Intraoperative epididymal sperm cryopreservation during microsurgical vasoepididymostomy for obstructive azoospermia: intracytoplasmic sperm injection outcomes from a single-center cohort

Introduction

Azoospermia is a significant cause of male infertility, with an estimated prevalence rate of approximately 1% in the general male population and 10–15% among infertile men. This condition is classified into two types: obstructive azoospermia (OA) and non-OA. OA, characterized by a physical blockage in the reproductive tract, is responsible for approximately 20–40% of azoospermia cases (1). The epididymis is the most common site of obstruction. Common causes of epididymal OA (EOA) include vasectomy, idiopathic factors, infections, iatrogenic interventions, and trauma.

For men with EOA, the combination of testicular sperm extraction (TESE) or percutaneous epididymal sperm aspiration (PESA) with intracytoplasmic sperm injection (ICSI) remains the predominant approach to achieving pregnancy through assisted reproductive technologies (ART). However, surgical sperm retrieval can result in complications including testicular devascularization, fibrosis, atrophy, hypogonadism, sexual dysfunction, and chronic pain (2,3). Moreover, ICSI treatment per cycle yields an average live birth rate of only 30–40% (4,5), and an initial failed cycle often leads to repeated TESE, which multiplies the physical trauma and economic burden for the patients.

Microsurgical vasoepididymostomy (MVE) is a viable surgical option for men with EOA. Post-operatively, patients have the potential to achieve pregnancy, either naturally or through ART utilizing sperm in the ejaculate. This procedure typically yields an average patency rate of approximately 72%, with a reported range from 50% to 84% (6,7). In cases where patency is not achieved post-surgery, sperm retrieval through TESE or PESA remains necessary for ART.

Intraoperative sperm collection and cryopreservation from the epididymal fluid or testicular tissue during MVE have been proposed (6,8). This approach provides patients with immediate access to sperm for future ART cycles, thereby avoiding the need for repeated surgical sperm retrieval procedures. Although recommended by several researchers, the practical efficacy and treatment outcomes of this strategy remain limited.

This study reports our clinical experience with intraoperative sperm cryopreservation during MVE and evaluates the subsequent ART outcomes using these cryopreserved samples in patients with OA. On the basis of this clinical experience, we aimed to test the hypothesis that cryopreserved epididymal sperm collected during MVE can serve as a clinically effective alternative to fresh ejaculated sperm for ICSI, thereby providing a back-up option for patients in whom MVE fails to restore patency. We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0297/rc).

Methods

Patients, study design and ethical approval

A retrospective study was conducted on 117 patients diagnosed with OA who underwent MVE combined with concurrent sperm cryopreservation at the Human Sperm Bank of Peking University Third Hospital (Beijing, China) between November 2022 and July 2025. OA was diagnosed by azoospermia on at least two semen analyses, normal serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and prolactin levels, normal testicular volumes (12–25 mL), and absence of abnormalities in the prostate, seminal vesicles, or ejaculatory ducts on transrectal ultrasound.

The study cohort comprised all ART cycles utilizing thawed epididymal sperm [thawed microsurgical vasoepididymostomy epididymal sperm (M-ES)] collected and cryopreserved during the initial MVE surgery. For comparison, two control cohorts were established for comparison from the same period: (I) a fresh ejaculated group, consisting of age-matched (1:10) cycles from patients undergoing ICSI with fresh ejaculated sperm; and (II) a thawed testicular sperm aspiration (TESA) group, consisting of cycles from patients with OA who underwent ICSI with thawed sperm previously obtained via TESA. The exclusion criteria were as follows: frozen-thawed oocyte cycles, necrozoospermia, teratozoospermia, non-OA, chromosomal abnormalities, and mild ovarian stimulation cycles. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Peking University Third Hospital (No. 2018S2-002), and written informed consent was obtained from all participants.

