Fresh or frozen testicular sperm? Guidance from an etiology-classified retrospective study on ICSI cycles for patients with azoospermia

Abstract

Using testicular sperm, men with azoospermia can father children. Sperm cryopreservation helps avoid the risk of cycle canceling because of the failure of testicular sperm retrieval. However, cryopreservation of testicular sperm can potentially impair sperm function. In addition, the different qualities of testicular sperm retrieved from men with various etiologies of azoospermia can lead to different intracytoplasmic sperm injection (ICSI) outcomes after sperm cryopreservation. A retrospective analysis was performed to elucidate the influence of frozen testicular sperm from men with different etiologies of azoospermia on assisted reproductive technology (ART) treatment outcomes. Testicular sperm retrieval rates were also compared. The fertilization rate was found to be influenced by testicular sperm cryopreservation in most cases, especially for patients with azoospermia factor c (AZFc) deletion, and embryo development was significantly impeded when frozen testicular sperm from Klinefelter syndrome (KS) patients were used. Moreover, ART treatment outcomes after embryo transfer were not significantly influenced by testicular sperm cryopreservation for any of the etiologies. In addition, significantly higher sperm retrieval rates were achieved in cryptorchidism, KS, and AZFc deletion patients, whereas the testicular sperm retrieval rate was significantly lower in patients with idiopathic nonobstructive azoospermia. Collectively, our findings suggest that except for patients with KS, testicular sperm retrieval prior to initiating ovarian stimulation of the patient’s partner should be considered the first-line approach. In addition, assisted oocyte activation should be performed in ICSI cycles using frozen testicular sperm, especially for patients with AZFc deletion.

INTRODUCTION

According to a report published by the World Health Organization (WHO), approximately one in six people of reproductive age experience infertility at some stage in their lives, and male factors contribute wholly or in part to 45.1% of infertility cases.1 Azoospermia, either obstructive or nonobstructive, is among the main causes of male infertility, and testicular sperm extraction (TESE) may be the last chance for some infertile men to father a child.

With the development of sperm cryopreservation technology, sperm retrieved via TESE can be frozen before initiating ovarian stimulation of the patient’s partner and can be thawed for intracytoplasmic sperm injection (ICSI) on the day of ovum retrieval. Testicular sperm retrieval prior to initiating ovarian stimulation was suggested as the first-line approach in patients with nonobstructive azoospermia (NOA).2 It can prevent the canceling of the cycle on the day of ovum retrieval in cases of failure to find available sperm in the testis, avoiding unnecessary risks for the partner from ovarian stimulation and ovum retrieval.2,3 In addition, considering the general live birth rate of ICSI, repeated ICSI cycles are sometimes needed.4,5 Testicular sperm cryopreservation, which helps avoid repeated testicular sperm sampling in subsequent ICSI cycles, is beneficial for patients with either obstructive azoospermia (OA) or NOA.2,5

However, cryopreservation of testicular sperm may lead to dysfunction of the sperm. Considering the physical difference between ejaculated sperm and testicular sperm, testicular sperm seem more sensitive to environmental stress. Testicular spermatozoa are extremely sensitive to cryodamage.6 In addition, changes in epigenetic patterns in cryopreserved testicular tissues have been reported.7 Although motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when the nuclei of nonviable spermatozoa are injected into oocytes,6 it is necessary to evaluate the effects of assisted reproductive technology (ART) treatment when cryopreserved testicular sperm are used in ICSI.

Accordingly, ICSI outcomes using cryopreserved testicular sperm have been evaluated repeatedly for years.2,5,8,9,10,11,12,13 There was no significant difference reported for the fertilization rate between ICSI cycles using fresh and frozen testicular sperm in most of these studies, but the results for the cleavage rate, pregnancy rate, and live birth rate were inconsistent. It is believed that the ICSI outcome is not affected by whether the testicular sperm used is fresh or frozen; however, the prerequisite is that motile sperm can be retrieved.2 Aizer et al.14 reported that the cumulative live birth rate was even better when cryopreserved testicular sperm were used.

Owing to the low quality of the sperm extracted from testicular biopsy, the cryopreservation of testicular sperm is controversial.2 Testicular sperm are more likely to lose vitality after cryopreservation. In some cases, the rare sperm retrieved from testicular biopsy can even be lost during cryopreservation. Therefore, some patients prefer to use fresh testicular sperm for ICSI, accepting the risk that sperm is not available on the day of ovum retrieval.

However, the structural imbalance of medical resources and population mobility overburdens high-tier hospitals in China.15 The schedules of operating rooms in these hospitals are always full, along with the overloading of ward beds. Moreover, the exact date for ovum retrieval is difficult to confirm in advance because of individual differences in follicle development. If patients choose to use fresh testicular sperm for ICSI, not only they are subject to the risk that no sperm is available, but also they are subject to the risk that no operating room or ward bed is available for them on the day of ovum retrieval.

To solve this problem, novel sperm cryopreservation technologies, along with novel sperm cryopreservation carriers, were introduced. A 0.25-ml straw is usually used as a testicular sperm cryopreservation carrier in our daily practice, but it is not suitable for preserving some of the rare sperm retrieved via TESE. At present, Cryopiece, a novel sperm cryopreservation carrier that is employed for individual sperm cryopreservation, is utilized for the cryopreservation of rare testicular sperm in clinical practice at our hospital (Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China).16 Rare sperm are not lost once preserved using the Cryopiece. With the help of this novel sperm cryopreservation carrier, our team achieved the first live baby delivery in China using individual cryopreserved testicular sperm.17 Moreover, the influence of using the Cryopiece for the cryopreservation of ejaculated sperm provided by healthy donors was evaluated. Compared with the use of 0.25-ml straws, the use of the Cryopiece resulted in significantly better recovery and retrieval rates after frozen ejaculated sperm were thawed.18

Further research focusing on the clinical application of the Cryopiece was also performed. The utilization of the Cryopiece for ejaculated or testicular sperm cryopreservation in ICSI results in similar ART outcomes, and the clinical application of the Cryopiece for rare ejaculated and testicular spermatozoa is beneficial.16 However, the benefits of the Cryopiece for testicular sperm compared with those of other cryopreservation carriers, especially for those derived from NOA patients, remain to be further evaluated.

Meanwhile, patients who undergo TESE have different etiologies. The common diagnoses for these patients include OA, Klinefelter syndrome (KS), azoospermia factor c region (AZFc) deletion, and cryptorchidism. Idiopathic NOA, which means that no definite or known relative factors contribute to the development of NOA, is also among the common diagnoses for these patients. Owing to the different etiologies, the quality of the testicular sperm retrieved from these patients varies greatly, suggesting that the influence of cryopreservation on these types of sperm is quite different. Therefore, the outcomes of ART using these types of sperm also vary greatly. However, few studies have focused on comparisons among different types of azoospermia. Although the clinical pregnancy rate was reported to be similar among different NOA etiologies,19 the influences of sperm cryopreservation in these cases were not discussed.

