Fresh versus frozen micro-TESE sperm and outcomes

Abstract

The use of fresh versus frozen spermatozoa in men with nonobstructive azoospermia (NOA) undergoing in vitro fertilization (IVF) has been a debated hot topic among reproductive specialists. Each approach presents distinct advantages and disadvantages, with fresh sperm typically showing superior sperm quality, while frozen sperm offers logistical flexibility and a reliable backup for repeated cycles. This review summarizes the latest advancements in sperm retrieval and cryopreservation techniques, providing practitioners with a comprehensive analysis of each option’s strengths and limitations. Comparative studies indicate that, although fresh sperm often has better quality metrics, cryopreservation methods such as vitrification have significantly improved postthaw outcomes, making frozen sperm a viable choice in assisted reproductive technologies (ART). The findings show comparable rates for fertilization, implantation, clinical pregnancy, and live birth between fresh and frozen microdissection testicular sperm extraction (micro-TESE) sperm in many cases, although patient-specific factors such as timing, cost-effectiveness, and procedural convenience should guide the final decision. Ultimately, the choice of using fresh or frozen sperm should align with the individual needs and conditions of patients. This tailored approach, supported by the latest advancements, can optimize ART outcomes and provide personalized reproductive care.

INTRODUCTION

Male factor infertility accounts for nearly 50% of all infertility cases. Although challenging to definitively identify the prevalence, Agarwal et al.1 reported that approximately 30 million males worldwide are diagnosed with male factor infertility. This condition is broadly categorized into pretesticular, testicular, and posttesticular conditions based on its underlying causes.2 When these factors severely impair spermatogenesis, it can result in azoospermia, the complete absence of sperm in the semen.

Azoospermia contributes to 10%–15% of all male infertility cases.3 Two-thirds of azoospermia cases are nonobstructive azoospermia (NOA), a condition where the testes fail to produce adequate sperm to result in no sperm in the semen.4 To address this, microdissection testicular sperm extraction (micro-TESE) has emerged as a valuable tool by carefully dissecting testicular tissue through a microsurgical approach and selecting more dilated seminiferous tubules among thin, flat tubules to retrieve sperm. Success rates can vary between 40% and 60% depending on multiple factors including patient characteristics, testicular histopathology, and the surgical techniques used.5,6 Sperm retrieval can be particularly challenging in cases of NOA, as there may be either too few or no spermatozoa available for intracytoplasmic sperm injection (ICSI), potentially leaving oocyte vitrification, ICSI with donor sperm, or cycle cancellation as alternative options.7

For couples undergoing assisted reproductive technology (ART) procedures, cryopreservation of testicular sperm before ovulation induction can be a beneficial strategy. However, research comparing fresh and frozen testicular sperm is limited and has shown conflicting results. Some studies have shown comparable success rates between freshly retrieved and frozen-thawed testicular spermatozoa,8,9,10 but others have suggested that frozen micro-TESE sperm could lead to poorer ICSI results.11,12 Thus, a comprehensive review of the existing literature on the efficacy of fresh versus frozen micro-TESE sperm can offer valuable insights to clinicians and researchers.

OVERVIEW AND PROCEDURAL DETAILS OF MICRO-TESE

Advancement in the field of ART has revolutionized the management of male factor infertility including several techniques involved in achieving superior reproductive outcomes. Micro-TESE is the gold standard surgical technique of sperm retrieval for men with NOA and was first introduced and described in 1999.13 With the assistance of an operating surgical microscope, searching for opaque, dilated seminiferous tubules might result in finding sperm in men with NOA.14,15

Micro-TESE is performed under general anesthesia. A midline incision in the testis exposes the parenchyma, granting access to seminiferous tubules. Optimally, the retrieved seminiferous tubules are assessed by laboratory technicians in the operating room for the presence or absence of spermatozoa to guide the surgeon. In a less optimal scenario, if laboratory technicians are not available to be in the operating room, dissected tubules are promptly placed in 3-(N-morpholino)propanesulfonic acid (MOPS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer media for transport to the embryology lab. In the lab, the retrieved tissue undergoes gentle washing with fresh buffer media. Sperm cells are extracted from the tubules under a microscope using fine needles or glass slides. This meticulous process is repeated until sufficient sperms are identified for ICSI or cryopreservation.16 For limited or dense tissue, enzymatic digestion with collagenase facilitates sperm extraction and minimizes tissue damage.17 Erythrocyte lysis buffer can further purify the sample by removing red blood cells.18 Sperm retrieved from micro-TESE can either be used fresh on the day of the procedure, or it can be cryopreserved for future use. Some considerations need to be given thought before deciding which approach to take. Likewise, different etiologies of NOA can be used as a prognostic factor on whether to use fresh or cryopreserved testicular sperm.19

Micro-TESE achieves a sperm retrieval rate approximately 1.5 times higher than conventional TESE (cTESE) and two times higher than testicular sperm aspiration (TESA).16 Specifically, micro-TESE has demonstrated a retrieval success rate of 47%, compared to 30% in cTESE, highlighting its effectiveness.20 Additionally, micro-TESE requires significantly less tissue excision, averaging only 4.65 mg of tissue removed versus 53.57 mg in cTESE. This reduced tissue removal not only limits surgical trauma but also helps preserve postoperative testosterone levels, reducing the likelihood of hormonal imbalances.20 However, while micro-TESE offers substantial benefits in terms of higher success rates and tissue conservation, it demands more specialized expertise, longer operating times, and generally incurs higher costs, underlining the need for skilled practitioners to optimize outcomes.

