidism history	2
mTESE interval (months)		8.85 ± 3.36
Female age (years)		31.55 ± 4.03
Male BMI (kg/m²)		25.67 ± 3.36
Female BMI (kg/m²)		22.42 ± 3.49
Right testicular volume (ml)		5.95 ± 3.85
Left testicular volume (ml)		6.05 ± 3.73
FSH (male, IU/L)		23.44 ± 16.47
LH (male, IU/L)		10.3 ± 8.08
Testosterone (male, nmol/L)		11.26 ± 4.43
AMH (male, ng/mL)		2.18 ± 0.97
Inhibin B (male, pg/mL)		41.70 ± 18.89
FSH (female, IU/L)		6.42 ± 2.62
E2 (female, pg/mL)		142.67 ± 59.50
AMH (female, ng/mL)		4.59 ± 2.93

Statistical presentation: Continuous variables are presented as mean ± standard deviation (SD) for normally distributed data and as median (interquartile range, IQR) for non-normally distributed data, based on the Shapiro–Wilk test for normality. All data were complete, and no missing values were observed. Units are specified for each variable.

BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; AMH, anti-Müllerian hormone; E2, estradiol.

Comparison of ICSI cycle outcomes

Table 2 compares the clinical outcomes of the two ICSI cycles—using cryopreserved versus fresh testicular sperm—in the 20 included patients. Compared with the cryopreserved group, the fresh sperm group showed significantly higher fertilization rates (86.7% vs. 62.7%, P = 0.0045) and average number of available embryos (3.50 ± 2.24 vs. 1.05 ± 0.60, P = 0.0005). The MII oocyte rate, calculated as the number of metaphase II oocytes divided by the number of oocytes retrieved, was similar between the two groups (82.8% vs. 79.6%, P = 0.831).The 2PN rate was also higher in the fresh group compared to the cryopreserved group (79.5% vs. 68.5%), with a difference approaching statistical significance (P = 0.094). Clinical pregnancy rate (55.0% vs. 15.0%, P = 0.021) and live birth rate (45.0% vs. 5.0%, P = 0.008) were both significantly higher in the fresh sperm group.

Table 2.

Comparison of clinical outcomes between ICSI cycles using cryopreserved and fresh testicular sperm.

Variable	Cryopreserved Sperm(n = 20)	Fresh Sperm(n = 20)	P-value
Number of oocytes retrieved (mean ± SD)	11.55 ± 6.50	12.30 ± 7.13	0.307
Number of MII oocytes (mean ± SD)	9.75 ± 6.54	10.65 ± 6.85	0.279
MII oocytes rate(%)	79.6	82.8	0.831
Fertilization rate (%)	62.7 ± 28.7	86.7 ± 16.5	0.0045
2PN rate (%)	68.5 ± 27.9	79.5 ± 24.3	0.094
Average number of available embryos (mean ± SD)	1.05 ± 0.60	3.50 ± 2.24	0.0005
Clinical pregnancy rate (%)	15.0	55.0	0.021
Live birth rate (%)	5.0	45.0	0.008

MII, metaphase II; 2PN, two-pronuclear; SD, standard deviation.Statistical analysis: Wilcoxon signed-rank test was used for continuous paired data; Exact McNemar’s test was used for paired binary outcomes (clinical pregnancy and live birth).

Comparison of sperm motility grade distribution

As shown in Table 3, there was a significant difference in the distribution of sperm motility grades between the two groups (P = 0.0003). In the cryopreserved group, progressive motility (PR) sperm accounted for only 20% (4/20), whereas in the fresh group, PR sperm accounted for as much as 75% (15/20), indicating that the freezing process significantly affected sperm motility status.

Table 3.

Comparison of sperm motility grade between cryopreserved and fresh testicular sperm.

Motility Grade	Cryopreserved Sperm (n)	Fresh Sperm (n)	P-value
PR	4	15	0.0003
NP	12	5
IM	4	0
Total	20	20

PR, progressive motility; NP, non-progressive motility; IM, immotility. Statistical analysis: Fisher’s exact test was used to compare the distribution of sperm motility grades between groups.

