Technologies to improve sperm retrieval in men undergoing micro-TESE for NOA

Abstract

Nonobstructive azoospermia (NOA) is considered the most challenging clinical scenario for infertile men and current treatments leave many men unsuccessful at being able to achieve a pregnancy with their partner using their own sperm. Microdissection testicular sperm extraction (micro-TESE) is the choice for men with NOA desiring to father children with their own gametes. Micro-TESE results in the highest numbers of sperm cells retrieved for use with in vitro fertilization/intracytoplasmic sperm injection. With suboptimal micro-TESE success rates of sperm retrieval and then pregnancy and live birth using the retrieved sperm with in vitro fertilization/intracytoplasmic sperm injection, advances to improve outcomes are necessary. This article comprehensively reviews the technologies investigated to date to improve the outcomes for men undergoing micro-TESE.

INTRODUCTION

There have been significant advances in diagnostic and therapeutic technology and techniques in the field of male infertility in the last four decades. Nonobstructive azoospermia (NOA) is arguably the most complex clinical scenario requiring the highest level of technology for both partners to achieve a pregnancy with their own gametes. The advent of microdissection testicular sperm extraction (micro-TESE) in the late 1990s by Dr. Schlegel significantly improved the opportunity for many couples with NOA male partners.1 Although this advancement has allowed for improved outcomes and opportunities for couples who previously would not have had such opportunities with sperm retrieval rates reported between 50% and 71% with micro-TESE, there remains a significant percentage of failure.1,2,3 This significant failure rate in not identifying sperm is a major challenge through an arduous, labor-intensive microsurgery, which may be lengthy, with operative time commonly approaching and exceeding 2 h under anesthesia.4 There are also many cases where micro-TESE must be performed bilaterally if sperm are not identified in the first testicle that is explored. Bilateral surgery naturally results in a more uncomfortable and difficult surgical recovery. Further advancements in technology to help improve outcomes are needed to improve sperm retrieval rates, decrease operative and anesthesia time, and decrease the need for bilateral surgery. This review discusses the advancements in technologies up to date that have been studied in efforts to improve outcomes in men with NOA undergoing micro-TESE (Table 1).

Table 1.

Advantages and disadvantages of novel techniques for performing microdissection testicular sperm extraction

Technology	Principles	Advantages	Disadvantages
Metabolite spectroscopy5	Metabolites signal intensity that is analyzed by MR spectroscopy correlates with the success of sperm retrieval in micro-TESE	Noninvasive and inexpensive screening method	Small clinical studies Not widely used in clinical practice
Stepwise mini-incision micro-TESE6	Modification of micro-TESE technique using a stepwise approach by creating three small incisions, connecting the incisions, then proceeding to standard micro-TESE and contralateral testis in case of a failure	Shorter operative time, less invasiveness, and comparable surgical sperm retrieval rates	Small clinical study No objective measure on lower percentage of parenchymal damage
Robot-assisted micro-TESE7,8,9	A tri-view feature with a video link from the andrology laboratory microscope Multiple imaging modalities from robotic consoles	Real-time observation from laboratory microscope	No clinical study yet Expensive

MR: magnetic resonance; micro-TESE: microdissection testicular sperm extraction

NOVEL TECHNIQUES FOR PERFORMING MICRO-TESE

Metabolite spectroscopy prior to micro-TESE

A technology that has been explored to help plan to optimize sperm retrieval rates for micro-TESE in men with NOA is the use of metabolite spectroscopy prior to micro-TESE. Karakus and Ozyurt5 studied the relationship between choline (Cho) signal intensity measured prior to micro-TESE and sperm retrieval rates in 10 men with NOA. The signal intensities of Cho, creatine, lactate, and lipids were analyzed by magnetic resonance (MR) spectroscopy before micro-TESE. Sperm were then retrieved in 5 out of 10 of these men. The levels of these metabolites were found to be significantly lower in patients in whom sperm were not retrieved via micro-TESE. The Cho and creatine signal intensities were comparable to those of a fertile control group, indicating that a high Cho metabolite prior to micro-TESE may improve the odds of success with micro-TESE.5 Although this provides interesting data, it is in a very limited number of men. Further larger studies are needed to validate this concept.

