Andrology laboratory techniques for micro-TESE/IVF/ICSI: a narrative review

Abstract

Since the early days of assisted reproductive technology (ART), the importance of sperm processing, employed to separate the motile, morphologically normal sperm from the semen, has been shown to be beneficial. The aim of the semen processing technique has been to remove seminal plasma and facilitate capacitation. Additionally, the presence of leukocytes, bacteria, and dead spermatozoa has been shown to be detrimental as it may cause oxidative stress that has an adverse effect on oocyte fertilization and embryo development. Hence, removal of leukocytes, bacteria, and dead spermatozoa is an important step of sperm processing for assisted reproduction. Currently, several sperm processing techniques have been evolved and optimized in the field of assisted reproduction. The requirements for in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and testicular sperm extraction (TESE) are different than those of intrauterine insemination (IUI). The yield of as many motile, morphologically normal sperm as possible is a prerequisite for the success of IVF insemination procedure. In ICSI, where injection of a single spermatozoon into the oocyte is performed by the embryologist, sperm selection techniques play a crucial role in the ICSI procedure. Finally, sperm retrieval in TESE samples with very low number of sperm may be challenging and requires extra care during sample processing. Additionally, sperm cryopreservation is necessary in TESE cases in order to avoid multiple biopsies.

INTRODUCTION

Sperm processing is a crucial step in assisted reproductive technology (ART). It aims to isolate healthy, motile sperm from semen while removing detrimental components such as dead sperm, bacteria, and white blood cells. This process not only facilitates capacitation, which is essential for sperm to fertilize an egg, but also minimizes oxidative stress that can hinder fertilization and embryo development.1,2 Different ART procedures, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and testicular sperm extraction (TESE), have unique sperm processing requirements. While IVF prioritizes a high yield of motile sperm, ICSI relies heavily on meticulous sperm selection techniques. TESE, which involves extracting sperm directly from the testicles, requires extra care due to the typically low sperm count and often necessitates cryopreservation to avoid repeated biopsies.

For the whole spermatozoa population, only a small fraction is susceptible of acquiring the capacity to achieve fertilization at a given time point under in vitro conditions. This observable intra-sample heterogeneity is surely determined by underlying differences among spermatozoa at the cellular and molecular levels. Using discontinuous Percoll gradient, Buffone et al.3 found at the different fractions (45%, 65%, and 90% of Percoll) that human spermatozoa possessed different characteristics regarding quality (motility and morphology) and capacity to show hyperactive motility and protein tyrosine phosphorylation in response to incubation under capacitating conditions.

This narrative review aims to provide an update on the various sperm separation methods in current practice, including their implications and importance for modern ART as well as an overview on different in vitro treatments of spermatozoa to improve their functional competence and to reduce detrimental effects. Moreover, this article aims to highlight the importance of the andrology laboratory and its contribution to the success of IVF, ICSI, and TESE cases.

SPERM SEPARATION/PROCESSING TECHNIQUE FOR IVF/ICSI

In in vitro condition, the ideal method of sperm separation must be fast, easy to perform, and cost-effective with the maximum sperm yield. It must also avoid causing any damage to the sperm or inducing non-physiological changes in separated sperm cells. Hence, it is mandatory in clinical practice to choose the appropriate method to achieve optimal yields of functionally competent sperm for conventional IVF insemination purposes. ICSI is a widely used ART that involves the injection of a single spermatozoon directly into the cytoplasm of an oocyte. The selection of the best sperm for ICSI is crucial for successful fertilization and embryo development, which can be affected by a range of factors including DNA damage, morphological abnormalities, and poor motility. Although technically IVF insemination and ICSI procedures are different and have specific clinical applications, sperm processing methods are similar for both procedures.

Simple wash and swim-up technique

This method is commonly employed for semen samples with good sperm concentration and motility to obtain a highly motile and morphologically normal sperm population, thus maximizing the chances of successful fertilization and embryo development. In this method after complete liquefaction, mixing of the ejaculate is performed with equal proportion of culture medium, and centrifugation is performed twice to remove the seminal plasma. Later, the motile cells are left to swim up from the pellet. The motile fraction of cells is used for IVF/ICSI procedures. Reduced centrifugal force (<500g) and fewer centrifugal steps are necessary to minimize damage caused by the formation of reactive oxygen species (ROS) from nonviable sperm and leukocytes,4 which can lead to DNA damage in sperm.5 The technique is very simple with less media consumption and relatively economical, but it can retain abnormal sperms along with the other seminal plasma components in the final pellet with the great risk of oxidative damage and imposes a higher risk for microbial contamination.