PRINCIPLES OF FRESH VERSUS FROZEN SPERM IN MICRO-TESE

In cases of NOA, fresh sperm taken from micro-TESE or cTESE has traditionally been preferred due to generally higher rates of fertilization and embryo quality than frozen sperm. Nevertheless, a recent study suggested that frozen sperm might offer comparable results with the advances and improvement of sperm cryopreservation technique.10 Frozen sperm has the additional benefit of alleviating pressures in coordinating sperm and ovum retrieval. Moreover, frozen samples from micro-TESE can support multiple ICSI cycles without the need for repeated testicular procedures, benefiting patients who may face physical or logistical challenges with fresh retrievals.21,22 Consequently, while both fresh and frozen sperm are effective, the choice ultimately depends on individual circumstances. Figure 1 is a schematic diagram for the workflow of fresh and frozen micro-TESE sperm in ART.

Figure 1.

Schematic diagram for the workflow of fresh and frozen micro-TESE sperm in ART. (a) In the fresh micro-TESE workflow, the procedure is performed on the same day as oocyte retrieval. Testicular tissue is surgically extracted, and viable sperms are identified and selected under a microscope. These sperms are immediately used for ICSI into the retrieved oocytes. Fertilized oocytes are then cultured to develop embryos, which are transferred to the uterus for implantation and potential pregnancy. (b) Conversely, the frozen micro-TESE workflow involves performing the micro-TESE procedure prior to oocyte retrieval, allowing for cryopreservation of the extracted sperm. After retrieval, viable sperms are identified, treated with cryoprotectants, and stored at freezing temperature. When the couple is ready for an IVF cycle, the sperms are thawed, and the best sperms are selected for ICSI. Fertilized oocytes are cultured into embryos and subsequently transferred to the uterus. Micro-TESE: microdissection testicular sperm extraction; NOA: nonobstructive azoospermia; ART: assisted reproductive technology; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization. This figure has been created with license in BioRender (https://BioRender.com/s71z006).

CRYOPRESERVATION TECHNIQUE FOR SPERM

Testicular sperm cryopreservation demands meticulous handling of membranes undergoing a delicate, fluid-to-gel transition. There are primarily two main types of sperm cryopreservation methods in popular use, which are conventional freezing and vitrification. Conventional freezing employs gradual temperature reduction and dehydration, followed by liquid nitrogen storage. To minimize cellular damage and preserve sperm viability, controlled cooling rates and cryoprotectants are crucial.23 Programmable freezers automate this process.24 Studies indicate that this method is useful for preserving sperm motility and viability in clinical settings, although some oxidative stress and structural changes may still occur during freezing and thawing.25,26,27

Vitrification, introduced in 2004 by Isachenko et al.,28 utilizes ultrarapid cooling, transforming cellular contents into a glass-like, ice-free state, which tackles a key problem with intracellular ice formation that can damage cell structures.29 This technique is effective in preserving sperm morphology and integrity, especially in cases with very low sperm counts, and is often applied experimentally in specialized clinical scenarios. Although vitrification reduces the risks of ice-induced damage, it requires precise control to prevent contamination and is still being refined for broader clinical application. Currently, vitrification of sperm is still considered experimental by the World Health Organization (WHO) 6^th Edition Manual for the examination and processing of human semen.30

Among frozen samples, vitrification appears to better preserve postthaw motility and minimize DNA fragmentation compared to conventional slow freezing. Patel et al.31 found that sperm vitrified without cryoprotectants had a progressive motility of 36.8%, which was notably higher than the 17.9% observed in conventional slow-frozen samples. DNA fragmentation was also lower in vitrified samples, with a fragmentation index of 9.7% compared to 20% in conventional freezing. Supporting this, another study demonstrated that vitrified sperm had structural integrity and acrosomal preservation closer to fresh samples, whereas conventional freezing led to higher rates of cytoskeleton damage and DNA fragmentation.32 Le et al.33 also noted that vitrification preserved normal morphology in 1.95% of sperm samples, slightly better than the 1.36% preserved by conventional freezing, though both methods resulted in a reduction compared to fresh sperm morphology.

FACTORS INFLUENCING TESTICULAR SPERM CRYOPRESERVATION FOR USE IN ICSI

Comparison between testicular sperm cryopreservation methods

Studies indicate that conventional slow freezing and vitrification each have unique impacts on sperm quality. Conventional freezing offers a controlled, gradual temperature reduction, beneficial for preserving motility but may result in structural damage due to ice formation. Vitrification, on the other hand, avoids ice formation, potentially preserving sperm integrity better in small volumes and exhibiting enhanced postthaw motility.34 While both methods achieve comparable levels of DNA fragmentation and morphology, conventional freezing suffers from sperm loss during manipulation, rendering it less suitable for samples with low sperm counts.23,35 Individual sperm vitrification, pioneered by Cohen et al.,36 offers an advantageous option for preserving limited-volume samples due to its rapid cooling and nonpermeable cryoprotectants. Notably, successful pregnancies using vitrified sperm in NOA cases have been reported. Despite this, the applicability of vitrification in larger samples is still under investigation.37,38,39

