Andrology laboratory technique for analysis of semen in men with azoospermia

Abstract

Discovery of spermatozoa during the 17^th century led to developing technologies for semen analysis in the early 1900s, and then, standard techniques were implemented during the 20^th century. Semen analysis has a pivotal role in the male infertility evaluation, and azoospermia is an important finding. Azoospermia is identified in 15% of infertile men. However, the accurate laboratory assessment of azoospermia poses certain technical challenges. Laboratories currently perform semen assessment with great variability; thus, a standard method should be used. Planning suitable management and determining the cause of infertility require a precise evaluation of azoospermia. This review aims to address the definition of azoospermia and highlight laboratory methods in the assessments of azoospermia. Basic methods such as centrifugation, repeat pellet analysis, and staining and advanced methods such as genetic testing and biomarkers have been discussed. These methods have helped in standardizing the protocol for accurate azoospermia assessments with less variability.

INTRODUCTION

Around 300 years ago, spermatozoa were first discovered by Johan Ham. He observed “animalcules” under the light microscope in human ejaculates.1,2,3 This finding was then confirmed by Anthony van Leeuwenhoek,1,3 and then, he described the general characteristics.2 In 1824, Jean-Louis Prévost and Jean-Baptiste-André Dumas provided evidence that eggs were fertilized by “animalcules” from the spermatic fluid.1,2 These “animalcules” were later termed “spermatozoa” whose absence led to infertility.

The number of cases of male factor infertility has been rising in recent years. Azoospermia, defined as the complete absence of sperm from the entire ejaculate, is the most severe type of male infertility.4 The term “azoospermia” and its diagnosis have been the focus of debate over the past decade. The definition was first put forth by Eliason in 1981 and was adopted by the World Health Organization (WHO) 5^th edition manual: “no spermatozoa are found in the sediment of a centrifuged sample”.5 A more thorough description was provided by the American Urological Association (AUA): “no sperm after centrifugation at 3000g for 15 min and examination of the pellet”.6 To enhance the likelihood of detection, rare sperm might be concentrated into a tiny pellet using centrifugation. This was shown to be important by Jaffe et al.7 They revealed that they were able to find sperm after centrifugation in 29 samples out of 140 samples that were considered azoospermic by routine semen analysis, and some of them were motile sperm. The purpose of semen pellet investigation is to detect the existence of a limited number of living sperm in the centrifuged pellet. The discovery of sperm in the centrifuged pellet is referred to as “cryptozoospermia”.5 Instead of being a clinical diagnosis, azoospermia and cryptozoospermia continue to be descriptions of the ejaculate. The semen analysis should be repeated with at least two semen samples obtained a few weeks apart to confirm azoospermia.4 It is especially crucial to accurately evaluate extremely low sperm counts to prevent classifying males who are severely oligospermic as azoospermic.

Azoospermia is identified in 15% of infertile men. This condition may occur due to impaired spermatogenesis, reproductive tract obstruction, or inadequate hormonal stimulation.8 Evaluation of physical and laboratory data, such as serum follicle-stimulating hormone (FSH) levels, bilateral vas deferens existence, testicular volume, and semen volume, is essential for formulating a management plan. Here, we focus on precise diagnostic methods that an andrologist/urologist must be aware of.

A literature search from Scopus and PubMed regarding azoospermia and laboratory techniques articles published until October 2023 was conducted and a summary was composed into a comprehensive narrative review of literature. A set of input terms were utilized to generate the search strategy: “centrifugation”, “cryptozoospermia”, “novel”, “semen analysis”, “laboratory technique”, “pellet analysis”, or “azoospermia”. A language limitation was set to only English literature.

GENERAL STEPS OF SEMEN ANALYSIS

According to the WHO 6^th edition manual,9 semen analysis (SA) consists of the following steps: (1) preexamination procedures and initial semen handling; (2) macroscopic evaluation (assessments of semen volume, pH, appearance, odor, liquefaction, and viscosity); and (3) microscopic evaluation (assessments of wet preparation, sperm concentration, sperm motility, sperm morphology, sperm vitality, and sperm pellet analysis).