However, a previous study has compared the efficacy of the simple wash method with traditional density gradient centrifugation method of sperm preparation and inferred that swim-up technique seems to offer improved reproductive outcomes.6 Another study investigated the effect of the simple wash method on sperm DNA fragmentation and inferred that it was safe and would not increase the sperm DNA fragmentation compared to the density gradient method.7 Overall, the simple wash method is a safe and effective alternative to the routinely used density gradient centrifugation method of sperm preparation for both IVF insemination technique and ICSI procedure. Considering the inconclusive published literature on superiority of swim-up technique over the density gradient method, there seems need for individualization based on clinic’s standard operating protocols (SOP) and patient-specific factors to decide on the choice of sperm preparation technique. Simple sperm wash technique is described in Figure 1.

Figure 1.

Pictorial depiction of simple sperm wash technique.

Migration-sedimentation

Direct swim-up from semen is applicable for sperm samples with average or good motility. On the other hand, migration-sedimentation is routinely used for samples with low motility.8 The technique relies on the swim-up movement along with natural settling of spermatozoa due to gravity. Although this technique is very cost-effective and recovers very clean fraction of highly motile spermatozoa, it is restricted to ejaculates with high sperm counts and motility. It has been shown that the migration sedimentation method significantly decreased the percentage of spermatozoa with normally condensed chromatin. The advantage of this technique is that it is a gentle method, and thus, the amount of ROS produced is not very significant. A disadvantage is that the specialized tubes needed for this technique are relatively expensive. A pictorial depiction of migration sedimentation technique is described in Figure 2.

Figure 2.

Pictorial depiction of migration sedimentation technique.

Density gradient centrifugation (DGC)

In DGC technique, semen sample is placed over a continuous or discontinuous density gradient and then centrifuged. Separation of sperm cells occurs based on density and motility; motile sperm cells will migrate to the bottom of the tube passing through the density gradients when subjected to centrifugation.

Motile sperm cells form a silky pellet at the bottom of the tube while immotile cells and debris get filtered and trapped between gradients.4

It is important to ensure that the gradients used in this method should not be toxic for spermatozoa and stable in solution, to avoid any pH alteration or osmolarity change. Ficoll and Percoll are the commonly used gradients to process the semen. Discontinuous gradients 80%–40% or 90%–45% are commonly used in andrology laboratory for semen processing.

Yield of motile sperm cells after density gradient centrifugation is largely influenced by conditions, such as sperm concentration, semen volume, number of gradient layers, centrifugal force applied, and centrifugation time.9 Caution needs to be exercised with centrifugal time and speed, as it may produce higher amount of ROS due to mechanical stress caused by centrifugation. The usual recommended centrifugation speed is 300–600g.

In comparison with swim-up method, this technique is less time-consuming and a method of choice for semen samples with very low sperm count. Nevertheless, additional cost due to use of density gradients and the need for learning curve to master the skill of layering the gradients need to be considered while choosing this processing technique. A pictorial depiction of density gradient centrifugation technique is described in Figure 3.

Figure 3.

Pictorial depiction of density gradient centrifugation technique.

The density gradient method has been widely employed in both research and clinical settings for sperm preparation prior to IVF/ICSI procedure. Studies performed on density gradient processed sample, found that this method effectively removed sperm with chromosomal abnormalities, thereby improving the chances of selecting spermatozoa with normal chromosomes for fertilization.10,11 Density gradient processing method helps in effectively selecting the high-quality spermatozoa with lower DNA fragmentation rates, resulting in improved fertilization and embryo development, and making it a suitable technique for sperm selection particularly in cases with severe male factor infertility cases. In spite of decades of research and clinical observations, there seems a need for further research to explore the efficacy of DGC technique for sperm processing to enhance the reproductive outcomes of IVF/ICSI cycles.

Magnetic activated cell sorting (MACS)

MACS technique has been used for sperm preparation for IVF and ICSI in assisted reproduction. The pictorial description of MACS technique is described in Figure 4, and the details of this technique are described in the advanced sperm preparation techniques.

