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Laboratory Evaluation of Azoospermia
Laboratory Evaluation of Azoospermia Grace M Centola, New York, NY, United States © 2018 Elsevier Inc. All rights reserved.
Introduction Azoospermia is defined as the complete absence of sperm cells (spermatozoa) in the ejaculate. Approximately 1% of all men suffer from azoospermia, with up to 15% of men found to have no sperm (Jarow et al., 1989; Oates, 2012; Gudeloglu and Parekattil, 2013). The causes of azoospermia are varied but include genetic (congenital), obstructive or nonobstructive, and unknown or idiopathic reasons. Genetic causes of azoospermia include hormonal abnormalities, Y chromosome microdeletions, absence of any sperm-producing cells (referred to as Sertoli cell only syndrome), and dysfunction of these sperm-producing cells (Gudeloglu and Parekattil, 2013). Obstructive azoospermia occurs in 15%–20% of azoospermic men and is caused by obstruction or blockage of the tubules or ducts that convey the sperm from the testicles to the outside. This includes ejaculatory dysfunction which prevents the semen from passing through the ducts and tubules during the ejaculatory process. Nonobstructive azoospermia occurs when the absence of sperm results from defects in sperm production. Congenital bilateral absence of the vas deferens (CBAVD) occurs when the vas deferens (the tube through which the sperm are transported) is missing from birth. Retrograde ejaculation, in which the semen is transported backward into the bladder rather than outward through the urethra, is also a form of nonobstructive azoospermia. In the case of retrograde ejaculation, sperm production is usually normal and the problem is often neurological, prohibiting antegrade or outward passage of sperm through the vas deferens. In most cases of retrograde ejaculation, sperm can be retrieved from the urine and used for assisted reproduction. Additional acquired causes of obstructive azoospermia include testicular trauma, infections including mumps orchitis, reproductive tract tumors, medications and recreational drug use, surgery and systemic diseases such as liver cirrhosis and kidney failure which prevent ejaculation through normally occurring ducts and tubules (Gudeloglu and Perkattil, 2013). A thorough history and physical exam, as well as hormone evaluation, and semen analyses are important steps in the evaluation and diagnosis of azoospermia and could assist in formulation of an appropriate treatment plan, if possible, for these individuals.
Laboratory Evaluation of the Azoospermic Male Azoospermia is diagnosed following microscopic examination of the semen. Generally two to three separate semen specimens will be examined by trained laboratory technologists. Each semen specimen should be collected 3–4 weeks apart and delivered to the laboratory within 30–45 min after ejaculation. Alternately, the specimens can be collected by masturbation in a private room provided at the laboratory facility. The laboratory technologist will then prepare a wet-mount slide of the well-mixed semen specimen by placing a drop of the semen on a glass slide and covering with a thin glass coverslip. The specimen smear on the slide is carefully examined using a light microscope for the presence of spermatozoa. If no sperm are found on the slide, a second slide will often be prepared using a new drop of well-mixed semen. Additionally, a semen smear can be preserved and stained to confirm the absence of sperm and permanently document the laboratory findings. If no sperm are found after scanning all areas of the slides, the semen can be centrifuged into a small pellet, and the pellet is examined for the presence of sperm (Jaffe et al, 1998; Corea et al, 2005; WHO, 1999). The pellet, containing all cellular components as well as noncellular debris, is then smeared onto a clean glass slide and carefully examined under the microscope for the presence of sperm. If even a low number of sperm is found, semen specimens may be frozen and stored for later use in assisted reproduction (Jaffe et al, 1998; Corea et al, 2005; WHO, 2010; Oates, 2012). When few sperm are found, the condition is referred to as severe oligozoospermia or cryptozoospermia (WHO, 2010; Gudeloglu and Parekattil, 2013). Centrifugation of a semen specimen is recommended in cases of post vasectomy semen analysis (WHO, 1999). The WHO (2010) laboratory manual and the American Urological Association (AUA) (Sharlip et al, 2017) suggest that centrifugation is not necessary to confirm the presence of rare nonmotile