Effects of the timing of testicular sperm retrieval on intracytoplasmic sperm injection outcomes
Article information
Abstract
Objective
This study aimed to evaluate reproductive outcomes according to the timing of testicular sperm retrieval.
Methods
The study included 282 infertile couples divided into three groups: group A (freeze-thawed testicular sperm extraction [TESE] sperm, n=233), group B (fresh TESE sperm collected 1 day before ovum pickup, n=22), and group C (fresh TESE sperm collected on the same day as ovum pickup, n=27). The indications for TESE were surgically uncorrectable azoospermia or ejaculation failure, often accompanied by medical comorbidities such as diabetes mellitus and spinal cord injury. The outcome parameters assessed were fertilization rates, embryo quality, and clinical pregnancy rates.
Results
The mean paternal age was 36.8±5.7 years, and the mean maternal age was 32.6±3.5 years. The mean duration of infertility was 2.9±1.8 years. The fertilization rates were 70.7%, 78.9%, and 73.0% for groups A, B, and C, respectively (p=0.047). The percentages of good-quality embryos were 68.2%, 65.3%, and 48.4%, respectively (p=0.007); specifically, the percentage of good-quality embryos was significantly lower in group C compared with the other two groups. Clinical pregnancy rates per transfer were similar at 51.1%, 50.0%, and 48.1% (p=0.958), with no differences observed in miscarriage rates.
Conclusion
Testicular sperm retrieval can be safely performed 1 day before ovum pickup, resulting in favorable fertility outcomes.
Introduction
Infertility affects approximately 15% of couples attempting pregnancy and has become a major health issue affecting millions of people of reproductive age worldwide. Males are solely responsible for 20% to 30% of infertility cases and contribute to 50% of cases overall [1]. Infertility affects approximately 15% of couples attempting pregnancy and has become a significant global health issue impacting millions of individuals of reproductive age. Male factors alone account for 20% to 30% of infertility cases and contribute to approximately 50% of all infertility cases overall [1]. Recent advances in assisted reproductive technology (ART), particularly intracytoplasmic sperm injection (ICSI), have significantly improved the treatment of severe male factor infertility. This procedure requires only a small number of viable sperm to achieve successful fertilization [2-4]. For male infertility due to surgically uncorrectable obstructive azoospermia (OA), such as congenital bilateral absence of the vas deferens (CBAVD), or refractory ejaculation failure unresponsive to medical or assisted ejaculation treatments, surgical sperm retrieval methods, notably testicular sperm extraction (TESE), are combined with ICSI [5]. Following surgical retrieval, embryologists process testicular tissue and sperm samples in the laboratory for fertilization purposes. Often, urologists perform TESE concurrently with the female partner’s ovum pickup procedure conducted by a gynecologist or fertility specialist. As TESE is relatively invasive, cryopreservation of excess testicular specimens is common practice, reducing the need for repeated procedures and allowing future use in subsequent ICSI cycles. Although most studies report favorable reproductive outcomes with frozen-thawed sperm, concerns remain regarding potential cryodamage [6]. Conducting TESE earlier may provide clinicians greater scheduling flexibility and offer embryologists adequate time to carefully select the optimal spermatozoa [7]. However, the practicality of earlier retrieval has not been thoroughly evaluated, and the majority of infertility clinics continue to perform sperm retrieval and ICSI procedures simultaneously [8-10]. The current study evaluated reproductive outcomes based on different timings of testicular sperm retrieval during ART, aiming to provide clinical guidance for improved coordination between medical departments.
Methods
We retrospectively analyzed the medical records of infertile couples who underwent TESE/ICSI for male infertility at a single fertility center from January 2011 to December 2019. The study was approved by Institutional Review Board of the CHA Gangnam Medical Center (IRB; GCI-21-07). Written informed consent was waived by the board due to a retrospective nature of our study. The inclusion criterion was TESE/ICSI cases where the male patients underwent TESE due to surgically uncorrectable OA or ejaculation failure, often accompanied by medical comorbidities such as diabetes mellitus and spinal cord injury. Patients with non-OA (NOA), genital infection, clinical varicocele, or other chronic diseases were excluded. The patients were divided into three groups according to TESE timing: group A (frozen-thawed sperm from prior TESE), group B (fresh TESE sperm collected 1 day before ovum pickup), and group C (fresh TESE sperm collected on the same day as ovum pickup).