Discussion

This study employed a paired design to retrospectively analyze the clinical outcomes of 20 patients with non-obstructive azoospermia (NOA) who underwent a second ICSI cycle using fresh testicular sperm following failure of a first ICSI attempt with cryopreserved testicular sperm. The results showed that the fresh sperm group had significantly higher fertilization rates, numbers of available embryos, clinical pregnancy rates, and live birth rates compared to the cryopreserved group, suggesting that performing a repeat microdissection testicular sperm extraction (mTESE) to obtain fresh sperm may serve as an effective rescue strategy for NOA patients with poor ICSI outcomes.

A key consideration in interpreting our findings is the rescue-focused cohort definition. By design, we included only men who experienced poor outcomes with a prior cryopreserved-thawed testicular sperm ICSI cycle and then underwent a second cycle using fresh sperm. This selection enriches for cases in which the initial frozen sample was likely suboptimal, and therefore the markedly low live birth rate observed in the cryopreserved cycle should not be extrapolated to the broader NOA population. In this context, our results do not suggest that fresh sperm is universally superior, but rather highlight the clinical utility of switching to fresh sperm as a targeted rescue strategy for selected patients with prior poor outcomes.

Further analysis revealed a significant difference in sperm motility distribution between the two cycles: only 20% of the cryopreserved cycles used sperm with progressive motility, compared to 75% in the fresh sperm group (P = 0.0003). This finding suggests that the cryopreservation-thawing process may lead to a marked reduction in sperm motility, and that sperm with higher motility may have stronger fertilization potential in ICSI, thereby improving pregnancy outcomes. The advantage of fresh testicular sperm in ICSI may be attributed to its superior biological properties. Previous studies have indicated that the cryopreservation-thawing process can damage sperm membrane integrity, mitochondrial function, and flagellar motility, thereby affecting fertilization capability and early embryo developmental potential^23,24. In addition, cryopreservation may increase sperm DNA fragmentationrates²⁵, further compromising embryo quality and pregnancy outcomes.

Sperm motility has been confirmed by multiple studies as an important predictive factor for ICSI outcomes. A retrospective cohort study involving 198 NOA patients found that the use of motile sperm for injection significantly improved fertilization rates (48% vs. 20%, P < 0.01) and clinical pregnancy rates (44% vs. 19%, P < 0.01), and was an independent predictor of pregnancy ou tcome (OR = 4.37, 95% CI: 1.61–11.85, P = 0.004)²⁶. Another retrospective study involving 113 NOA patients and a total of 128 mTESE procedures provided additional support from the perspective of sperm motility. The study showed that the sperm retrieval rate was significantly higher in the fresh group (67.7%) compared to the cryopreserved group (45.3%), and that 85.7% of fresh sperm samples exhibited good motility suitable for ICSI, whereas only 52.7% of thawed sperm samples retained motility after freezing⁸. These data indicate that the cryopreservation-thawing process indeed reduces sperm motility, thereby indirectly affecting ICSI outcomes.

Although the findings of this study highlight the advantages of using fresh testicular sperm, published studies remain somewhat controversial regarding ICSI outcomes between fresh and cryopreserved testicular sperm. Some studies have reported no significant differences in fertilization and clinical pregnancy rates between the two. A systematic review and meta-analysis showed comparable fertilization and clinical pregnancy rates between ICSI cycles using fresh versus cryopreserved testicular sperm¹⁴. Similarly, a study published in 2024 by Lala Suleymanova and colleagues reported no significant differences in pregnancy outcomes when using cryopreserved-thawed or fresh mTESE-derived testicular sperm for ICSI in NOA patients¹¹.

Meanwhile, other studies have clearly supported the clinical advantages of using fresh testicular sperm. A study published by Zhang et al. in 2021 found that using fresh micro-TESE sperm for ICSI significantly improved clinical pregnancy and live birth rates²⁰. Similarly, a large retrospective study published in 2023 by Okuyama et al., which included 801 male infertility patients, also demonstrated that compared to cryopreserved testicular sperm, the use of fresh sperm combined with fresh oocytes significantly enhanced fertilization, pregnancy, and live birth rates, further emphasizing the clinical value of fresh sperm¹⁷.

The inconsistency among these study findings may be attributed to differences in study design, sample composition, cryopreservation and thawing techniques, as well as laboratory procedures. This study adopted a paired design, which effectively controlled for inter-individual variability, thereby enhancing the accuracy and reliability of the comparisons.