sperm, suggesting that careful slide examination of the raw semen may be sufficient (Korthorst et al., 2010). Furthermore, Steward and colleagues (2008) concluded that examination of an uncentrifuged, raw semen specimen is sufficiently reliable for identifying > 100,000 sperm. Since centrifugation might interfere with sperm motility, the AUA suggests that clinically relevant numbers of sperm can be identified without centrifugation (Sharlip et al, 2017). Nonetheless, if no sperm are observed after careful examination of the raw semen specimen (duplicate slides as mentioned above), it is the standard and acceptable laboratory procedure to examine a centrifuged semen specimen for the presence or absence of sperm in order to correctly and reliably report a diagnosis of azoospermia. Two other important parameters of the semen analysisdsemen volume and pHdcan provide useful information as to the cause of azoospermia (Oates, 2012). Approximately 10% of the ejaculate volume is contributed by the testicles, 70% by the seminal vesicles, and 20% from the prostate gland. The seminal vesicles provide alkaline fluid, while the prostate gland provides acidic fluid. Hence, the semen is alkaline since the seminal vesicles provide the majority of the fluid in the normal ejaculate (Oates, 2012). A low volume, low pH (acidic), azoospermic semen specimen is primarily made up of fluid from the prostate gland, suggesting absence of the seminal vesicles, bilateral absence or blockage of the vas deferens, or bilateral blockage of the ejaculatory ducts (Oates, 2012).
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Encyclopedia of Reproduction, 2nd edition, Volume 5
https://doi.org/10.1016/B978-0-12-801238-3.64837-7
Semen Analysis j Laboratory Evaluation of Azoospermia
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Alternately, in an azoospermic male, if the semen volume is normal, and the semen is alkaline, the seminal vesicles are presumably functional and duct obstruction is not a consideration. A blockage may be at a level closer to the testicles, or sperm production might be compromised in the testicle. Careful examination of centrifuged semen with a diagnosis of azoospermia can, in the case of normal volume and alkaline semen, confirm testicular failure. If even a few sperm are found in an ejaculate, testicular production of sperm is confirmed, albeit reduced, and blockage of the ducts, or absence of the vas deferens would not be possible (Oates, 2012). Precise laboratory examination of both raw and centrifuged semen, measurement of semen volume and pH, along with evaluation of complete medical history, physical exam and hormone analyses, can confirm a diagnosis of azoospermia or complete absence of sperm in the ejaculate. Following such a diagnosis, a treatment algorithm can be individualized for each patient for purposes of reproduction.
References Corea, M., Campagnone, J., & Sigman, M. (2005). The diagnosis of azospermia depends on the force of centrifugation. Fertility and Sterility, 83(4), 920–922. Gudeloglu, A., & Parekattil, S. J. (2013). Update in the evaluation of the azoospermic male. Clinics, 68(S1), 27–34. Jaffe, T. M., Kim, E. D., Hoekstra, T. H., & Lipshultz, L. I. (1998). Sperm pellet analysis: A technique to detect the presence of sperm in men considered to have azoospermia by routine semen analysis. Journal of Urology, 159, 1548–1550. Jarow, J. P., Espeland, M. A., & Lipshultz, L. I. (1989). Evaluation of the azoospermic patient. Journal of Urology, 142(1), 62–65. Korthorst, R. A., Consten, D., & Van Roijen, H. J. (2010). Clearance after vasectomy with a single semen sample containing < than 100 000 immotile sperm/mL: Analysis of 1073 patients. BJU International, 10511. Oates, R. A. (2012). Evaluation of the azoospermic male. Asian Journal of Andrology, 14(1), 82–87. Sharlip, I. D., Belker, A. M., Honig, S., Labrecque, M., Marmar, J. L., Ross, L. S., Sandlow, J. I., & Sokal, D. C. (2017). American Urological Association GuidelinedVasectomy. http://www.auanet.org/education/guidelines/vasectomy.cfm. Steward, B., Hays, M., & Sokal, D. (2008). Diagnostic accuracy of an initial azoospermic reading compared with results of post-centrifugation semen analysis after vasectomy. Journal of Urology, 180, 2119. World Health Organization. (1999). WHO laboratory manual for the examination and processing of human semen (4th edn.). Geneva: World Health Organization. World Health Organization. (2010). WHO laboratory manual for the examination and processing of human semen (5th edn.). Geneva: World Health Organization.