Figure 4.

Pictorial depiction of magnetic-activated cell sorting (MACS).

Microfluidics (MF)

Microfluidic sperm selection technique is a newer sperm preparation technique designed to prepare motile sperm for IVF and ICSI in assisted reproductive technology procedures. MF technique separate sperm based on their motility. They create a microenvironment using micro channels or micro-pores in a filter. The device takes advantage of the unique characteristics of sperm cells, such as their swimming ability and structural differences, to separate and isolate the most viable and motile sperm. This improves the chances of successful embryo development and ultimately increases the likelihood of a successful pregnancy for couples undergoing IVF or ICSI treatments.15 Further details are described below in the segment of advanced sperm preparation methods. A pictorial depiction of MF sperm sorting technique is described in Figure 5.

Figure 5.

Pictorial depiction of microfluidics sperm sorting technique.

ADVANCED SPERM PREPARATION METHODS

MACS

MACS is a technology that uses magnetic beads coated with antibodies to separate spermatozoa based on their surface antigens. This method can selectively isolate spermatozoa with high DNA integrity and capacitation status, resulting in higher fertilization rates and better embryo development. MACS have been shown to be effective in selecting spermatozoa with low DNA fragmentation in infertile men and hence can be beneficial for sperm selection during ICSI process.16 MACS separates apoptotic spermatozoa from non-apoptotic spermatozoa using an apoptotic marker known as phosphatidyl serine expressed by the spermatozoa membrane. Annexin V has a strong affinity for phosphatidyl serine. Colloidal superparamagnetic beads (approximately 50 nm in diameter) are conjugated to highly specific antibodies to annexin V and used to separate dead and apoptotic spermatozoa by MACS.

Spermatozoa prepared by density gradient centrifugation followed by MACS have a higher percentage of motility, a higher percentage viability, and a lower expression of apoptotic markers than spermatozoa prepared by DGC alone.12 Annexin V-negative spermatozoa have a higher motility, lower caspase activation, lower membrane mitochondrial potential disruption, lower amounts of DNA damage, and higher oocyte penetration capacity than annexin V-positive spermatozoa13 and associated with higher cleavage and pregnancy rates than spermatozoa selected by DGC in oligoasthenozoospermic cases.14 The technique is rapid, convenient, and noninvasive, resulting in optimal recovery with reliable and consistent outcomes, but the drawback of this method is that it needs to be used in conjunction with other techniques such as DGC to remove the other substances (round cells and semen debris), and this technique is relatively expensive.

Microfluidic devices

Microfluidics gained significant attention in recent years for its potential to improve the success rates and efficiency of ICSI procedures. Microfluidic devices employ hydrodynamic forces to sort motile spermatozoa for ICSI.17,18 The device demonstrated high sperm motility recovery and improved fertilization rates compared to conventional methods.18 Another study describes a microfluidic device designed to select spermatozoa with high DNA integrity for ICSI. The device utilized specific channel geometries and surface coatings to separate sperm based on DNA fragmentation levels. The use of this device resulted in improved embryo quality and pregnancy rates.19 They report several advantages over conventional methods, such as shorter processing time, higher precision, and lower sample volume requirement. However, microfluidics sperm sorting is relatively a new concept and still awaits clinical validation.

Piezo-ICSI

Piezo-ICSI is a modified form of ICSI that uses a Piezo micromanipulator to inject the spermatozoa into the oocyte. This technique showed better blastocyst quality and pregnancy rates compared with the conventional technique.20 A significant delay in sperm chromatin decondensation and oocyte activation events was observed after conventional ICSI.21 Piezo-ICSI has been reported to be effective in cases of poor semen quality and previous ICSI failure.21,22 A pictorial depiction of Piezo-ICSI technique is described in Supplementary Figure 1 ^{(154.3KB, tif)} .

Sperm selection by hyaluronic acid (HA)

HA is a glycosaminoglycan present in the cumulus cells surrounding the oocyte. The mature and high-quality spermatozoa have a high affinity for HA. The binding of sperm reflects the heat shock protein A2 (HSPA2) chaperone activity, which is linked to a series of cytoplasmic and nuclear events in cellular maturation.23 This technique is commercially available as physiological ICSI (PICSI) technique or physiological ICSI technique. HA-bound spermatozoa have been shown to have higher DNA integrity and fertilization rates compared to conventionally prepared spermatozoa. A study has reported improved clinical outcomes with HA-selected spermatozoa in ICSI.24 A pictorial depiction of sperm selection by HA binding technique is described in Supplementary Figure 2 ^{(41.5KB, tif)} .