Types of cryoprotectants and vitrification devices

The choice of cryoprotectants (e.g., glycerol and ethylene glycol) significantly affects postthaw sperm quality. Glycerol is commonly used for conventional freezing due to its efficacy in reducing ice crystal damage.40 Cryoprotectants with high molecular weight agents such as sucrose and trehalose minimize toxicity and osmotic stress,41 and combinations of various cryoprotectants offer potential for reducing toxicity.23 Using carrier devices such as zona pellucida raises concerns about heterologous DNA introduction, and nonbiological carriers like straws are efficient but limit single sperm isolation.42 Cryopreservation of surgically retrieved sperms in a droplet facilitates single sperm recovery but poses handling challenges.43 Closed cryopreservation systems like cell sleepers achieve good success rates but have needle breakage risks.44 The recently developed sperm vitrification device (VD) allows individual sperm vitrification, and it has been successfully used clinically.45 The rate of freezing and warming also influences the sperm recovery postthawing. Rapid freezing and thawing minimizes ice crystal formation, leading to optimum sperm survival.46,47 Alternative cryoprotectants like dimethyl sulfoxide (DMSO) are often paired with specific devices in vitrification (e.g., Cryopiece) to support ultrafast freezing and optimal cell preservation. This factor is essential for avoiding oxidative stress and maintaining motility postthawing.48

Biological changes in frozen sperm

Cryopreservation reduces sperm vitality, motility, and cytoskeletal integrity.12 Additionally, cryopreservation-induced oxidative stress leads to DNA fragmentation, potentially compromising embryo development.49 This oxidative damage, triggered by excess reactive oxygen species (ROS) attacking sperm membranes, can ultimately impact fertilization success.50 Furthermore, even the thawing process itself can negatively influence sperm quality.51 Thus, understanding and mitigating the functional and structural alterations associated with cryopreserved testicular sperm remains crucial for optimizing NOA-ART outcomes.

Patient-specific factors and sperm quality considerations

Conditions such as Klinefelter syndrome and cancer treatments can substantially influence outcomes in testicular sperm cryopreservation for ICSI. Men with Klinefelter syndrome, who typically have nonmosaic karyotypes (47,XXY), face challenges with azoospermia, which requires specialized approaches for successful sperm retrieval. Studies show that the outcomes of ICSI using testicular sperm from these patients are comparable between fresh and cryopreserved samples, although the process can be complex due to the lower baseline fertility in Klinefelter patients.52,53

Cancer treatments, such as chemotherapy and radiation, are another significant factor, as they can lead to severe oligospermia or even permanent azoospermia. Testicular tissue cryopreservation is often advised before treatment to safeguard reproductive potential, especially since posttreatment sperm quality is typically lower than that in healthy controls.54,55 Patient-specific factors like these highlight the need for individualized approaches in cryopreservation strategies and underscore the importance of early fertility counseling and preservation options.

LABORATORY PROCEDURES IN PROCESSING MICRO-TESE SPERM FOR ICSI

Sperm preparation and cryopreservation methodology

Upon receiving the biopsied tissue samples, the embryologist promptly rinses them with sperm wash media to eliminate blood residues. Subsequently, the tissue is gently teased using two glass slides or syringes fitted with bent needles. The tissue is then finely minced using round bottom tubes. The resulting mixture is then transferred to a conical tube for further processing. If necessary, an erythrocyte lysis buffer can be used to obtain a blood-free sample. These samples can be processed using either the simple wash method or the density gradient, as previously detailed by Esteves and Varghese.56

For future cryopreservation, an equal measure of the sperm cryopreservation medium is added to the processed sample’s final pellet. This resultant mixture is subsequently divided into multiple vials. These vials then undergo a refrigeration period of 20–30 min before being exposed to liquid nitrogen vapor, maintained at 10 inches, for 1 h.57 The final step involves submerging the vials into liquid nitrogen, thus ensuring their long-term preservation.

ICSI dish setup for testicular sperm

Given the limited availability of sperm in testicular biopsy cases, a practical approach involves dispensing numerous tiny droplets of processed sperm within an ICSI dish. This strategy aids in the convenient identification of viable sperm. In addition, a few droplets of buffer media are distributed to facilitate micromanipulation. At the same time, a few drops of polyvinylpyrrolidone (PVP) are dispensed to regulate the suction pressure within the ICSI injection needle. To maintain stable osmolality, the setup is then covered with tissue culture oil.

Sperm selection for ICSI

After the sperm is processed and free of seminal plasma, sperm is selected for injection at 400× magnification using the inverted microscope attached to the ICSI machine.58 This helps the embryologist select viable motile sperm devoid of any abnormalities. At this stage, if the sperm shows no signs of viability or motility, additives that enhance sperm motility, such as theophylline or pentoxifylline, may be used.

Role of theophylline and pentoxifylline

Ebner et al.59 noted that theophylline can stimulate sperm motility, facilitating swifter and more precise sperm selection. A similar effect of pentoxifylline on testicular sperm has been reported by Kovačič et al.60 This effect, in turn, can potentially enhance fertilization rates and overall pregnancy outcomes. In alignment with the manufacturer’s standard operating procedure, theophylline can be added to sperm droplets to assist in sperm selection.