Preexamination procedures and initial semen handling consist of several procedures: giving information to the patients regarding how to collect and transport the semen samples, the ideal abstinence period, names of patients, the date of sample taking, and the completeness of the sample.9 Brief history of the patient’s condition and medication history should be considered because some conditions and medications can affect the number of sperm in the ejaculate.10 Sometimes, in the laboratory orders, clinicians write a brief of information regarding the patients condition and history, but it is also advisable for the laboratorians to ask directly to the patients to make sure about their condition and history. The patient’s identity must be put on the container, and it needs to be double checked. Errors in these steps may lead to a false finding of azoospermia if the sample has been collected improperly.

In macroscopic evaluation, the assessed parameters included sample volume, ejaculate pH, appearance, odor, liquefaction, and viscosity. For sample volume, the WHO 6^th edition manual states the reference limit for semen volume as 1.4 ml.9 An abnormally low value may be the result of incomplete semen collection, agenesis of the Wolffian ducts, obstruction in the male reproductive system at the level of the ejaculatory ducts, hormonal abnormalities such as testosterone deficiency, or partial retrograde ejaculation.11,12 It can also happen when the collected sample is incomplete; in this case, the fraction that was not collected should be reported since such an analysis may be unreliable. It should be noted which fraction was lost. The first fraction is rich in sperm, and then, the number of sperm is decreasing in the next sequential fraction, and the last fraction is mainly comprised of fluid from the seminal vesicles.6 The SA should be repeated with a complete sample collected. Complete sample collection with low semen volume and no sperm found is most commonly due to congenital bilateral absence of the vas deferens (CBAVD), and occasionally due to ejaculatory duct obstruction. Furthermore, the WHO states that the pH of the semen sample should be higher than 7.2. The final pH of the semen sample is a combination of alkaline seminal vesicle secretions and acidic prostatic secretions.13 If the pH is below 7.2, it suggests the possibility of seminal vesicle aplasia, obstruction in the ejaculatory duct, or contamination with urine. Conversely, if the pH is above 8, it may be due to chronic prostatitis or related to poor sample handling.13 pH paper is commonly used to measure pH and should have a range from 6 to 8 with small intervals for accuracy. The color standard should always be calibrated.5,9,14 Once the sample liquefies, the pH should be measured as soon as feasible; ideally, this is within 30–60 min. A delay of pH testing may lead to an inaccurate result due to the loss of CO₂. Finally, with regard to the sample’s appearance, odor, liquefaction, and viscosity, the WHO states that semen will typically coagulate after ejaculation due to proteins from the seminal vesicles and subsequently liquefy under the effect of prostatic enzymes. In the case of azoospermia due to agenesis of seminal vesicles, the semen will not coagulate and will always remain liquid.

In addition, in the event of azoospermia, the microscopic examination consists just of wet preparation and sperm pellet analysis, even though assessments of sperm concentration, motility, morphology, vitality, and sperm preparation are all included in the entire set of microscopic evaluation. Two wet preparations must be made for double check. If no spermatozoa are seen, then the replicate should be assessed in the same manner. If the replicate confirms the absence of spermatozoa, the entire ejaculate should be centrifuged to confirm azoospermia.5,9 It is generally accepted that the term azoospermia can only be used if no spermatozoa are found in the sediment of a centrifuged sample. It is important to keep in mind that: (1) centrifugation at 3000g for 15 min is unable to sediment all spermatozoa from a sample, and (2) whether spermatozoa are identified within a pellet is dependent on the centrifugation time and speed as well as how much of the pellet is analyzed. Viewing samples under low magnification gives a summary of the sample, indicating the presence or absence of spermatozoa. Assessing the wet preparation under higher magnification permits assessment of the “round cells” that are present (leukocytes and immature sperm) and cells other than spermatozoa (e.g., epithelial cells).

METHODS OF DIAGNOSING AZOOSPERMIA IN LABORATORY

One of the methods used to assess azoospermia in the laboratory is sperm pellet analysis. Sperm pellet analysis is performed by centrifuging the entire ejaculate. Centrifugation is used to concentrate rare sperm from the semen into a small pellet, which may indicate the occurrence of cryptozoospermia. The prognosis of men with cryptozoospermia is much different from those with true azoospermia, so this is an important distinction to make.

The steps of sperm pellet analysis start with centrifuging a well-mixed semen sample. After that, the supernatant is discarded, and the pellet is resuspended. The wet preparations for assessment require one or two samples (10 μl each). A phase-contrast microscope is used to assess the wet preparate, and observation should be in a zig-zag pattern of examination.

