Abstract
Intracytoplasmic sperm injection (ICSI) with cryopreserved testicular spermatozoa retrieved via testicular sperm aspiration (TESA) or microdissection testicular sperm extraction (micro-TESE) is an effective treatment for azoospermia and cryptozoospermia. The current research on testicular sperm cryopreservation has primarily focused on comparing ICSI outcomes between fresh and frozen-thawed testicular spermatozoa. Another focus of these researches is evaluating treatment efficacy differences between ICSI using spermatozoa from patients with obstructive azoospermia (OA) versus non-obstructive azoospermia (NOA). However, few studies have investigated ICSI outcomes among patients with OA, NOA, and cryptozoospermia. This retrospective cohort study analyzed the data from 688 patients at Sichuan Human Sperm Bank (Chengdu, China) between September 2021 and April 2024, comparing cryopreservation efficacy, sperm utilization rate, ICSI outcomes, and delivery outcomes of cryopreserved testicular sperm suspensions in OA group (n = 542), NOA group (n = 114), and cryptozoospermia group (n = 32). Post-thaw analysis revealed that the median sperm viability before and after freezing, as well as the viability ratio, did not differ among the three groups (all P > 0.05). Sperm utilization in NOA group (60.5%) was significantly higher (both P < 0.0001) compared with cryptozoospermia group (31.3%) and OA group (19.9%), while no significant difference was found between OA and cryptozoospermia groups (P > 0.05). Of 162 couples undergoing ICSI, there were no differences in fertilization, clinical pregnancy, delivery, and miscarriage rates among patients with OA, NOA, and cryptozoospermia (all P > 0.05). In the cryptozoospermia group with five newborns, all were full-term with normal birth weights, but this group had the highest maternal pregnancy complication rate.
Keywords: cryptozoospermia, intracytoplasmic sperm injection, non-obstructive azoospermia, obstructive azoospermia, testicular sperm cryopreservation
INTRODUCTION
Azoospermia is defined as the complete absence of spermatozoa after at least two rounds of semen centrifugation.1 This condition affects approximately 1% of the general male population and accounts for 10%–15% of all male infertility cases.2 Azoospermia is primarily categorized into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA).2 In OA patients, a physical blockage in the reproduction ducts prevents spermatozoa entering the ejaculate, despite normal testicular sperm production.3 In contrast, NOA, the most severe form of male infertility, is characterized by severely impaired sperm production in testes. Cryptozoospermia is predominantly caused by spermatogenic disorders; in cryptozoospermia patients, spermatozoa are not visible in fresh semen samples but can be detected in the centrifuged pellet, which differs from azoospermia.1
Since the advent of intracytoplasmic sperm injection (ICSI), a revolutionary-assisted reproductive technique that fertilizes oocytes with a single testicular spermatozoon, new treatments have emerged for NOA, OA, and cryptozoospermia.4 Today, testicular sperm aspiration (TESA) and microdissection testicular sperm extraction (micro-TESE) have become the standard methods for sperm extraction in patients with OA and NOA, respectively.5,6 Numerous studies have established the efficacy of TESA-ICSI and micro-TESE-ICSI in treating male infertility associated with azoospermia.7,8,9,10,11 Moreover, for patients with cryptozoospermia who fail to achieve successful ICSI outcomes using ejaculated spermatozoa, TESA-ICSI and micro-TESE-ICSI offer alternative pathways to live birth.12
Insemination is not possible if no spermatozoa is available on the day of ICSI; however, testicular sperm cryopreservation addresses this issue. The clinical potential of testicular sperm cryopreservation was first validated in 1996, when Hovatta et al.13 achieved a live birth from frozen-thawed testicular spermatozoa via ICSI. Subsequently, Brook et al.14 reported the first attempt to establish a freezing protocol for human testicular cell suspensions, which greatly advanced the field. An increasing number of studies have demonstrated no difference in the rates of fertilization, high-quality embryos, clinical pregnancy, or miscarriage between fresh and frozen-thawed testicular spermatozoa collected from patients with azoospermia or cryptozoospermia and subjected to ICSI.9,15 Additionally, cryopreservation also mitigates the risks of repeat testicular biopsies and alleviates the emotional, physical, and financial tolls associated with failed sperm retrieval during oocyte collection.