Stepwise mini-incision micro-TESE

The actual surgical technique has been evaluated as another potential avenue for improving micro-TESE outcomes. Zhang et al.6 published a retrospective study assessing 665 men with NOA who underwent a stepwise mini-incision micro-TESE technique versus the 365 men with NOA who underwent standard micro-TESE. The stepwise mini-incision technique consisted of three small incisions of nearly 1 cm each, made in the equatorial region of the testis, which were examined under an operating microscope for more dilated seminiferous tubules to determine the presence of sperm. If no sperm were isolated, the 3 incisions were connected and transversely transected to proceed to a more standard micro-TESE, and this technique was performed in the contralateral testis if the first testis failed to reveal sperm. The primary goal was to minimize testicular parenchyma damage if sperm could be isolated through the first 3 mini-incisions. This study demonstrated shorter operative time, less invasiveness, and equivalent surgical sperm retrieval (SSR) rates when comparing mini-incision micro-TESE to standard micro-TESE cases.6 Although this study demonstrates some potential gains with this technique, it does not address if it truly minimizes parenchymal damage by any measurable parameter, which is one of the proposed advantages of the mini-incision micro-TESE technique.

Robot-assisted micro-TESE

The idea of robots assisting humans in tasks in society has been of great interest for decades. The idea of robots performing surgery for us has been of interest, and although that has not come to fruition at this time, it has been very commonplace for robots to assist surgeons with procedures every day across the country. The da Vinci system was initially developed for use in gross surgical procedures, and its use has advanced to the microsurgical platforms. It has primarily been used for microsurgical vasectomy reversal and varicocele repair.7,8,9 One of the potential advantages of the robotic platform in micro-TESE that has been described is the use of a tri-view feature with a video link from the andrology laboratory microscope to observe the tissue being evaluated microscopically in real-time to improve operative efficiency and to determine when to move to other areas of the testicular parenchyma to retrieve seminiferous tubules for assessment. As imaging modalities are developed, the use of multiple imaging modalities in robotic consoles may hold promise for improving outcomes.9 This seems to be primarily demonstrating proof of concept of feasibility, rather than improvement in objective outcomes with the current robotic technology that is available.

TECHNOLOGY TO IMPROVE IDENTIFICATION OF SPERMATOZOA AND SPERM SELECTION IN VIVO AND IN VITRO

Germ cell-specific proteins

One study used techniques of targeted proteomics, fluorescence microscopy, and flow cytometry to evaluate proteins specific to the testicle on the basis of the progression of germ cell differentiation. Spermatozoa acrosomes and tails expressed the late germ cell-specific proteins (ASPX)_HUMAN and A-kinase anchoring protein 4 (AKAP4)_HUMAN, respectively. In men with NOA, rarely, but morphologically intact AKAP4⁺/ASPX⁺/Hoechst⁺ cells were identified in semen pellets via flow cytometry. This finding may be useful as a noninvasive diagnostic test prior to micro-TESE in men with NOA.10 The lack of access to technology such as targeted proteomics, fluorescence microscopy, flow cytometry for use in a clinical setting, and the complexity of flow cytometry limits the feasibility of the use of this technology in clinical practice (Table 1).

Multiphoton microscopy

Multiphoton microscopy utilizes a near-infrared laser to induce autofluorescence to illuminate tissue in real-time, obviating the need for exogenous labels and processing of tissue processing. The ability of multiphoton microscopy to identify areas of spermatogenesis in the testes was assessed in a rat model. Rat testicular tissue was fixed and seminiferous tubular nuclei and F-actin were fluorescently labeled, and multiphoton microscopy was performed without sectioning the tissue. Normal rat tissue was compared to a cryptorchid rat model. Terminal deoxyribonucleotidyl transference deoxyuridine phosphate nick end labeling (TUNEL)-stained images were compared. The seminiferous epithelium was observed without sectioning of tissues by fluorescent probes and multiphoton microscopy. Active spermatogenesis could be observed by labeling F-actin either with or without fixation.11 Another rat model study used multiphoton microscopy to assess the stages of spermatogenesis in neonatal, pubertal, and adult rat testis tissues. Sperm DNA fragmentation was also assessed in tissues from sperm of different intensities to assess the possible risk of photo damage. The study reported that multiphoton microscopy was able to identify the stage of spermatogenesis in fresh testicular tissue without the need for exogenous labels. Tubules harboring sperm had lower ranges of autofluorescence than did Sertoli cell-only (SCO) tubules. There was very minimal sperm DNA fragmentation at the laser intensities required for this assessment.12 An ex vivo human tissue study used multiphoton microscopy to correlate areas of spermatogenesis with histology.13 In this study, 7 men with either normal or abnormal spermatogenesis had tissue from testicular biopsies imaged by multiphoton microscopy, and then the results were subsequently correlated with histology in a blinded fashion. An 86% concordance rate was reported between multiphoton microscopy and histology. The seminiferous tubules in NOA men were smaller than those in controls which also correlated histologically as well.13 Although this offers appeal with no need for exogenous labels or tissue processing, clinical in vivo studies involving the translation of these data are needed to prove that multiphoton microscopy can be used in real-time during micro-TESE to aid in the identification of sperm.

