Introduction
Infertility is a complex and emotionally challenging medical condition affecting approximately 10% to 15% of couples globally [1]. Male factor infertility, which is solely responsible for 20% to 30% of cases and contributes to 50% of all infertility cases [2,3], may result from problems with semen quantity, semen quality, or the structure of the male genital tract [4].
Oligozoospermia, which is characterized by a low sperm concentration, accounts for about 10% of male factor infertility cases [5]. Cryptozoospermia, a severe form of oligozoospermia, is defined by the World Health Organization as the presence of isolated sperm cells in the ejaculate that can only be detected through extensive microscopic examination or after centrifugation [6].
Previously, men with severe oligozoospermia or cryptozoospermia had few fertility options, primarily relying on donor sperm. However, the advent of intracytoplasmic sperm injection (ICSI) now allows men with extremely low sperm counts or cryptozoospermia to achieve biological parenthood [7]. Various techniques have been developed to retrieve sperm from men with severe oligozoospermia, cryptozoospermia, and azoospermia. These techniques include testicular sperm extraction (TESE), microsurgical TESE (micro-TESE), testicular sperm aspiration (TESA), microepididymal sperm aspiration, and percutaneous epididymal sperm aspiration (PESA). These methods facilitate the direct collection of sperm from the epididymis or testicle [8,9]. The sperm obtained can then be used for ICSI in assisted reproductive technology (ART) procedures.
The decision between using ejaculated and testicular sperm as the optimal source remains inadequately characterized. Testicular sperm may theoretically provide reduced DNA fragmentation by circumventing genomic material degradation during epididymal transit [10,11]. Conversely, the sequential washing and centrifugation processes necessary to isolate sperm in men with cryptozoospermia or severe oligozoospermia could elevate the production of reactive oxidative species, potentially degrading sperm quality and adversely affecting ART outcomes [12].
Previous reports have suggested that using testicular sperm in ICSI results in higher pregnancy and live birth rates, as well as lower miscarriage rates, particularly in cases of severe oligozoospermia and cryptozoospermia [13,14]. However, a meta-analysis has contested these findings, demonstrating no significant difference in pregnancy rates from ICSI between testicular and ejaculated sperm in couples with cryptozoospermia [12].
In the context of ICSI, embryologists face a crucial decision regarding the selection of the sperm source—whether to use testicular or ejaculated sperm. This choice is important because it directly affects the success rates of ICSI procedures and, consequently, the outcomes of fertility treatments.
In recent years, there has been a growing interest in finding an optimal predictor to assist embryologists in their decision-making process. Identifying reliable markers that can effectively distinguish between cases where testicular sperm retrieval or ejaculated sperm is more appropriate offers significant potential to improve the efficiency and success rates of ICSI procedures. Additionally, it could reduce the emotional, physical (including risks of vascular injury, hematoma, infection, fibrosis, or even hypogonadism), and economic burdens often associated with testicular sperm retrieval surgeries. This study aimed to explore the predictive factors involved in recommending testicular sperm retrieval versus the use of ejaculated sperm in managing severe male infertility.
Methods
1. Study design and population
This study was conducted with 75 infertile men diagnosed with idiopathic severe oligozoospermia (a sperm count less than 5 million/mL) or cryptozoospermia (characterized by the presence of few sperms in the ejaculate, detectable only after centrifugation of a semen sample) at the Shahid Beheshti fertility clinic in Isfahan, Iran. All patients underwent standard chromosome analysis (karyotype) and screening for Yq microdeletions to rule out well-known causes of cryptozoospermia and severe oligozoospermia. We excluded couples where the female partner was over 40 years old and either partner had a body mass index (BMI) above 25 kg/m², those with no sperm retrieval from fine-needle aspiration (FNA), those with a correctable male factor (e.g., varicocele, semen infection, exposure to a gonadotoxin, or hypogonadism), those with fewer than six metaphase II oocytes retrieved on ovum pick-up day, and those with severe oocyte abnormalities, including coarse granulation, smooth endoplasmic reticulum clusters, and vacuolization. The study received ethical approval from the Ethics Committee of Isfahan University of Medical Sciences (IR.ARI.MUI.REC.1402.040). Moreover, informed consent was obtained from all participants.
