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. 2025 Apr 25;27(3):307–310. doi: 10.4103/aja202529

Diagnostic algorithm in men suspected with nonobstructive azoospermia

Mahdi Bazzi 1, Matthew Chabot 1,✉, Amarnath Rambhatla 1, Eric Chung 2
PMCID: PMC12112923  PMID: 40275557

Abstract

This review focuses on the diagnostic algorithm for nonobstructive azoospermia (NOA), a significant male factor contributing to infertility. NOA, characterized by the absence of sperm in the ejaculate, requires a systematic diagnostic approach to identify reversible conditions, genetic factors, and prognosis for achieving pregnancy. The diagnostic pathway involves semen analysis and a comprehensive evaluation for hormonal deficiencies, anatomical abnormalities, and genetic factors. The importance of medical history, physical examination, endocrine evaluation, imaging, and genetic testing is emphasized. This review highlights the significance of differentiating NOA from obstructive azoospermia (OA) and outlines key considerations for effective management, including surgical sperm retrieval and assisted reproductive techniques. Testicular biopsy is discussed as a definitive method to distinguish obstructive cases from nonobstructive cases, providing valuable prognostic information. Overall, a thorough and systematic diagnostic approach is essential for the effective management of men suspected with NOA, offering insights into potential treatment options and reproductive outcomes.

Keywords: follicle-stimulating hormone receptor, luteinizing hormone, nonobstructive azoospermia, semen

INTRODUCTION

Infertility is a complex condition affecting the reproductive system and is characterized by the inability to achieve pregnancy after 12 months of regular unprotected sexual intercourse.1 Among infertility cases, approximately 20% can be solely attributed to male factors, while in 30%–40% of cases, male factors contribute partially.2 This review specifically delves into one significant male factor associated with infertility, nonobstructive azoospermia (NOA), and outlines the diagnostic pathway to identify this condition.

The incidence of azoospermia in men undergoing infertility workup ranges from 5% to 15%, with the majority stemming from nonobstructive causes.3,4 NOA is defined as the absence of sperm in the ejaculate due to impaired spermatogenesis. This impairment can result from primary testicular failure or a deficiency in the hypothalamic–pituitary–testicular axis (HPT axis) responsible for the production of gonadotropins and downstream testosterone synthesis and spermatogenesis.

When assessing men with NOA, it becomes crucial to focus on identifying reversible or treatable conditions, genetic factors that may be passed on to offspring, and the prognosis for achieving pregnancy. Determining the etiology of azoospermia serves as a guide for treating physician in devising an appropriate management strategy.

Nonobstructive causes encompass hormonal deficiencies, anatomical abnormalities including varicocele, genetic factors, and idiopathic causes.5 Unraveling these not only aids in understanding the condition but also influences decision-making in the pursuit of effective management.

MAKING THE DIAGNOSIS

Azoospermia is diagnosed when no spermatozoa are detected in a semen analysis. When azoospermia is suspected, it is crucial to centrifuge the semen sample to differentiate between true azoospermia from cryptospermia, where a few sperm may be identified in the centrifuged pellet.6 Centrifugation increases the detection of sperm under microscopy by up to 35%, emphasizing the importance of this step for an accurate azoospermia diagnosis.7

Distinguishing between azoospermia and crypotspermia is significant due to the distinct prognosis associated with each condition. Once azoospermia is confirmed, a second semen analysis should be performed to validate the initial finding. The optimal timing for the repeat semen analysis has not been firmly established, but obtaining a follow-up test after 70 days, which is the time it takes for spermatogenesis, is deemed reasonable.2,5 If both samples confirm azoospermia, an evaluation must commence to differentiate obstructive azoospermia (OA) from NOA. In addition, one can look for the presence of immature germ cells, spermatids, or spermatocytes in the ejaculate, in addition to a through medical history, physical examination, and relevant laboratory testing, to confirm the diagnosis of NOA over OA.

In cases of low ejaculate volume <1.4 ml, considerations should extend to the possibility of ejaculatory duct obstruction or retrograde ejaculation.1 In both cases, the volume of seminal fluid would be low, whereas in cases of NOA, the seminal fluid is not affected. If low ejaculate volume <1.4 ml is identified on initial semen analysis, a postejaculate urinalysis at the time of repeat semen analysis should be obtained to rule out possible retrograde ejaculation. If there is a high clinical suspicion for an obstructive process or retrograde ejaculation in the setting of normal semen volume, a post-ejaculate urinalysis can be obtained in these situations as well. In this review, we focus on the diagnostic pathway for NOA (Figure 1).

Figure 1.