Despite these advances, the current scope of research remains limited. Investigations into testicular sperm cryopreservation have focused mainly on differences in ICSI outcomes between fresh and frozen-thawed testicular spermatozoa.9,15,16,17,18,19,20 Another focus of these researches is the comparison of ICSI outcomes with testicular spermatozoa from patients with OA and NOA.21,22,23,24,25 Studies on cryptozoospermia have mainly compared ICSI outcomes between testicular and ejaculated spermatozoa,15,26,27 and few have directly compared ICSI outcomes with testicular spermatozoa from patients with OA, NOA, or cryptozoospermia.
The present study aimed to compare the cryopreservation efficacy, sperm utilization rate, ICSI, and delivery outcomes of cryopreserved testicular sperm suspensions among patients with OA, NOA, and cryptozoospermia.
PATIENTS AND METHODS
Patients
This retrospective study included 688 patients with azoospermia or cryptozoospermia who underwent cryopreservation of testicular cell suspensions extracted by TESA and micro-TESE at the Sichuan Human Sperm Bank (Chengdu, China) between September 2021 and April 2024. All patients underwent a systematic clinical evaluation by an experienced andrologist, including physical examination and ultrasound examination of the genitalia to evaluate the anatomy of the testes, seminal vesicles, prostate, distal vasa deferentia, and ejaculatory ducts. Karyotyping and Yq microdeletions were examined by an experienced laboratory technician. At least two semen analyses were performed to diagnose azoospermia and cryptozoospermia before TESA and micro-TESE. The diagnostic criteria for the azoospermia and cryptozoospermia are in accordance with the 5th edition of World Health Organization (WHO) guidelines for human semen analysis.1 After ejaculatory disorders and retrograde ejaculation were ruled out, azoospermia is defined as the complete absence of spermatozoa after at least two rounds of semen centrifugation. In cryptozoospermia patients, spermatozoa are not visible in fresh semen samples but can be detected in the centrifuged pellet, which differs from azoospermia. Azoospermia can be further classified into OA and NOA. Patients with OA have normal testicular volume, epididymal duct dilatation or vas deferens agenesis, and normal spermatogenic function. NOA is diagnosed when the criteria for OA are not met.28 Ultimately, the patients were divided into OA group (n = 542), NOA group (n = 114), and cryptozoospermia group (n = 32). The collected clinical data comprised the patient age, body mass index (BMI), marital status, duration of infertility, disease type, testicular sperm-related data, ICSI outcomes, and delivery outcomes.
Ethical approval
This study was approved by the Ethics Committee of West China Second University Hospital of Sichuan University (approval No. 20230297). All participants provided written informed consent for all procedures, study inclusion, and the public disclosure of data involved in this study.
Testicular sperm extraction
All patients underwent TESA or micro-TESE performed by an experienced andrologist, as previously described.29,30 For TESA, the testis was pierced with a side hole needle, and tissue was extracted using suction into the attached 20-ml syringe when the needle had reached the appropriate depth to maintain negative pressure.29 For micro-TESE, the testicular tissue was exposed through a transverse incision and observed at 10× magnification with an operating microscope (Carl Zeiss AG, Baden-Württemberg, Germany) to identify seminiferous tubules with normal spermatogenic function.30 The testicular tissue was collected into a 1.5-ml Eppendorf tube with 0.9% NaCl and sent to the laboratory for testing.
Preparation and freeze-thawing of testicular cell suspensions
The tissue was mechanically minced with two needles (attached to 1-ml syringes) on sterile slides to release the spermatozoa from the seminiferous tubules. Spermatozoa were identified using an inverted microscope at 400× magnification (Olympus, Tokyo, Japan). If spermatozoa were observed, the fresh testicular tissue was sectioned and prepared as a testicular suspension with gradient in vitro fertilization (G-IVF) plus fertilization medium (Vitrolife, Göteborg, Sweden). Subsequently, testicular suspensions were mixed with pentoxifylline-supplemented cryoprotectant at a ratio of 1:1 (v:v) and incubated for 5 min in an incubator at 37°C with 5% carbon dioxide (CO2). After combining 10-µl aliquots of testicular suspension with 10 µl of 0.5% eosin solution on a microscope slide, sperm vitality was assessed at 400× magnification. Another 10-µl aliquot was prepared for a 22 mm × 22 mm coverslip. The total sperm count (TSC) was defined as the aggregate number of spermatozoa counted in ten high-power fields under 400× magnification. Next, 100 μl of the testicular suspension was loaded into 300-μl CBS High Security Semen Straws (Cryo Bio System, Normandy, France), which were heat-sealed at both ends and rapidly frozen at 5 cm above the liquid nitrogen surface for 10 min. Subsequently, the straws were placed in liquid nitrogen at −196°C for storage. The recovery straws were thawed in a 37°C water bath for 2 min, and the testicular suspension was then transferred to a 1.5-ml Eppendorf tube and placed immediately in an incubator at 37°C with 5% CO2 for 15 min. Finally, testicular sperm vitality was assessed, and the TSC was calculated. The sperm samples were frozen and thawed in accordance with the standard sperm freezing and thawing protocol of our institution.
