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.

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).

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.

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).

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.