Centrifugation time and speed can increase the likelihood of identifying spermatozoa and this can differ based on whether the sample is being used for diagnostic purposes or intracytoplasmic sperm injection (ICSI).7,15 Many studies have been conducted to identify the ideal centrifugation force and duration to ascertain there are no spermatozoa samples suspected of azoospermia. These investigations ranged from 600g for 10 min up to 3000g for 15 min with various results. The WHO and AUA recommended 3000g for 15 min as the standard centrifugation force.5,9,15,16,17 Nevertheless, some research suggests that the likelihood of discovering sperm in an “azoospermic” specimen is not increased by centrifugal force >1000g. The International Organization for Standardization (ISO) standard recommends using 1000g for 15 min.18 Centrifugation power and duration used in various guidelines for sperm pellet analysis are shown in Table 1.9,15,17,18,19,20,21

Table 1.

Centrifugation power and duration used in various guidelines and studies for sperm pellet analysis

Guideline and study	Centrifugation process
	Power (g)	Duration (min)
AUA 201017	Maximum: 3000	15
ASRM 201819	3000	15
WHO 6th edition manual 20219	3000	15
EAU 202320	3000	15
CUA 202321	3000	15
Corea et al.15 2005	Minimum: 1000 Maximum: 3000	15
ISO 23162:202118	1000	15

AUA: American Urology Association; ASRM: American Society of Reproductive Medicine; CUA: Canadian Urological Association; EAU: European Association of Urology; WHO: World Health Organization; ISO: International Organization for Standardization

This method is reasonably simple and inexpensive, and it could save an azoospermic man from testicular sperm extraction. Given that increased centrifugation force can result in the generation of reactive oxygen species (ROS), it is best to minimize centrifugal force on a sperm sample whenever possible.22 ROS are linked to sperm membrane damage, which may have an impact on sperm quality. A minimum centrifugation at 1000g for 15 min should be followed by an inspection of the pellet to distinguish between severe oligozoospermia and azoospermia.15

It is recommended to repeat testing to accommodate for biological variability as well as intra- and inter-observer variability when analyzing semen samples.23 In some cases, sperm might be found in the semen analysis of azoospermic patients during serial analysis.24 The optimal timing of the repeat test has not been established but should be at least 2 weeks after the first test.5,25

Another method that can help assess azoospermia is biochemistry analyses of the semen sample. Seminal plasma is a mix of secretions from multiple parts of the male reproductive system.26 Analyzing the biochemical properties of semen and its constituent parts offers useful, noninvasive biomarkers for assessing the performance of various accessory sex glands and seminiferous tubules that contribute to semen production.

Biochemistry of seminal plasma is marked as an optional examination, and its usefulness is widely acknowledged on theoretical grounds. Acid phosphatase, citric acid, and zinc are known products from the secretions of the prostate. Fructose, proteins, and prostaglandins originate from the seminal vesicles.27 Absence of fructose in seminal plasma may be a sign of hypoplasia of the seminal vesicles or blockage of the ejaculatory duct.23,27,28 The epididymis produces neutral alpha-glucosidase (NAG) in the normal ejaculate.26 As a result, the four previously described markers are capable of assessing various male reproductive duct components. According to Guerin et al.,29 NAG activity was reduced in azoospermia patients. Sandoval et al.30 showed increased activity of NAG in nonobstructive azoospermia (NOA) men compared to activity of NAG seen in men with obstructive azoospermia.

Until now, the WHO only published their references on fructose, NAG, and zinc.5,9 The protocol of this assessment requires that the semen samples need to be centrifuged for 10 min at 1000g. The supernatant seminal plasma is quickly and carefully recovered and put to freeze at −20°C. It is necessary to ascertain the fructose and NAG concentrations in the retained seminal plasma. However, biochemical testing may be misleading when not properly performed,23 and it is seldom practiced systematically.27

