Microbiological investigation in male infertility: a practical overview

Indications.

Sperm culture enables the detection of micro-organisms that are responsible for infection in one or more male accessory sex glands and allows an aetiological diagnosis to be made. Therefore, the cultural examination of the ejaculate is useful in screening for male infertility. The search must be extended to all micro-organisms that may alter sperm function (La Vignera et al., 2011; Leterrier et al., 2011).

Micro-organisms to be searched for.

The microbiological investigation of semen must focus on the following micro-organisms:

1. Gram-negative bacteria (Enterobacteriaceae such as E. coli, Klebsiella species, Proteus, Serratia, Pseudomonas species, etc.) recognized as ‘certain pathogens’; micro-organisms considered to be ‘likely, occasional pathogens’ (Gram-positive bacteria, such as Enterococcus species and S. aureus; obligate anaerobes) or ‘possible, controversial pathogens’ (coagulase-negative germs, such as Staphylococcus haemolyticus, Staphylococcus epidermidis and Mycoplasma) for prostate infection;

2. according to clinical indications and the partner’s clinical history, sperm culture may be extended to the aetiological agents of sexually transmitted diseases (Trichomonas vaginalis and fungi);

3. in patients with previous antibiotic therapy, sperm culture should be extended to germs promoting superinfection (Pseudomonas and fungi).

Sampling methods.

In the pre-analytical phase, information must be given to patients regarding the collection procedures (masturbation), the length of sexual abstinence (3–4 days) and abstention from chemo-antibiotic treatment for at least 1 week prior to sample collection. It is essential not to contaminate the sample during or after collection. For this reason, collection must be preceded by accurate hand and external genitalia scrubbing. The patient should urinate prior to sperm collection in a sterile container.

The microbiological search must simultaneously involve the first urine flow and the seminal fluid to better differentiate a UTI from an infection of the seminal tract. The first urinary stream also allows for the detection of T. vaginalis and N. gonorrhoeae (after centrifugation), which are rarely isolated from semen due to the presence of substances that inhibit their growth. The sample must reach the laboratory within 1 h from collection. If this is not possible, it should be collected on the premises provided by the laboratory.

Cultural examination.

Culture focuses on fungi, aerobic organisms, Gardnerella vaginalis and mycoplasmas. Some bacteriostatic properties of the seminal plasma are related to the production of bactericides (by Gram-positive species and N. gonorrhoeae). Before sowing on the selective specific medium, 1 ml of the semen sample should be diluted (1 : 10) with sterile saline and centrifuged at 1500 r.p.m. for 15 min. After removing the supernatant, the sediment should be sown using 10 µl calibrated loops. This procedure increases the cultural sensitivity because it concentrates bacteria in the cell pellet and eliminates the seminal plasma that could exert an inhibitory effect on bacterial growth (Vicari et al., 1986). Culture media, conditions and incubation times are summarized in Table 2. To detect U. urealyticum and Mycoplasma hominis, the culture media used are similar to those described for urethral swabs (see below).

Identification and sensitivity.

The indications are the same as those described for the urethral swabs. Any aerobic Gram-positive or Gram-negative micro-organisms present in a concentration greater than 10³ c.f.u. ml⁻¹ must be identified and tested for antibiotic resistance. If the culture has three or more bacteria, the investigation should be stopped, as this may indicate polymicrobial flora of contamination. For the isolation of urogenital mycoplasmas, 72 h of incubation should be performed to observe minimal changes in these micro-organisms.

Indications.

The following signs may be indicative for taking a urethral swab:

1. patients are less than 35 years of age with a history of sexual promiscuity, recurrent UTI, sexually transmitted infection, partner’s infection (Trichomonas vaginalis), presence of symptomatic urethral discharge with more than four granulocytes per microscopic field (×1 000 magnification) or more than 15 neutrophils (×400 magnification) in the microscopic examination of the first urinary stream sediment;

2. patients are more than 35 years of age with or without risk factors, infertility, clinical manifestations of epididymo-orchitis (an extreme degree of MAGI complicated by ascending infection) and symptoms of persistent leukocytospermia, as well as those patients who appear to be poor responders to recent antibiotic treatment for uropathogenic bacteria.

Methods of conservation and transport of the urethral swabs.