MVE and sperm collection

MVE was performed following the technique previously described by Hong et al. (8). During the procedure, the lumen of the epididymal tubule was gently incised longitudinally between two suture needles. The epididymal fluid was immediately collected and subjected to microscopic evaluation at 400× magnification to identify the presence of spermatozoa. If motile sperm (defined as >3–5 progressively motile sperm per high-power field) were observed, additional fluid was collected for cryopreservation. (Ideally, epididymal fluid should be assessed using an inverted microscope, enabling direct cryopreservation of samples containing adequate numbers of high-quality spermatozoa. Unfortunately, due to the lack of necessary equipment at the time, the sperm used for microscopic observation could not be frozen in our facility). In cases where the sperm in the epididymal fluid did not meet the cryopreservation criteria, a testicular biopsy was subsequently performed to obtain tissue for TESE.

Epididymal fluid processing

Epididymal fluid was collected using a 40 µL sterile capillary glass tube and immediately diluted in 1 to 1.5 mL of G-MOPS PLUS medium (Cat. 10130; Vitrolife, Gothenburg, Sweden). The diluted sample was then transferred to the laboratory at 4 ℃. Sperm parameters (count, motility, and morphology) were assessed under a phase-contrast microscope (Nikon TS100; Nikon, Tokyo, Japan) at 200× magnification using a Makler counting chamber.

Testicular tissue processing

A segment of testicular tissue (approximately 2–3 mm × 2–3 mm × 5 mm) was harvested and immersed in 1 to 1.5 mL of G-MOPS PLUS medium. The tissue sample was transferred to the laboratory at 4 ℃. Upon arrival, the seminiferous tubules were minced using two sterile 1 mL syringe needles to release sperm, creating a homogeneous cell suspension in a 35 mm sterile cell culture dish (Corning Inc., Corning, NY, USA). The suspension was initially examined under an inverted microscope (Nikon TS100) to verify the presence of sperm and to determine the required number of cryovials. If necessary, it was then either centrifuged and resuspended or diluted to achieve the desired sperm concentration in a final volume of 0.5 mL per vial. Following this step, and prior to cryoprotectant addition, the sample was mixed thoroughly, and a 10-µL aliquot was taken for assessment of sperm concentration, motility, and morphology using a Makler chamber under a phase-contrast microscope (Nikon TS100).

Sperm cryopreservation

Subsequently, the processed samples (from either epididymal fluid or testicular tissue) were mixed with a sperm cryoprotectant (Cat. RBC-1015; RUBAI, Liaocheng, China) in a 1:1 ratio. The mixture was aliquoted into 2 mL cryogenic vials (1 mL per vial; Cat. 430659; Corning, Suzhou, China). For cryopreservation, the vials were suspended in liquid nitrogen vapor at 3–5 cm above the liquid nitrogen surface for 10 minutes, then plunged directly into and stored in liquid nitrogen tanks at the Human Sperm Bank of Peking University Third Hospital until required for subsequent ART cycles.

ART and data collection

In cases where patency was not achieved following vasoepididymostomy, cryopreserved sperm samples were utilized for subsequent ART cycles. All ART procedures, including ovarian stimulation, oocyte retrieval, ICSI, and embryo transfer (ET), were conducted in our center according to standard clinical protocols (9).

The following data were retrospectively collected from the couples’ medical records: parental age, female and male body mass index (BMI), duration and type of infertility, baseline serum FSH, estradiol (E₂), LH, and anti-Müllerian hormone (AMH) levels, antral follicle count (AFC), ovarian stimulation protocol, hormone levels on trigger day (E₂, progesterone, LH), total gonadotropin (Gn) dosage, endometrial thickness, number of oocytes retrieved, laboratory parameters, and clinical outcomes following ET.

Outcome measures and definition

Reproductive outcomes were assessed using standardized laboratory and clinical parameters. The fertilization rate was calculated as the number of fertilized oocytes divided by the number of metaphase II (MII) oocytes used for ICSI. Embryonic development was evaluated through the day-3 cleavage rate (defined as the number of cleaved embryos divided by the number of fertilized oocytes) and the good-quality embryo rate (defined as the percentage of embryos with 5–8 cells, <30% fragmentation, and even-sized blastomeres, divided by the number of fertilized and cleaved embryos).