On ovum pick-up day, the 75 infertile men were equally divided into three groups based on sperm concentrations: cryptozoospermia (group 1, n=25), 0.1–1 million/mL (group 2, n=25), and 1–5 million/mL (group 3, n=25). Patients within each group underwent ICSI, which involved the insemination of sibling oocytes using either ejaculated spermatozoa or testicular spermatozoa obtained by FNA (Figure 1).
2. Semen processing
Semen samples were collected through masturbation following 3 to 5 days of sexual abstinence. After collection, the samples were allowed to liquefy for at least 15 minutes at 37 °C prior to analysis. Sperm concentration, motility, and morphology were assessed in these fresh semen samples. In cases of oligozoospermia, the liquefied semen was mixed with 2 mL of sperm washing media (Sperm wash; Origio) and centrifuged for 5 minutes at 3,000 ×g. The supernatant from the upper part of the sample was then discarded, and 0.5 mL of sperm wash medium was added to obtain a usable sperm sample.
In cases of cryptozoospermia, where initial analysis fails to detect spermatozoa, the entire semen sample is subjected to centrifugation at an relative centrifugal force of 1,000 ×g. Following centrifugation, the semen precipitate is divided into several droplets of culture medium on the injection plate. This is followed by a meticulous, extended search for viable motile spermatozoa for ICSI.
3. Ovarian stimulation
All participants followed a standardized ovulation induction regimen, which included six doses of recombinant follicle-stimulating hormone (Gonal-F) at 150 IU/day, administered via subcutaneous injections starting on the second day of their menstrual cycle. Additionally, they received intramuscular human menopausal gonadotropin (Menogon; Ferring Pharmaceuticals A/S) at 75 IU/day beginning on the fourth day of the menstrual cycle and continuing until the trigger day. Transvaginal sonography was initiated on day 7 of the menstrual cycle and was performed every other day thereafter. Cetrotide (Merck Serono) at 250 μg/day was administered subcutaneously once at least three mature follicles (≥14 mm) were confirmed, and administration continued until the trigger day. In cases where three or more mature follicles (≥17 mm in diameter) were identified via transvaginal sonography, a 0.2 mg subcutaneous injection of the gonadotropin-releasing hormone analog triptorelin (Decapeptyl) was prescribed. Oocyte retrieval occurred 35 to 36 hours after human chorionic gonadotropin administration, using a 17-gauge ovum aspiration needle (Prince) under general anesthesia.
4. Intracytoplasmic sperm injection
When possible, a viable sperm exhibiting normal morphology was selected from either washed semen precipitates or processed testicular tissues in a culture medium. This sperm was carefully retrieved using an injection pipette containing a small amount of poly-vinyl-pyrrolidone (PVP). The selected sperm was then washed within the PVP droplet to remove any surrounding debris particles that could potentially harm the oocyte and the resulting embryo. The ICSI procedure was conducted under a warmed-stage microscope (Nikon) at ×200 magnification, utilizing a Hoffman optic system equipped with hydraulic micromanipulation (Eppendorf).
5. Variables and outcome measures
The primary outcome measure was the fertilization rate, specifically the formation of two-pronuclei zygotes. Fertilization was assessed 16 to 18 hours following the microinjection process. Secondary outcomes encompassed the rates of cleavage, the production of high-quality day 3 embryos, and blastocyst formation, which was determined by the number of blastocysts obtained on day 5.
Cleavage embryos were evaluated 72 hours post-fertilization based on their morphology, the amount of fragmentation, and the number of blastomeres. The grading categories were defined as follows: grade A embryos, the highest quality, have 7–9 blastomeres and no more than 20% cytoplasmic fragmentation. Grade B embryos also contain 7–9 blastomeres but exhibit over 20% cytoplasmic fragmentation. Grade C embryos have 4–6 blastomeres with up to 20% fragmentation. Grade D embryos, the lowest quality, possess 4–6 blastomeres with more than 20% fragmentation [15].
6. Fine-needle aspiration technique
Testicular sperm retrieval was carried out using either unilateral or bilateral FNA, performed under local anesthesia. All procedures were conducted by the same surgeon, following the method previously described. First, the scrotal skin was thoroughly cleansed with a 10% povidone-iodine solution and then covered with sterile drapes. Each testis was manually stabilized, with the epididymis and vas deferens positioned posteriorly. Following this, each testis underwent two aspirations at three distinct sites—upper, middle, and lower sections—using a larger 18-gauge disposable butterfly needle attached to a 60 mL Luer-Lock syringe.