Swabs taken to detect fungi or aerobic bacteria must be transported to the laboratory in a specific medium and stored at room temperature for no more than 24 h. The swab used to detect T. vaginalis must be maintained in the appropriate transport medium/culture (37 °C) for 15 min and then must be sent promptly to the laboratory. The swab used to detect Mycoplasma/Ureaplasma must be placed in a vial containing the transport medium and can be stored at room temperature for 8 h or at 2–8 °C for 36 h. The swab used to detect C. trachomatis must be collected and stored following the guidelines depending on the detection method used. Finally, the material obtained from the swab to detect N. gonorrhoeae should be sown without delay on a Thayer–Martin agar plate kept at room temperature (or better, pre-warmed to 37 °C) and incubated immediately. Alternatively, the swab may be sent to the laboratory as soon as possible in a specific transport medium without glycerophosphate. In Stuart–Amies medium at room temperature, gonococci survive for 12 h. Once sown, the medium must be incubated at 37 °C in an atmosphere of 5 % CO₂ for 48 h.

Analytical phase: pathogens to be examined.

When urethritis from sexually transmitted diseases is suspected, the presence of the following pathogens should be evaluated: C. trachomatis (observed in 23–55 % of non-gonococcal urethritis), U. urealyticum, M. hominis and N. gonorrhoeae. T. vaginalis and fungi are rarely the cause of urethritis; they should be taken into account particularly in cases of persistent symptoms, partner infection, dysuric symptoms and/or post-antibiotic administration balanoposthitis.

If other micro-organisms that are considered ‘certain’ (Gram-negative bacteria, such as Enterobacteriaceae) or ‘possible’ (Gram-positive bacteria, such as Streptococcus faecalis, Haemophilus species, Streptococcus species, etc.) pathogens for the urinary tract are detected in the urethral swab, then these should be searched for in the seminal fluid. These acquire real significance only in the presence of a high microbial load associated with other laboratory signs of infection/inflammation (increased polymorphonuclear cells, symptoms of irritation; see diagnostic criteria for MAGI).

Identification phase.

(Isemberg, 1998; Schiefer, 1998; Murray et al., 2003; Vesić et al., 2007; Orellana et al., 2009.)

For the bacteriological examination, the slide for microscopic examination must be prepared by rotating the swab on the slide and drying the material in the air. The dried glass slide will be inserted into the transport container and can be stored at room temperature until delivery to the laboratory. It is important that it is prepared immediately after collection and not after the swab has been sent to the laboratory. The slide should be subjected to Gram staining and observed at ×400 and ×1000 magnifications. The identification of fungi, leukocytes and microbial species, which are distinct based on their morphology and Gram staining, must be expressed using a semiquantitative system (+, ++, +++). The observation of typical Gram-negative intracellular diplococci is strongly indicative for gonorrhoea; all suspected cases must be confirmed by culture.

To detect T. vaginalis if a bacterial culture is not possible, a fresh dark field microscopic examination should be performed. In this case, the secretions should be collected with a swab and placed in a tube containing 1 ml of saline solution kept at 37 °C. The fresh slide must be examined immediately to avoid the disappearance of Trichomonas motility that, together with the morphology, is one of its specific features. Culture is a more sensitive method of detection than the microscopic analysis of trophozoites.

The microbiological culture examination of the urethral swab aims to detect aerobic bacteria, fungi, N. gonorrhoeae, G. vaginalis and urogenital mycoplasmas (Table 3).

To detect U. urealyticum and M. hominis, the buffer must be dissolved in a transport/culture medium. Three drops of this medium should be seeded on solid A7 medium (Vázquez et al., 1995), which will be incubated at 37 °C for 48–72 h in anaerobic or microaerophilic conditions. To set up a possible antibiogram, the test tubes containing the remaining medium can be refrigerated for a few days or frozen at −80 °C.

Bacterial culture is the ‘gold standard’ to detect T. vaginalis both for sensitivity and specificity. The exudate, collected with a sterile swab, should be sown immediately after collection in 5 ml CPLM Trichomonas broth (Patel et al., 2000) preheated to 37 °C containing streptomycin (1000 mg ml⁻¹), penicillin (1000 U ml⁻¹), chloramphenicol (50 g ml⁻¹) and sterile horse serum (50 ml l⁻¹). Incubation is performed at 37 °C for 5 days with fresh microscopic observations performed daily, beginning on the second day of incubation.

Anaerobic micro-organisms should not be routinely sought out, but other micro-organisms observed during Gram staining in the setting of an increase in polymorphonuclear cells and associated symptoms (Haemophilus species, Streptococcus species, etc.) should be investigated.