The following criteria were used to assess pregnancy outcomes: clinical pregnancy was confirmed by a positive serum β-human chorionic gonadotropin (β-hCG) test between 14 and 21 days post-embryo transfer with subsequent ultrasonographic visualization of one or more gestational sacs. Early miscarriage was defined as pregnancy loss before 12 weeks of gestation, while ectopic pregnancy was defined as extrauterine implantation. The multiple gestation rate was calculated as the percentage of clinical pregnancies with two or more intrauterine sacs.

Statistical analysis

All statistical analyses were performed using SPSS (version 26.0; IBM Corp., Armonk, NY, USA). All analyses were based on complete-case data, and no missing values were present for the key variables included in the analyses. Continuous data are presented as mean ± standard deviation (SD), and categorical data as number (percentage). The normality of data distribution was assessed using the Shapiro-Wilk test. Based on this assessment, group comparisons for normally distributed variables were performed using one-way analysis of variance (ANOVA), while the Kruskal-Wallis test was employed for non-normally distributed variables. Associations between categorical variables were examined using the Chi-squared test, with Fisher’s exact test applied when any expected cell count was <5. Covariates for multivariable models were selected based on baseline imbalances (P<0.05). Variables such as fertilization rate and embryo quality were not included to avoid overadjustment. Additionally, it should be noted that the Gn dosage was log-transformed before the adjustment. The results are reported as adjusted odds ratios (aORs) with corresponding 95% confidence intervals (CIs). A two-sided P value <0.05 was considered statistically significant. To account for potential within-patient correlation from repeated ART cycles, we also performed a sensitivity analysis restricted to the first cycle per patient.

Results

Between November 2022 and July 2025, a total of 117 patients with OA underwent fertility preservation during MVE. The cryopreserved sperm were sourced from epididymal fluid (n=73) and testicular tissue (n=44). The clinical characteristics of the cohort are summarized in Table 1. Surgical procedures included bilateral MVE in 46 patients (39.3%), unilateral MVE in 28 patients (23.9%), and 43 (36.8%) patients who were deemed ineligible for anastomosis during surgery.

In subsequent ART treatments, 29 patients used thawed epididymal sperm for 43 ovarian stimulation cycles and 27 frozen ET (FET) cycles, while 6 patients used thawed testicular sperm for 7 stimulation cycles and 1 FET cycle (Figure 1). Among the 35 patients who used frozen sperm for ICSI-ET, 7/46 (15.2%) were from the bilateral anastomosis group, 7/28 (25.0%) from the unilateral anastomosis group, and 21/43 (48.8%) from the non-anastomosis group.

Figure 1 The flowchart of current fertility preservation and subsequent use of cryopreserved sperm in MVE at the Center for Reproductive Medicine of Peking University Third Hospital. ES, epididymal sperm; ET, embryo transfer; FET, frozen embryo transfer; IVF, in vitro fertilization; MVE, microsurgical vasoepididymostomy; TS, testicular spermatozoa.

Data from the 6-patient testicular sperm subgroup are presented descriptively only and were not included in any comparative statistical analyses. The primary analytic cohort (thawed M-ES group) consisted of the 29 patients who used cryopreserved epididymal sperm for ICSI. Their outcomes were benchmarked against two reference groups: Fresh ejaculated (n=430 cycles) and thawed TESA (n=301 cycles).

Baseline characteristics differed across the three groups (Table 2). Relative to the reference groups, the thawed M-ES group had lower female BMI (21.31±2.40 vs. 22.96±3.47 and 22.63±3.49 kg/m², P=0.006), AFC (9.76±5.51 vs. 12.58±6.36 and 13.23±6.52, P<0.001) and a lower proportion of antagonist protocol use (67.4% vs. 77.7% and 80.4%, P=0.01). The fresh ejaculated group had a significantly lower rate of primary infertility (68.1% vs. 79.1% and 81.4%, P=0.002), while the thawed TESA group had lower maternal and paternal ages, shorter infertility duration, and lower total Gn dosage compared to the fresh ejaculated group (all P<0.05).