Intracytoplasmic morphologically selected sperm injection (IMSI)

IMSI is a technique used for selecting the best sperm based on their morphology using high-magnification microscopy. This method was first introduced in 2001, allowing the motile-sperm organelle morphology examination (MSOME) using differential interference contrast (DIC) microscopy enhanced by high magnification.25 The final magnification that can be achieved ranges from 6000× to 10 000×, facilitating the visualization of both major and minor sperm defects and especially the presence of sperm head vacuoles.

Meta-analysis of 15 randomized controlled trials involving 1901 couples found that IMSI was associated with a higher clinical pregnancy rate and live birth rate compared to conventional ICSI. IMSI allows for a more detailed and accurate examination of sperm morphology, enabling the selection of sperm with better characteristics that might otherwise be missed with conventional ICSI.26 The advantage of the technique includes improved pregnancy rates and increased accuracy in selection of better-quality sperms, whereas it is time-consuming, causing potential damage to sperms due to phototoxicity and only limited evidence is available to use IMSI as a routine clinical practice. More research is needed to establish its efficacy, safety, and cost-effectiveness. A pictorial depiction of the IMSI technique is described in Supplementary Figure 3 ^{(89.4KB, tif)} .

Hyperspectral microscopy

Hyperspectral microscopy is a powerful imaging technique that has gained attention in recent years for its potential applications in reproductive medicine. It allows for the capture of high-resolution images with high spectral resolution, providing information on both the spatial and spectral properties of samples in contrast to the conventional sperm selection using bright-field microscopy.27 Hyperspectral microscopy has the potential to overcome these limitations by providing additional information on the chemical composition of the sperm by capturing images at different wavelengths.28 Although the technique is promising for sperm selection in ICSI, it needs further research to determine its efficacy and safety. One limitation is that it requires specialized equipment and expertise, which may limit its widespread use in clinical practice.

However, this method may not always identify subtle defects in sperm morphology or chromatin abnormalities that may affect fertilization or embryo development. A pictorial depiction of fluorescence lifetime imaging microscopy (FLIM) technique is described in Supplementary Figure 4 ^{(85.1KB, tif)} . Table 1 is an overview of various sperm selection techniques with their brief principle, advantages, and disadvantages.

Table 1.

Summary of various semen processing techniques applied during intracytoplasmic sperm injection

Sperm selection method	Principle	Advantage	Disadvantage
DGC	Separates sperm based on density	Removes debris and dead sperm, selects sperm with better motility, morphology, and chromatin integrity	May result in the loss of viable sperm during selection, time-consuming
Swim-up method	Sperm swim to the top of a culture medium	Selects motile sperm without the need for centrifugation	May not remove all debris or dead sperm
HA binding	Selects sperm based on ability to bind to HA	Selects sperm with better DNA integrity and fertilization potential	May not select sperm with the best motility or morphology
MACS	Selects sperm based on surface markers	Can select sperm based on specific characteristics, such as DNA fragmentation or acrosome status	May not be widely available and requires specialized equipment and expertise

MACS: magnetic-activated cell sorting; HA: hyaluronic acid; DGC: density gradient centrifugation

EFFECT OF SPERM PREPARATION METHODS ON DNA FRAGMENTATION

Sperm DNA fragmentation is a major factor of male infertility. It has been hypothesized that in vitro handling of sperm for assisted reproduction can induce sperm DNA fragmentation and alter reproductive outcomes. The exposure of spermatozoa to immature germ cells and leukocytes during sperm processing may increase the production of ROS, which can lead to oxidative stress and sperm DNA fragmentation.29 Additionally, long centrifugation time has a detrimental effect in ROS production and as consequence sperm DNA damage.30 On comparing the effectiveness of swim-up and DGC, it was found that both methods were effective in reducing DNA fragmentation, whereas DGC was more effective in removing sperms with high levels of DNA damage.30 Furthermore, advanced sperm selection techniques such as MACS, IMSI, and PICSI have been developed as a means to reduce sperm DNA fragmentation31 MACS eliminates apoptotic sperm and has been shown to reduce sperm DNA fragmentation32 PICSI, a sperm selection method based on hyaluronan binding on mature sperm, is efficient in cases of advanced SDF.33 Studies have shown that DGC and MACS before sperm cryopreservation in cancer patients were the most effective methods for reducing DNA fragmentation.34