Introduction Azoospermia is defined as the complete absence of sperm cells (spermatozoa) in the ejaculate. Approximately 1% of all men suffer from azoospermia, with up to 15% of men found to have no sperm (Jarow et al., 1989; Oates, 2012; Gudeloglu and Parekattil, 2013). The causes of azoospermia are varied but include genetic (congenital), obstructive or nonobstructive, and unknown or idiopathic reasons. Genetic causes of azoospermia include hormonal abnormalities, Y chromosome microdeletions, absence of any sperm-producing cells (referred to as Sertoli cell only syndrome), and dysfunction of these sperm-producing cells (Gudeloglu and Parekattil, 2013). Obstructive azoospermia occurs in 15%–20% of azoospermic men and is caused by obstruction or blockage of the tubules or ducts that convey the sperm from the testicles to the outside. This includes ejaculatory dysfunction which prevents the semen from passing through the ducts and tubules during the ejaculatory process. Nonobstructive azoospermia occurs when the absence of sperm results from defects in sperm production. Congenital bilateral absence of the vas deferens (CBAVD) occurs when the vas deferens (the tube through which the sperm are transported) is missing from birth. Retrograde ejaculation, in which the semen is transported backward into the bladder rather than outward through the urethra, is also a form of nonobstructive azoospermia. In the case of retrograde ejaculation, sperm production is usually normal and the problem is often neurological, prohibiting antegrade or outward passage of sperm through the vas deferens. In most cases of retrograde ejaculation, sperm can be retrieved from the urine and used for assisted reproduction. Additional acquired causes of obstructive azoospermia include testicular trauma, infections including mumps orchitis, reproductive tract tumors, medications and recreational drug use, surgery and systemic diseases such as liver cirrhosis and kidney failure which prevent ejaculation through normally occurring ducts and tubules (Gudeloglu and Perkattil, 2013). A thorough history and physical exam, as well as hormone evaluation, and semen analyses are important steps in the evaluation and diagnosis of azoospermia and could assist in formulation of an appropriate treatment plan, if possible, for these individuals.
Laboratory Evaluation of the Azoospermic Male Azoospermia is diagnosed following microscopic examination of the semen. Generally two to three separate semen specimens will be examined by trained laboratory technologists. Each semen specimen should be collected 3–4 weeks apart and delivered to the laboratory within 30–45 min after ejaculation. Alternately, the specimens can be collected by masturbation in a private room provided at the laboratory facility. The laboratory technologist will then prepare a wet-mount slide of the well-mixed semen specimen by placing a drop of the semen on a glass slide and covering with a thin glass coverslip. The specimen smear on the slide is carefully examined using a light microscope for the presence of spermatozoa. If no sperm are found on the slide, a second slide will often be prepared using a new drop of well-mixed semen. Additionally, a semen smear can be preserved and stained to confirm the absence of sperm and permanently document the laboratory findings. If no sperm are found after scanning all areas of the slides, the semen can be centrifuged into a small pellet, and the pellet is examined for the presence of sperm (Jaffe et al, 1998; Corea et al, 2005; WHO, 1999). The pellet, containing all cellular components as well as noncellular debris, is then smeared onto a clean glass slide and carefully examined under the microscope for the presence of sperm. If even a low number of sperm is found, semen specimens may be frozen and stored for later use in assisted reproduction (Jaffe et al, 1998; Corea et al, 2005; WHO, 2010; Oates, 2012). When few sperm are found, the condition is referred to as severe oligozoospermia or cryptozoospermia (WHO, 2010; Gudeloglu and Parekattil, 2013). Centrifugation of a semen specimen is recommended in cases of post vasectomy semen analysis (WHO, 1999). The WHO (2010) laboratory manual and the American Urological Association (AUA) (Sharlip et al, 2017) suggest that centrifugation is not necessary to confirm the presence of rare nonmotile sperm, suggesting that careful slide examination of the raw semen may be sufficient (Korthorst et al., 2010). Furthermore, Steward and colleagues (2008) concluded that examination of an uncentrifuged, raw semen specimen is sufficiently reliable for identifying > 100,000 sperm. Since centrifugation might interfere with sperm motility, the AUA suggests that clinically relevant numbers of sperm can be identified without centrifugation (Sharlip et al, 2017). Nonetheless, if no sperm are observed after careful examination of the raw semen specimen (duplicate slides as mentioned above), it is the standard and acceptable laboratory procedure to examine a centrifuged semen specimen for the presence or absence of sperm in order to correctly and reliably report a diagnosis of azoospermia. Two other important parameters of the semen analysisdsemen volume and pHdcan provide useful information as to the cause of azoospermia (Oates, 2012). Approximately 10% of the ejaculate volume is contributed by the testicles, 70% by the seminal vesicles, and 20% from the prostate gland. The seminal vesicles provide alkaline fluid, while the prostate gland provides acidic fluid. Hence, the semen is alkaline since the seminal vesicles provide the majority of the fluid in the normal ejaculate (Oates, 2012). A low volume, low pH (acidic), azoospermic semen specimen is primarily made up of fluid from the prostate gland, suggesting absence of the seminal vesicles, bilateral absence or blockage of the vas deferens, or bilateral blockage of the ejaculatory ducts (Oates, 2012).