In fresh ET cycles, primary clinical outcomes—including clinical pregnancy, early miscarriage, ectopic pregnancy, and multiple pregnancy rates—did not differ significantly among the three groups (Table 2). A multivariate logistic regression analysis was performed to identify factors associated with clinical pregnancy. After adjustment for potential confounders, the source of sperm was not an independent predictor of clinical pregnancy (Table 3). To account for potential within-patient correlation from repeated ART cycles, we performed a sensitivity analysis restricting the analysis to the first ovarian stimulation cycle and the first ET cycle per patient (thawed M-ES: n=29; fresh ejaculated: n=428; thawed TESA: n=243 for fresh ET cycles). The results remained consistent with the primary analysis: fertilization (0.75±0.22 vs. 0.74±0.24), cleavage (0.99±0.03 vs. 0.99±0.06), and clinical pregnancy rates (41.2% vs. 44.3%) were comparable between the thawed M-ES and fresh ejaculated groups (Tables S1,S2).

Baseline characteristics and clinical outcomes were compared among the three groups in FET cycles. The thawed TESA group exhibited significantly younger maternal (30.76±4.73 vs. 32.93±3.51 and 32.52±4.45 years, P<0.001) and paternal ages (32.43±5.50 vs. 34.49±4.24 and 35.37±6.93 years, P<0.001) and a greater number of embryos transferred (1.52±0.50 vs. 1.41±0.49 and 1.37±0.49, P=0.04) compared to both the fresh ejaculated group and thawed M-ES group. The thawed M-ES group had a significantly higher proportion of natural cycles for endometrial preparation (51.9% vs. 31.5% and 41.1%) than the other two groups. Despite these baseline differences, key clinical outcomes—including clinical pregnancy, early miscarriage, and ectopic pregnancy rates—remained similar across the groups. The multiple pregnancy rate was significantly higher in the thawed TESA group compared to the fresh ejaculated group (22.1% vs. 7.8%, P=0.007) (Table 4). Logistic regression analysis confirmed that sperm source was not an independent predictor of clinical pregnancy in FET cycles (Table 5).

Discussion

To the best of our knowledge, this is the first study to evaluate the strategy of routine fertility preservation during MVE. Our findings indicate that the clinical outcomes of ICSI using cryopreserved epididymal sperm retrieved during MVE are comparable to those achieved with fresh ejaculated sperm or cryopreserved testicular sperm from TESA.

In cases where MVE fails to restore patency, subsequent sperm retrieval procedures (such as TESA or micro-TESE) can impose financial burden and lead to additional surgical trauma. To circumvent the need for such secondary interventions, we performed sperm cryopreservation at the time of MVE using sperm retrieved from epididymal fluid or testicular tissue.

In our cohort, the utilization rate of cryopreserved sperm, which may inversely correlate with surgical success, was 15.2% (7/46) in patients with bilateral anastomosis, 25.0% (7/28) in those with unilateral anastomosis, and 48.8% (21/43) in patients who did not undergo anastomosis. Overall, the utilization rate was 29.9% (35/117). This relatively low utilization rate likely reflects favorable patency outcomes, consistent with our previously reported patency rates of 68% (bilateral) and 40% (unilateral) (8). Nevertheless, several factors should be considered when interpreting this utilization data. First, some patients returned to our center to use their cryopreserved sperm; others with unsuccessful MVE likely opted for TESA-ICSI at local institutions due to geographic distance and economic considerations, which may have lowered the observed utilization rate. Second, given the variability and often extended duration (1.5–12 months) required to achieve patency, certain patients remain under postoperative observation and may eventually use their frozen samples. Despite these caveats, intraoperative sperm cryopreservation clearly provides a critical safeguard for MVE surgical patients, particularly for those who are intraoperatively deemed ineligible for vasoepididymostomy, as reflected by the 48.8% utilization rate in the non-anastomosis subgroup.