A further culture method involves the use of a gallery with domes that contain substrates for micro-organism identification as well as lyophilized antibiotics. These domes are rehydrated with the transport/culture medium which has been inoculated with the sample that has been tested. The domes are then covered with paraffin oil to ensure anaerobic conditions and incubated at 37 °C for 24 h. Cultures in liquid medium provide better results than those sown directly on solid medium (due to the dilution of potential inhibitors and the breaking of immune complexes, etc.). PCR can also be used to specifically highlight genital mycoplasmas that are difficult to cultivate, such as Mycoplasma genitalium and Mycoplasma fermentans.

Indications.

The Meares–Stamey test is indicated when one or more of the following conditions occur:

1. the presence of one or more of the following recurring symptoms (associated with prostatitis): onset in the post-ejaculatory phase of prostate-perineal, scrotal-testicular, penile, vesical or suprapubic tenesmus (Zermann et al., 1999); prostatorrhoea, often associated with voiding disorders and occasional haemospermia;

2. sperm culture with repeated negative results;

3. persistent leukocytospermia, even after antibiotic therapy;

4. ultrasonographic findings of prostate-vesiculitis [prostatic lobe asymmetry, multiple hypoechoic areas (acinar stagnation)]; vesicular anteroposterior diameter (>14 mm) with polycyclic areas resistant to first therapeutic measures.

Contraindications.

The Meares–Stamey test should not be performed in cases of acute prostatitis (acute infection, abscess-like), which are almost always due to a bacterial infection, and the pathogens involved are typical of the micro-organisms of the urinary tract.

Sampling procedure.

The patient should have a full bladder, must not have ejaculated for 2 days and must not have taken antibiotics in the previous month. The patient should collect the first 10 ml of urine (VB1) and then another 10 ml of urine (VB2). At this point, a prolonged prostate massage is performed at both gland lobes and the prostatic secretion is collected by trying to facilitate the escape from the urethral meatus by pressing the penis from the base to the glans (EPS). The patient should then collect another 10 ml of urine (VB3) and thereafter, he can empty his bladder completely.

Microscopic examination.

A drop of EPS is subjected to Giemsa staining. The presence of more than ten leukocytes per microscopic field (×400 magnification) and/or the presence of leukocyte aggregates is indicative of prostatitis. A bacterial count in EPS and VB3 at least ten times higher than in VB1 and VB2 suggests that the germs are of prostatic origin. A higher load in VB2 than in VB3 suggests that bacteriuria should be treated with drugs such as nitrofurantoin that do not penetrate to the prostate. The test should then be repeated.

Bacterial culture.

Samples should be sown in the shortest possible time on selective and non-selective media after 1 : 100 dilution in sterile saline solution and inoculation of 100 µl by spatulation. To detect fungi, aerobic organisms, G. vaginalis and urogenital mycoplasmas, the procedures for seminal culture should be performed.

Identification, antibiogram and C. trachomatis research.

The procedures for seminal culture should be performed.

Indications.

This investigation is useful for integrating the urethral swab, but has limited indications in patients with a history/clinical presentation of the following: phimosis, hypospadias, diabetes mellitus, recurring balanoposthitis and persistent UTI. Patients with balanitis exhibit glans irritation and inflammation, which is often extended to the foreskin (balano-). Due to the high frequency of occasional balanitis, irritation due to the simple mechanical effects of maceration and poor sanitation in the balanopreputial region should be excluded first. After exclusion of these forms of balanitis, other cases worthy of further investigation are:

1. cases in the purulent stage, where the balanitis may be secondary to urethral infection that may go unnoticed (these cases of balanitis are often located at the meatus). It is useful to take into consideration the following micro-organisms: N. gonorrhoeae, mycoplasmas, T. vaginalis, G. vaginalis, C. albicans, group A and B Streptococcus and S. aureus (Horner & Taylor-Robinson, 2011);

   1. balanitis associated with skin (condylomata acuminata or plana) or erythematous-vesicular lesions. In these cases, the organisms responsible for balanitis that should be investigated are Herpes simplex virus (HSV-1, HSV-2) and HPV.

Sampling procedure.

Sampling should be performed with nylon plugs from the balanopreputial sulcus. Many swabs should be used as there are different types of tests to be performed. The patient should not use antibiotics or antifungal topical or systemic therapy for 3–4 days prior to the test. Samples should be stored and transported as described for the urethral swab.

Bacterial culture.

For the cultivation of fungi, aerobic organisms, N. gonorrhoeae and G. vaginalis, the material is seeded and incubated. The culture media used for U. urealyticum and M. hominis detection are the same as those described for the urethral swabs.