ICSI outcome assessment
The indicators of embryo development included fertilization rate, cleavage rate, blastocyst formation rate, and high-quality embryo rate. The fertilization rate referred to the rate of 2 pronuclei fertilization in all mature MII stage oocytes. The cleavage rate was the proportion of cleaved embryos in fertilized oocytes. The blastocyst formation rate was the percentage of blastocysts from cleaved embryos. The high-quality embryo rate was the proportion of embryos classified as “high quality” in the total number of evaluable embryos.
The clinical outcomes were the rates of biochemical pregnancy, clinical pregnancy, miscarriage, and delivery per embryo transfer cycle. Clinical pregnancy was confirmed by the detection of a fetal heartbeat in the intrauterine gestational sac during transvaginal ultrasonography at 5–6 weeks of gestation. The clinical pregnancy rate was the proportion of cycles with clinical pregnancy per total number of embryo transfer cycles; the biochemical pregnancy rate was the percentage of embryo transfer cycles resulting in a positive serum β-human chorionic gonadotropin test (indicating implantation) but without clinical evidence of a gestational sac per total number of embryo transfer cycles; the miscarriage rate was the percentage of embryo transfer cycles that ended in spontaneous fetal loss before 20 weeks of gestation; and the delivery rate was the proportion of cycles that resulted in a live delivery from the total number of embryo transfer cycles.
Delivery outcome assessment
The delivery outcomes comprised the number and sex of newborns, pregnancy duration, birth weight, maternal complications, and neonatal outcomes. A full-term birth refers to the delivery of a fetus at 37–41 weeks; and 6 days of gestation and preterm birth refers to the delivery of a fetus before 37 completed weeks of gestation. Low birth weight was designated as <2500 g and macrosomia as ≥4000 g, both independent of gestational age. Maternal complications during pregnancy, childbirth, or the postpartum period refer to adverse medical events that affected the health of the mother, including pre-eclampsia, gestational diabetes, postpartum hemorrhage, and other conditions. Neonatal outcomes were assessments of the health and survival of infants during the first 28 days of life, including the Apgar score, neonatal intensive care unit admission, and congenital anomalies.31
Statistical analyses
Statistical analyses were conducted by using GraphPad Prism (version 9.5; GraphPad Software, San Diego, CA, USA). Continuous variables were assessed for normality from the Shapiro–Wilk test. For non-normally distributed continuous variables, group differences were evaluated using the Kruskal–Wallis test for three-group comparisons and the Mann–Whitney U test for two-group comparisons. Data are reported as the median (interquartile range [IQR]). Categorical variables (e.g., miscarriage rate) were compared using the Chi-square test when all expected frequencies were greater than 5; otherwise, Fisher’s exact test was applied. A two-sided P < 0.05 was considered statistically significant.
RESULTS
Characteristics of the diagnostic groups
A total of 688 patients underwent cryopreservation of testicular spermatozoa retrieval by TESA (598, 86.9%) and micro-TESE (90, 13.1%). The cohort comprised 542 (78.8%) patients with OA, 114 (16.6%) with NOA, and 32 (4.7%) with cryptozoospermia. There was no significant difference (all P >0.05) among the three groups in the patient age, BMI, and infertility duration (Table 1).
Table 1.