To minimize potential bias related to female factor infertility, we excluded TESE/ICSI cases in which the female partner had a poor ovarian response (age ≥40 years, ≤3 retrieved oocytes, ≥3 previous in vitro fertilization [IVF] cycles, or clear uterine or tubal pathology).
1. Testicular sperm extraction
Patients underwent open TESE procedures as previously described [11]. Under local anesthesia, the scrotum was incised to expose the testis. A small incision was made in the tunica albuginea, and seminiferous tissue (approximately 5×5×5 mm) was retrieved. The testicular tissue sample was then placed in a Petri dish, and seminiferous tubules were compressed gently using fine forceps to extract sperm cells. The presence of sperm cells was confirmed microscopically at 200–400× magnification. In preparation for ICSI, the sperm suspension was transferred into a Falcon tube and centrifuged at 1,400 rpm for 5 minutes. In cases of early TESE, a 1-day overnight culture was performed. Ham’s F10 medium supplemented with 20% serum protein substitute (SPS) was used for culture.
2. Freezing–thawing method for testicular tissue
Testicular tissue cryopreservation was performed according to previously described methods [12]. Briefly, testicular tissues were frozen after confirming the presence of motile testicular spermatozoa. If motile spermatozoa were absent or insufficient, a 1-day culture was performed using Ham’s F10 medium supplemented with 20% SPS. Testicular tissues were mixed at a 1:1 ratio with sperm freezing medium (Origio) in a 2-mL sperm cryovial. The samples underwent sequential incubation at room temperature for 10 minutes and at −20 °C for 30 minutes. They were then exposed to liquid nitrogen (LN2) vapor for approximately 15 minutes before plunging into LN2.
Thawing was carried out immediately before oocyte retrieval. Samples were thawed in a 37 °C water bath for 3 minutes, then washed with Ham’s F10 medium supplemented with 10% SPS and centrifuged at 1,400 rpm for 5 minutes to remove the cryoprotectant. After discarding the supernatant, the sperm pellet was resuspended in 0.2 mL of sperm medium.
3. Ovarian stimulation and intracytoplasmic sperm injection
Ovarian stimulation and oocyte retrieval procedures were performed following standard protocols. Briefly, female partners underwent ovarian stimulation using recombinant follicle-stimulating hormone (FSH) combined with either a gonadotropin-releasing hormone (GnRH) antagonist or a GnRH agonist. Oocyte retrieval occurred 36 hours after administering human chorionic gonadotropin (hCG). ICSI was performed at 200× magnification under an inverted microscope (TE-2000-U; Nikon) using two positioning manipulators, as previously described [13]. Selected spermatozoa were placed in a drop containing 10% polyvinylpyrrolidone and immobilized. Prior to injection, laser-assisted zona pellucida thinning was conducted with a laser pulse duration of 100 μs (ZILOS-tk class I laser; Hamilton Thorne Research). A mature oocyte was secured with a holding pipette, and sperm was injected via an injection pipette.
4. Evaluation of fertilization, embryo development, and pregnancy
Fertilization and oocyte degeneration were assessed at 16 to 18 hours post-ICSI. Fertilized embryos were transferred into drops of fresh cleavage medium and cultured during the cleavage stage, followed by transfer to drops of blastocyst medium for the blastocyst stage. All incubation occurred at 37 °C with 6% CO2 and 5% O2 in incubators (HERAcell 240; Thermo Fisher Scientific). Embryo quality was graded based on the percentage of fragmentation and the number and size of blastomeres. Grade 3 and 4 embryos were classified as good-quality embryos, as previously described [14]. Embryo quality assessments were independently performed by two experienced embryologists. Pregnancy was confirmed by measuring serum β-hCG levels 12 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac with a detectable fetal heartbeat at 5 to 7 weeks of gestation.