Apart from fructose and NAG, there are several seminal biochemistry parameters that are experimental. Two such markers for the identification of spermatogenesis are inhibin B and anti-Müllerian hormone (AMH). They also serve as markers for the degree of spermatogenesis disruption in NOA men. The absence of sperm cells in the semen of azoospermic men is correlated with a considerable reduction in seminal plasma AMH.26 Inhibin B levels were shown to be higher in azoospermic specimens with maturation arrest at later phases of spermatogenesis than in those devoid of any germ cells.31 According to this finding, semen contains sensitive biomarkers, such as inhibin B and AMH, which may indicate the functional status of the seminiferous epithelium. Seminal plasma is mixed with an antiprotease mixture (complete antiprotease) in tablet form to measure inhibin B and AMH in semen. The enzyme-linked immunosorbent assay (ELISA) sandwich procedure was utilized to ascertain the levels of AMH and inhibin B.26 Seminal inhibin B and AMH evaluations are inexpensive, but they are not frequently carried out, and more research is necessary before they could potentially be used clinically. They are noninvasive spermatogenesis predictors, and it is advisable to utilize them to determine azoospermia. Semen findings and its clinical implications are shown in Table 2.14,18,32,33

Table 2.

Semen findings and its clinical implications14,18,32,33

Semen finding	Clinical implication
Macroscopic parameters
Less opacity in seminal fluid	Possibility of azoospermia
Low volume of seminal fluid	Possibility of ejaculatory duct obstruction or agenesis seminal vesicles and/or male reproductive tract; possibility of incomplete semen collection
Low pH (<7.2)	Possibility of ejaculatory duct obstruction or agenesis seminal vesicles/and male reproductive tract
Microscopic parameters
Presence of precursor cells	Rules out obstruction
Sperm pellet analysis
Presence of spermatozoa in sperm pellet analysis	Cryptozoospermia
Biochemistry
Low fructose	Possibility of ejaculatory duct obstruction or agenesis seminal vesicles
Low NAG	Epididymal obstruction

NAG: neutral alpha-glucosidase

ADVANCEMENTS IN LABORATORY TECHNIQUES FOR ASSESSING AZOOSPERMIA

Aside from the two methods mentioned above, the staining method can also be used to improve the detection of sperm in sperm pellet analysis. One of them is nuclear fast and picroindigocarmine (NF-PIC) staining. The earliest report on the use of NF-PIC staining following centrifugation to identify sperm in an azoospermic sample dates back to 1998.34 Sequential dilution of semen samples has shown the value of the NF-PIC staining method in detecting sperm at low concentrations.

The steps of performing the NF-PIC staining consist of: (1) fixation of the sample-smeared slide using ethanol; (2) each slide has an NF solution applied to it for 15 min, after which the NF solution is removed; (3) a few drops of PIC staining are applied to the NF-stained slide for 30 s; (4) rinsing the slide with ethanol, and then let the slide air dry; (5) sealing the slide with Cytoseal; and (6) evaluation using high magnification (objective 100×) with oil immersion.35

Using this technique under a microscope, spermatozoa can be more easily identified because their heads stain red and their tails are green. The outcomes are stated as follows: (1) no sperm seen: this occurs when no sperms are visible following staining; or (2) sperm observed (whether separate tails or just the heads of sperm are visible). This technique is particularly useful in identifying a small number of sperm in the semen sample such as in men who have cryptozoospermia. This is an easy-to-use, quick approach that works in any laboratory. As an extra measure in the assessment of azoospermia, it might be suggested.35

As aforementioned, the centrifugation process plays an important role in semen pellet analysis. Previous studies researched modifications in the power of spin and the duration of the centrifugation process. Apart from power and duration, they used the same “swing-out” centrifuge device. In recent years, there have been few studies that tried using another centrifugation device to compare the efficacy to the standard method proposed by the WHO. These strategies can potentially be used to help improve the accuracy of detecting azoospermia.

One of the modifications is researched using cytocentrifugation (Cytospin). This method is done using a cytospin device. By this technique, the sample is sedimented into a tiny (32 mm²), discernible area. Shandon Diagnostics Ltd. (Cambridge, United Kingdom) was the original developer of cytospin, which Thermo Scientific eventually purchased. This device is appropriate for sediment analysis-based cell concentration in a variety of fluids. This device is processing the sample in the CytoFunnel Filter with the centrifugation force of 500g for 10 min. A control slide is also needed for quality control, using samples from healthy men.35 Even though this method requires the particular equipment, it is quick and easy to use.