While conventional post-thaw survival analysis is indispensable for assessing cryopreservation efficacy, clinical outcomes are the ultimate metric for validating its success. Therefore, we evaluated the ART outcomes achieved with sperm cryopreserved during MVE, selecting two control groups: one using fresh ejaculated sperm, and the other using frozen-thawed testicular sperm from patients with OA who underwent TESA. This study design enables a direct comparison of embryological and clinical outcomes between different sperm sources and provides evidence to guide the use of epididymal or testicular sperm cryopreservation in this setting.

The debate on reproductive outcomes between fresh and cryopreserved sperm remains unresolved, with some studies favoring the use of fresh sperm (10), while others, particularly those concerning testicular sperm, report comparable pregnancy, live birth, and neonatal outcomes following ICSI (11-14). In cases of epididymal sperm, several studies have confirmed that frozen-thawed samples yield similar fertilization and clinical pregnancy rates to their fresh counterparts (15,16). Furthermore, the use of epididymal sperm in ICSI does not appear to increase the risk of adverse neonatal outcomes or congenital malformations (17,18), providing further evidence to support the cryopreservation of epididymal sperm retrieved during MVE.

Consistent with previous evidence, our study found no significant differences in primary clinical pregnancy outcomes across various sperm sources, whether in fresh or frozen-thawed ET cycles. However, we observed that the use of frozen-thawed testicular sperm yielded significantly lower rates of fertilization, cleavage, and good-quality embryos compared to fresh ejaculated sperm. This finding contrasts with previous reports (19-21), which suggested equivalent fertilization potential between testicular and ejaculated spermatozoa, but aligns with the observations of Namath et al. (22). We hypothesize that this discrepancy may be attributed to limitations in our retrospective data, which did not allow for the exclusion of patients with OA who might also have coexisting spermatogenic impairment (e.g.,​ non-OA); it is well-established that such defects are linked to lower fertilization rates in non-OA compared to those with severe oligozoospermia (23) or pure OA (24). Another plausible explanation is the lower yield of motile sperm in testicular tissue compared to ejaculated semen or epididymal fluid. It was demonstrated that there was a positive correlation between sperm motility and fertilization rate and embryo quality (25,26). Despite these plausible explanations, persistently conflicting literature on the efficacy of testicular sperm demands further well-designed studies to definitively elucidate its potential for fertilization and embryo development.

A study conducted by Lewin et al. analyzed data from 214,649 ICSI cycles, which included 199,818 with ejaculated sperm, 5,646 with epididymal sperm, and 9,185 with testicular sperm. The study demonstrated that cycles utilizing epididymal sperm achieved higher implantation, clinical pregnancy, and live birth rates than those utilizing testicular sperm. In contrast, no statistically significant differences were observed in outcomes between fresh and cryopreserved sperm samples in surgical retrieval cycles (27). Integrating our findings with this evidence, we propose the following clinical strategy for fertility preservation in men undergoing MVE: prioritize cryopreservation of epididymal fluid when a sufficient number of motile sperm are available. This approach minimizes surgical trauma, streamlines the procedure, and may potentially lead to superior ART outcomes compared to the use of testicular sperm. Nevertheless, testicular sperm cryopreservation remains a viable alternative when motile sperm are absent or scarce in the epididymal fluid. Although this single-center experience requires validation, the described workflow is reproducible in centers with basic andrology and ART infrastructure.

This study has several limitations. First, its retrospective nature and the inclusion of only those patients who returned to our center for ART may have introduced selection bias. Second, the number of cycles using thawed testicular sperm was small (n=7), which precluded their inclusion in any comparative analyses; these cases were therefore presented descriptively only. Third, although we adjusted for baseline differences in multivariable analyses, residual confounding due to the non-randomized design cannot be fully excluded. Fourth, live birth outcomes were not uniformly captured across all cycles, which limits the clinical interpretability of our findings. Consequently, further studies with larger, prospective, and more comprehensively matched cohorts are needed to validate these results.