Raman spectroscopy

Raman spectroscopy uses vibratory technology to reveal chemical structures by relying on scattered light patterns which are emitted when a diode laser contacts a sample.14 Raman spectroscopy has been utilized in a murine model of busulfan-induced NOA to identify seminiferous tubules that harbor sperm versus SCO tubules compared to a control group treated with a placebo saline dose rather than busulfan. The testicular tissues of the NOA group and the control placebo group were assessed by Raman spectroscopy and then subsequently confirmed by histopathology. Raman peaks were more intense in tubules of men with ongoing spermatogenesis than in tubules of men with SCO, demonstrating the ability to distinguish the 2 groups with a sensitivity of 91.2% and a specificity of 82.9% by this technology.15 Translation from animal studies to human clinical studies is necessary before this technology would be considered for clinical use.

Full-field optical coherence tomography

Full-field optical coherence tomography institutes while light interference microscopy to create rapid high-resolution tomographic Images. The use of this technique offers the technical advantage of not requiring processing or staining of the fresh tissue. When using a light source without a laser, it should not induce cellular mutagenesis or photo damage of cells. Testicular tissues were extracted from a busulfan-induced NOA rat model to compare to testicular tissues extracted from a normal control rat model, and ex vivo full-field optical coherence tomography was performed. Spermatogenesis was successfully identified within the seminiferous tubules and the NOA model exhibited seminiferous tubules heterogeneous in size and shape, while the fertile control exhibited uniform tubular shape and size.16 This technology offers the appeal of not necessitating processing or staining of fresh tissue; however, well-performed human clinical studies are needed prior to any consideration for use in clinical practice.

Confocal fluorescence microscopy

The use of fluorescein isothiocyanate (FITC)-conjugated mouse anti-human acrosomal Hs-14 antibody to label sperm was established in 2009. The label was injected at sites of exposed seminiferous tubules in 13 fertile and 8 sterile mice, and multiphoton microscopy was successful at visualizing sperm with fluorescent labels.17 Smith et al.18 published a study using a busulfan-induced NOA mouse model in which FITC-labeled antibody against Hs-14, an intra-acrosomal protein, was microinjected in a retrograde fashion through the rete testis to deliver the label into the seminiferous tubules. In vivo fiberoptic confocal fluorescence microscopy was performed in the anesthetized model to detect foci of spermatogenesis. Immunofluorescence microscopy was performed on the seminiferous tubules that were extracted from the areas of detection of sperm by confocal microscopy to confirm the presence of sperm after the tissue was squash crushed.18 Translation from animal model studies to human clinical studies is necessary prior to consideration of this technology for clinical use.

ORBEYE 4K-three-dimensional (3D) microscope

The use of an operative microscope was first introduced in the field of urology for microsurgical vasovasostomy in the 1970s.19 The remaining challenges associated with the operative microscopy include poor ergonomics, a narrow field of view, and the need for frequent repositioning. In 2017, the 4K-3D ORBEYE video microscope (Olympus, Center Valley, PA, USA) was launched. The ORBEYE is composed of a 4K-3D exoscope attached to a semirobotic arm that is positioned over the surgical field at 26× magnification and with autofocus features. This device offers ergonomics that are natural and nonstatic. Ergonomics and surgeon comfort have been demonstrated in several studies.20,21 The ORBEYE was shown to perform at a high level during micro-TESE.20 The financial investment in such a microscope limits its accessibility to most microsurgeons.

Intraoperative Doppler ultrasound, contrast-enhanced Doppler, and grayscale ultrasound