Every testicular puncture involved multiple rapid, precise in-and-out movements until the flow of yellowish fluid stopped or bloody fluid appeared. The tubing was then clamped with artery forceps to maintain negative pressure. As the needle was slowly withdrawn from the testis and scrotal skin, intact seminiferous tubules protruding from the puncture site were extracted. These tissues were carefully cut using sharp scissors and placed in Petri dishes using two pairs of fine tweezers. After each puncture, the contents were washed in warmed sperm wash media to remove red blood cells. The washed tubules were then minced using two insulin syringes on a sterile plate containing warmed medium. Subsequently, cell lysate and debris were removed by centrifugation (at 300 ×g for 5 minutes) to obtain a pellet containing viable spermatozoa suitable for ICSI [16].
7. Statistical analysis
The variables were presented as mean±standard deviation and as percentages. We assessed the normality of the variables using the Kolmogorov-Smirnov test. Differences in means between two variables were determined using the Student t-test, while the chi-square test was employed for comparing proportions. All analyses were conducted using IBM SPSS ver. 24 (IBM Co.), and p-values less than 0.05 were considered statistically significant.
Results
Baseline characteristics of the study groups are presented in Table 1. Karyotype analysis was normal (46XY) in all patients. No Y chromosome microdeletions were detected in the study groups. Bilateral FNA was performed in four patients, while the rest underwent unilateral FNA. Sperm was successfully recovered in 90.36% (75/83) of the men overall, including 100% (25/25) of the men in group 2 (0.1–1 million/mL), 75.75% (25/33) of the men in the cryptozoospermia group, and 100% (25/25) of the men in group 3 (1–5 million/mL). There were no significant differences between groups in mean female age, male age, male BMI, female BMI, female anti-Müllerian hormone levels, testicular volume, male follicle-stimulating hormone levels, male luteinizing hormone levels, and total testosterone. Details regarding the outcomes of ICSI using ejaculated sperm and testicular sperm in each group are shown in Table 2. The use of testicular sperm demonstrated better outcomes across multiple parameters in two specific groups: group 2 (0.1–1 million/mL) and the cryptozoospermia group. These better results were particularly evident in the fertilization rate, cleavage rate, high-quality embryo rate, and blastocyst rate. Conversely, a contrasting trend was observed in the group characterized by sperm concentrations ranging from 1–5 million/mL. Within this cohort, the use of testicular sperm was associated with a decline in the aforementioned parameters, namely, the fertilization rate (48.53% vs. 71.69%, p<0.001), cleavage rate (75.85% vs. 91.92%, p=0.007), high-quality embryo rate (36.54% vs. 65.93%, p=0.001), and blastocyst rate (3.16% vs. 10.06%, p=0.001).
Discussion
Determining the predictive factors that influence the choice between using testicular or ejaculatory sperm for ICSI is crucial in managing severe male infertility. Our findings indicate distinct patterns in the use and outcomes of testicular sperm retrieval based on sperm concentration. Specifically, we noted improved results across multiple parameters, including fertilization rate, embryo formation rate, high-quality embryo rate, and blastocyst rate, in the testicular sperm subgroup of group 2 (0.1–1 million/mL) and the cryptozoospermia cohort. These improved outcomes highlight the potential advantages of using testicular sperm in cases with sperm concentrations below 0.1 million/mL or in the presence of cryptozoospermia. The improved outcomes associated with testicular sperm in cryptozoospermia patients align with findings from previous studies. Hauser et al. [17] conducted a comparative analysis of outcomes between testicular and ejaculated sperm for ICSI treatment in patients with cryptozoospermia. Their study showed consistently higher rates of fertilization, high-quality embryos, implantation, and pregnancy with testicular sperm. In addition, Cui et al. [18] found that using testicular sperm for ICSI in cryptozoospermia patients led to higher rates of top-quality embryos, embryo implantation, pregnancy, and successful births compared to using ejaculated sperm. Furthermore, the most recent systematic review and meta-analysis, which included 578 patients with cryptozoospermia undergoing 761 ICSI cycles (541 with ejaculated sperm, 153 with fresh testicular sperm, and 67 with frozen-thawed testicular sperm), concluded that testicular sperm is superior to ejaculated sperm for ICSI in males with cryptozoospermia [14]. These observations suggest that testicular sperm exhibits superior quality compared to ejaculated sperm, likely due to less acquired DNA damage. This damage can occur during transit through the epididymis and the extensive processing that semen samples from men with cryptozoospermia undergo for ICSI use, potentially increasing oxidative stress levels and affecting sperm quality. Conversely, in another study, Amirjannati et al. [19] demonstrated comparable fertilization rates and embryo quality when comparing testicular and ejaculated spermatozoa in 19 cases of cryptozoospermia. This discrepancy among studies may be due to variations in patient populations, methods of extracting testicular sperm, procedures for freezing and thawing sperm, and the skill level and availability of laboratory technicians to process the samples. Differences in outcome measures and confounding factors related to patient history, such as underlying causes of oligozoospermia and uterine conditions among the baseline population, may also contribute to the observed variations. However, our data support the use of testicular sperm in men diagnosed with cryptozoospermia, reinforcing the rationale outlined in previous studies.