Figure 1

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A flow chart demonstrating the diagnostic pathway for NOA. OA: obstructive azoospermia; NOA: nonobstructive azoospermia; LH: luteinizing hormone; FSH: follicle-stimulating hormone; SA: semen analysis; MRI: magnetic resonance imaging.

HISTORY AND PHYSICAL EXAMINATION

In patients with NOA, obtaining a thorough infertility history is paramount. One should begin by establishing a timeline for the couple’s attempts to conceive, including details about prior fertility, previous treatments, and sexual history. Equally crucial is gathering information about the partner, such as age, ovarian reserve, menstrual cycle regularity, and the presence of conditions such as polycystic ovarian disease that may impart fertility potential.

Identifying childhood illnesses affecting testicular health, such as viral orchitis or cryptorchidism, as well as a history of prior infections, trauma to the genitourinary tract, genitourinary surgery, exposure to chemotherapy, radiation, toxins, exogenous testosterone, anabolic steroids, or other medications, is essential.5 Additionally, one should inquire about family history regarding infertility or genetic abnormalities. The culmination of this information aids clinicians in building a differential diagnosis for the underlying cause of azoospermia.

Conducting a physical examination is equally integral to evaluating men with NOA. The clinician must focus on assessing testicular size and consistency, the presence of vasa deferentia and epididymides, penile abnormalities, the existence of varicoceles, palpable cystic structures during a prostate examination, tanner stage of genitals, and secondary sexual characteristics indicative of endocrine abnormalities. Normal testicular size falls within the range of 16–20 ml, with testes smaller than 15 ml or a long axis <4.6 cm more likely to exhibit problems with spermatogenesis and NOA. Testicular atrophy can stem from both primary and secondary testicular failure.5 Identifying an absent vas can differentiate between anatomical disruptions in the sperm transport pathway consistent with OA and genetic abnormalities and syndromes. Palpable masses during a digital rectal examination may suggest cystic structures causing ejaculatory duct obstruction.

ENDOCRINE EVALUATION

Understanding the endocrine evaluation of men with NOA involves recognizing the self-regulating HPT axis with a positive and negative feedback loop system. The hypothalamus releases gonadotropin-releasing hormone (GnRH) in pulsatile rhythmic secretions, which acts on the anterior pituitary to increase the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into systemic circulation.8 LH acts on Leydig cells to produce testosterone, while FSH acts on Sertoli cells to promote spermatogenesis and the production of inhibin B within the testicle. Testosterone is converted peripherally into estradiol, and it is testosterone, estradiol, and inhibin B which modulate negative feedback inhibition on both the hypothalamus and pituitary to decrease the secretion of gonadotropins.9 There is no supporting evidence that evaluating for testosterone derivatives such as free testosterone will be beneficial in the evaluation of NOA. Checking a total testosterone is usually sufficient. The concentration of intratesticular testosterone is around 100 times greater than that of serum testosterone in the average male and is in the bioavailable form.10,11

A minimum endocrine evaluation for men with NOA includes assessing testosterone and FSH levels. Intratesticular testosterone is crucial for sperm maturation, and elevated FSH supports spermatogenesis. In addition, baseline serum LH, prolactin, and estradiol levels should be obtained at the time of initial evaluation to complete the hormonal evaluation. FSH levels >7.6 mIU ml−1 and a testicular long axis length <4.6 cm suggest NOA. Elevated gonadotropins and low testosterone indicate testicular failure consistent with NOA, while low LH, FSH, and testosterone levels suggest hypogonadotropic hypogonadism.

In addition to hormonal testing, there may be a role to test for byproducts of accessory sexual glands such as the epididymis, prostate, and seminal vesicle. These glands provide products that play a necessary role in sperm motility. Biochemical assays may be done to test for neutral α-glucosidase (NAG), L-carnitine, or glycerophosphocholine, which comes from the epididymis. One can also test for fructose or prostaglandins from the seminal vesicles, or citric acid, zinc, or glutamyl transpeptidase or acid phosphatase from the prostate. Deficiencies in these byproducts may change the differential diagnosis from a problem with spermatogenesis to a problem with the accessory sexual glands, consistent with OA rather than NOA. However, it is important to remember that many of these biochemical assays are experimental and outside the standard of routine care.