The ability of the resistive indices of intratesticular arteries to assess pathological spermatogenesis was investigated via color Doppler ultrasound. This study suggested that men with pathological spermatogenesis demonstrated a greater resistive index (>0.6) than men with normal spermatogenesis. There may be an opportunity to apply this approach to men with NOA and to optimize the outcomes of micro-TESE.22 Contrast-enhanced ultrasound (CEUS) offers the advantage of visualizing the testicular vasculature and assisting in the identification of the areas with the greatest perfusion which may be the most likely to harbor sperm. CEUS was used to guide micro-TESE in 187 testicular units of 120 men with NOA. The surgical sperm retrieval rates were compared between the areas with the highest perfusion and those with the lowest perfusion. The study assessed arrival time, the time from contrast injection to visualization of the first contrast bubbles in the testicle, as well as the time to peak intensity after injection of the contrast agent, and the peak intensity, which represents the maximum number of bubbles in the capillaries, as predictors of outcomes. The study suggested using contrast-enhanced ultrasound preoperatively can be advantageous in identifying the highest areas of intratesticular perfusion. In the regions of high perfusion if an arrival time of ≤27 s, a time-to-peak intensity of ≤45 s, or a peak intensity of ≥11 dB are demonstrated, the patient would be considered as a good candidate for micro-TESE with high expectations for SSR.23 In a retrospective study of 806 men who underwent micro-TESE, grayscale ultrasound imaging was performed to assess seminiferous tubule size. The results were found to be predictive of sperm retrieval with micro-TESE in patients with a size ≥250 µm; however, the sperm retrieval rate was only 29.8%.24 Another study suggested that grayscale ultrasound can identify dilated seminiferous tubules within the testis prior to micro-TESE for surgical planning.25

Intraoperative laser Doppler-assisted TESE

A study including 12 patients with NOA examining 40 biopsies from 20 testicles underwent perfusion mapping using color Doppler ultrasound, with areas of different levels of perfusion being marked with needles. After incising the testes, laser Doppler flowmeter examination was performed with biopsies taken for retrieval of sperm as well as for histology. Retrieved sperm were prepared for clinical use with intracytoplasmic sperm injection (ICSI). From the 40 biopsies, the tissue was analyzed for the presence of sperm, quantification of sperm, and assessment of sperm quality. Sperm quality and quantity are positively correlated with regions of greater perfusion.26 Higher powered studies to confirm these findings would be useful.

Preoperative magnetic resonance imaging (MRI)

MRI has been evaluated for its ability to evaluate testicular volume, apparent diffusion coefficient, and the magnetization transfer ratio as potential predictive factors for SSR with micro-TESE in men with NOA.27 Larger testicular volumes on MRI were positively correlated with successful SSR, and a higher apparent diffusion coefficient and magnetization transfer ratio were negatively correlated with success.28 The significant financial cost of MRI may be a deterrent for the use of this preoperative diagnostic tool.

Artificial intelligence (AI)

The use of AI to enhance sperm selection has gained traction. AI is a computer technology with the ability to process and complete human-like tasks. This approach is particularly useful for interpreting large sets of data when a small margin of error is required. Parameters for the selection of sperm such as motility and morphology may be evaluated and characterized by AI. Although this technology holds promise, there is not currently the ability for AI to correlate information with sperm retrieval.14

Deep convoluted neural networks

Wu et al.29 studied on a computer-aided sperm analysis (CASA) system that was developed to use deep learning to automate the identification of sperm in TESE samples. A total of 702 testicular biopsy samples were collected from 30 patients. The images were normalized, passed through glare filters, and subjected to diffraction correction. A deep object detection network was then trained on this dataset. The results were then subsequently compared to those of embryologists at detecting sperm with the findings via deep learning-based technology improving, which has improved the efficiency and the isolation of sperm in testicular biopsy samples.29

MR spectroscopy

Ntorkou et al.30 reported on the use of proton MR spectroscopy in the testes of men with NOA to detect testicular metabolite differences among different histological stages of NOA and as a predictor of the presence of sperm prior to micro-TESE. This prospective study revealed reduced normalized concentrations of total choline, myo-inositol, and lipids in the testes of 40 men with NOA in comparison to 50 age-matched controls. In NOA men with histological diagnoses of maturation arrest or a SCO pattern, there were significantly lower normalized concentrations of macromolecules and lipids in comparison to those with a histologic diagnosis of hypospermatogenesis. Moreover, men with NOA who failed to have sperm retrieved at the time of micro-TESE were also found to have significantly higher normalized concentrations of glutamate. MR spectroscopy may be used to gain testicular metabolic data prior to micro-TESE.30

Narrow band-imaging (NBI) system

NBI uses white light filtered into blue and green wavelengths that are absorbed by hemoglobin and penetrate the surface of tissue. An animal study evaluated 5 rats that were administered busulfan to create a NOA model. As it has been demonstrated in human studies that areas of higher levels of perfusion within the testis are correlated with areas of spermatogenesis in men with NOA, NBI was used in a rat model of NOA to identify areas of spermatogenesis via visualization of microvasculature within the rat testis.31 This would need to be replicated in human clinical studies to gain traction for potential use in clinical practice.