5. Statistical analysis
Statistical analysis was performed using SPSS ver. 25.0 (IBM Corp.). Data are presented as means±standard deviations. One-way analysis of variance was used to compare patient characteristics among the three groups, and the chi-square test was utilized to compare reproductive outcomes. A p-value of less than 0.05 was considered statistically significant.
Results
Our study included 282 infertile couples who required TESE/ICSI procedures to achieve pregnancy. The patients were divided into three groups as follows: group A (frozen-thawed sperm from prior TESE, n=233), group B (fresh TESE sperm collected 1 day before ovum pickup, n=22), and group C (fresh TESE sperm collected on the same day as ovum pickup, n=27).
The decision to perform early or same-day TESE depended on clinical scheduling flexibility of the urologist, with early TESE being the preferred approach. Same-day TESE was performed when clinical schedules did not permit early TESE, and also included cases involving male partners who could not successfully provide semen on the ovum pickup day due to temporary erectile dysfunction or ejaculation failure. Patients primarily underwent open TESE procedures under local anesthesia, although intravenous general anesthesia was occasionally used. No patients experienced significant adverse effects related to the TESE procedure.
Baseline characteristics, including male and female ages, baseline FSH levels in women, mean FSH levels in men, etiology of infertility, and types of ovulation induction, showed no significant differences among the three groups (Table 1). The mean paternal age was 36.8±5.7 years, and the mean maternal age was 32.6±3.5 years. The mean duration of infertility was 2.9±1.8 years.
Characteristics of the study groups
The total numbers of ICSI cycles conducted in each group were as follows: 2,168 cycles in group A, 194 cycles in group B, and 185 cycles in group C. The embryologic characteristics of these ICSI cycles are detailed in Table 2. Regarding reproductive outcomes, fertilization rates were 70.7%±4.0%, 78.9%±4.15%, and 73.0%±2.67% in groups A, B, and C, respectively (p=0.047). The percentages of good-quality embryos were 68.2%±0.78%, 65.3%±0.85%, and 48.4%±0.86% in groups A, B, and C, respectively (p=0.007), indicating a significantly lower percentage of good-quality embryos in group C. Clinical pregnancy rates per embryo transfer were comparable at 51.1%, 50.0%, and 48.1%, respectively (p=0.958). Additionally, there were no differences among the groups in miscarriage rates (Table 3).
Embryologic data of the intracytoplasmic sperm injection cycles
Reproductive outcomes in the groups
Discussion
In cases of male infertility caused by surgically uncorrectable OA, such as CBAVD or high-level vasal obstruction, and refractory ejaculation failure unresponsive to medical therapy or assisted ejaculation methods, TESE/ICSI is the only viable option for pregnancy. Assisted reproduction via TESE/ICSI necessitates a highly coordinated team effort involving the urology, IVF laboratory, and obstetrics and gynecology departments. Our findings revealed no differences in reproductive outcomes between the early TESE group and the same-day TESE group. Only a few previous studies have investigated the clinical benefits of early TESE [15,16]. One prior study reported comparable fertilization and clinical pregnancy rates between TESE performed on the day prior to oocyte retrieval and TESE performed on the same day [7].
In most infertility clinics, surgical sperm retrieval (TESE) typically coincides with the day of oocyte pickup. Although sperm motility is regarded as the most reliable indicator for selecting suitable sperm for ICSI, only a small fraction of testicular spermatozoa typically show motility immediately following TESE. One study reported observing sperm motility in just 13% of samples immediately after TESE, increasing to 76% after 24 hours of incubation [17]. This limited immediate motility can complicate the process of selecting optimal sperm within the restricted timeframe available in the IVF laboratory. Our study demonstrates the clinical feasibility of performing sperm retrieval earlier, before oocyte pickup, thereby providing embryologists sufficient time to carefully identify the most suitable sperm. At our center, early TESE is the preferred practice. This approach offers clinicians scheduling flexibility by allowing TESE procedures to be conducted either the day before or on the day of ovum pickup. We believe this approach would be particularly advantageous in clinics handling high volumes of ART procedures, where efficient coordination between clinical and laboratory departments is critical.