Another modification of the centrifugation method is ultracentrifugation, which was developed by Monteiro et al.,36 in 2013. This technique spins samples at a high speed (2000g for 15 min) in the microcentrifuge device and claims to be as effective as cytospin but is much simpler and less expensive and provides results with intact spermatozoa and minimal cellular debris.36,37

A combination of cytocentrifugation and NF-PIC staining has also been researched, with promising results. Sharma et al.38 reported that combining the cytocentrifugation method and NF-PIC staining is superior to the existing method described by the WHO for finding rare sperm. The result of the study shows that rare sperm can be identified in 23.9% of samples that have been labeled as azoospermic according to the WHO manual.

In recent years, there have been several advances in azoospermia examination, such as biomarker detection in seminal plasma. Biomarkers that can identify spermatogenesis are being investigated. Genetic biomarkers can be found in the seminal plasma and assessed by specific examination to help differentiate between obstructive and nonobstructive azoospermia. These biomarkers consist of messenger ribonucleic acids (mRNAs) carrying specific genes such as DEAD-box helicase 4 (DDX4), semenogelin 1 (SEMG1), and transmembrane gamma-carboxyglutamic acid protein 4 (TMG4).39,40 DDX4 is known to originate from the testicle, SEMG1 originates from the seminal vesicle, and TGM4 is secreted from the prostate.39,41,42 Among these three biomarkers, DDX4 can be used as a marker of spermatogenesis and is proven to be the most reliable in distinguishing between obstructive and nonobstructive azoospermia.39,40 In this procedure, RNA must be extracted from the seminal plasma, and then augmented, before it can be detected.

If there is a complete obstruction such as in CBAVD, the seminal plasma is found to be cell-free and absent of DDX4. In a partial obstruction, DDX4 can be found in the seminal plasma.39,40 In NOA, DDX4 is absent in complete Sertoli cell-only syndrome (SCOS).39 The existence of these markers in the seminal plasma gives an implicit, meaning that spermatogenesis is present in the testis and proceeding with sperm retrieval is reasonable.39,40,41,42 This procedure could be classified as a noninvasive screening tool and should also be confirmed by histopathology in selected cases.41

Aside from mRNAs, other biomarkers that can be detected are testicular-expressed proteins. In recent years, a list of testicular- and epididymal-expressed proteins was identified by comparing the seminal plasma of healthy and fertile men before and after the vasectomy procedure.43 The seminal plasma of nonobstructive azoospermic patients is then compared in the list. Among the list, there is a protein named testis-expressed protein 101 (TEX101), which is a testicular germ cell-specific protein and shed into seminal plasma.44 TEX101 is located on the plasma membrane of immature germ cells and testicular spermatozoa.45 TEX101 was used for noninvasive assessment of azoospermia types (obstructive or nonobstructive).44,45

TEX101 is assessed using mass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry. These assessments are used to characterize the TEX101 protein as an analyte in seminal plasma, and then the TEX101 level was measured using ELISA.44 Different NOA subtypes can be identified by measuring the seminal plasma levels of TEX101.44,45 Normal spermatogenesis is indicated by values of 120 ng ml⁻¹ or greater; hypospermatogenesis or maturation arrest is linked to levels of 5–120 ng ml⁻¹, while SCOS may be indicated by levels below 5 ng ml⁻¹.

Although it is still being studied, the clinical implication is that azoospermic patients can now be selected, which will be beneficial if surgical sperm retrieval is needed.

CONCLUSION

Azoospermia is the most challenging problem encountered in male infertility. The term “azoospermia” describes the condition in which spermatozoa are absent from the ejaculate. One of the challenges in male infertility is accurately and reliably assessing azoospermia. For accurate assessment of azoospermia, the semen analysis must be performed according to the WHO manual or ISO standards followed by pellet analysis. In this review, we have summarized the utility of newer methods, such as cytospin, NF-PIC staining, seminal biochemical markers, cell-free seminal mRNA assessments, and testicular-expressed protein. These methods are recommended as useful additional steps in this matter. Future research should compare more advanced centrifugation and staining procedures and equipment to uncover novel advances in identifying the most effective methodology for azoospermia laboratory assessments.

AUTHOR CONTRIBUTIONS

All authors are involved in concepting, writing, reviewing, and revising this manuscript, and read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.