Ultrasonically actuated silicon microprobe-based testicular tubule metrology

Micro-TESE surgical technique is based on identifying slightly more dilated seminiferous tubules that are more likely to harbor sperm than the thin, flat seminiferous tubules comprising the majority of the testicular parenchyma in men with NOA. Therefore, technologies to help identify the more dilated tubules beyond simple microscopic visualization may help improve the microsurgeons’ ability to achieve successful results. An animal study assessed the use of an ultrasonically actuated silicon microprobe-based testicular tubule assay.32 Microfabrication of a silicon microprobe integrated with an ultrasonic horn actuator and gauges for strain levels was performed. Axial force-sensitive polysilicon strain gauges integrated into the microprobe detected the boundaries between seminiferous tubules enabling the measurement of individual tubule diameters. Rat experiments confirmed the ability of the microprobe to estimate the average tubule diameter of the sperm harboring tubules and to sense the presence of larger tubules buried among thinner tubules.32 Translational human studies are necessary prior to consideration for clinical use.

SPERM SORTING/IDENTIFICATION TECHNOLOGY FOR MICRO-TESE SPECIMENS

Following surgical sperm retrieval, additional laboratory techniques need to be performed to identify and sort the highly functional sperm from nonfunctional sperm and other debris for in vitro fertilization (IVF)/ICSI.33 This is essential as testicular spermatozoa oftentimes are low in numbers and have poor to no motility. Following this step, sperm can be retrieved that have suitable quality for assisted reproductive technology (ART) use and thereby increase the chance of ART success. Sperm sorting and identification can be performed using mechanical, enzymatic, or other novel and more advanced techniques (Table 2).

Table 2.

Advantages and disadvantages of technology to improve identification of spermatozoa and sperm selection in vivo and in vitro

Technology	Principles	Advantages	Disadvantages
Germ cell-specific proteins10	Evaluate proteins specific to the testicle on the basis of the progression of germ cell differentiation	Noninvasive diagnostic test	Limited availability of flow cytometry Difficult Small clinical study
Multiphoton microscopy11,12,13	Near-infrared laser to induce autofluorescence to illuminate tissue and identify areas of spermatogenesis	Real-time	No clinical study yet Incorrect photo power may result in the damage of spermatozoa
Raman spectroscopy14,15	Creating a chemical footprint that relies on scattered light patterns to differentiate between seminiferous tubules that have sperm and those that do not	Noninvasive diagnostic test	No clinical study yet Unknown safety profile in human samples
Full-field optical coherence tomography16	Rapid high-resolution tomographic images created from light interference microscope to identify spermatogenesis within seminiferous tubules	Do not induce cellular mutagenesis or photo damage of cells	No clinical study yet
Confocal fluorescence microscopy17,18	Visualization of sperm with fluorescent labels	Rapid sperm detection	No clinical study yet Limited availability of equipment Unknown safety profile in human samples
ORBEYE 4K-3D microscope20,21	4K-3D exoscope that is attached to a semirobotic arm that is positioned over the surgical field at 26× magnification and with autofocus features	Ergonomics and surgeon comfort	Expensive, cost-efficacy unknown
Intraoperative Doppler ultrasound22	Visualizing the testicular vasculature	Easy Noninvasive	Limited clinical studies
Contrast-enhanced Doppler23	Use resistive indices of intratesticular arteries to assess pathological spermatogenesis	Easy Noninvasive	Limited clinical studies
Grayscale ultrasound24,25	Visualization of dilated seminiferous tubule	Easy Noninvasive	Limited clinical studies
Intraoperative laser Doppler-assisted TESE26	Laser Doppler flowmeter examination was performed to assess levels of perfusion in the exposed tissue during TESE	Easy Large availability of equipment	Limited clinical studies
Preoperative MRI27,28	Diffusion coefficients and the magnetization transfer ratio in MRI as potential predictive factors for SSR	Easy Large availability of equipment	Expensive, cost-efficacy unknown
Artificial intelligence14	Interpreting large sets of data to develop information with sperm retrieval	Different datasets may have in different result (difficult generalizability)	Limited clinical studies
Deep convoluted neural networks29	A CASA system that was developed to automate the identification of sperm in TESE	May improved the efficiency	Limited clinical studies
MR spectroscopy30	Metabolites signal intensity that is analyzed by MR spectroscopy correlates with the success of sperm retrieval in micro-TESE	Noninvasive and inexpensive screening method	Small clinical studies Not widely used in clinical practice
Narrow band-imaging system31	Identify areas of higher levels of perfusion within the testis using white light filtered into blue and green wavelengths	Simple Low cost Relatively safe	No clinical study yet
Ultrasonically actuated silicon-microprobe-based testicular tubule metrology32	Identifying slightly more dilated seminiferous tubules using microfabrication of a silicon microprobe	Noninvasive May avoid the need of large incision with a scalpel to examine the tissue	No clinical study yet