Hypo-osmotic (HOS) test

In cases where processed sperm samples lack visible motility, assessing sperm viability can be accomplished through the HOS test. This test entails subjecting sperm cells to a hypoosmotic solution, like HEPES buffer medium, inducing live and intact sperm cells to swell and exhibit robust motility.61 Subsequently, HOS-positive sperm cells are chosen for the ICSI procedure.

Tail curling test

Another technique to assess sperm viability is the “curling test” or “tail flexibility test”, wherein the injection needle is delicately drawn across the sperm’s tail. Alternatively, this test can be conducted using a diode laser, where a brief laser beam is directed at the sperm’s tail. If the sperm tail curls as a response to either method, it indicates its viability for subsequent micromanipulation. Although the tail curling test is not as widely recognized as other viability tests, it has been utilized in certain specialized laboratories to assess sperm functionality. This method helps embryologists confirm the sperm’s responsiveness, which is crucial for successful ICSI outcomes.62

Role of intracytoplasmic morphologically selected sperm injection (IMSI)

Conventional ICSI sperm selection is performed at 400× magnification. An alternative, IMSI, as proposed by Bartoov et al.,63 uses ultrahigh magnification (>6000×). This technique involves meticulous sperm evaluation, focusing on specific organelles like the acrosome, postacrosomal lamina, neck, mitochondria, tail, and nucleus. The aim is to pinpoint sperms with optimal morphology for micromanipulation.

Role of artificial oocyte activation (AOA) in ICSI with micro-TESE sperm

Individuals with impaired spermatogenesis often have less physiologically mature testicular sperm, potentially limiting their ability to trigger oocyte activation. Lower fertilization rates with ICSI have been consistently observed when utilizing sperm retrieved from testicular surgeries.64 AOA with calcium ionomycin has emerged as a potential solution. AOA has shown promise in cases with a history of poor fertilization rates or complete fertilization failure.65,66 A recent publication highlighted enhanced fertilization rates in patients with NOA, wherein micro-TESE sperm combined with AOA was employed for ICSI.67

For optimal outcomes utilizing cryopreserved testicular sperm, the synergistic combination of IMSI with theophylline supplementation emerges as the most promising technique.68 IMSI’s meticulous, high-magnification selection of spermatozoa with optimal morphology specifically addresses the potential oocyte activation limitations inherent in testicular sperm. Theophylline supplementation, through its motility-enhancing properties, facilitates swift and precise sperm selection, potentially leading to improved fertilization and pregnancy rates. This integrated approach, when combined with established techniques such as ICSI and viability assessment, presents the most compelling evidence-based strategy for maximizing success with cryopreserved testicular sperm, particularly in complex cases.

ICSI AND SPERM QUALITY OUTCOMES WITH FRESH VERSUS FROZEN TESTICULAR SPERM

In 1997, the first published comparative study on the use of fresh and cryopreserved sperm in men with NOA was reported by Friedler et al.69 Since the advent of the micro-TESE technique in 1999,13 various studies have researched the outcomes of fresh and frozen sperm retrieved via micro-TESE specifically. Although collective evidence showed comparable outcomes, a few studies have noted conflicting results.

Tavukcuoglu et al.10 compared fresh and frozen micro-TESE sperm in 82 IVF cycles (43 with fresh and 39 with frozen sperm) in men with NOA undergoing ICSI. The fertilization rates were nearly identical at 44.79% for fresh and 46.76% for frozen sperm, indicating no significant differences in fertilization efficiency between the two groups. Similarly, good embryo quality rates (58.1% for fresh vs 51.3% for frozen), embryo transfer (1.60 for fresh vs 1.59 for frozen), and clinical pregnancy outcomes (44.2% for fresh vs 43.6% for frozen) were comparable between the two groups. The study concluded that cryopreservation of testicular sperm was beneficial for reducing procedural redundancy without compromising outcomes.10

Karacan et al.70 retrospectively analyzed 337 ICSI cycles, including fresh sperm retrieved on the day of or the day before oocyte retrieval and frozen-thawed sperm. Fertilization rates were 70.7% for fresh sperm, 68.7% for sperm retrieved a day prior, and 67.3% for frozen sperm. Clinical pregnancy rates were 31.3%, 30.9%, and 25.5%, respectively, while LBRs were 28.9%, 28.5%, and 23.2%, respectively. While the outcomes suggested slightly better results with fresh sperm, the differences were not statistically significant. The study emphasized the reliability of frozen sperm in cases where fresh retrieval is not feasible.70

Nagawkar Perlov et al.71 evaluated 93 embryo transfer cycles using fresh (46 cycles) or frozen-thawed (47 cycles) micro-TESE sperm in men with NOA. Fertilization rates were comparable between the groups. However, embryo quality rates were significantly higher in the fresh sperm group compared to the frozen group (83.3% vs 50%). Comparisons of clinical pregnancy, implantation, or LBRs were also comparable. Neonatal outcomes were also similar, such as birth weight or gestational complications. These findings reinforce the equivalence of fresh and frozen sperm in clinical and obstetrical outcomes.71

