Author's personal copy Urol Clin N Am 35 (2008) 157–171 Assessing Sperm Function Ashok Agarwal, PhD, HCLD*, Frances Monette Bragais, MD, Edmund Sabanegh, MD Reproductive Research Center, Glickman Urological and Kidney Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA Every male infertility work-up should start collection. Two separate samples should be ana- with the basics: a good history, physical exami- lyzed. These samples should be not less than 7 nation, and at least two semen analyses. Through- days apart [1,2]. The duration of the abstinence out the past 50 years or so that it has been in should be constant if possible, because each addi- existence, the semen analysis largely has remained tional day can add as much as 25% in sperm con- unchanged. This basic test is inexpensive, non- centration [3]. Lubricants should be avoided, as invasive, and remains the cornerstone of the they may interfere with motility results. Coitus in- infertility evaluation. As advances are made, terruptus often leads to inaccurate results, as the however, other tests are introduceddnot to sup- first part of the ejaculate that contains most of plant or replace this testdbut rather to delve the sperm may be lost. A clean, sterile container further into the specific causes of male infertility. should be used as a receptacle. A complete list Just like any other aspect in the dynamic field of of the guideline is provided in the World Health medicine, this role of the semen analysis has been Organization (WHO) laboratory manual for ex- challenged, its validity questioned, and its tech- amination of human semen and sperm–cervical niques scrutinized. mucus interaction [4]. This article reviews basic semen tests and new fertility tests that are providing great insights to Semen collection the rapidly developing understanding of male Semen specimens can be produced in various infertility. Finally, promising new tests under ways. At times patients will require assistance. development are mentioned with their potential Masturbation in a clinical setting. This is the clinical applications. recommended procedure where the collection is done in a private room in the same facility where the semen will be analyzed. The glans and the The basic test: the semen analysis penis should be cleaned with a wet paper towel Collection and timing (soap should be avoided). Lubricant use is dis- couraged but if needed should not be applied to Suboptimal sperm collection remains a fre- the glans. The container should be provided by quent cause of error in the semen analysis. It the laboratory to avoid contamination or spermi- should be emphasized to patients that there cidal effects. The main advantage of this collection should be 2 to 7 days of sexual abstinence before method is its simplicity, noninvasiveness, and inexpensiveness. Optimal specimens, however, may be difficult to procure for some men who are uncomfortable providing a sperm specimen in * Corresponding author. Reproductive Research, this environment [1]. Glickman Urological and Kidney Institute, 9500 Euclid Avenue, A19.1, Cleveland, OH 44195. Masturbation with assistance. Some men may not E-mail address: [email protected] (A. Agarwal). be able to achieve adequate erection and 0094-0143/08/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ucl.2008.01.012 urologic.theclinics.com Author's personal copy 158 AGARWAL et al ejaculation. Assistance can be provided for by samples without spermatozoa in an initial assess- oral medications such as PDE5 inhibitors given 30 ment should be centrifuged at 2000 g for 10 minutes to 60 minutes before collection. Cavernosal and and reexamined for the presence of sperm. If subcutaneous injections are less popular but no sperm is visible at this point, further centrifuga- possible options to administer to patients who tion and microscopic examination at 3000 g for have erectile dysfunction. Seminal pouches that 15 minutes are advised. There should be repeated do not contain any spermicides also can be used centrifugation and sperm counting performed and allow the patient to engage in sexual activity before azoospermia can be reported in a single should he be incapable or uncomfortable pro- semen analysis [8]. The assessment of motility and ducing specimens by masturbation. morphology is an acquired skill for the medical technologist requiring both didactic lectures and Vacuum erection devices. These can be used to practical experience. Quality control testing is obtain erection by creating a vacuum around the a critical component of an accurate semen analysis penis generating a pressure differential that fills and often is underemphasized in nonspecialized the corpora with blood. A constrictive band is laboratories [6,9]. It is therefore crucial