TESE: testicular sperm extraction; micro-TESE: microdissection TESE; MR: magnetic resonance; MRI: magnetic resonance imaging; SSR: surgical sperm retrieval; CASA: computer aided sperm analysis; 3D: three-dimensional

Enzymatic tissue digestion

Intraoperative wet preparation analysis of testicular tissue samples after cutting and putting into suspension may still result in unidentifiable sperm. To increase the possibility of finding sperm, enzymatic digestion in the laboratory can be performed. Aydos et al.34 in 2005 studied testicular tissue samples from 177 men with NOA that were retrieved by micro-TESE. After mechanical searching through shredded biopsy fractions under the microscope for around 30 min, sperm were detected in only 36% of cases. With the additional enzymatic digestion by DNase and collagenase type IV, sperm were successfully detected in 23% more cases, yielding a total of 57% rate sperm recovery rate.34 This rate is similar to a previous study in 1998 that revealed sperm in seven out of 27 cases where spermatozoa were not detected in the initial mechanical search.35 The addition of enzymatic digestion was considered not only to increase the sperm recovery rate but also to shorten searching time, minimize cellular damage, and maintain clinical pregnancy rates compared to those who only undergo mechanical searching to isolate sperm.34

A larger study by Ramasamy et al.12 analyzed 1054 men with NOA retrospectively. After extensive mechanical searching, if no sperm were found, the testicular tissue samples were processed overnight to allow the enzymatic digestion to occur. Sperm were then searched for 1 h on the next day. In this study, an additional 7% sperm detection rate was obtained after employing enzymatic tissue dissection, resulting in a total sperm retrieval rate of 59.5%. They also found that the detection of germ cells intraoperatively could predict successfully finding sperm in laboratory processing.12

Microfluidics: dielectrophoretic cell sorting, spiral channel sorting, and pinched flow fractionation

Microfluidics technology is a technique that utilizes fluid physical dynamics in a various range of channels to assist in rapid and efficient sperm sorting. The majority of this technology relies on sperm motility, but recently, many novel microfluid techniques have been developed to isolate sperm rapidly from other cells irrespective of motility. In conventional microfluid devices, the channel is straight to allow a parallel laminar flow with two streams. In this approach, motile sperm will gather in one specific area or stream, separated from other cells, due to the attraction from substances, temperature differences, and others depending on the principles of microfluid technique used. Microfluidic sperm sorting that relies on motility includes micro-obstacle course, chemotaxis, linear velocity/stream swim over, swimming into microchannel, and thermotaxis.36

Finding nonmotile sperm is sometimes needed in cases of patients with NOA, especially to reduce the probability of sperm loss as the system heavily depends on sperm motility. More importantly, in the sample from TESE, the goal is also to find several nonmotile but viable spermatozoa. To be able to sort viable nonmotile sperm, one technique that can be used is dielectrophoretic cell sorting. While it uses a similar two-laminar stream design in the devices, there is an external energy source that directs cells along the field gradients. With the differences in cell size and charges, sperm can be isolated away from other debris. A study has shown that the head and tail of sperm have a unique and independent electrical frequency corresponding to the negative and positive forces from dielectrophoretic.37 Promising results were reported in previous studies which were mainly in animal studies. Huang et al.38 reported an increase of fertilization by about 5% in the group that used positive dielectrophoretic force from indium tin oxide glass electrodes to manipulate mouse sperm and oocytes. Microfluidic dielectrophoretic sorting has also successfully been used to sort bovine Y and X- chromosome sperm in a proof-of-concept study.39 Further research is needed to optimize dielectrophoretic technology for automated human sperm sorting.

The other technique is using spiral microfluidic channel. The idea is that the fluid inertia within the spiral channel causes cells to move laterally until equilibrium is reached. Differences in shapes and sizes of sperm and other cells result in separation in the outlet channel.36 This technique successfully separated sperm from white blood cells in pyospermia samples, with an end result of 83% of sperm cells collected.40 Despite being fairly inexpensive, there was a concern about leaking in the system as a result of high pressures.