IMAGING

Imaging studies serve as adjunctive measures in the NOA evaluation and are more commonly performed in evaluation of OA. Transrectal ultrasound may reveal dilated seminal vesicles, ejaculatory ducts, or prostatic cysts, but should only be considered when ejaculatory duct obstruction is strongly suspected.12,13 Scrotal ultrasound can be used to measure testicular volume and to identify varicoceles but is not routinely recommended. Scrotal ultrasonography can also identify testicular microlithiasis, which is associated with low sperm retrieval rates in men with NOA.14,15 In patients with hypogonadotropic hypogonadism and an elevated prolactin level, MRI can be utilized to identify possible pituitary tumors.16

GENETIC TESTING

Azoospermia may have a genetic origin, and approximately 15% of men with NOA have cytogenetic abnormalities. Karyotype and Y chromosome microdeletion tests should be performed as these can be both diagnostic and prognostic with implications on future offspring.

The most common genetic cause of infertility is Klinefelter syndrome (47,XXY), and approximately 10% of men with NOA have a nonmosaic form.17 Phenotypic features classically seen include gynecomastia, increased height, and delayed or no virilization with small testes. However, men with this genetic feature can have a normal phenotypic appearance.18 Only 8% of men with Klinefelter syndrome will have sperm in their ejaculate. The majority of these men will have NOA, and approximately 50% will have sufficient mature sperm identified with a sperm retrieval procedure.19

Karyotype testing can also reveal chromosome structural abnormalities. Male carriers of Robertsonian translocations involving chromosomes 13, 14, 21, and 22 may experience abnormal sperm counts, infertility, and miscarriage.20,21 These translocations cause azoospermia due to an increase in the frequency of disomic and diploid spermatocytes.22

The Y chromosome, which determines the male gender, has a region in its long arm that plays a significant role in sperm formation. This region is known as the azoospermia factor (AZF), compromising three subregions (AZFa, AZFb, and AZFc) with 26 genes involved in spermatogenesis. Microdeletions in these subgroups can cause failure of spermatogenesis and are the second most common genetic cause of azoospermia, affecting 5%–10% of men with NOA.15 Testing for these microdeletions can also be useful for prognosis. Men with complete microdeletions in the regions of AZFa or AZFb should not undergo sperm retrieval procedures as this condition is associated with no spermatogenesis.23 AZFc deletions are associated with successful sperm recovery in about 60% of cases, but this abnormality can be transmitted to male embryos. In this scenario, preimplantation genetic testing can be employed to select embryos without this abnormality.

Kallmann syndrome is another genetic disease that can cause NOA. This syndrome affects 1 in 30 000 males and is found in 1%–2% of men being evaluated for infertility.24 Features of this disease include hypogonadotropic hypogonadism due to the failure of pituitary hormonal secretion, resulting in the failure of testicular/spermatogenesis stimulation. Men with this syndrome also suffer from anosmia. Genes KAL1 or fibroblast growth factor receptor 1 (FGFR1) are involved in Kallmann syndrome and can be tested when suspected. The importance of genetic testing in men with NOA can be highlighted with this abnormality. If a diagnosis is established, treating these patients with human chorionic gonadotropin (hCG) and recombinant follicle-stimulating hormone (rFSH) or Menopur (rLH and rFSH) can stimulate spermatogenesis.25

Cystic fibrosis can cause azoospermia due to bilateral absence of the vas deferens. Although this is considered a cause of OA, it can be checked for if there is a high degree of suspicion based on physical examination by screening for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.26

One important consideration during genetic testing is counseling couples on the transmission of genetic abnormalities to offspring.

TESTICULAR BIOPSY/SPERM EXTRACTION

Sperm extraction procedures for men with NOA include conventional testicular sperm extraction (TESE) with single or multiple incisions, microdissection TESE (micro-TESE), testicular sperm aspiration (TESA), and fine needle aspiration (FNA) mapping mainly to help identify the areas of spermatogenesis within the testicle.27

The most definitive method to distinguish between NOA and OA is through a testicular biopsy, though this is not routinely preformed during the initial evaluation of azoospermia. It is generally reserved for equivocal cases when it is difficult to distinguish between OA and NOA based on history, examination, and laboratory evaluation. At the time of TESE, a portion of tissue may be sent for histologic analysis as a formal biopsy specimen. TESE and testicular biopsy are often synonymously used. If normal spermatogenesis is identified, the diagnosis of OA is made. If tissue analysis reveals pathologic or absent spermatogenesis, a diagnosis of NOA is made. Histologic analysis also provides prognostic information which can help guide future sperm retrieval procedures, particularly if sperm are not identified on initial specimen evaluation and a repeat sperm extraction procedure is planned. Sperm retrieval rates are generally around 10%–21% for repeat sperm extraction procedures after failed micro-TESE, and usually hypospermatogenesis is associated with a more favorable outcome.28,29

CONCLUSION

The diagnostic algorithm for NOA is a multifaceted process that necessitates a systematic and thorough approach. This review has underscored the importance of accurately identifying NOA through comprehensive evaluation, including semen analysis, hormonal assessment, genetic testing, imaging studies, and, when indicated, testicular biopsy. By differentiating NOA from OA and other potential causes of male infertility, clinicians can effectively tailor management strategies to each patient’s specific needs.