In this study, a retrospective analysis was conducted, and the ART treatment outcomes of infertile couples with different male etiologies were compared on the basis of the use of fresh or cryopreserved testicular sperm in their ICSI cycles, along with the testicular sperm retrieval rates of patients with different etiologies who underwent TESE. The aim is to provide evidence for supporting recommendations on which type of sperm, fresh testicular sperm or frozen testicular sperm, should be chosen for specific ART treatments.

PARTICIPANTS AND METHODS

Participants

Clinical information was obtained from the Department of Andrology and Reproductive Medicine Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine. The Research and Ethics Committee of Shanghai General Hospital approved this study (Approval No. Shanghai General Hospital Ethics Review [2020]34). All the participating patients signed informed consent forms. The data involved in the present study were authorized for disclosure.

Enrollment

This study included couples who received ART treatment using their own testicular sperm at Shanghai General Hospital. From September 2020 to March 2025, a total of 591 follicular puncture cycles in which testicular sperm were used were performed. After the couples whose first follicle puncture cycles were performed before September 2020 were excluded, a total of 494 follicular puncture cycles were included, with 143 follicular puncture cycles (followed by 149 embryo transfer cycles for 140 couples) using fresh testicular sperm and 351 follicular puncture cycles (followed by 398 embryo transfer cycles for 290 couples) using cryopreserved testicular sperm.

These couples were further divided into groups according to the diagnosis of the men. Six groups, namely the KS group, AZFc deletion group, OA group, cryptorchidism group, idiopathic NOA group, and other factors group, were established for cycles involving either fresh testicular sperm or cryopreserved testicular sperm. Notably, the “cryptorchidism group” mainly consisted of couples in which the men suffered from cryptorchidism or had a history of cryptorchidism, while couples in which the men had undescended testes or gliding testes were also included in this group because the spermatogenesis dysfunction in all of these men was caused mainly by the abnormal position of the testis. The “other factors group” mainly consisted of couples in which the men had a history of mumps orchitis, varicocele, retrograde ejaculation, or anejaculation. The number of included patients who suffered from each of these diseases was too small to be analyzed individually using statistical tools; thus, they were included in the “other factors group” to determine their contribution to the general outcomes.

Moreover, the records of all TESE surgeries performed at Shanghai General Hospital during the period from September 2020 to March 2025 were collected for testicular sperm retrieval rate analysis. These records were also grouped according to the diagnoses of the patients.

Testicular sperm retrieval and cryopreservation

Male patients suffering from azoospermia (including NOA and OA), retrograde ejaculation, or anejaculation underwent TESE treatment after clinical evaluation. General anesthesia was performed, and seminiferous tubules that potentially contained sperm were collected from the patient’s testis via testicular biopsy. These seminiferous tubules were dissected using ophthalmic scissors and a 1-ml syringe. After confirming the presence of testicular sperm via microscopic examination, these testicular specimens were sent to the laboratory for ICSI or cryopreservation.

Testicular sperm can sometimes be retrieved via testicular sperm aspiration (TESA) for patients with OA or abnormal ejaculation.

In most cases (269 out of 290 cases), testicular sperm were cryopreserved using 0.25-ml straws (transparent PETG 0.25-ml sperm straw; Catalog No. 010261; Cryobio System, L’Aigle, France). In brief, the testicular suspension was mixed with a cryoprotective agent (CPA; Cryosperm; Catalog No. 10670010F; Origio, Malov, Denmark) at a ratio of 1:1 and drawn into the straws; then, the straws were heat-sealed, placed in a straw holder, and fumigated with liquid nitrogen vapor for at least 10 min before being transferred to liquid nitrogen for preservation.

When the number of spermatozoa in the testicular sample was extremely low (i.e., the estimated quantity was no more than 50), individual sperm cryopreservation was performed using a Cryopiece as the carrier. Briefly, the testicular suspension was gradient centrifuged to remove tissue fragments and loaded in a Petri dish (Catalog No. 1006; Falcon, Durham, NC, USA) as microstrips. CPA was mixed with modified HTF medium (Catalog No. 90126; Irvine Scientific, Santa Ana, CA, USA) supplemented with 5% serum substitute supplement (Catalog No. 99193; Irvine Scientific) at a ratio of 1:1 and loaded as drops onto a Cryopiece (1 μl for each drop). Afterward, the Cryopiece was placed in the Petri dish. The microstrip and Cryopiece were covered with light mineral oil (OVOIL; Catalog No. 10029; Vitrolife, Vastra Frolunda, Sweden). An ICSI needle (Catalog No. MIC-35-35; CooperSurgical, Trumbull, CT, USA) was used to transfer the motile sperm from the microstrips to the CPA drops individually. After the spermatozoa were transferred, the Cryopiece was removed from the oil, the oil was carefully drained, and the sample was blotted with tissue and placed in a cryogenic vial (Catalog No. 430659; Corning, Corning, NY, USA). The cryogenic vial with the Cryopiece was fumigated with liquid nitrogen vapor at 5 cm above the liquid–air interface of the liquid nitrogen for at least 10 min, after which it was stored in liquid nitrogen.

Spermatozoa thawing, ICSI, and assisted oocyte activation (AOA)

The straws were thawed in a water bath at 37°C for 3 min. Then, the sealed ends of the straws were cut, and the contents were gently blown out of the straws and dropped into prewarmed recovery solution.

To thaw the sperm cryopreserved in the Cryopiece, light mineral oil in a Petri dish was preheated in an incubator at 37°C, after which the Cryopiece was rapidly removed from the cryogenic vial and placed in the warmed oil. The frozen drops were thawed within seconds.

Fresh or thawed sperm samples were loaded on Petri dishes and covered with 3 ml of light mineral oil. Afterward, the spermatozoa were individually selected and placed in a 20-µl microdroplet containing G-Gamete medium (Catalog No. 10126; Vitrolife). Selected motile spermatozoa in the microdroplet were used for ICSI. In some cases, after the patient provided written informed consent, immotile thawed spermatozoa were used for ICSI when no motile spermatozoa or too few motile spermatozoa were found.