Laboratory parameters comparison among obstructive azoospermia, non-obstructive azoospermia, and cryptozoospermia groups
| Parameter | OA | NOA | Cryptozoospermia | P |
|---|---|---|---|---|
| Patient (n) | 542 | 114 | 32 | - |
| Male age (year), median (IQR) | 30 (28–34) | 31 (28–34) | 32.5 (29–36) | 0.067 |
| Male BMI (kg m−2), median (IQR) | 24 (21–27) | 23 (21–26) | 25 (23–26) | 0.3424 |
| Duration of infertility (year), median (IQR) | 2 (1–4) | 2 (1–4) | 3 (2–4) | 0.0804 |
| TSC | ||||
| Pre-freeze (n), median (IQR) | 30 (21–41) | 21 (12–36) | 25 (17–35) | <0.0001* |
| Post-thaw (n), median (IQR) | 18 (13–24) | 13 (8–22) | 16 (10–21) | 0.0005* |
| Recovery rate (%), median (IQR) | 61.0 (49.0–75.0) | 60.0 (48.5–83.5) | 63.0 (54.0–70.0) | 0.8100 |
| Vitality (%), median (IQR) | ||||
| Pre-freeze | 74.0 (66.0–79.0) | 74.0 (65.0–80.0) | 74.0 (64.0–80.0) | 0.9590 |
| Post-thaw | 42.0 (36.0–49.0) | 44.0 (39.0–48.5) | 42.0 (34.5–52.0) | 0.4794 |
| Vitality ratio | 58.0 (50.0–67.0) | 59.0 (53.5–66.0) | 63.0 (57.0–69.0) | 0.1009 |
| Straws (n), median (IQR) | ||||
| Cryopreserved straws | 3 (2–5) | 3 (2–4) | 3 (2–5) | 0.3497 |
| Utilized straws | 1 (1–1) | 1 (1–2) | 1 (1–2) | 0.7671 |
| Utilization rate (%) | ||||
| Sperm utilization rate | 19.9 | 60.5 | 31.3 | <0.0001* |
| Straw utilization rate, median (IQR) | 33.3 (20.0–50.0) | 33.3 (25.0–50.0) | 33.3 (20.0–50.0) | 0.5335 |
*Significant difference among the three groups. P-values for intergroup comparisons were as follows. Pre-freeze TSC: OA vs NOA, P<0.0001; OA vs cryptozoospermia, P=0.0379; NOA vs cryptozoospermia, P=0.3861. Post-thaw TSC: OA vs NOA, P=0.0002; OA vs cryptozoospermia, P=0.1757; NOA vs cryptozoospermia, P=0.2863. Sperm utilization rate: OA vs NOA, P<0.0001; OA vs cryptozoospermia, P=0.5460; NOA vs cryptozoospermia, P<0.0001. -: not available; IQR: interquartile range; BMI: body mass index; TSC: total sperm count; OA: obstructive azoospermia; NOA: non-obstructive azoospermia
Quality assessment of pre-freeze and post-thaw testicular spermatozoa
Post-thaw TSC and vitality were assessed in 619 out of 688 samples. The OA group exhibited the highest median TSC prior to freezing. The median pre-freeze TSC was 16.7% lower in the cryptozoospermia group (P = 0.0379) and 30.0% lower in the NOA group (P < 0.0001) compared to the OA group. Similarly, the median post-thaw TSC was higher in the OA group than that in the NOA group (P = 0.0002) but similar to that in the cryptozoospermia group (P > 0.05), as shown in Table 1. Median TSC values were comparable between the NOA and cryptozoospermia groups both before freezing and after thawing. The sperm recovery rate (post-thaw TSC/pre-freeze TSC) was comparable among all three groups. Similarly, median sperm vitality did not differ significantly among the groups (all P > 0.05) in either pre-freeze or post-thaw samples. Additionally, the vitality ratio (post-thaw vitality/pre-freeze vitality) was also comparable across the groups (Table 1).
Testicular spermatozoa cryopreservation results
Cryopreserved testicular spermatozoa were used for ICSI treatment in 187 patients (27.2%). The sperm utilization rate (the proportion of patients using cryopreserved sperm for ICSI) was significantly higher in the NOA group compared with those in the OA and cryptozoospermia groups (both P < 0.0001), while no significant difference was found between OA group and cryptozoospermia group (P > 0.05). The median numbers of cryopreserved and utilized straws were similar in all three diagnostic groups. The straw utilization rate (the proportion of cryopreserved straws used per patient) was also comparable among the groups (Table 1).