Conclusions

In conclusion, this study demonstrates that cryopreserved epididymal sperm collected during MVE can achieve ICSI outcomes comparable to those of fresh ejaculated sperm. These findings support the integration of routine epididymal sperm cryopreservation into the MVE surgical workflow as a reliable back-up strategy for patients at risk of surgical failure.

Based on our data, we propose a practical algorithm: when motile sperm are identified in the epididymal fluid, cryopreserve epididymal sperm as the primary fertility preservation option; reserve testicular sperm cryopreservation for cases where epididymal sperm are absent or insufficient. This approach minimizes additional surgical interventions while optimizing ART outcomes.

Prospective multi-center studies with larger cohorts and complete follow-up are warranted to confirm the generalizability of these findings and to assess live birth outcomes.

Acknowledgments

We truly appreciate all the medical staff working in the Center for Reproductive Medicine, Peking University Third Hospital, including the doctors, nurses, and lab technicians for their invaluable contributions to patient care, laboratory procedures, and data management that made this study possible.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0297/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0297/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0297/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 22275197), the National Key Research and Development Program of China (No. 2022YFC2703004), the Capital’s Funds for Health Improvement and Research (No. 2022-2-4094), and the Peking University Third Hospital Clinical Cohort Construction Project (No. BYSYDL2023016).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0297/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Peking University Third Hospital (No. 2018S2-002), and written informed consent was obtained from all participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Minhas S, Boeri L, Capogrosso P, et al. European Association of Urology Guidelines on Male Sexual and Reproductive Health: 2025 Update on Male Infertility. Eur Urol 2025;87:601-16. [Crossref] [PubMed]
2. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia? A systematic review. Hum Reprod Update 2007;13:539-49. [Crossref] [PubMed]
3. Zohdy W, Shah R, Ho CCK, et al. Changes in Testosterone Levels Following Surgical Sperm Retrieval in Men with Non-Obstructive Azoospermia: Systematic Review and Meta-Analysis. World J Mens Health 2025;43:523-38. [Crossref] [PubMed]
4. Wang Y, Li R, Yang R, et al. Intracytoplasmic sperm injection versus conventional in-vitro fertilisation for couples with infertility with non-severe male factor: a multicentre, open-label, randomised controlled trial. Lancet 2024;403:924-34. [Crossref] [PubMed]
5. Kong F, Wang Y, Li R, et al. Assisted reproductive technology in China: introduction to the special issue. Hum Reprod 2023;38:ii1-2. [Crossref] [PubMed]
6. Wang Z, Wang X, Song C, et al. The pregnancy outcomes in patients with epididymal obstructive azoospermia after microsurgical vasoepididymostomy: a systematic review and meta-analysis. Front Med (Lausanne) 2023;10:1186729. [Crossref] [PubMed]
7. Wang SY, Fang YY. Outcomes of microsurgical vasoepididymostomy using intussusception technique: a systematic review and meta analysis. Sci Rep 2023;13:3340. [Crossref] [PubMed]
8. Hong K, Zhao LM, Xu SX, et al. Multiple factors affecting surgical outcomes and patency rates in use of single-armed two-suture microsurgical vasoepididymostomy: a single surgeon's experience with 81 patients. Asian J Androl 2016;18:129-33. [Crossref] [PubMed]
9. Zhang L, Wang YY, Zheng XY, et al. Novel predictors for livebirth delivery rate in patients with idiopathic non-obstructive azoospermia based on the clinical prediction model. Front Endocrinol (Lausanne) 2023;14:1233475. [Crossref] [PubMed]