68
Encyclopedia of Reproduction, 2nd edition, Volume 5
https://doi.org/10.1016/B978-0-12-801238-3.64837-7
Semen Analysis j Laboratory Evaluation of Azoospermia
69
Alternately, in an azoospermic male, if the semen volume is normal, and the semen is alkaline, the seminal vesicles are presumably functional and duct obstruction is not a consideration. A blockage may be at a level closer to the testicles, or sperm production might be compromised in the testicle. Careful examination of centrifuged semen with a diagnosis of azoospermia can, in the case of normal volume and alkaline semen, confirm testicular failure. If even a few sperm are found in an ejaculate, testicular production of sperm is confirmed, albeit reduced, and blockage of the ducts, or absence of the vas deferens would not be possible (Oates, 2012). Precise laboratory examination of both raw and centrifuged semen, measurement of semen volume and pH, along with evaluation of complete medical history, physical exam and hormone analyses, can confirm a diagnosis of azoospermia or complete absence of sperm in the ejaculate. Following such a diagnosis, a treatment algorithm can be individualized for each patient for purposes of reproduction.
References Corea, M., Campagnone, J., & Sigman, M. (2005). The diagnosis of azospermia depends on the force of centrifugation. Fertility and Sterility, 83(4), 920–922. Gudeloglu, A., & Parekattil, S. J. (2013). Update in the evaluation of the azoospermic male. Clinics, 68(S1), 27–34. Jaffe, T. M., Kim, E. D., Hoekstra, T. H., & Lipshultz, L. I. (1998). Sperm pellet analysis: A technique to detect the presence of sperm in men considered to have azoospermia by routine semen analysis. Journal of Urology, 159, 1548–1550. Jarow, J. P., Espeland, M. A., & Lipshultz, L. I. (1989). Evaluation of the azoospermic patient. Journal of Urology, 142(1), 62–65. Korthorst, R. A., Consten, D., & Van Roijen, H. J. (2010). Clearance after vasectomy with a single semen sample containing < than 100 000 immotile sperm/mL: Analysis of 1073 patients. BJU International, 10511. Oates, R. A. (2012). Evaluation of the azoospermic male. Asian Journal of Andrology, 14(1), 82–87. Sharlip, I. D., Belker, A. M., Honig, S., Labrecque, M., Marmar, J. L., Ross, L. S., Sandlow, J. I., & Sokal, D. C. (2017). American Urological Association GuidelinedVasectomy. http://www.auanet.org/education/guidelines/vasectomy.cfm. Steward, B., Hays, M., & Sokal, D. (2008). Diagnostic accuracy of an initial azoospermic reading compared with results of post-centrifugation semen analysis after vasectomy. Journal of Urology, 180, 2119. World Health Organization. (1999). WHO laboratory manual for the examination and processing of human semen (4th edn.). Geneva: World Health Organization. World Health Organization. (2010). WHO laboratory manual for the examination and processing of human semen (5th edn.). Geneva: World Health Organization.