The significance of history-taking and physical examination cannot be overstated, as they provide valuable insights into potential etiologies and guide further diagnostic steps. Endocrine evaluation plays a crucial role in understanding the underlying hormonal imbalances that may contribute to NOA, while genetic testing offers essential diagnostic and prognostic information, aiding in counseling patients and their partners regarding fertility treatment options and the transmission of genetic abnormalities.

Imaging studies serve as adjunctive tools in the evaluation of NOA, helping to identify structural abnormalities that may impact fertility. Additionally, testicular biopsy, although typically reserved for sperm extraction procedures, offers valuable prognostic information regarding spermatogenesis severity and the likelihood of successful sperm retrieval.

Overall, a comprehensive diagnostic approach is essential for effectively managing men suspected of NOA, offering valuable insights into potential treatment options and reproductive outcomes. By implementing this diagnostic algorithm, clinicians can optimize patient care, improve treatment success rates, and ultimately help couples achieve their goal of conception.

AUTHOR CONTRIBUTIONS

MB provided analysis of the included studies and drafting of the manuscript. MC provided critical revision of the manuscript and the figure. AR provided conception of the design of the review, interpretation of included studies, and critical revision of the manuscript. EC provided conception of the design of the review, critical revision of the manuscript, and final approval for submission. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

REFERENCES

  • 1.World Health Organization. Infertility. Available from: https://www.who.int/news-room/fact-sheets/detail/infertility . [Last accessed on 2024 Jan 24]
  • 2.Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, et al. Incedence and main causes of infertility in a resident population (1,850,000) of three French regions (1988-1989) Hum Reprod. 1991;6:811–6. doi: 10.1093/oxfordjournals.humrep.a137433. [DOI] [PubMed] [Google Scholar]
  • 3.Jarow JP, Espeland MA, Lipshultz LI. Evaluation of the azoospermic patient. J Urol. 1989;142:62–5. doi: 10.1016/s0022-5347(17)38662-7. [DOI] [PubMed] [Google Scholar]
  • 4.Mazzilli F, Rossi T, Delfino M, Sarandrea N, Dondero F. Azoospermia: incidence, and biochemical evaluation of seminal plasma by the differential pH method. Panminerva Med. 2000;42:27–31. [PubMed] [Google Scholar]
  • 5.Male Infertility Best Practice Policy Committee of the American Urological Association, Practice Committee of the American Society for Reproductive Medicine. Report on evaluation of the azoospermic male. Fertil Steril. 2006;86:S210–5. doi: 10.1016/j.fertnstert.2006.08.030. [DOI] [PubMed] [Google Scholar]
  • 6.Schlegel PN. Causes of azoospermia and their management. Reprod Fertil Dev. 2004;16:561–72. doi: 10.10371/RD03087. [DOI] [PubMed] [Google Scholar]
  • 7.Ron-El R, Strassburger D, Friedler S, Komarovski D, Bern O, et al. Extended sperm preparation:an alternative to testicular sperm extraction in non-obstructive azoospermia. Hum Reprod. 1997;12:1222–6. doi: 10.1093/humrep/12.6.1222. [DOI] [PubMed] [Google Scholar]
  • 8.Gan EH, Quinton R. Physiological significance of the rhythmic secretion of hypothalamic and pituitary hormones. Prog Brain Res. 2010;181:111–26. doi: 10.1016/S0079-6123(08)81007-2. [DOI] [PubMed] [Google Scholar]
  • 9.Chu Z, Takagi H, Moenter SM. Hyperpolarization-activated currents in gonadotropin- releasing hormone (GnRH) neurons contribute to intrinsic excitability and are regulated by gonadal steroid feedback. J Neurosci. 2010;30:13373–83. doi: 10.1523/JNEUROSCI.1687-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Oates RD. The natural history of endocrine function and spermatogenesis in Klinefelter syndrome:what the data show. Fertil Steril. 2012;98:266–73. doi: 10.1016/j.fertnstert.2012.06.024. [DOI] [PubMed] [Google Scholar]
  • 11.Jarow JP, Zirkin BR. The androgen microenvironment of the human testis and hormonal control of spermatogenesis. Ann N Y Acad Sci. 2005;1061:208–20. doi: 10.1196/annals.1336.023. [DOI] [PubMed] [Google Scholar]