that pa- placed at the base, however, to maintain erection, tients are referred to a laboratory that can provide and this can inhibit the flow of semen with reliable results. This may eliminate the need for ejaculation. repeated tests and in the end allow the clinician to Vibratory stimulation and electroejaculation. Me- make an accurate and cost-effective diagnosis [9]. chanical/vibratory stimulation may be used for Clinical laboratories engaged in diagnostic work patients who have suffered spinal cord injury (if in the reproductive field in the United States are the spinal cord lesion is T8 and above) [5]. Rectal accredited by agencies such as the College of probe electrostimulation (RPE) may induce ejacu- American Pathologist (CAP). They follow rigor- lation by stimulation of the efferent fibers of the ous procedures and protocols and provide superior hypogastric plexus. Precautions for autonomic test results over a laboratory without any external dysreflexia should be performed while doing these inspection of its records and protocols. procedures, as some patients with high spinal cord lesions can have life-threatening hypertension [1]. Standard procedures The semen sample should be examined within Technical aspects of the semen analysis 1 hour of production and receipt in the labora- It should be emphasized that nonspecialized tory. Some of the semen parameters can be laboratories often will have inadequate equipment affected by a delay in assessment. Motility de- and inexperienced personnel to perform the semen creases significantly after 2 hours and progres- analysis. Semen analysis is one of the few manu- sively diminishes afterwards while reactive oxygen ally performed examinations remaining in medical species (ROS) level increases. Ideally, semen is laboratories, and ideally it should be performed in placed in a 37 C gently shaking incubator for 30 an experienced laboratory [6]. There is no reliable minutes to allow liquefaction and mixing. The and cost-effective automation. Experienced labo- semen analysis characteristics can be classified ratories often will use the Neubauer chamber into three groups: macroscopic, microscopic and (Zeiss, Jena, Germany) as recommended by WHO physiologic. for sperm counting. This requires careful dilution and frequent cleaning. Incorrect use can increase Macroscopic chamber depth, producing erroneous results [6]. Table 1 lists the five macroscopic measure- Counting chambers, such as the Makler (Sefi ments in a standard sperm analysis. These param- Medical Instruments, Haifa, Israel), that do not eters have remained fairly constant, with the require dilution are also subject to the same vari- normal values remaining relatively unchanged ation [7]. Disposable counting chambers (Cell Vu since the inception of the semen analysis. Some (Millennium Sciences, New York), Microcell variation in macroscopic parameters (ie, liquefac- (Conception Technologies, San Diego, California)) tion) is relatively common and has little clinical are fairly inexpensive and offer less exposure of significance, although it also can be found in ac- the clinician to bodily fluids by eliminating the cessory gland dysfunction [4,10]. The specifics of cleaning process. The availability of an appro- how the tests are conducted (for all variables) priate centrifuge also can be crucial. Semen are found in the WHO manual [2]. Author's personal copy ASSESSING SPERM FUNCTION 159 Table 1 Macroscopic parameters of the semen analysis and clinical significances of some of the abnormal values Parameters Normal values Abnormalities Clinical significance pH 7.8 Acidic: !6.5–7 w/ low volume and noncoagulation: congenital absence of the vas Ejaculatory duct obstruction Partial retrograde ejaculation Coagulation/liquefaction Coagulates and liquefies within No coagulation Congenital absence of the 20 mins at room 37 seminal vesicles Prolonged liquefaction Poor prostatic secretions Color Whitish-gray; pearl-white Yellowish color Jaundice, carotenemia, drugs Reddish brown Haematospermia secondary to urethral bleeding or inflammation of the seminal vesicles, but other GU tumors will need work-up to be excluded Viscosity 4 mm threading O6 mm Of importance when associated No threading with low motility Volume 2–4 mL 0 (aspermia) Retrograde ejaculation !2 mL (hypospermia) Incomplete collection Partial retrograde ejaculation Short duration of sexual abstinence O6 mL Prolonged sexual abstinence Microscopic milliliter) and sperm count (number of sperm/ Microscopic examination of the semen in ejaculate) is conducted after liquefaction. Dilution essence assesses spermatogenesis. This part of of the semen is required if a Neubauer counting the semen analysis is subject to technical error, chamber is used, while Makler