AOA was performed in some cases by exposing the oocytes to 10 μmol l⁻¹ ionomycin (Catalog No. I9657; Sigma-Aldrich, St. Louis, MO, USA) for 10 min after ICSI. Briefly, a 4-well round bottom plate (Catalog No. 14444; Nunc, Rosklide, Denmark) was filled with 500 µl of IVF plus (Catalog No. 10136; Vitrolife); then, 5 µl of ionomycin (1 mmol l⁻¹) was added to each well to prepare a 10-μmol l⁻¹ ionomycin solution. The injected oocytes were transferred to ionomycin medium 1 h after ICSI and incubated for 10 min at 37°C. The oocytes were then sequentially washed in the other three wells to remove residual ionomycin. The AOA-treated oocytes were cultured in G-1 plus medium (Catalog No. 10128; Vitrolife), and fertilization was assessed 16 h to 18 h after ICSI. AOA was performed in 54 out of 290 cases using cryopreserved testicular sperm and in 28 out of 140 cases using fresh testicular sperm.

For participants who had no sperm available on the day of ovum retrieval, cryopreservation of the oocyte or utilization of a donor’s sperm for ICSI was performed depending on the couple’s preferences.

Fertilized oocytes (two pronuclei [2PN], one pronucleus [1PN], and two polar bodies [2PB]) were cultured in G-1 plus medium (Catalog No. 10128; Vitrolife) in an incubator at 37°C with 6% CO₂, 5% O₂, and 89% N₂. Embryos were transplanted on day 2 or 3.

ICSI and embryo transfer were conducted according to a standard procedure that is widely followed in clinical practice. The detailed procedure is described in the studies by Küpker et al.3 and Liu et al.19

Ovarian stimulation and pregnancy outcomes

Ovarian stimulation was achieved using a gonadotropin-releasing hormone (GnRH) antagonist (Cetrorelix; Merck Serono, Boston, MA, USA), recombinant follicle-stimulating hormone (FSH; Gonal-F; Merck Serono), and human menopausal gonadotropin (hMG; Livzon Pharmaceutical Group Inc., Zhuhai, China). Ultrasound-guided follicular puncture was performed 36 h after the injection of human chorionic gonadotropin (hCG; Livzon Pharmaceutical Group Inc.). Harvested oocytes were denuded enzymatically with 2-hydroxyethyl (HEPES)-buffered medium containing hyaluronidase (Catalog No. 90101; Irvine Scientific) and mechanically by pipetting with a commercial glass pipette. The selected spermatozoa were injected into mature metaphase II (MII) oocytes. Biochemical pregnancy was confirmed by β-human chorionic gonadotropin (β-hCG) positivity in the blood or urine 2 weeks after embryo transfer. Clinical pregnancy was confirmed 4–6 weeks after embryo transfer by ultrasonography on the basis of the presence of a gestational sac and a fetal heartbeat in the uterine cavity.

Statistical analyses

PASW Statistics for Windows, version 17.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. Continuous variables were presented as the mean ± standard deviation (s.d.), and Student’s t-test was used to assess differences between the two groups. The Chi-square test was used to compare categorical variables, which are presented as frequencies and percentages. The value of P < 0.05 was considered to indicate statistical significance.

Calculation of the parameters

The methods for calculating the involved parameters are listed below: normal fertilization rate (%) = n (2PN zygotes)/n (MII oocytes used) × 100%; 2PN cleavage rate (%) = n (cleavages of 2PN zygotes)/n (2PN zygotes) × 100%; proportion of good-quality embryos (%) = n (good-quality embryos)/n (valid embryos) × 100%; proportion of 2PN cleavages developing into valid embryos (%) = n (valid embryos)/n (cleavages of 2PN zygotes) × 100%; proportion of 2PN cleavages developing into good-quality embryos (%) = n (good-quality embryos)/n (cleavages of 2PN zygotes) × 100%; proportion of MII oocytes developing into valid embryos (%) = n (valid embryos)/n (MII oocytes used) × 100%; proportion of MII oocytes developing into good-quality embryos (%) = n (good-quality embryos)/n (MII oocytes used) × 100%; transferable embryos per cycle = n (cycles with transferable embryos)/n (follicle puncture cycles); no pregnancy cycle rate (%) = n (cycles without pregnancy)/n (embryo transfer cycles) × 100%; biochemical pregnancy rate (%) = n (cycles with biochemical pregnancy)/(n [embryo transfer cycles] − n [cycles waiting for final outcomes]) × 100%; miscarriage rate (%) = n (cycles with miscarriage)/(n [embryo transfer cycles] – n [cycles waiting for final outcome]) × 100%; live birth rate (per follicle puncture cycle, %) = n (cycles with live births)/(n [follicle puncture cycles] – n [cycles with transferable embryos without embryo transfer] – n [cycles waiting for final outcome]) × 100%; and live birth rate (per embryo transfer cycle, %) = n (cycles with live births)/(n [embryo transfer cycles] – n [cycles waiting for final outcome]) × 100%.

RESULTS

Patients in the AZFc deletion group, KS group, and cryptorchidism group achieved higher testicular sperm retrieval rates via TESE

During the period from September 2020 to March 2025, a total of 2183 TESE surgeries were performed at Shanghai General Hospital. The overall mean testicular sperm retrieval rate was 36.9%. Among these surgeries, 441 were in the OA group, 200 were in the KS group, 145 were in the cryptorchidism group, 51 were in the AZFc deletion group, 1036 were in the idiopathic NOA group, and 310 were in the other factors group. Unsurprisingly, the testicular sperm retrieval rate in the OA group was 100.0%. Thus, patients in the OA group were excluded from further analysis. Among the remaining groups, the testicular sperm retrieval rates in the AZFc deletion group, KS group, and cryptorchidism group were significantly greater than the general testicular sperm retrieval rate (all P < 0.01; Table 1). The general testicular sperm retrieval rate (the overall mean rate after exclusion of patients from the OA group) was 21.0%, while the testicular sperm retrieval rate in the AZFc deletion group was the highest (45.1%), followed by that in the KS group (42.5%) and the cryptorchidism group (31.0%). The testicular sperm retrieval rate of the idiopathic NOA group was significantly lower (only 15.3%; P < 0.01), whereas no significant difference was detected between the rate in the other factors group and the general rate (P > 0.05), as shown in Table 1.

Table 1.