Suleymanova et al.72 analyzed 223 men with NOA, comparing fresh micro-TESE sperm (152 cases) and frozen-thawed sperm (71 cases). Fertilization was achieved in 208 cycles, and no significant differences were noted in fertilization rates, pregnancy rates, cleavage-stage embryo quality, or LBRs between fresh and frozen sperm groups. This study highlighted that cryopreservation of micro-TESE sperm did not adversely affect key reproductive outcomes, supporting its utility for repeat ICSI cycles or when immediate fresh use is unfeasible.72

Zhang et al.73 analyzed 338 men with NOA who underwent micro-TESE and subsequent ICSI cycles. Among these, 222 patients with 234 cycles used fresh sperm (Group A), while 116 patients with 110 cycles used frozen sperm (Group B). The study found no statistical differences in fertilization rates (47.68% vs 44.25%), embryo transfer (1.64 vs 1.65), and embryo quality rates (52.13% vs 53.75%) between the groups. However, the use of fresh sperm showed a significantly higher clinical pregnancy rate (52.3% vs 40.5%) and higher LBR (48.7% vs 34.5%) when compared to frozen sperm, suggesting a potential advantage of fresh sperm in achieving favorable pregnancy outcome.73

Another study involved 40 patients undergoing 40 cycles with fresh sperm and another 30 patients undergoing 30 cycles with frozen sperm. It was found that there were similar outcomes in fertilization and embryo rates between the two groups. However, the use of frozen sperm showed a significantly higher miscarriage rate (23.81% vs 0, P < 0.05) and lower birth rate (50% vs 75%, P < 0.05) when compared to fresh sperm. This study also suggests the advantage of fresh sperm in ICSI outcomes for NOA patients.74

All the evidence above regarding reproductive outcomes of fresh versus frozen sperm remains mixed, with some studies favoring fresh sperm for higher fertilization, implantation, and LBRs, and others showing comparable outcomes with frozen sperm. The female age, as one of the important factors that can affect reproductive outcomes, was also addressed in the studies above. Almost all of the studies show no significant difference between the mean female age of the fresh versus frozen sperm group, with all of them reporting a female mean age of less than 35 years. Many literatures have reported that an age of over 35 years for women is a crucial factor of decreased fertilization and pregnancy rates.75,76,77 Studies have also highlighted the impact of female age and the number of oocytes available to ICSI outcomes in NOA patients.75,76,77 Thus, the studies compared above have minimalized the risk of lower reproductive outcomes due to the female age factor. The various comparisons of reproductive outcomes between the use of fresh versus frozen sperm have been summarized in Table 1.

Table 1.

Comparisons of reproductive outcomes between fresh and frozen micro-TESE sperm for men with NOA

Study	Sperm origin	Fertilization rate (%)	Embryo transfer	Good embryo quality rate (%)	Clinical pregnancy rate (%)	Live birth rate (%)	Miscarriage rate (%)
Tavukcuoglu et al.10 2013	Fresh	44.79	1.6	58.1	44.2	37.2	-
	Frozen	46.76	1.59	51.3	43.6	30.8	-
Karacan et al.70 2013	Fresh	67.2	1.9	-	29.2	27.2	6.8
	Frozen	64.7	2.1	-	23.8	21.4	10
Zhang et al.73 2021	Fresh	47.68	1.64	52.13	52.3*	48.7*	6
	Frozen	44.25	1.65	53.75	40.5	34.5	14.9
Zhang et al.74 2021	Fresh	65.69	1.93	51.59	75	75*	0*
	Frozen	60.12	1.60	45.54	75	50	23.81*
Nagawkar Perlov et al.71 2024	Fresh	59	-	83.3*	87	72.2	-
	Frozen	62	-	50	80.9	75	-
Suleymanova et al.72 2024	Fresh	94.7	-	-	50	55.3	-
	Frozen	90.1	-	-	43.7	54.8	-

^*P<0.05, statistical significance achieved for outcomes of fresh vs frozen thawed micro-TESE sperm. Micro-TESE: microdissection testicular sperm extraction; NOA: nonobstructive azoospermia; -: not available

When comparing the quality of sperm used, a study by Sawaid Kaiyal et al.78 found that fresh sperm had better outcomes compared to frozen sperm in parameters such as motility, viability, and DNA integrity. They reported that while 67.7% of fresh micro-TESE samples were motile and usable for ICSI, frozen-thawed samples had a lower usability rate at 45.3%, reflecting a substantial loss in quality postthaw.78 Additionally, frozen sperm generally exhibits higher DNA fragmentation compared to fresh sperm, which has minimal structural damage since it avoids the freezing and thawing stressors that contribute to fragmentation and cellular injury.79

CONSIDERATIONS IN THE USE OF FRESH VERSUS FROZEN TESTICULAR SPERM

Timing of fresh versus frozen testicular sperm use

Timing is a critical factor when deciding between fresh and frozen sperm, especially concerning synchronization with the female partner’s cycle. Using fresh sperm requires the male partner to undergo a micro-TESE procedure on the day of oocyte retrieval, which introduces uncertainty for the female partner. If no viable sperm are found during the micro-TESE, the ICSI procedure may need to be canceled or postponed, requiring additional coordination and potentially creating emotional and logistical stress for both partners.