From a clinical perspective, for NOA patients who experience ICSI failure using cryopreserved testicular sperm, the decision to reuse frozen samples should be carefully evaluated. For patients with poor sperm motility or low sperm retrieval success rates, undergoing a second micro-TESE to obtain fresh testicular sperm may serve as an important strategy to improve reproductive outcomes. However, this rescue approach must also be weighed in terms of cost-effectiveness and ethical acceptability. Repeat mTESE entails a second invasive operation, additional anesthesia exposure, perioperative risks, and a substantial financial burden for the couple, and may not be justified in patients with a very poor overall prognosis or limited resources. Therefore, we believe that a second mTESE should be reserved for carefully selected couples in whom the expected incremental gain in live-birth probability reasonably justifies the added surgical and economic costs compared with alternative options, such as reusing previously frozen sperm, discontinuing treatment, or considering donor sperm. Ethically, decisions regarding repeat mTESE should be embedded in thorough counselling about realistic chances of success, potential complications, and available alternatives, in order to support informed and autonomous decision-making and to avoid futile or disproportionately burdensome interventions.

This study has several limitations. First, the 20 NOA patients included in this analysis were selected from a total of 322 men who underwent mTESE during the study period, among whom 131 (40.7%) had successful sperm retrieval. The small paired sample size largely reflects the strict inclusion criteria and the rarity of the specific clinical scenario addressed here—namely, poor or failed ICSI outcomes using a particular cryopreserved-thawed testicular sperm sample followed by a subsequent cycle using freshly retrieved sperm. While this design enhances the clinical relevance of our findings for this rescue setting, it also introduces substantial selection bias and limits statistical power, particularly for subgroup analyses. Therefore, our results should be interpreted as supporting fresh testicular sperm as an individualized rescue option in selected patients with prior poor outcomes, rather than evidence of universal superiority over cryopreserved sperm in unselected NOA populations. In addition, we did not assess sperm DNA fragmentation index or refined sperm morphology, both of which may influence ICSI outcomes. Larger multicenter prospective studies with standardized laboratory protocols and broader NOA cohorts are warranted to validate these observations and to further clarify the contribution of sperm motility and cryopreservation-related damage to ICSI outcomes.

Conclusions

This study demonstrates that in men with non-obstructive azoospermia who previously experienced ICSI failure using cryopreserved testicular sperm, switching to fresh testicular sperm through repeat microdissection testicular sperm extraction (mTESE) significantly improves fertilization, embryo development, clinical pregnancy, and live birth outcomes. The findings highlight that cryopreservation may impair sperm motility and fertilization potential, and that the use of fresh sperm can restore reproductive success in selected patients. Clinically, these results emphasize the importance of individualized treatment planning—particularly for patients with poor prior ICSI outcomes—by considering a second mTESE to obtain fresh sperm as a viable rescue strategy. This approach provides valuable guidance for optimizing ART protocols in severe male infertility.

Abbreviations

NOA

Non obstructive azoospermia

ICSI

Intracytoplasmic sperm injection

mTESE

Microdissection testicular sperm extraction

ART

Assisted reproductive technology

BMI

Body mass index

FSH

Follicle-stimulating hormone

LH

Luteinizing hormone

E2

Estradiol

AMH

Anti-Müllerian hormone

2PN

Two pronuclei

PR

Progressive motility

NP

Non-progressive motility

IM

Immotility

β-hCG

Beta-human chorionic gonadotropin

SD

Standard deviation

OR

Odds ratio

CI

Confidence interval

WHO

World Health Organization

Author contributions

Lingxiao Zhang contributed to conceptualization, methodology, investigation, data curation, formal analysis, original draft writing, and supervision. Yanlin Ma was responsible for supervision and critical review and editing of the manuscript. Qi Li contributed to statistical analysis, data validation, and manuscript review and editing. Tingting Wu participated in investigation, data curation, and resource management. Huaye Chen was involved in providing clinical resources and performing clinical procedures. Yanhua Huang contributed to data curation and patient management.

Funding

This study was funded by the Hainan Province Science and Technology Special Fund (ZDYF2022SHFZ312) and supported by the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202310). Additional support was provided by the Hainan Province Clinical Medical Center.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This retrospective study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Hainan Medical University (approval number: 2023-KYL-264). The requirement for written informed consent was waived by the Ethics Committee due to the retrospective nature of the study.