Samuel et al.41 in 2020 also studied spiral channel sorting with a modification. Some of the features of the microfluid system in that study were the use of low magnitude and short period of force in the spiral channels; therefore, it minimized the risk of DNA damage and oxidative stress. It also had enrichment steps to the sample at the end by omitting excessive media of the sperm. They found that more than 13 sperm per minute could be detected using this technique, which was almost 9-fold higher than the conventional processing. Nevertheless, they still found that at a certain high concentration of debris and cells other than spermatozoa, the enrichment step was prone to leak. Therefore, further optimization of system prototyping is still needed.41

Pinched flow fractionation is another microfluidic technology that uses fluid to align the cells into one side wall as it streams along the microchannel with a pinched segment. Afterward, the fluid flow profile will be spread out in the channel and the cells will be separated based on their density and diameter. The process can be modified by adjusting the stream flow, shape of microchannel, and the location of outlets to collect the separated cells. Berendsen et al.42 reported successful retrieval of 95% of spermatozoa in a sample that only contained 2.5% of spermatozoa. Moreover, up to 90% of red blood cells were effectively separated using pinched flow fractionation technology. This technique has also been proven effectively separate sperm from epithelial cells at a low cost in the case of sexual assault.43 Further research is needed to evaluate the use of this technique to select sperm after TESE, particularly because there is some concern regarding mechanical clogging.36

Fluorescence-activated cell sorting (FACS)

FACS is a type of flow cytometry that actively sorts cells based on the detected fluorescent labeling on the surface of the cells. As each cell population has specific fluorescent antibodies or DNA labels, FACS can effectively sort a single cell population without any interference from the other cells. Multiple cell populations can typically be separated using FACS at the same time, increasing experimental diversity and efficiency. Nevertheless, the use of FACS for sperm sorting from tissue taken after micro-TESE has not been studied extensively. A pilot study of 10 patients (2 specimens of normal semen samples from cadavers and the remaining from micro-TESE testicular tissue) by Mittal et al.44 in 2017 used DNA-stains To-Pro-3 of SYTO17. FACS was then employed to select spermatozoa, which later was confirmed using standard light microscopy. Four out of 8 patients who underwent micro-TESE had spermatozoa retrieved, including those with maturation arrest who had a negative micro-TESE. More research is needed to provide clearer evidence on the use of FACS for sorting spermatozoa beyond this small pilot sutdy.44

Magnetic-activated cell sorting (MACS)

Similar to FACS, MACS used antibody-coated magnetic beads that bind to antigens on the sperm cell surface to sort cells. In a recent meta-analysis of five prospective studies, the use of MACS for sperm selection was shown to significantly increase pregnancy rates with a risk ratio of 1.50 (95% confidence interval [CI]: 1.14–1.98) when compared with density gradient centrifugation (DGC) and swim-up techniques, but there were no differences on implantation and miscarriage rates.45 Nevertheless, although MACS seems to be a feasible choice for isolating undamaged sperm, its effectiveness is restricted to a generally uniform sperm sample and has not been investigated for nonmotile sperm from micro-TESE.36 More well-designed studies are needed to confirm the efficacy and safety of MACS. Clinicians should exercise caution when recommending the use of MACS for ART. It is important to have a thorough discussion with each couple, providing them with relevant information regarding the limited evidence and lack of consensus on the impact of MACS on results.46

Physiological ICSI (PICSI), zeta sperm selection, and SpermSlow

Several other advanced sperm selection techniques are available, such as hyaluronic acid-based sperm selection and zeta sperm selection. Hyaluronic acid is the primary component of the extracellular matrix at the outer layer of oocytes. Mature spermatozoa with intact plasma membrane, complete cytoplasmic extrusion, and nuclear maturation can bind and digest the hyaluronic acid to reach oocyte and fertilize. Hyaluronic acid is a more natural and physiological alternative in sperm selection technology. PICSI and SpermSlow are two systems that incorporate hyaluronic acid for selecting sperm. PICSI employs a hyaluronic acid culture dish, in which the mature spermatozoa will bound to the bottom of the dish. In contrast, SpermSlow is a viscous medium containing hyaluronic acid. In this medium, mature spermatozoa appear slow. A study found that both PICSI and SpermSlow result in comparable efficiency in selecting functional spermatozoa that subsequently result in a good-quality embryo rate.47 Nevertheless, the evidence is still very limited and needs further research.48

Zeta sperm selection is based on the charge in the surface of sperm. Normally differentiated negatively charged sperm are rapidly separated and collected to a specific chamber as a result of the applied current. Compared to conventional ICSI, the use of zeta sperm selection can result in an increased live birth rate based on a single study which requires higher-level evidence to confirm.48 Moreover, the use of zeta potential was also reported to be associated with increased good-quality embryos and pregnancy rates when combined with DGC technique compared to DGC alone. Both techniques seem to have the same efficacy in selecting sperm with intact DNA integrity and also with sperm telomeres lengths.49