Testicular sperm retrieval rates in different groups

Parameters	General (OA cases were excluded)	KS	AZFc deletion	Cryptorchidism	Idiopathic NOA	Other factors
Age (year), mean±s.d.	30.8±4.0	30.0±4.0	31.2±3.4	30.7±4.1	30.8±4.0	31.0±4.1
Sperm retrieved (cycle), n	365	85	23	45	159	53
Age (year), mean±s.d.	31.1±4.2	29.1±3.2**	31.5±3.3	30.8±3.9	31.5±4.0*	33.1±5.4**
Sperm not retrieved (cycle), n	1377	115	28	100	877	257
Age (year), mean±s.d.	30.7±4.0	30.6±4.3	30.9±3.5	30.7±4.2	30.7±4.0	30.5±3.6
Sperm retrieval rate (observation), %	21.0	42.5**	45.1**	31.0**	15.3**	17.1
Sperm retrieval rate (expectation), %a	-	23.2	21.6	21.7	18.9	20.4

^*P<0.05; ^**P<0.01. ^aThe Chi-square test was applied, and cases involving OA were excluded from the general data. -: expectation value is not available for this term; s.d.: standard deviation; OA: obstructive azoospermia; KS: Klinefelter syndrome; AZFc: azoospermia factor c; NOA: nonobstructive azoospermia

It was reported that in general, age does not adversely affect sperm retrieval via microdissection TESE,20 but in patients with nonmosaic KS, it has been reported that age is a limiting factor for successful sperm retrieval.21 Another study revealed that younger men with KS more frequently had germ cell-containing testis histology; however, these findings were not associated with increased odds of sperm retrieval.22

In the present study, the ages of the patients whose sperm were retrieved from the testis significantly differed from those whose sperm were not retrieved in the KS group (P < 0.01), idiopathic NOA group (P < 0.05), and other factors group (P < 0.01). However, the average ages of these patients did not differ markedly. The average age of the patients in the KS group whose sperm were retrieved was approximately 1.5 years younger than that of those whose sperm were not retrieved. The average age of the patients in the idiopathic group whose sperm were retrieved was approximately 0.8 years older than that of those whose sperm were not retrieved. The average age of the patients in the other factors group whose sperm were retrieved was approximately 2.6 years older than that of those whose sperm were not retrieved (Table 1). In general, it seems that age is not a determining factor for predicting the sperm retrieval rate.

General grouping information

Since some patients undergo repeated follicular punctures and embryo transfers depending on the ART outcomes, the numbers of follicular puncture cycles and embryo transfer cycles are not essentially equal to the included sample size. The sample size and the number of cycle types in each group are listed in detail in Table 2.

Table 2.

Information on the included sample size and the number of each type of cycles

Group	Sample size (couples), n	Age of the husband (year), mean±s.d.	Age of the wife (year), mean±s.d.	Follicular puncture cycle ( n )	Embryo transfer cycle ( n )
General
Fresh sperm	140	31.0±4.8	29.1±3.7	143	149
Frozen sperm	290	31.3±4.3	29.8±3.8	351	398
OA
Fresh sperm	41	31.8±6.3	30.0±4.5	42	42
Frozen sperm	104	31.2±4.2	29.6±4.0	131	160
KS
Fresh sperm	41	29.1±3.0	27.4±2.8	41	50
Frozen sperm	24	30.0±3.8	28.5±3.9	27	27
AZFc deletion
Fresh sperm	8	32.4±4.4	30.3±2.1	9	9
Frozen sperm	9	30.8±3.6	29.1±2.2	9	9
Cryptorchidism
Fresh sperm	7	31.3±4.3	29.9±3.2	8	8
Frozen sperm	20	29.8±3.9	28.7±3.2	23	28
Idiopathic NOA
Fresh sperm	33	31.6±4.4	29.5±4.0	33	29
Frozen sperm	105	31.4±4.2	30.0±3.8	130	137
Other factors
Fresh sperm	10	32.2±3.8	29.5±2.2*	10	11
Frozen sperm	28	33.6±5.0	31.8±3.2	31	37

^*Significant difference, P<0.05. s.d.: standard deviation; OA: obstructive azoospermia; KS: Klinefelter syndrome; AZFc: azoospermia factor c; NOA: nonobstructive azoospermia

As mentioned previously, the number of included patients who had conditions, such as mumps orchitis, varicocele, retrograde ejaculation, anejaculation, or other diseases that would cause azoospermia, was too small to be analyzed individually using statistical tools. Thus, they were included in the “other factors group” to determine their contribution to the general outcomes. The 10 couples in the other factors group for which fresh testicular sperm were used for ICSI included 3 men with anejaculation, 2 men who had previously developed mumps orchitis, 1 man who had been diagnosed with varicocele, and 4 men who experienced retrograde ejaculation. Moreover, the 28 couples in the other factors group who used frozen testicular sperm for ICSI included 5 men with anejaculation, 4 men who had previously developed mumps orchitis, 9 men who had been diagnosed with varicocele, 6 men who experienced retrograde ejaculation, 1 man who had been diagnosed with azoospermia caused by chemotherapy, 1 man who had a testicular tumor, 1 man who had difficulty with semen procurement, and 1 man who had been diagnosed with erectile dysfunction.

As shown in Table 2, the age (mean ± s.d.) of the women in all the included couples was 29.8 ± 3.8 years. The age (mean ± s.d.) of the women who used fresh testicular sperm for ART was 29.1 ± 3.7 years, whereas that of the women who used cryopreserved testicular sperm for ART was 29.8 ± 3.8 years. No significant difference in the age of the women was observed between the group using fresh testicular sperm and the group using frozen testicular sperm (P > 0.05). When the participants were grouped according to their partners’ diagnosis, no significant difference could be observed in terms of age, regardless of whether they chose fresh testicular sperm for ART treatment or not, except in the other factors group (all P > 0.05, except for the other factors group, for which P < 0.05). In terms of the other factors group, the age (mean ± s.d.) of the women who used fresh sperm for ART was 29.5 ± 2.2 years, while that of the women who used frozen sperm was 31.8 ± 3.2 years.

Moreover, the age (mean ± s.d.) of the men in all the included couples was 31.2 ± 4.4 years. The age (mean ± s.d.) of the men who used fresh testicular sperm for ART was 31.0 ± 4.8 years, while that of the men who used cryopreserved testicular sperm for ART was 31.3 ± 4.3 years. No significant difference in the age of the men was observed between the group using fresh testicular sperm and the group using frozen testicular sperm (P > 0.05). When grouped according to the diagnosis, no significant difference could be observed in the men’s age, regardless of whether they chose fresh testicular sperm for ART treatment (all P > 0.05).

The normal fertilization rate of cycles using testicular sperm was influenced by sperm cryopreservation

In general, the normal fertilization rate of the cycles using fresh testicular sperm was significantly greater than that of the cycles using cryopreserved testicular sperm (P < 0.01; Table 3). When the men were grouped according to their diagnosis, the normal fertilization rate was significantly greater when fresh testicular sperm was used in the OA group (P < 0.01), KS group (P < 0.05), and AZFc deletion group (P < 0.01), as shown in Table 3.

Table 3.