In contrast, frozen sperm allows more flexibility in scheduling, as the sample is prepared and available before ovarian stimulation, eliminating the need for precise timing on the day of retrieval. Asanad et al.79 support this, noting that frozen samples can better accommodate scheduling requirements, thus offering a less disruptive treatment process for both partners. Thus, a frozen micro-TESE backup sample can prevent unnecessary procedure cancellation in case of an unsuccessful fresh micro-TESE on the day of the assisted reproductive procedure.80 Additionally, as all laboratory procedures are time-sensitive, any unexpected challenges during a fresh micro-TESE could result in delays that could impact the entire day’s schedule in the laboratory.

Expertise of surgeon and andrology laboratory staff

The expertise of both the surgeon and the andrology laboratory team is an essential factor to consider when deciding on a treatment plan for testicular sperm extraction with micro-TESE, followed by ICSI. The skill and experience of the surgeon can influence outcomes micro-TESE, especially in determining the quality and viability of retrieved sperm for use. Studies have shown that the sperm retrieval rate (SRR) significantly increases with surgical expertise, with Ishikawa et al.81 reporting improved SRRs after the first 100 procedures, followed by Dabaja and Schlegel82 reporting additional gains after 500 cases.

Skilled surgeons are more adept at identifying dilated seminiferous tubules, which are crucial for successful sperm retrieval in NOA patients. These differences may affect the choice between fresh and frozen sperm. For example, fresh sperm retrieval during ICSI often requires fewer viable sperm, allowing for a less extensive surgical procedure and minimizing testicular tissue damage.83 In contrast, the viability of frozen-thawed sperm is typically reduced to approximately 33%, necessitating a higher initial yield during retrieval.84

High proficiency ensures efficient sperm retrieval in adequate numbers while also striving to preserve as much as normal testicular architecture, enabling flexibility in choosing between fresh or frozen sperm without compromising outcomes. These findings highlight the importance of surgeon experience in optimizing SRR as well as mitigating the procedural challenges associated with cryopreservation and thawing processes.

The availability and expertise of andrology laboratory staff significantly influence the optimization of sperm discovery during micro-TESE procedure, directly affecting outcomes. High-performing ART laboratories emphasize sufficient staffing levels to manage complex procedures, ensuring quality and efficiency. For instance, Sciorio et al.85 noted that inadequate staffing often leads to rushed procedures, reduced concentration, and increased risk of errors, ultimately compromising outcomes.

The technical expertise of embryologists in processing surgically retrieved sperm, whether fresh or frozen, is critical. Specifically, testicular sperm ICSI is a more technically challenging procedure than conventional ICSI, and it requires the embryologist to have a high level of skill and experience. Embryologists with prior experience in dealing with similar complex cases may be better suited for ICSI with frozen micro-TESE.

Either use of fresh or frozen sperm requires skill and expertise on both sides. Fresh micro-TESE sperm retrieval cycles often demand immediate laboratory processing, requiring the presence of skilled staff to promptly identify viable sperm and ensure their timely use. Conversely, the cryopreservation of micro-TESE sperm involves precision in freezing techniques to minimize DNA damage and maintain postthaw viability, highlighting the necessity of staff adept in advanced cryobiology techniques. A study by Knudtson et al.86 revealed that consistent laboratory practices, including meticulous sperm preparation and cryopreservation protocols, contribute to optimal fertilization and LBRs, irrespective of sperm preservation method. These findings once again underline the importance of maintaining adequate, well-trained andrology staff to support either fresh or frozen micro-TESE sperm use, ensuring the best possible reproductive outcomes for patients.

Sample quality

The quality of the sperm sample is essential in deciding between fresh and frozen sperm, as cryopreservation can impact the quality of the sperm sample used. Poor-quality sperm samples may be more susceptible to damage during the freezing process, leading to lower survival rates after thawing. Cryopreservation significantly decreases sperm motility, viability, and morphology.29,87,88 Ozkavukcu et al.87 found a strong link between reduced motility and decreased viability. Studies showing cryopreservation-induced DNA damage suggest correlations with reduced fertilization rates, increased pregnancy loss, and other negative reproductive outcomes.89,90,91 In such cases, a fresh micro-TESE may be necessary to obtain enough sperm for ICSI.

Cost-effectiveness

Cost considerations are substantial when choosing between fresh and frozen micro-TESE sperm, as failed fresh retrievals often lead to additional costly procedures. Asanad et al.79 highlights that if viable sperm is not found during a fresh micro-TESE, repeat surgeries may be required, driving up out-of-pocket costs. In contrast, frozen micro-TESE serves as a cost-effective “backup” option, reducing the need for additional retrievals by providing a stored sperm source. Additionally, frozen micro-TESE minimizes logistical and financial complications by eliminating the need for exact synchronization with oocyte retrieval, thus allowing greater scheduling flexibility and reducing cycle cancellations.92 This approach not only lowers costs but also streamlines treatment planning, making it a practical and patient-centered choice.