  • 12.Meacham RB, Hellerstein DK, Lipshultz LI. Evaluation and treatment of ejaculatory duct obstruction in the infertile male. Fertil Steril. 1993;59:393–7. doi: 10.1016/s0015-0282(16)55683-0. [DOI] [PubMed] [Google Scholar]
  • 13.Jarow JP. Transrectal ultrasonography of infertile men. Fertil Steril. 1993;60:1035–9. doi: 10.1016/s0015-0282(16)56406-1. [DOI] [PubMed] [Google Scholar]
  • 14.Xu C, Liu M, Zhang FF, Liu JL, Jiang XZ, et al. The association between testicular microlithiasis and semen parameters in Chinese adult men with fertility intention:experience of 226 cases. Urology. 2014;84:815–20. doi: 10.1016/j.urology.2014.03.021. [DOI] [PubMed] [Google Scholar]
  • 15.Barda S, Hauser R, Mano R, Savin Z, Molad-Hayo Y, et al. Testicular microlithiasis defines a subgroup of azoospermic men with low rates of sperm retrieval. Int J Urol. 2022;29:65–8. doi: 10.1111/iju.14717. [DOI] [PubMed] [Google Scholar]
  • 16.Layman LC. Hypogonadotropic hypogonadism. Endocrinol Metab Clin North Am. 2007;36:283–96. doi: 10.1016/j.ecl.2007.03.010. [DOI] [PubMed] [Google Scholar]
  • 17.Groth KA, Skakkebæk A, Høst C, Gravholt CH, Bojesen A. Clinical review:Klinefelter syndrome – a clinical update. J Clin Endocrinol Metab. 2013;98:20–30. doi: 10.1210/jc.2012-2382. [DOI] [PubMed] [Google Scholar]
  • 18.Oates RD, Lamb DJ. Genetic aspects of infertility. In: Lipshultz LI, Howards SS, Niederberger CS, editors. Infertility in the Male. New York: Cambridge University Press; 2009. pp. 251–27. [Google Scholar]
  • 19.Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, et al. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia:testicular histology can predict success of sperm retrieval. J Urol. 1999;161:112–6. [PubMed] [Google Scholar]
  • 20.Pastuszek E, Kiewisz J, Kulwikowska PM, Lukaszuk M, Lukaszuk K. Sperm parameters and DNA fragmentation of balanced chromosomal rearrangements carriers. Folia Histochem Cytobiol. 2015;53:314–21. doi: 10.5603/fhc.a2015.0032. [DOI] [PubMed] [Google Scholar]
  • 21.Suganya J, Kujur SB, Selvaraj K, Suruli MS, Haripriya G, et al. Chromosomal abnormalities in infertile men from southern India. J Clin Diagn Res. 2015;9:GC05–10. doi: 10.7860/JCDR/2015/14429.6247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Godo A, Blanco J, Vidal F, Sandalinas M, Garcia-Guixé E, et al. Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers. Reprod Biomed Online. 2015;31:79–88. doi: 10.1016/j.rbmo.2015.04.003. [DOI] [PubMed] [Google Scholar]
  • 23.Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, et al. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod. 2003;18:1660–5. doi: 10.1093/humrep/deg348. [DOI] [PubMed] [Google Scholar]
  • 24.Sonne J, Leslie SW, Lopez-Ojeda W. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. [[Last updated on 2024 Dec 11]]. Kallmann syndrome. Available from: https://www.ncbi.nlm.nih.gov/books/NBK538210/ [PubMed] [Google Scholar]
  • 25.Sokol RZ, Lipshultz LI, Howards SS, Niederberger CS. Infertility: Endocrine Evaluation in the Male. New York: Cambridge University Press; 2009. pp. 199–214. [Google Scholar]
  • 26.Hotaling J, Carrell DT. Clinical genetic testing for male factor infertility: current applications and future directions. Andrology. 2014;2:339–50. doi: 10.1111/j.2047-2927.2014.00200.x. [DOI] [PubMed] [Google Scholar]
  • 27.Kanto S, Ichioka K, Sato Y, Uchino Y, Tanaka T, et al. Revisiting non-obstructive azoospermia:is there a best way to retrieve testicular sperm? Reprod Med Biol. 2025;24:e12632. doi: 10.1002/rmb2.12632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Heller CH, Clermont Y. Kinetics of the germinal epithelium in man. Recent Prog Horm Res. 1964;20:545–75. [PubMed] [Google Scholar]
  • 29.Elbardisi H, Bakircioglu E, Liu W, Katz D. Second chance in fertility:a comprehensive narrative review of redo micro-TESE outcomes after initial failure. Asian J Androl. 2024 doi: 10.4103/aja202446. DOI:10.4103/aja202446. [Online ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar]

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