Influence of testicular sperm cryopreservation on fertilization, cleavage, and embryo development in different groups

Group	Normal fertilization rate a (%)	2PN cleavage rate a (%)	Proportion of good-quality embryos a (%)	Proportion of 2PN cleavages developing into valid embryos a (%)	Proportion of 2PN cleavages developing into good-quality embryos a (%)	Proportion of MII oocytes developing into valid embryos a (%)	Proportion of MII oocytes developing into good-quality embryos a (%)
General
Fresh sperm	64.5 (60.8)	98.6 (98.2)	53.9 (48.9)	82.9 (81.8)	44.7 (40.0)	52.7 (48.8)	52.3 (23.9)
Frozen sperm	59.2 (60.8)**	98.0 (98.2)	46.5 (48.9)**	81.2 (81.8)	37.8 (40.0)**	47.2 (48.8)**	21.9 (23.9)**
OA
Fresh sperm	76.4 (68.4)	99.1 (98.6)	57.0 (49.9)	82.4 (80.1)	47.0 (40.0)	62.4 (54.3)	35.6 (26.9)
Frozen sperm	66.0 (68.4)**	98.4 (98.6)	47.3 (49.9)**	79.3 (80.1)	37.5 (40.0)**	51.5 (54.3)**	24.4 (26.9)**
KS
Fresh sperm	62.9 (59.7)	99.0 (99.0)	49.0 (47.2)	81.1 (77.6)	39.7 (37.1)	50.5 (45.8)	24.8 (21.9)
Frozen sperm	54.6 (59.7)*	98.8 (99.0)	45.3 (47.2)	70.9 (77.6)*	32.1 (37.1)	38.2 (45.8)**	17.3 (21.9)*
AZFc deletion
Fresh sperm	52.4 (40.0)	100.0b	57.4 (53.8)	85.5 (81.3)	49.1 (43.8)	44.8 (32.5)	25.7 (17.5)
Frozen sperm	26.3 (40.0)**	100.0b	44.4 (53.8)	72.0 (81.3)	32.0 (43.8)	18.9 (32.5)**	8.4 (17.5)**
Cryptorchidism
Fresh sperm	46.2 (53.0)	100.0 (99.0)	50.0 (42.7)	98.0 (92.8)	49.0 (39.6)	45.2 (48.7)	22.6 (20.8)
Frozen sperm	55.6 (53.0)	98.8 (99.0)	40.3 (42.7)	91.1 (92.8)	36.7 (39.6)	50.0 (48.7)	20.1 (20.8)
Idiopathic NOA
Fresh sperm	62.0 (58.5)	96.1 (96.8)	55.8 (49.7)	80.8 (82.5)	45.1 (41.0)	48.1 (46.7)	26.9 (23.2)
Frozen sperm	57.5 (58.5)	97.0 (96.8)	48.0 (49.7)	83.0 (82.5)	39.8 (41.0)	46.3 (46.7)	22.2 (23.2)
Other factors
Fresh sperm	62.9 (56.6)	100.0 (98.6)	54.9 (47.6)	86.3 (86.0)	47.4 (40.9)	54.3 (48.0)	29.8 (22.9)
Frozen sperm	54.0 (56.6)	97.9 (98.6)	43.9 (47.6)	85.9 (86.0)	37.7 (40.9)	45.4 (48.0)	19.9 (22.9)*

^*P<0.05; ^**P<0.01. ^aResults are presented in the form of “observation (statistical expectation)”. The Chi-square test was applied, and the frequency was converted into a percentage. ^bExpectation value is not available for this term. OA: obstructive azoospermia; KS: Klinefelter syndrome; AZFc: azoospermia factor c; NOA: nonobstructive azoospermia; 2PN: two pronuclei; MII: metaphase II

Although no significant difference could be observed (P > 0.05), the normal fertilization rates of the idiopathic NOA group and the other factors group were also lower than the statistical expectation when frozen testicular sperm were used. Only in the cryptorchidism group, the use of cryopreserved testicular sperm achieved a better normal fertilization rate, but the difference was not statistically significant (P > 0.05; Table 3).

The general normal fertilization rate of all the included cycles was approximately 60.8%, while the OA group achieved the highest statistical expectation of normal fertilization rate (68.4%) and that of the AZFc deletion group was the lowest (40.0%), as shown in Table 3.

The 2PN cleavage rate was not influenced by whether the testicular sperm used were cryopreserved

The general 2PN cleavage rate of the included cycles was approximately 98.2%. No significant difference could be observed between the cycles using fresh testicular sperm and those using cryopreserved sperm (P > 0.05). When the men were grouped according to their diagnosis, no significant difference was observed (Table 3). The 2PN cleavage rate of the idiopathic NOA group was the lowest (approximately 96.8%), while that of the AZFc deletion group was the highest (100.0%).

Development of valid embryos in the KS group was influenced by testicular sperm cryopreservation

Except for the KS group, no significant difference in the number of valid embryos generated from 2PN cleavages was detected, regardless of the type of testicular sperm used (all P > 0.05, except that of the KS group, in which P < 0.05; Table 3).

The proportion of 2PN cleavages developing into valid embryos decreased significantly in the KS group when cryopreserved testicular sperm were used (P < 0.05). In the KS group, the proportion of 2PN cleavages that developed into valid embryos in response to fresh testicular sperm was nearly 10% greater than that in response to cryopreserved sperm.

However, owing to the influence of frozen testicular sperm on the fertilization rate, a significant decrease in the number of valid embryos generated from MII oocytes was detected not only in the KS group but also in the AZFc deletion group and OA group when cryopreserved testicular sperm were used (all P < 0.01). Moreover, the overall number of valid embryos generated from MII oocytes decreased significantly when cryopreserved testicular sperm were used (P < 0.01; Table 3).

The overall proportion of MII oocytes that developed into valid embryos was approximately 48.8%, while the OA group achieved the highest overall proportion of MII oocytes that developed into valid embryos (approximately 54.3%), and that of the AZFc deletion group was the lowest (approximately 32.5%).

The proportion of good-quality embryos was influenced by testicular sperm cryopreservation in the OA group

In general, the proportion of good-quality embryos decreased significantly when cryopreserved testicular sperm were used (P < 0.01). When the patients were grouped according to the men’s diagnoses, the proportion of good-quality embryos decreased significantly in the OA group when frozen testicular sperm were used (P < 0.01; Table 3).

The general proportion of good-quality embryos was 48.9%; the AZFc deletion group had the highest proportion of good-quality embryos (approximately 53.8%), and the cryptorchidism group had the lowest proportion (approximately 42.7%).

Although the proportion of good-quality embryos was significantly influenced by testicular sperm cryopreservation only in the OA group (P < 0.01), the observed proportion of good-quality embryos was generally lower when cryopreserved testicular sperm were used in the other factors group. In addition, owing to the significant decrease in the fertilization rate when cryopreserved testicular sperm were used (P < 0.01), the percentage of MII oocytes developing into good-quality embryos in most of these groups also decreased significantly (P < 0.01 for the OA group and AZFc deletion group; P < 0.05 for the KS group and the other factors group). Only the cryptorchidism group and idiopathic NOA group were not significantly influenced by the use of cryopreserved testicular sperm (both P > 0.05; Table 3).