FROZEN SPERM AND ICSI OUTCOMES

The use of frozen sperm in conjunction with ICSI has become increasingly popular due to its numerous advantages. The availability of frozen sperm allows for flexibility of scheduling, preservation of fertility, and the opportunity to maximize pregnancy outcomes. The use of fresh or frozen semen has demonstrated comparable reproductive outcomes. A large retrospective study found that frozen sperm ICSI cycles achieved similar live birth rates as fresh sperm ICSI cycles.35 This study also indicates that there is no increased risk of congenital abnormalities or adverse perinatal outcomes associated with frozen sperm ICSI.35

The quality of frozen-thawed sperm can vary depending on various factors. Cryopreservation techniques, such as slow freezing or vitrification, and the use of cryoprotectants play a crucial role in maintaining sperm quality.36 Despite the success of frozen sperm with ICSI, certain limitations should be acknowledged. The availability of frozen sperm may be limited in certain situations, and the cost of long-term storage can be a consideration for some individuals. Furthermore, more studies are needed to evaluate the long-term effects of frozen sperm on offspring health and development.

LABORATORY ASPECTS OF HANDLING MICROSURGICAL TESTICULAR SPERM EXTRACTION (MICRO-TESE) SAMPLE FOR ICSI PROCEDURE

In NOA cases successful sperm retrieval is obtained37,38,39 with 55% from TESE40 and 63% from micro-TESE.37,41 Micro-TESE yields 50-fold–70-fold less tissue than conventional TESE42,43 where it focuses on the tissue containing active spermatogenesis.44,45,46 A major challenge with sperm retrieval in NOA is that majority of the sperm obtained are nonmotile and compromised in quality which requires special care and great caution while processing such sample in order to select sperm with better fertilizing potential or advanced techniques are used to select only viable spermatozoa for the ICSI procedure47 in the IVF laboratory.

In the past, in order to achieve better outcome, it was suggested to perform surgical sperm retrieval the day before oocyte retrieval which improves sperm motility by incubating the sample in culture medium. However, the in vitro incubation has to be limited to 48 h to avoid scrotal skin bacterial contamination, even when strict sterile laboratory condition is maintained.47 Nevertheless, with advent of improved cryopreservation techniques of testicular tissue, it is now possible to perform a micro-TESE randomly and freeze the sample and later use for ICSI. The outcomes of ICSI with fresh or frozen micro-TESE have been equivocal and encouraging. Details of freezing of testicular samples are dealt in detail further in this review.

TESE AND MICRO-TESE SAMPLE PREPARATION IN IVF LABORATORY

The next step after obtaining the TESE or micro-TESE sample is to release the spermatozoa from the tissue and to remove the red blood cells (RBCs) in order to facilitate the detection of sperm. The most common methods for the separation of spermatozoa from the seminiferous tubules are mechanical, while the removal of RBCs is usually achieved using an erythrocyte lysis buffer.

Mechanical methods

All the laboratory steps are performed under sterile conditions. Different mechanical methods may be employed such as rough shredding, vortexing, fine mincing, squeezing, crushing in a grinder with pestle, scissor cutting, scalpel tear, and 26 G needle dissection.48 Once the tissue reaches the laboratory, it is processed under the stereozoom microscope. The seminiferous tubules are mechanically minced until no intact tubules are visualized and examined under an inverted microscope. If no sperm are seen or insufficient numbers of sperm for ICSI are found, the other portion of testicular specimen is biopsied. All specimen homogenates are processed by centrifugation at 750g for 5 min49 and the pellet is used for ICSI procedure. DGC is also used to avoid immature germ cells and RBCs in the final fraction.49

Erythrocyte lysis buffer (ELB)

When the testicular specimen is highly contaminated with red blood cells, the mechanically dissected homogenate sample is treated with 1–2 ml of ELB50 which does not affect the sperm viability. The mixture is incubated at room temperature for 5 min followed by adding pre-warmed 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) medium to dilute ELB and centrifuged again. The supernatant is discarded completely, and the pellet is re-suspended with 0.5–1 ml of prewarmed HEPES media and used for ICSI.