During in vitro culture, sperm cells become vulnerable to damage by reactive oxygen species, a risk that increases with extended incubation time [18,19]. Additionally, the sperm preparation process in ART can induce exogenous stresses, potentially increasing sperm DNA fragmentation [20]. Prolonged in vitro incubation can also induce oxidative stress in oocytes, negatively impacting fertilization outcomes [21]. The optimal recommended timing for sperm injection in ICSI is within 40 hours after hCG administration to avoid oocyte aging and lower fertilization rates. A previous study suggested performing oocyte denudation at least 2 to 3 hours post-retrieval to maximize fertilization and implantation rates, emphasizing that sperm injection should promptly follow denudation [22]. However, same-day TESE often necessitates prolonged incubation for acquiring sufficient sperm motility. Early TESE could minimize potential oocyte aging by reducing the duration of incubation required for sperm motility acquisition, particularly benefiting ICSI cases involving poor oocyte quality.
Cryopreservation of testicular sperm is commonly practiced to avoid repeated invasive procedures; however, concerns regarding potential cryodamage to sperm cells persist. Several prior studies have reported no significant differences in fertilization, pregnancy, or miscarriage rates between fresh and frozen-thawed testicular sperm [23,24]. Nevertheless, previous studies included confounding factors, such as cases of NOA [25]. Sperm from NOA patients inherently possess defects originating from impaired spermatogenesis, and increased sperm DNA fragmentation has been documented in these cases [26]. The freezing-thawing process tends to be more harmful to NOA-derived sperm compared to sperm from OA patients. We believe this difference could adversely influence fertilization and embryo development outcomes, thus becoming a significant confounding factor in earlier studies. Concerns still persist regarding the possibility of decreased fertility due to cryofreezing. In our study, while there were no differences in clinical pregnancy or miscarriage rates between fresh sperm groups, the frozen-thawed sperm group had fewer good-quality embryos. Therefore, fresh sperm use could be considered as the preferable first option, although it necessitates repeated invasive surgical procedures.
Some limitations of our study should be acknowledged. First, the subgroups involving fresh sperm retrieval (groups B and C) were relatively small (n=22 and n=27, respectively), potentially limiting statistical power. Further studies with larger sample sizes would strengthen our conclusions. Second, although our single-center design minimized heterogeneity in ICSI protocols across infertility centers, it might limit generalizability to other clinics with different patient populations or procedural protocols. To minimize confounding effects associated with NOA sperm, our study exclusively included OA cases with normal spermatogenesis. Future well-designed multi-center studies are warranted to validate our findings. Third, the sperm motility data were not comprehensively addressed, representing a significant limitation. Notably, a substantial portion of the same-day TESE cases included delayed procedures due to unexpected temporary erectile dysfunction preventing timely semen collection. Consequently, embryologists often had insufficient time to thoroughly assess sperm motility, sometimes necessitating the selection of immotile sperm for rapid ICSI to minimize oocyte aging during in vitro incubation. Further studies with accurate data, including sperm motility acquisition time post-TESE, would provide valuable insights. Although both fresh and frozen TESE sperm result in similar reproductive outcomes in ICSI for male factor infertility, we recommend fresh sperm as the initial option, with early TESE preferable to same-day retrieval.
In conclusion, our findings support the early TESE approach for couples undergoing ICSI due to male factor infertility. Fresh sperm retrieval performed the day before ovum pickup yields reproductive outcomes comparable to same-day TESE. Early retrieval provides embryologists with sufficient time for meticulous sperm selection and offers clinicians greater scheduling flexibility for surgical procedures.
Notes
Conflict of interest
Seung-Hun Song is an associate editor of the journal, but he was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts.
Author contributions
Conceptualization: THL, SHS. Methodology: THH, JKP, DHS. Formal analysis: JKP, DHS, WHL. Data curation: WHL, YH. Project administration: WHL, YEK, SRL. Visualization: THH, SHS. Validation: JKP, THL, SRL. Investigation: THH, YEK, YH. Writing-original draft: THH, JKP. Writing-review & editing: THL, SHS. Approval of final manuscript: THH, THL, SHS.