The general proportion of MII oocytes developing into good-quality embryos was approximately 23.9%, whereas the OA group had the highest general proportion of MII oocytes developing into good-quality embryos (approximately 26.9%), and the AZFc deletion group had the lowest proportion (17.5%).

Fertilization and embryo development were not influenced by testicular sperm cryopreservation in the cryptorchidism group and the idiopathic NOA group

In the cryptorchidism and idiopathic NOA groups, the normal fertilization rate, cleavage rate, proportion of MII oocytes or 2PN cleavages developing into valid embryos, proportion of MII oocytes or 2PN cleavages developing into good-quality embryos, and proportion of good-quality embryos did not significantly differ between frozen testicular sperm and fresh testicular sperm (all P > 0.05; Table 3).

The cycles with transferable embryos decreased significantly in the KS group when cryopreserved testicular sperm were used

Although the proportions of MII oocytes developing into good-quality embryos and valid embryos were significantly influenced by the use of cryopreserved testicular sperm in the OA (both P < 0.01), AZFc deletion (both P < 0.01), and KS groups (P < 0.05 and P < 0.01, respectively), it did not significantly influence whether there were transferable embryos for transplantation in each follicle puncture cycle, except for those in the KS group (both P > 0.05, except that of the KS group, in which P < 0.05), as shown in Table 4. In the KS group, the use of cryopreserved testicular sperm decreased the proportion of cycles with transferable embryos by approximately 16.1%.

Table 4.

Influence of testicular sperm cryopreservation on embryo transfer and corresponding outcomes in different groups

Group	Transferable embryos per cycle a (%)	No pregnancy cycle rate a (%)	Biochemical pregnancy rate a (%)	Miscarriage rate a (%)	Live birth rate (per follicle puncture cycle, %) a	Live birth rate (per embryo transfer cycle, %) a
General
Fresh sperm	92.3 (90.1)	48.3 (46.3)	13.1 (11.7)	3.8 (6.7)	34.0 (37.1)	27.7 (31.1)
Frozen sperm	89.2 (90.1)	45.5 (46.3)	11.3 (11.7)	7.6 (6.7)	38.2 (37.1)	32.3 (31.1)
OA
Fresh sperm	95.2 (94.8)	47.6 (47.0)	5.4 (12.3)	5.4 (9.7)	43.3 (36.4)	35.1 (28.7)
Frozen sperm	94.7 (94.8)	46.9 (47.0)	13.9 (12.3)	10.8 (9.7)	34.7 (36.4)	27.2 (28.7)
KS
Fresh sperm	97.6 (91.2)	54.0 (50.6)	15.9 (13.0)	6.8 (5.8)	22.6 (28.3)	15.9 (21.7)
Frozen sperm	81.5 (91.2)*	44.4 (50.6)	8.0 (13.0)	4.0 (5.8)	36.4 (28.3)	32.0 (21.7)
AZFc deletion
Fresh sperm	100.0 (83.3)	22.2 (38.9)	42.9 (31.3)	0 (6.3)	28.6 (18.8)	28.6 (18.8)
Frozen sperm	66.7 (83.3)	55.6 (38.9)	22.2 (31.3)	11.1 (6.3)	11.1 (18.8)	11.1 (18.8)
Cryptorchidism
Fresh sperm	87.5 (93.5)	50.0 (44.4)	0 (3.1)	0b	33.3 (55.6)	33.3 (46.9)
Frozen sperm	95.7 (93.5)	42.9 (44.4)	3.8 (3.1)	0b	61.9 (55.6)	50.0 (46.9)
Idiopathic NOA
Fresh sperm	81.8 (85.3)	48.3 (45.2)	15.4 (9.2)	0 (5.9)	29.2 (38.1)	26.9 (34.9)
Frozen sperm	86.2 (85.3)	44.5 (45.2)	7.9 (9.2)	7.1 (5.9)	40.0 (38.1)	36.5 (34.9)
Other factors
Fresh sperm	90.0 (87.8)	45.5 (43.8)	9.1 (14.6)	0 (2.1)	55.6 (42.5)	45.5 (35.4)
Frozen sperm	87.1 (87.8)	43.2 (43.8)	16.2 (14.6)	2.7 (2.1)	38.7 (42.5)	32.4 (35.4)

^*P<0.05. ^aResults are presented in the form of “observation (statistical expectation)”. The Chi-square test was applied, and the frequency was converted into a percentage. ^bExpectation value is not available for this term. OA: obstructive azoospermia; KS: Klinefelter syndrome; AZFc: azoospermia factor c; NOA: nonobstructive azoospermia

Notably, although the use of cryopreserved testicular sperm decreased the proportion of cycles with transferable embryos by nearly 33.3% in the AZFc deletion group, which was greater than that in the KS group, the difference was not significant (P > 0.05). This may be due to the limited number of patients included in the AZFc deletion group, which is a limitation of our work.

Transplantation of embryos derived from cryopreserved testicular sperm does not influence ART outcomes

After embryo transfer, no significant differences were observed in the pregnancy or miscarriage rates, regardless of whether the embryos were derived from frozen testicular sperm (both P > 0.05; Table 4). No significant difference could be observed in the live birth rate per cycle between the use of fresh testicular sperm and cryopreserved testicular sperm in any of the groups (all P > 0.05; Table 4).

The general live birth rate was approximately 37.1% for each follicle puncture cycle and 31.1% for each embryo transfer cycle. The cryptorchidism group achieved the highest live birth rate (approximately 55.6% for each follicle puncture cycle and 46.9% for each embryo transfer cycle) and that of the AZFc deletion group was the lowest (approximately 18.8% for each follicle puncture cycle and each embryo transfer cycle).

ART outcomes were not satisfactory when the Cryopiece was applied for testicular sperm cryopreservation

Among the 290 couples for whom frozen testicular sperm was used for ICSI, 21 couples underwent sperm cryopreservation using a Cryopiece, but only 2 of these 21 couples had a live birth. Considering that the use of sperm derived from men with different etiologies results in different ART outcomes, the 21 couples were further grouped according to the men’s diagnoses. Among the 21 couples, 12 were in the idiopathic NOA group, 4 were in the KS group, 3 were in the AZFc deletion group, and 2 were in the other factors group. Owing to the limited sample size in the KS, AZFc deletion, and other factors groups for statistical analysis, the analysis of the ART treatment outcomes using the Cryopiece as a cryopreservation carrier was performed only on the idiopathic NOA group. Only 1 live birth was recorded when the Cryopiece was utilized in the idiopathic NOA group. The fertilization rate, number of transferable embryos for transplantation in each follicle puncture cycle, and live birth rate per follicle puncture cycle decreased significantly when the Cryopiece was utilized (all P < 0.01, except that of the live birth rate per follicle puncture cycle, for which P < 0.05), whereas the biochemical pregnancy rate increased significantly (P > 0.05), as shown in Supplementary Table 1 and 2.