MICROFLUIDIC SPERM SORTING FOR TESTICULAR RETRIEVED SPERM

The non-motile spermatozoa population in testicular retrieved sperm makes the routine microfluidic laminar flow technique inefficient. The rapid and efficient sperm isolation from TESE samples were obtained by spiral microfluidic system separating sperm from RBCs and cellular debris,51,52 using inertial forces followed by removal of excess media by means of a hollow fiber membrane designed for mammalian cell isolation without causing any detrimental effect on spermatozoa viability, morphology and DNA integrity. However, it can lead to sperm loss while processing, especially in the case of samples containing very few sperm and there is a need for determining the viability of sperm postisolation.

FLUORESCENCE-ACTIVATED CELL SORTING

Fluorescent-activated cell sorting when compared with standard sample TESE processing, this new technique can help identify rare sperms easily and bring about technical feasibility.53 Selection of viable spermatozoa is based on labeling with fluorophore-conjugated antibodies from seminal fluid when irritated with laser.54 However, the method is time-consuming, and the impact of sperm viability due to fluorophores and antibodies limits its usage.

SPERM SELECTION TECHNIQUES FOR ICSI IN TESE AND MICRO-TESE SAMPLES

In NOA cases, the spermatozoa obtained by TESE are usually scarce, morphologically abnormal, and with low or no motility. As a result, the detection of viable and competent spermatozoa for injection is challenging. Several sperm selection techniques have been developed to facilitate the selection of sperm for ICSI in TESE and micro-TESE samples.

ENZYMATIC DIGESTION METHOD

In scenarios where limited tissue samples are obtained as a part of testicular sperm aspiration, mechanical extraction or dissection is challenging and can damage the sperms.55 Injecting such immotile sperm significantly decreased the fertilized rate.56,57 Chemical compounds such as pentoxifylline, caffeine, and theophylline are phosphodiesterase inhibitors (PDE) inhibiting cyclic nucleotide phosphodiesterase which breaks down cyclic adenosine monophosphate (cAMP) to 5’-AMP.58,59 Approximately 3 μl of SpermMobil commercially available product (GM501 SpermMobil Gynemed, Sierksdorf, Germany) is added to the sperm droplet in the ICSI dish and incubated for 10 min. However, there are safety concerns for using this chemical compound which may have toxic effects on embryo development,60 but there has not been definitive evidence of anomalies in the offspring from either theophylline or pentoxifylline.61

LASER-ASSISTED SPERM SELECTION

In this technique, a direct laser shot is made at the tip of the sperm tail for approximately 2 ms. The viable sperm is identified by curling of tail following laser shot. Aktan et al.62 has reported increased fertilization and live birth rates compared with other randomly selected sperm. An advantage of the laser-assisted sperm selection is that it is less time-consuming without any chemical treatment involved. A major concern of the procedure is equipment cost and skilled laboratory personnel to perform it.

BIREFRINGENCE SPERM SELECTION USING POLARIZATION MICROSCOPY

The technique involves selecting viable mature sperms based on its birefringence characteristics such as orderly arrangement of nucleoprotein filaments in the protoplasmic texture of the nucleus and acrosomal complex. This aids the light wave to split into unequally reflected waves by an optically anisotropic medium (mature sperm). Significantly higher pregnancy rates were obtained when comparing with Hypo-Osmotic-Swelling-Test (HOST) in TESE-ICSI cases.63 In order to conclude, this technique lacks larger studies, and the cost of the equipment required for this technique is high. A pictorial depiction of sperm birefringence is described in Supplementary Figure 5 ^{(70.2KB, tif)} .

RAMAN SPECTROSCOPY-ASSISTED SPERM RETRIEVAL

Raman spectroscopy provides information about the internal structure of molecules through chemical fingerprints by laser scattering technique.64 This method assists in identifying seminiferous tubules with spermatogenesis in testes of humans and rats by Raman spectroscopy.65,66,67 Based on the current data, a probe specifically designed for this purpose is not fully developed. Further safety studies of this method are required to know about the laser-induced testicular tissue damage.