ICSI results
ICSI data were available for 162 of 187 couples using post-thaw testicular spermatozoa, corresponding to 186 ICSI cycles and 225 embryo transfer cycles. Female age and BMI were similar in the three groups. The fertilization rate, high-quality embryo rate, and blastocyst formation rate were comparable among the groups. Clinical pregnancy, miscarriage, and delivery rates also did not differ significantly among the groups (all P > 0.05; Table 2).
Table 2.
Comparison of intracytoplasmic sperm injection outcomes from frozen-thawed testicular spermatozoa among OA, NOA, and cryptozoospermia groups
| Parameter | OA | NOA | Cryptozoospermia | P |
|---|---|---|---|---|
| Patient (n) | 85 | 67 | 10 | - |
| Female age (year), median (IQR) | 30 (27–32) | 30 (26–32) | 29 (26–32) | 0.8070 |
| Female BMI (kg m−2), median (IQR) | 22 (20–24) | 22 (20–24) | 24 (20–25) | 0.3677 |
| ICSI cycles (n) | 90 | 79 | 17 | - |
| Injected oocytes (n) | 822 | 716 | 128 | - |
| Fertilization rate (%), median (IQR) | 75.0 (61.2–87.5) | 70.6 (50.0–84.2) | 61.5 (44.4–91.7) | 0.1780 |
| High-quality embryos rate (%), median (IQR) | 44.4 (20.0–71.4) | 35.1 (20.0–57.9) | 40.0 (25.0–75.0) | 0.5625 |
| Blastocyst formation rate (%), median (IQR) | 55.6 (33.3–80.0) | 60.4 (34.4–100.0) | 50.0 (25.0–75.0) | 0.4310 |
| Transfer cycles (n) | 109 | 97 | 19 | - |
| ET cycles (n) | 44 | 40 | 8 | 0.9857 |
| FET cycles (n) | 65 | 57 | 11 | - |
| Embryos transferred (n) | 148 | 130 | 29 | - |
| Biochemical pregnancy rate (% per ET) | 9.3 | 8.3 | 8.7 | 0.8484 |
| Clinical pregnancy rate (% per ET) | 49.5 | 60.8 | 47.4 | 0.2203 |
| Miscarriage rate (% per ET) | 9.2 | 7.2 | 15.8 | 0.4812 |
| Delivery rate (% per ET) | 22.9 | 36.1 | 26.3 | 0.1116 |
| Ongoing pregnancy (n) | 9 | 17 | 1 | - |
-: not available; IQR: interquartile range; BMI: body mass index; OA: obstructive azoospermia; NOA: non-obstructive azoospermia; ICSI: intracytoplasmic sperm injection; ET: embryo transfer; FET: frozen embryo transfer
Delivery outcomes
Sixty-five couples gave birth to 73 newborns (38 males and 35 females), comprising 57 singletons and 8 twins. In the cryptozoospermia group, all 5 newborns were male singletons. Preterm births occurred in five cases each in the OA and NOA groups, whereas all 5 births in the cryptozoospermia group were full-term. The OA and NOA groups had 9 and 8 low-birth-weight newborns, respectively, while all newborns in the cryptozoospermia group had a birth weight ranging from 2.5 kg to 4.0 kg. Eleven pregnancy complications were observed across the study groups: 4 in the OA group (3 cases of gestational diabetes mellitus and 1 case of gestational hypertension), 6 in the NOA group (3 cases of gestational diabetes mellitus, 2 cases of gestational hypertension complicated with diabetes, and 1 case of intrahepatic cholestasis of pregnancy), and 1 in the cryptozoospermia group (gestational diabetes mellitus), as shown in Table 3.
Table 3.