Supplementary Table 1.

Influence of Cryopiece application on fertilization, cleavage, and embryo development

Carrier type	Sample size (couples)	Age of the husband (years), mean±s.d.	Age of the wife (years), mean±s.d.	Normal fertilization rate a (%)	2PN cleavage rate a (%)	Proportion of good-quality embryos a (%)	Proportion of 2PN cleavages developing into valid embryos a (%)	Proportion of 2PN cleavages developing into good-quality embryos a (%)	Proportion of MII oocytes developing into valid embryos a (%)	Proportion of MII oocytes developing into good-quality embryos a (%)
Others	93	31.3±4.3	29.9±3.9	60.9 (57.5)	97.2 (97.0)	48.0 (48.0)	83.0 (83.0)	39.9 (39.8)	49.1 (46.3)	23.6 (22.2)
Cryopiece	12	32.4±3.2	31.1±3.4	15.0 (57.5)**	86.7 (97.0)	45.5 (48.0)	84.6 (83.0)	38.5 (39.8)	11.0 (46.3)**	5.0 (22.2)**

^**Significant difference, P<0.01; ^aResults are presented in the form of “observation (statistical expectation).” The Chi-square test was applied, and the frequency was converted into a percentage. 2PN: two pronuclei; MII: metaphase II; s.d.: standard deviation

Supplementary Table 2.

Influence of Cryopiece application on embryo transfer and corresponding outcomes

Carrier type	Sample size (couples)	Age of the husband (years), mean±s.d.	Age of the wife (years), mean±s.d.	Transferable embryos per cycle a (%)	No pregnancy cycle rate a (%)	Biochemical pregnancy rate a (%)	Miscarriage rate a (%)	Live birth rate (per follicle puncture cycle) a (%)	Live birth rate (per embryo transfer cycle) a (%)
Others	93	31.3±4.3	29.9±3.9	89.8 (86.2)	44.7 (44.5)	6.6 (7.9)	7.4 (7.1)	43.3 (40.0)	37.2 (36.5)
Cryopiece	12	32.4±3.2	31.1±3.4	50.0 (86.2)**	40.0 (44.5)	40.0 (7.9)*	0.0 (7.1)	9.1 (40.0)*	20.0 (36.5)

^*Significant difference, P<0.05; ^**Significant difference, P<0.01; ^aResults are presented in the form of “observation (statistical expectation). The Chi-square test was applied, and the frequency was converted into a percentage. s.d.: standard deviation

DISCUSSION

Previous studies have reported no significant difference in the fertilization rate between ICSI cycles using fresh and frozen testicular sperm.2,5,8,9,10,11,12,13 In our study, a significant difference was observed in the normal fertilization rate, which decreased when cryopreserved testicular sperm were used in most cases, even though in most of these cases, only motile sperm were chosen for ICSI. This difference may be caused by the different standards for counting fertilized oocytes. In our study, only normal fertilized oocytes with 2PNs and secondary PB extrusion were counted; while in some studies,5,8 it was not clear what types of fertilized oocytes were counted. In some studies, only 2PN zygotes were counted as fertilized oocytes, and the sample sizes were too small to achieve statistical significance; only a decreasing trend in the fertilization rate was observed in these studies.2,10,11 Another factor that affects the fertilization rate is the man’s diagnosis. Different fertilization rates were observed when testicular sperm derived from men with different diagnoses were used. Furthermore, whether testicular sperm cryopreservation influences the fertilization rate also seems to be related to the man’s diagnosis (Table 3). However, the diagnoses of the men were not incorporated into the analyses in any of the previous studies.

When the cleavage rate and the subsequent development of embryos were considered, it seems that testicular sperm cryopreservation did not influence most cases except for those in the KS group. Only in the KS group, the proportion of 2PN cleavages developing into valid embryos decreased significantly when cryopreserved testicular sperm were used (Table 3). Furthermore, since no significant difference could be observed in the live birth rate between the cycles using fresh testicular sperm and frozen testicular sperm after embryo transfer (Table 4), the use of frozen testicular sperm does not influence the live birth rate as long as there are transferable embryos.

Thus, our data indicated that the key to achieving a high live birth rate is to obtain an adequate number of valid embryos. Since the use of frozen testicular sperm results in a decrease in the fertilization rate in most cases, the use of AOA is recommended when frozen testicular sperm are used. In particular, AOA is strongly recommended for couples in which the man has AZFc deletion, as the normal fertilization rate of the AZFc deletion group decreased by nearly 26.1% when frozen testicular sperm were used. The lower fertilization rate caused by the use of frozen testicular sperm in the AZFc deletion group may be due to poor sperm quality. Testicular sperm retrieved from AZF deletion patients usually have poor morphology, especially in terms of the head. Such sperm are more likely to be injured during cryopreservation, impairing the integrity of the biological membrane, such as that of the acrosome. The loss of oocyte-activating factors from the acrosome ultimately leads to a failure of fertilization.

It could also be determined that the development of embryos using testicular sperm derived from KS patients is significantly impeded by sperm cryopreservation. Only in the KS group, the number of valid embryos generated from 2PN cleavage decreased significantly when frozen testicular sperm were used, approximately 10.2% lower than when fresh testicular sperm were used. A decrease in the number of valid embryos may ultimately lead to a significant decrease in the number of transferable embryos for transplantation in each follicle puncture cycle. Although the present results were not identical to those in Barro’s study, which showed that fertilization, implantation, clinical pregnancy, and live birth rates were all higher with fresh testicular spermatozoa,23 we also concluded that the use of fresh sperm would result in better ICSI outcomes for KS patients than the use of cryopreserved sperm. It has been reported that X chromosome deactivation and DNA methylation are abnormal during the development of germ cells in patients with KS,24 but whether these abnormalities contribute to the inhibition of embryo development after sperm cryopreservation is unclear. Does abnormal epigenetic activity reduce the stability of the genome in germ cells and increase their fragility during sperm freezing? This possibility remains to be verified and is an essential topic for further investigation. To ensure the proper development of embryos in ART treatment for KS patients and to obtain more valid embryos, the use of fresh testicular sperm is recommended. In addition, the significantly higher testicular sperm retrieval rate in the KS group indicates that the risk of no sperm being retrieved during TESE surgery is relatively low, which also supports the use of fresh sperm for KS patients via the retrieval of testicular sperm on the day of ovum retrieval.