Regarding patients diagnosed with oligozoospermia, the data are also conflicting. Kendall Rauchfuss et al. [4] showed that using TESE-derived sperm did not enhance ICSI outcomes compared to ejaculated sperm in the absence of specific conditions such as azoospermia, recurrent in vitro fertilization failure, or abnormal DNA fragmentation index (DFI), particularly in patients with an average sperm concentration of 6.1 million/mL. Conversely, several studies have reported improved fertilization rates with testicular sperm compared to ejaculated semen, even after freeze-thaw cycles [17,20].
In our study, men were categorized based on their sperm concentration, as research has indicated that the severity of oligozoospermia can adversely affect ICSI outcomes. We observed that patients with a sperm concentration greater than 1 million/mL experienced higher fertilization, cleavage, and high-quality embryo rates when using ejaculated sperm compared to testicular sperm. Additionally, a greater number of blastocysts were produced using ejaculated sperm. In contrast, for patients with a sperm concentration of less than 1 million/mL, the results were completely opposite. Thus, it appears that testicular sperm is more effective in patients with cryptozoospermia and those with sperm concentrations below 1 million/mL, while ejaculated sperm is preferable for patients with concentrations above 1 million/mL.
Currently, the literature lacks sufficient data on the stratification of oligozoospermia to determine whether to recommend TESA/PESA or the use of ejaculated sperm for improved ICSI outcomes. Our study, however, aims to establish a relative cutoff point for this decision based on sperm concentration. A notable strength of our study is its use of a sibling oocyte model, which allows for a direct comparison of outcomes between the two sperm retrieval methods within the same patient. This method is particularly beneficial as it reduces the impact of interpatient variability, a significant confounding factor in studies of this nature. In a related study, Alharbi et al. [21] investigated the outcomes of ICSI with TESA in males diagnosed with severe oligozoospermia and cryptozoospermia. Patients were divided into four groups based on sperm concentration: cryptozoospermia, <0.1, 0.1–1, and 1–5 million/mL. Their findings showed that the group with sperm concentrations between 0.1 and 1 million/mL had the highest pregnancy rate, suggesting that patients within this range might benefit more from TESA than those in other groups. Conversely, our study did not evaluate pregnancy outcomes, which could be seen as a limitation. Additionally, DNA fragmentation was not examined in our study due to the inability to perform the test in some cases because of extremely low sperm concentrations. However, it is important to note that during the most recent DFI examination conducted around the time of the operation, over 90% of the eligible patients showed abnormal levels of DNA fragmentation. Despite these limitations, DNA fragmentation testing can offer valuable additional insights for identifying couples who might benefit more from using testicular sperm. Ultimately, our study focused exclusively on sperm concentration, neglecting the assessment of sperm morphology and motility, which are crucial sperm characteristics. However, our findings did indicate that patients benefiting from ejaculatory sperm exhibited better sperm morphology and motility compared to those benefiting from TESA. These aspects, however, require further investigation in future studies.
In conclusion, our study underscores the importance of considering sperm concentration as a predictive factor in recommending TESA versus the use of ejaculated sperm for managing severe male infertility. Testicular sperm retrieval may provide substantial benefits in cases of lower sperm concentrations or cryptozoospermia. However, its utility may be limited in scenarios where sperm concentrations exceed 1 million/mL. Future research should focus on identifying the predictive factors that influence the choice of sperm retrieval techniques, with the goal of optimizing treatment outcomes for couples undergoing assisted reproduction.
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