Delivery outcomes among OA, NOA, and cryptozoospermia groups
| Parameter | OA | NOA | Cryptozoospermia |
|---|---|---|---|
| Newborn (n) | 30 | 38 | 5 |
| Singleton | 20 | 32 | 5 |
| Multiple pregnancy | 5 | 3 | 0 |
| Sex | |||
| Male | 14 | 19 | 5 |
| Female | 16 | 19 | 0 |
| Pregnancy duration | |||
| Full-term (≥37 weeks) | 25 | 33 | 5 |
| Preterm (<37 weeks) | 5 | 5 | 0 |
| Birthweight (g) | |||
| 1501–2500 | 9 | 8 | 0 |
| 2500–4000 | 18 | 27 | 5 |
| >4000 | 3 | 3 | 0 |
| Complication (n) | 4 | 6 | 1 |
OA: obstructive azoospermia; NOA: non-obstructive azoospermia
We conducted a comprehensive post hoc power analysis using G*Power (version 3.1; available at: https://www.gpower.hhu.de; last accessed on June 22, 2025) for the laboratory parameters and ICSI outcomes. For the laboratory parameters (n = 688), the post hoc power analysis confirmed that the sample size provided >95% power to detect moderate-sized group differences. Regarding the ICSI outcomes, with a total sample size of 162 couples (OA: 85, NOA: 67, and cryptozoospermia: 10) and α = 0.05, the statistical power values were 0.832, 0.906, and 0.927, respectively; all exceeded the acceptable threshold of 0.80.
DISCUSSION
Cryopreservation of testicular spermatozoa extracted by TESA or micro-TESE is an important intervention for treating male infertility, including cryopreserved testicular seminiferous tubules and testicular cell suspension. In our study, we selected testicular cell suspension freezing, which could help embryologists markedly shorten the time required for ICSI treatment. As expected, owing to the impairment of testicular spermatogenic function, the TSC of spermatozoa was lower in the NOA group than those in the other two groups both before and after cryopreservation. To determine whether testicular sperm quality and cryotolerance differed among the OA, NOA, and cryptozoospermia groups, we evaluated the vitality of testicular spermatozoa before and after cryopreservation, and the analysis showed no significant intergroup difference. The average post-thaw testicular sperm vitality exceeded 42% across all three diagnostic groups, which is consistent with the 40% reported in a previous study.32
Although sperm banks play a crucial role in preserving fertility options for men with infertility, research on the storage and utilization of cryopreserved testicular sperm straws remains limited. In the present study, we found that the median number of cryopreserved straws per patient was three. This finding aligns closely with a previous study that reported a median of four straws,33 suggesting a degree of standardization in clinical practice regarding sperm cryopreservation. Our data demonstrated that an average of one straw was utilized per patient, leaving a substantial reserve of cryopreserved spermatozoa. This reserve serves a dual purpose, allowing for future ICSI attempts after treatment failure and offering patients a second chance at fertility without the need for additional sperm retrieval procedures. The reserve also caters to patients planning for second-child fertility needs, ensuring that they have readily available spermatozoa when desired. This strategy not only enhances patient convenience but also minimizes repeat testicular trauma from additional sperm retrieval procedures, thereby reducing the associated risks and costs.
An important finding of the present study was the decrease in the sperm utilization rate across the diagnostic groups: NOA group (60.5%), cryptozoospermia group (31.3%), and OA group (19.9%). This disparity may be attributed to several factors. First, owing to the profound testicular dysfunction inherent in NOA,34 the sperm recovery rate in patients with NOA is approximately 50%,35 which is lower than the rate of greater than 95% observed in patients with OA.36 When testicular sperm are retrieved on the day of oocyte retrieval for ICSI, the risk of sperm acquisition failure is high in patients with NOA. Consequently, in our experience, a high proportion of patients with NOA opt to use cryopreserved testicular spermatozoa as a strategic safeguard against such procedural risks. In contrast, given that the testes of patients with OA contain a relatively large number of spermatozoa, physicians in reproductive centers might suggest extracting fresh testicular spermatozoa on the day of oocyte retrieval, in these patients. Second, for patients with NOA undergoing micro-TESE, the technical complexity of the procedure and its high associated costs are an incentive to cryopreserve the maximum feasible number of spermatozoa during the initial surgery and subsequently use the cryopreserved testicular spermatozoa. Third, unlike patients with NOA, some patients with OA or cryptozoospermia have alternative treatment options. The European Association of Urology has stated that microsurgical vasovasostomy or epididymovasostomy may be performed for patients with azoospermia caused by epididymal or vasal obstruction whose female partners have a good ovarian reserve.37 The recovery period following this procedure typically delays the requirement for ICSI with cryopreserved testicular spermatozoa. For patients with cryptozoospermia, freshly ejaculated sperm are often used on the day of oocyte retrieval for ICSI, reducing reliance on cryopreserved samples and contributing to the lower utilization rates in this patient cohort. A 30-year Israeli sperm bank follow-up reported that the utilization rates for cryopreserved testicular sperm were 67% for patients with OA and NOA.33 The lower utilization rate observed in the present study (19.9%–60.5%) may be attributed to the shorter follow-up duration (3 years). Therefore, a long-term follow-up (≥10 years) is essential to accurately assess cryopreserved testicular sperm utilization trends and incorporate the findings into clinical guidelines.