FUTURE ASPECTS

Several other routine processing techniques for ejaculate sample need to be investigated for their efficiency with testicular sperms.68 In order to overcome the limitations with the existing techniques, studies are carried out such as assessing the viability/motility of sperm obtained from spiral microfluidics combining with short-term culture69 and sperm DNA integrity interrogation noninvasively by Raman spectroscopy.70,71 Several recent studies in rats focused on distinguishing the seminiferous tubules with or without sperm in real-time with multiphoton microscopy (MPM) based on fluorescence difference in rats72 and full-field optical coherence chromatography using white light interference microscopy without any contrast or fixative.73 A novel approach on ICSI has been developed based on FLIM. FLIM is a powerful imaging technique that allows for the detection of molecular interactions and changes in the micro-environment of cells. FLIM has been explored for its use in ICSI to improve the selection of viable sperm and the success of fertilization. By detecting changes in the fluorescence lifetime of molecules within the sperm, FLIM can provide information on the metabolic activity of the sperm, such as the levels of nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD), which are indicators of sperm viability.74 While FLIM shows promise for sperm selection in ICSI, further research is needed to establish its efficacy, safety, and cost-effectiveness in the clinical setting.

Sperm freezing for TESE samples

Sperm extraction is a major indication for sperm freezing. In several ART clinics, the TESE procedure is scheduled on the day of egg retrieval in order to avoid using a frozen-thawed sample. Planning the TESE procedure on the day of oocyte retrieval poses a concern of backup plans. Sperm recovery is not always possible, and in such situations, cryopreservation of oocytes is considered. After oocyte cryopreservation, many patients never come back to use them with donor sperms, and hence, they are never used, which makes it a redundant intervention. An alternative option is to plan sperm extraction in advance and freeze the sample. This allows the couple to avoid the emotional, physical, and financial investment of the ovarian stimulation and egg retrieval in case of failure to find sperm cells, and other options such as the use of a sperm donor may be explored by the couple, and in many cases with IUI rather than IVF. Even in cases that TESE is synchronized with the oocyte retrieval, the surplus TESE sample not used for the ICSI on the day of oocyte retrieval/ICSI may be cryopreserved to avoid multiple sperm retrieval attempts in the future. The ICSI outcomes in men with NOA are comparable between fresh and frozen testicular sperm, and sperm cryopreservation should be considered even when very few spermatozoa are retrieved.75

Conventional slow freezing

The slow freezing method is a technique that has been traditionally employed for sperm freezing at ART clinics and is suitable for fresh ejaculates, surgically retrieved sperm, or testicular tissue. It involves the mixing of sperm with cryoprotectant that serves to lower the freezing temperature and prevent ice crystal formation. A very common cryoprotectant is TEST-yolk. It is added dropwise to the sample until a ratio 1:1 sperm to the freezing solution and the mixture is left in room temperature. Then, the mixture is loaded to cryovials or straws. The next step is to gradually lower the temperature, using a programmable freezer. Another option is to place the straws or vials at −20°C for 20 min and then liquid nitrogen vapor for 60 min. Finally, the vials or straws are transferred to liquid nitrogen at −196°C for storage. The major risks of the cryopreservation process are cell injury because of intracellular and extracellular ice crystal formation and osmotic damage due to extensive cell shrinkage or swelling. The use of high solubility molecules named cryoprotectants protects the cells from cryoinjury. Cryoprotectants are categorized as permeable, such as glycerol, ethylene glycol, and dimethyl sulfoxide and nonpermeable molecules that are sugar compounds.

Vitrification

Vitrification is an alternative process used to freeze sperm, where liquid is solidified into an amorphous or glassy state rapidly. In 2019, Isachenko et al.76 introduced vitrification without the use of cryoprotectants. Benefits of this method include the simplicity of equipment and the avoidance of the cytotoxicity of cryoprotectants. The vitrification technique has been found to be superior to slow freezing in terms of total and progressive motility.77 On the other hand, vitrification technique has several limitations. According to most vitrification protocols, a small volume of sperm is loaded to the vitrification device, to achieve higher cooling rates. This fact makes the freezing of large volumes of sperm not possible when employing the vitrification procedure. Additionally, direct contact of the sample with liquid nitrogen increases the potential risk of disease transmission. These problems may be overcome by the continuous evolution of vitrification carriers and the development of new strategies to avoid contamination. Currently, sperm vitrification is considered experimental by the 6^th edition of the WHO manual.78