10. Mantravadi KC, Martinez M, Rahmat FA, et al. Fresh versus frozen micro-TESE sperm and outcomes. Asian J Androl 2025;27:399-408. [Crossref] [PubMed]
11. Nagawkar Perlov SS, Deri N, Eldar-Geva T, et al. Comparison of obstetrical and neonatal outcomes between fresh versus frozen-thawed testicular sperm derived from microTESE. J Assist Reprod Genet 2024;41:2681-90. [Crossref] [PubMed]
12. Wu S, Zhao J, Wu Y, et al. Comparison of pregnancy and neonatal outcomes of intracytoplasmic sperm injection performed with frozen versus fresh testicular sperm. Transl Androl Urol 2022;11:472-9. [Crossref] [PubMed]
13. Amer M, Fakhry E. Fresh vs frozen testicular sperm for assisted reproductive technology in patients with non-obstructive azoospermia: A systematic review. Arab J Urol 2021;19:247-54. [Crossref] [PubMed]
14. Suleymanova L, Bayram H, Dönmez Çakıl Y, et al. Pregnancy outcomes in patients with non-obstructive azoospermia undergoing micro-TESE: comparison of fresh vs. frozen-thawed testicular sperm. J Assist Reprod Genet 2024;41:3399-404. [Crossref] [PubMed]
15. Kuznyetsova I, Moskovtsev SI, Ng S, et al. Permeable cryoprotectants-free vitrification of human TESE, PESA and OAT spermatozoa: clinical outcomes. Syst Biol Reprod Med 2025;71:54-60. [Crossref] [PubMed]
16. Liu H, Xie Y, Gao L, et al. Impact on using cryopreservation of testicular or epididymal sperm upon intracytoplasmic sperm injection outcome in men with obstructive azoospermia: a systematic review and meta-analysis. J Assist Reprod Genet 2020;37:2643-51. [Crossref] [PubMed]
17. Fedder J, Loft A, Parner ET, et al. Neonatal outcome and congenital malformations in children born after ICSI with testicular or epididymal sperm: a controlled national cohort study. Hum Reprod 2013;28:230-40. [Crossref] [PubMed]
18. Jin L, Li Z, Gu L, et al. Neonatal outcome of children born after ICSI with epididymal or testicular sperm: A 10-year study in China. Sci Rep 2020;10:5145. [Crossref] [PubMed]
19. Klami R, Tomás C, Mankonen H, et al. ICSI outcome after microdissection testicular sperm extraction, testicular sperm aspiration and ejaculated sperm. Reprod Biol 2024;24:100825. [Crossref] [PubMed]
20. Arafa M, AlMalki A, AlBadr M, et al. ICSI outcome in patients with high DNA fragmentation: Testicular versus ejaculated spermatozoa. Andrologia 2018; [Crossref] [PubMed]
21. Abhyankar N, Kathrins M, Niederberger C. Use of testicular versus ejaculated sperm for intracytoplasmic sperm injection among men with cryptozoospermia: a meta-analysis. Fertil Steril 2016;105:1469-1475.e1. [Crossref] [PubMed]
22. Namath A, Flannagan K, Le K, et al. Comparing blastulation of surgically retrieved sperm with ejaculated sperm in donor oocyte cycles. Fertil Steril 2025;124:436-42. [Crossref] [PubMed]
23. Demir B, Arikan II, Bozdag G, et al. ICSI outcome of patients with severe oligospermia vs. non-obstructive azoospermia. Clin Exp Obstet Gynecol 2012;39:141-3. [PubMed]
24. Romano M, Cirillo F, Ravaioli N, et al. Reproductive and obstetric outcomes in TESE-ICSI cycles: A comparison between obstructive and non-obstructive azoospermia. Andrology 2025;13:159-68. [Crossref] [PubMed]
25. Vogiatzi P, Pouliakis A, Sakellariou M, et al. Male Age and Progressive Sperm Motility Are Critical Factors Affecting Embryological and Clinical Outcomes in Oocyte Donor ICSI Cycles. Reprod Sci 2022;29:883-95. [Crossref] [PubMed]
26. Park YS, Lee SH, Song SJ, et al. Influence of motility on the outcome of in vitro fertilization/intracytoplasmic sperm injection with fresh vs. frozen testicular sperm from men with obstructive azoospermia. Fertil Steril 2003;80:526-30. [Crossref] [PubMed]
27. Lewin J, Lukaszewski T, Sangster P, et al. Reproductive outcomes after surgical sperm retrieval in couples with male factor subfertility: a 10-year retrospective national cohort. Fertil Steril 2023;119:589-95. [Crossref] [PubMed]