ROLE OF THE INTRATESTICULAR MICROBIOME AS A POSSIBLE TARGET IN NOA

Microbiome has been studied recently in association with a number of physiological processes including hormone production and spermatogenesis. Seminal microbiome characteristics are different between men with idiopathic NOA, OA, and normal fertility. Campbell et al.50 found that Proteobacteria and Firmicutes were underrepresented in men with NOA compared to men with normal fertility. Eschericia, Shigella, Sneathia, and Raoutella were also significantly different between the two groups.50 A study by Chen et al.51 also found that the semen of men with NOA and OA had less microbes compared to those of healthy controls. While the Proteobacteria and Actinobacteria were decreased in azoospermic men, Chen et al.51 found an increase in Firmicutes and Bacteroidetes in these subsets of men.

Using a testicular specimen, it was found that the sample of negative sperm at micro-TESE had an increase of Actinobacteria, a decrease in Firmicutes and Clotridia, as well as a complete absence of Peptoniphilus asaccharolyticus.52 In a recent systematic review and meta-analysis of sperm function and male infertility, it was found that Ureaplasma urealyticum, Enterococcus faecalis, Mycoplasma hominis, and Prevotella negatively impacted semen parameters, whereas Lactobacillus appeared to protect sperm quality.53

Alteration of the microbiome in the semen of men with NOA results in changes in the metabolic pathways including a decrease of microbial degradation of toluene, increased metabolism of fructose or mannose, and the direct harmful effect of microbes on the sperm cells. Certain microbes in men with azoospermia will add an increased risk of metabolic, infectious, and immune diseases.54 With the incongruence of the findings, more research is still needed for a robust and conclusive finding that can facilitate development of therapy for infertile men, including those to improve micro-TESE outcome for azoospermic men.

Aside from the seminal microbiome, gut microbiota also played a great role in spermatogenesis and also sex hormones, including testosterone and estrogen. Gut microbiome plays a role in the metabolism of dihydrotestosterone and testosterone. It can result in extremely high free levels of the most potent androgens. Additionally, gut microbiomes can provide a supportive environment of spermatogenesis by regulating testicular immunity and the integral structure of testes. Men with normal and abnormal semen parameters had a different microbiota in the intestinal and genitourinary.55

Cao et al.56 discovered that the gut microbial profiling of men with NOA showed significantly higher levels of Bacteroides vulgatus and Streptococcus thermophilus. Conversely, the control group had higher levels of Bacteroides thetaiotaomicron and Parabacteroides sp. CT06. Additionally, they discovered a significant association between specific bacteria species, such as Acinetobacter johnsonii, and clinical indicators such as testosterone and follicle-stimulating hormone (FSH).56

A study in mice found that an alteration of gut microbiota through fecal microbiota transplantation can improve spermatogenesis, especially for its concentration and motility.57 Further research is needed if we can utilize the characteristics of microbiome for creating probiotics or even for diagnosis or prognosis efforts.

LIKELY FUTURE DIRECTIONS

The future directions of micro-TESE technology will include an improvement in surgical techniques and tissue processing. High-resolution optics can enable a higher precision, better visualization, and lower complications during micro-TESE.14 A 4K-3D videomicroscope may also be developed for micro-TESE procedure. The 4K-3D videomicroscope resulted in nonsignificantly shorter operative time and better ergonomics for operating room staff in varicocele repair.58 More research on its efficacy for micro-TESE should be conducted. Technological advancements in surgery should also be coupled with high levels of sample preparation and sperm selection in the laboratory. Aside from the techniques that have been described, AI can be developed to assist in sperm identification and selection for better outcome of ART.59

Limitations to the current review include that the animal studies included are not translatable to human results and need further validation, as well as a number of the studies discussed include a limited number of cases and should be validated with larger studies.

CONCLUSIONS

An unacceptable percentage of men with NOA who undergo micro-TESE fail to have sperm retrieved, and when sperm is retrieved, ART success rates are suboptimal. Technological advancements are needed to help alleviate these challenges. Although a diverse spectrum of technological advances have been described in this review, none have come to the forefront of the science with efficacy that has allowed for implementation into clinical practice as standard of care or to the level to be included in societal guidelines for best practice. Further research is needed to advance this most challenging of areas in reproductive care.

AUTHOR CONTRIBUTIONS

PKK contributed to design, literature search, and writing the manuscript. WA performed literature search and wrote the manuscript. GMP contributed to critical review and revision. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.