Single sperm freezing

The conventional sperm freezing methods are not suitable for freezing and storing individually selected sperm. Single sperm freezing is an emerging technique that is ideal in cases of very limited sperm such as TESE samples. The technique was first developed in 1997 when Cohen et al.79 succeeded in cryopreserving single human spermatozoa in an empty zona pellucida. Since then, several nonbiological carriers have been developed that may contain a small number of sperm such as cryoloops, cell sleepers, culture dishes, and cryotops.80 Under the inverted microscope, a single or very few spermatozoa are transferred to the vitrification device in a small droplet of sperm washing and sperm freezing medium. Subsequently, the device is plunged directly into liquid nitrogen. A novel carrier that has been designed specifically for the vitrification of a small number of sperm is the sperm vitrification device (sperm VD). It is an efficient and simple carrier that allows a recovery rate of 96% post-thaw.81 After thawing, the sperm VD may be placed inside the ICSI dish, facilitating the process and reducing the searching time for motile spermatozoa. The novel CRYOPIECE is another new carrier, suitable for the preservation of testicular seminiferous tubules and testicular cell suspensions82 that has been shown to possess high recovery rate and retrieval rate after thawing.83

ARTIFICIAL INTELLIGENCE (AI) AND SPERM SELECTION FOR OPTIMAL REPRODUCTIVE OUTCOMES

Prior to concluding this review article, a short note on AI is essential. AI has emerged as a powerful tool in various fields, including ART. In the context of ICSI, AI technology has shown promising applications in sperm preparation. Traditional methods of sperm assessment and selection rely on manual evaluation, which can be subjective and time-consuming. AI-powered sperm sorting systems automate the procedure using machine learning algorithms by training on large datasets of successful fertilization outcomes. This intelligent sorting enables improved embryo quality and increased pregnancy rates.84 By monitoring and analyzing sperm samples during preparation, AI can detect abnormalities, such as clumping or debris and provide real-time feedback to optimize the process. This ensures consistent and reliable sperm quality for ICSI procedures.

While AI shows great promise in sperm preparation for ICSI, several challenges need to be addressed. Large-scale validation studies are required to confirm the reliability of AI-based systems. Moreover, ethical considerations regarding data privacy and algorithm transparency must be carefully addressed for widespread adoption.

CONCLUSION

There is room for optimization of sperm processing and selection while taking into consideration the impact of oxidative stress of sperm in fertilization, cleavage, and implantation rates. The use of more mild and sophisticated techniques such as MACS may benefit IVF outcomes even in female factor cases. Moreover, the design of new sperm vitrification devices and the addition of antioxidants in the cryopreservation media may improve sperm recovery rates after thawing and improve laboratory results in frozen TESE cases.

Overall, the selection of the andrology techniques to be used should be tailored to each individual case, taking into account patient-specific characteristics, the expertise and comfort of the laboratory team with the technique, and the potential use of pharmacological interventions. This facilitates thorough evaluation, and examination of male partners in infertile couples to ensure a suitable approach is taken.

The data presented in this review indicate the various advancements currently taking place in sperm processing, and selection methods can have a major impact on pregnancy outcomes and live birth rates. It is a laborious and time-consuming task for the embryologist to select the most promising sperm for ICSI. Hence, the recent machine learning approaches combined with data processing capabilities can potentially avoid the challenges associated with current sperm intervention and selection procedures in the future with more robust well-done studies to indicate the application of this technology.

AUTHOR CONTRIBUTIONS

KCM has helped writing the basic and advanced sperm processing techniques. CA has helped writing the processing of testicular sperm and their freezing techniques. FRP has helped in mentoring, designing, and proofreading. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Figure 1

Pictorial depiction of Piezo-ICSI technique. ICSI: intracytoplasmic sperm injection.

Supplementary Figure 2

Pictorial depiction of sperm selection by hyaluronic acid binding technique. PICSI: physiological ICSI.

Supplementary Figure 3

Pictorial depiction of intracytoplasmic morphologically selected sperm injection (IMSI) technique.

Supplementary Figure 4

Pictorial depiction of fluorescence lifetime imaging microscopy (FLIM) technique.

Supplementary Figure 5

Pictorial depiction of sperm birefringence with polarized light.