Studies by Berger et al.93 and Liu et al.94 provide additional insight into the implications of failed fresh micro-TESE sperm retrievals, which are rare but impactful. When sperm retrieval fails, the female partner may face cycle cancellation, leading to avoidable financial costs and increased health risks from repeated ovarian stimulation and oocyte retrieval. Additionally, in regions where regulatory restrictions prohibit the use of donor sperm, couples are limited in their options and may experience increased pressure to rely on fresh retrieval attempts or repeat procedures if prior fresh micro-TESE fails. Consequently, frozen micro-TESE sperm not only reduces the direct costs associated with repeated retrievals but also helps circumvent regulatory challenges, thereby enhancing both cost-efficiency and accessibility of treatment.

Female partner age and number of oocytes retrieved

The age of the female partner plays an important role in determining reproductive outcomes such as fertilization rates, embryo formation, and live birth. Studies consistently demonstrate a decline in oocyte quality and ovarian reserve with advancing age, leading to reduced success rates. Wang et al.95 observed that clinical pregnancy rates dropped from 64.7% in women under 32 years to 24.2% in those over 40 years, highlighting the substantial impact of age. In addition, Cherouveim et al.96 reported that women undergoing ICSI with frozen sperm were on average older (36.7 years) compared to those using fresh sperm (34.5 years) and have lower clinical pregnancy rates (9.4% vs 13.0%, P < 0.001).

Despite these differences, more newer studies have found no significant interaction between the use of fresh or frozen micro-TESE sperm and LBRs,71,72 indicating that outcomes were independent of sperm preservation method when adjusted for female age and ovarian reserve. These findings highlighted that while female age is a critical determinant of ART success, the choice between fresh and frozen sperm can yield comparable outcomes, particularly when female reproductive factors are optimized.

The number of oocytes retrieved during ovarian stimulation also contributes to the success of reproductive outcomes. Studies have demonstrated that LBRs increase with the number of oocytes retrieved up to an optimal range but plateau or decline beyond a certain threshold due to diminishing embryo quality or increased risks like ovarian hyperstimulation syndrome (OHSS). For instance, Bahadur et al.97 reported that LBR peaked at 35.3% when 16–25 oocytes were retrieved and decreased significantly with very high oocyte yields (≥26), where LBR dropped to 18.7%. Similarly, Polyzos et al.98 observed a steady increase in cumulative LBRs with higher oocyte numbers, reaching 70% with ≥25 oocytes, although fresh LBRs tended to plateau beyond seven oocytes, influenced by increased freeze-all cycle rates.

However, in the context of fresh versus frozen micro-TESE sperm use, there has been no studies up-to-date that directly investigates the success rate of reproductive outcomes between the two groups with the number of retrieved oocytes.

Logistical and infrastructure requirements

Logistical and infrastructure requirements play a critical role in determining whether fresh or frozen micro-TESE sperm should be utilized. Adequate facilities for sperm retrieval, processing, and storage are essential to ensure optimal outcomes. A study by Frederick et al.99 highlight the need for coordinated systems that include specialized fertility preservation teams and dedicated laboratory facilities.

Centers with comprehensive infrastructure are needed to handle either fresh or frozen sperm. The use of fresh sperm requires immediate processing and advanced embryology support to identify and utilize viable sperm ICSI. Similarly, the process of cryopreservation in frozen sperm use requires robust storage capabilities, precision freezing protocols, and expertise in thawing procedures to maintain sperm viability and DNA integrity. Additionally, Alviggi et al.100 emphasize the significance of integrating ART services within broader reproductive healthcare systems to ensure timely and seamless access to both fresh and frozen sperm options.

Facilities lacking infrastructure for fresh sperm use may rely on frozen sperm to overcome logistical constraints, such as delays in scheduling procedures or unavailability of personnel. These findings underscore that well-developed logistical frameworks are essential for optimizing ART outcomes and providing flexibility in sperm preservation methods tailored to patient needs.

ADVANTAGES AND DISADVANTAGES OF FRESH AND FROZEN SPERM

The use of fresh or frozen testicular sperm for IVF/ICSI cycle has its advantages and disadvantages (Table 2). It depends on the clinical scenarios, which should benefit the infertile couple. This will guide the clinicians on which to choose to achieve superior reproductive outcomes.

Table 2.

Advantages and disadvantages of the use of fresh or frozen testicular sperm for men with nonobstructive azoospermia79,87,92,93

Category	Fresh sperm		Frozen sperm
	Advantage	Disadvantage	Advantage	Disadvantage
Availability at the time of ICSI procedure	Immediate use possible; suitable for rare, delicate sperm	Risk of not finding viable sperm at the time of retrieval; potential for cancelled cycles	Ensures availability before ICSI; acts as a backup if fresh retrieval fails	Potential damage during freezing–thawing process
Sperm quality	Avoids freezing–thawing damage, generally higher motility and viability, no fear of losing motility	Limited by sperm quality at retrieval	Allows pre-evaluation of sperm quality; allows embryologists to identify viable sperm	Freezing can reduce motility and viability
Procedure timing	Immediate ICSI if sperm is found, suitable for synchronized cycles	Scheduling challenges if coordination is needed	Allows better scheduling and planning; avoids scheduling conflicts (e.g., operating room and urologist availability)	Requires precise planning for cryopreservation and thawing
Stress and logistics	Reduces need for multiple procedures; less stress if sperm is found and used immediately	Stress and uncertainty if no viable sperm is found (even if female partner has been prepared/stimulated)	Confirms sperm availability, reducing stress; practical for busy centers; avoids the male partner having surgery on the same day as oocyte retrieval, avoiding the challenge of postcare for both partners	Additional steps for freezing, storage, and thawing
Cost and efficiency	Lower costs if successful on first attempt	Financial loss if no viable sperm is found, leading to cancelled procedures	Avoids multiple retrievals, practical for busy IVF centers	Costs associated with freezing, storage, and potential additional procedures
Clinical outcomes	Often better in cases with high motility and viability; higher reported motility and viability	Variability in clinical outcomes; risk of no viable sperm	Provides a backup in case fresh retrieval fails, comparable pregnancy rates in many cases; avoids postmaturity oocyte damage	Some studies report lower pregnancy rates and higher miscarriage rates

IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection

The decision to use fresh or frozen testicular sperm for ART in men with NOA relies on several factors that have been discussed above.20 These include the expertise of the surgeon, cryopreservation protocols of the IVF centers, anticipated difficulty sperm retrieval, scheduling conflicts, availability of viable sperm before oocyte retrieval, favorable histopathology, fear of future failure of finding sperm, and potential risk during the freeze–thaw process. These factors should be considered before deciding which sperm should be used for IVF/ICSI cycle. In a survey among the members of the Society for Study of Male Reproduction, 6 out of 10 respondents primarily performed micro-TESE and froze sperm.79 On the contrary, the majority (59.3%) still prefer fresh micro-TESE in an ideal practice. The cost, scheduling, and the doctor’s availability are the most reported barriers for the use of both fresh and frozen testicular samples.

CRITICS OF CURRENTLY AVAILABLE LITERATURE ON FROZEN VERSUS FRESH SPERM

Although most of the evidence showed comparable results using fresh and frozen testicular sperm for ICSI cycles in men with NOA, it is limited by small sample sizes to derive definitive conclusions on reproductive outcomes. Most of the included studies were retrospective cases. This limits the ability to control unknown factors that might affect the outcomes. Since the results of IVF/ICSI cycles do not only depend on the nature of the testicular sperm, other confounding factors should also be evaluated and eliminated such as the impact of female factors (e.g., age), quality of embryo, and lifestyle behaviors (e.g., smoking, alcohol, drug, and other systemic diseases) as these may also influence the outcomes. A well-designed controlled study should be conducted to arrive at definitive evidence. In addition, this will improve ICSI outcomes. Strict inclusion and exclusion criteria are needed in future studies.

FUTURE DIRECTIONS AND CLINICAL RECOMMENDATIONS

Innovations such as the Cryopiece system are designed to maximize postthaw viability, especially in cases with limited sperm.48 The Cryopiece provides a controlled, contamination-free environment that supports higher motility rates in postthaw samples, which is especially beneficial for men with oligospermia or low-volume samples. Recent studies emphasize that the Cryopiece system improves outcomes by preserving motility and structural integrity, offering an edge over conventional freezing methods.45,48 Additionally, advances in vitrification devices have made it feasible to preserve high-quality sperm samples with minimal cryoprotectant exposure, maintaining DNA integrity and motility better than traditional slow freezing. This technological progress enables fertility centers to better address the needs of patients with severe male factor infertility or those undergoing treatments that jeopardize fertility, making it possible to preserve even small, fragile sperm samples for later use.

In addition, newer technologies have been developed to improve sperm quality and ART outcomes.91 Sperm birefringence evaluation allows the detection of sperm with intact DNA and greater reproductive potential viability without affecting the other semen parameters.101 This showed positive impact when applied to sperm selection to ameliorate ICSI outcomes. In addition, there are several attempts to improve the identification of sperm during micro-TESE. Machine learning has been deployed to improve the diagnosis and treatment of assisted reproduction.102 Artificial intelligence algorithm was developed to increase the probability of finding viable sperm for men with NOA.103,104 Deep learning has been utilized to directly detect sperm from testicular tissues. Machine learning methods were applied to aid the decision support system in predicting the presence or absence of sperm in men with NOA.105 Identification and morphological classification of the sperm can be improved with the complementary roles of deep convolutional neural networks and object detection architectures.79 These approaches will increase the success rate and decrease the time required during sperm retrieval in the future. More extensive and prospective studies should be conducted to arrive at definitive recommendations for men with NOA.

CONCLUSION

The micro-TESE technique, coupled with advancements in sperm selection and cryopreservation, has significantly improved fertility outcomes or men with NOA. In cryopreservation, conventional slow freezing and vitrification each present unique advantages, with vitrification generally offering better postthaw motility and lower DNA fragmentation than slow freezing. However, up until now, the reproductive outcomes of fresh compared to frozen sperm remain inconclusive. The choice between fresh and frozen sperm should ultimately be personalized, taking into account individual patient circumstances, logistical considerations, and clinical expertise. Before undergoing the procedure, patients should be provided with thorough informed consent and a clear explanation of both options including the benefits and limitations of each approach.

AUTHOR CONTRIBUTIONS

KCM contributed in conceptualization, original draft preparation, literature search, data curation, and bibliography. MM contributed in conceptualization, original draft preparation, and bibliography. FAR contributed in conceptualization, original draft preparation, literature search, data curation, and bibliography. AA contributed in original draft preparation, literature search, table, and image visualization. MS and PB contributed in conceptualization, original draft preparation, and supervision. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.