The cryptozoospermia, OA, and NOA groups achieved similar ICSI outcomes from the use of cryopreserved testicular spermatozoa, including fertilization, high-quality embryo, blastocyst formation, clinical pregnancy, miscarriage, and delivery rates. Several previous studies have also reported similar ICSI outcomes between OA and NOA groups.21,22,28 However, few studies have directly compared the ICSI outcomes between these three distinct patient groups, and our study attempts to identify these similarities. We observed an interesting phenomenon regarding the delivery outcomes that all newborns in the cryptozoospermia group were male singletons delivered at full term with normal birth weights, but the cryptozoospermia group had the highest incidence of pregnancy complications. In contrast, newborns of patients with azoospermia had higher prevalence of preterm birth and low birth weight, and the mothers had less pregnancy complications. These findings reveal the potential differences in the associations between different types of male fertility disorders and delivery outcomes. This information may be valuable for obstetricians and reproductive specialists. However, mechanistic research must be combined with large-sample analysis to provide accurate clinical counseling and management of female partners who become pregnant with cryopreserved testicular spermatozoa from patients with OA, NOA, and cryptozoospermia.
The present study has several limitations. First, the retrospective study design comes with inherent selection bias and limitations. The non-random patient inclusion caused baseline imbalances: men older than 35 years accounted for 14.2% of the OA group, 14.9% of the NOA group, and 25.0% of the cryptozoospermia group. Furthermore, the follow-up data were incomplete, with dropout rates of 21.3% in the OA group, 2.9% in the NOA group, and 0 in the cryptozoospermia group, primarily owing to patients receiving ICSI at external centers. Treatment heterogeneity, including varied sperm retrieval techniques and non-standardized female ovulation induction protocols, further confounded the causal interpretation of outcomes. Unfortunately, the follow-up duration was insufficient to evaluate accurately the long-term utilization rate and clinical outcomes. Finally, the study groups exhibited substantial numerical imbalances (542 patients in the OA group, 114 in the NOA group, and 32 in the cryptozoospermia group). However, the relatively small sample size in the cryptozoospermia group aligns with clinical practice, as most patients diagnosed with cryptozoospermia prioritize using ejaculated spermatozoa for ICSI over invasive procedures such as TESA or micro-TESE. Despite these limitations, the present study contributes important findings that address critical gaps in the literature. Our findings require verification in prospective multicenter studies with a longer follow-up duration and a larger, more balanced sample size.
In summary, this study conducted a systematic and comprehensive analysis of the cryopreservation efficacy, sperm utilization rate, ICSI outcomes, and delivery outcomes of cryopreserved testicular spermatozoa from patients with OA, NOA, and cryptozoospermia. Our results show that patients with these conditions can achieve similar ICSI outcomes. We recommend that patients consider testicular sperm cryopreservation during the first TESA or micro-TESE for potential future use. These findings offer reassurance to patients with male infertility and clinicians selecting optimal sperm retrieval strategies.
AUTHOR CONTRIBUTIONS
JYX performed the conceptualization and methodology and wrote the original draft. BL, SSL, and XFL participated in the investigation and reviewed and edited the manuscript. CL, WRZ, YL, DML, and LJY performed the data curation and formal analysis. YX and FPL provided project administration, resources, and supervision. All authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
ACKNOWLEDGMENTS
This study was supported by the Sichuan Science and Technology Program (2024NSFSC0647) and the Health Commission of Sichuan Province Medical Science and Technology Program (24SYJS01). We would like to express our sincere gratitude to Dr. Cheng Lu (West China Second University Hospital, Sichuan University, Chengdu, China) for consultations related to the methodology of this research.
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