For couples in other groups, however, in addition to the risk of failure to retrieve testicular sperm on the day of ovum retrieval mentioned previously, some other factors also limit the use of fresh testicular sperm in the ICSI cycle. One of these factors is limited medical resources. As mentioned previously, high-tier hospitals are overburdened in China because of imbalanced medical resources and population mobility,15 and the operating room schedule is always full in these hospitals. Moreover, the ward beds are always full. Owing to the individual differences in follicle development after ovarian stimulation, not until 2 days or 3 days before the day of ovum retrieval could the doctor confirm the exact date of the operation; however, the reservation of the operating room and ward bed becomes quite difficult with such short notice. This means that the surgery would probably be delayed without additional efforts to negotiate with related departments. Therefore, TESE surgery on the day of ovum retrieval should be avoided if possible.

Although the decrease in fertilization rates when frozen testicular sperm were used resulted in a general decrease in the percentage of valid embryos, considering that the live birth rate is not influenced by testicular sperm cryopreservation, using frozen testicular sperm for ICSI treatment in most cases is acceptable. The use of frozen testicular sperm is therefore strongly recommended, especially for patients with idiopathic NOA, because in the idiopathic NOA group, the testicular sperm retrieval rate is low, and the influence of testicular sperm cryopreservation on the fertilization rate and embryo development is limited.

Another factor concerning the clinical use of frozen testicular sperm is the application of a novel carrier, the Cryopiece. As mentioned previously, the Cryopiece was developed to preserve as much rare sperm as possible. For a long time, because of their superior performance relative to that of other carriers, 0.25-ml straws were the only carrier utilized for testicular sperm cryopreservation in our center (Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine). However, in some cases, when TESE specimens in which sperm were extremely rare, were cryopreserved using 0.25-ml straws, and were then thawed for ICSI, no sperm could be found. These ICSI cycles would have to be canceled if the couples refused to use donor sperm. The oocytes prepared for ICSI were discarded or cryopreserved for another ICSI cycle according to the couples’ preferences. Usually, the men would choose to undergo another TESE to find sperm again, but the cost is high. Therefore, as soon as the Cryopiece was developed, it was applied in testicular sperm cryopreservation to preserve TESE specimens. Using the Cryopiece, cryopreserved sperm can always be retrieved after thawing. Although the utilization of the Cryopiece is beneficial for clinical rare sperm cryopreservation for ICSI,16 the application of the Cryopiece for testicular sperm cryopreservation was not satisfactory in the present study, with a further decrease in the fertilization rate and transferable embryos in each follicle puncture cycle, leading to a lower live birth rate in the idiopathic NOA group (Supplementary Table 1). Moreover, although a comparison of Cryopiece application was not performed in the AZFc deletion group because of the limited sample size, the use of the Cryopiece may have contributed greatly to the low fertilization rate in the AZFc group when frozen testicular sperm were used, as one third of the frozen sperm in the AZFc group was cryopreserved using the Cryopiece. This may be due to the low quality of these sperm, which had to be preserved using the Cryopiece, while sperm specimens that could be cryopreserved using other carriers, such as 0.25-ml straws, are usually of better quality. More cases would help elucidate the influence of Cryopiece application on the fertilization rate using testicular sperm derived from AZFc deletion patients, but it will take time to collect an adequate number of cases. However, despite the potential negative effect of Cryopiece application in testicular sperm cryopreservation, the Cryopiece is the only option for some patients to preserve their sperm. If these rare sperm specimens are not preserved using the Cryopiece, these patients are more likely to lose the possibility of ART treatment because of the loss of sperm during cryopreservation. Thus, the application of the Cryopiece in the cryopreservation of rare testicular sperm is still beneficial. Further work is necessary to focus on improving the method for preserving such rare testicular sperm specimens and their utilization in ICSI treatment. Whether the poor ICSI outcomes when using sperm cryopreserved by the Cryopiece are caused by the lower quality of sperm that must be preserved using the Cryopiece remains to be elucidated. A well-designed cohort of cases is needed for further analysis of the significance of Cryopiece application in testicular sperm cryopreservation in future work.

Moreover, it should be noted that the findings in our study are limited. The cases included in our study were divided into different groups according to the diagnoses of the men, making the sample sizes in some of the groups extremely small. Although some of these groups were combined into the “other factors group” for statistical analysis, the sample sizes in some of the groups, such as the AZFc deletion group and cryptorchidism group, were still small. The limitation of sample size in our work inevitably influenced the detection of small effects and exposed the statistical results to the possibility of being affected by outliers, resulting in inaccurate and unstable conclusions.

In addition, in some of the groups, the number of cases in which frozen or fresh sperm were used was unequal. The difference in sample size between the two groups was caused by the actual clinical proportion of patients classified into these groups. An extreme imbalance between the two groups can lead to biased results. Usually, the solutions for addressing such problems include oversampling, undersampling, threshold shifting, and cost or weight adjustment.25,26 However, all these solutions essentially incorporate human interventions.

As a retrospective study, the number of available cases for inclusion is somewhat limited by the actual number of patients available. We would like to show the nature of the objective data; thus, human interventions were restricted in the present work, ensuring that the results are true and reliable. In addition, it is generally accepted that bias is generated when the imbalance ratio exceeds 4:1. Rebalancing is needed when the imbalance ratio is >5,27 and the imbalance ratios in our work do not exceed this value. Therefore, the results we achieved here are acceptable. Certainly, to achieve more accurate and stable statistical results, it is necessary to include more cases, but this requires considerable time. Designing a well-designed cohort with a longer period of observation would be helpful in our future work.

In summary, our findings suggest that couples in which men suffer from NOA caused by KS should consider using fresh testicular sperm in their ICSI cycles. For men with other forms of azoospermia, especially those with idiopathic NOA, the initiation of testicular sperm retrieval prior to ovarian stimulation of the patient’s partner should be considered as the first-line approach.

AUTHOR CONTRIBUTIONS

ZJZ, LHM, and ZL designed the work. ZJZ and WC drafted the manuscript. ZJZ, WC, CZD, and FRB collected the clinical data. ZJZ and LHM processed the raw data and performed the analysis. ZJZ, CZD, and FRB retrieved testicular sperm during TESE surgery and cryopreserved the sperm. PL and ZL administrated the TESE surgery. XJH, LNZ, SJ, and WC administrated the ART treatment. All authors agreed to be 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, and read and approved the final manuscript.

COMPETING INTEREST

The author ZL is a member of the Editorial Board of Asian Journal of Andrology, but he has not been involved in the peer-review or decision-making process for this manuscript. The authors declare no other competing interests.