For the identification of samples positive for C. trachomatis, the ImmunoComb® (Orgenics, Yvane, Israel) was used, which is a quantitative indirect enzyme immunoassay for the qualitative determination of IgA antibodies to C. trachomatis in semen. Anti-C. trachomatis IgA antibodies detect the active status in acute chronic and recurrent C. trachomatis infection (Clad et al., 1994). This particular assay includes a card with projections, each of which is sensitized with two reactive goat antibodies to human IgA (internal control) and inactivated antigens of C. trachomatis, as well as containing a developing plate consisting of 6 rows. The developing plate was left for an incubation period (37°C, 5% CO2, 20 min), after which 25 µl of semen was pipetted into row A (antigen-antibody reaction row) of the developing plate and left for 40 minutes at room temperature. After the reaction period of 40 minutes, the projections were left for 2 minutes in row B (first wash), followed by insertion into row C (alkaline phosphatase-labeled goat anti-human IgA antibody solution) to allow for the binding of the conjugate. After 20 minutes, the comb was immersed for 2 minutes in row D followed by row E, both of which are washing solutions. The projections were then inserted and left for 10 minutes into row F (colour reaction) which consists of a chromogenic substrate solution containing 5-bromo -4-chloro-3-indoyl phosphate and nitro blue tetrazolium. After the reaction period, the comb was inserted into row E for 1 minute to halt the reaction.
19
To analyse the result, the intensity of the colour reaction on each projection was observed in comparison to the positive control and samples positive for C. trachomatis were identified by the developed blue stain marking.
Figure 2: ImmunoComb® quantitative indirect enzyme immunoassay for the detection of C. trachomatis
20
Semen sample: liquefaction period (37°C, 5% CO2, 30 min)
Acrosome reaction
200 µl of seminal plasma frozen in liquid
N+ (-196°C) Viability Routine semen analysis (WHO, 2010) PMN ELISA assay Objective 2:
Examine the effects of N. gonorrhoea, C. trachomatis and T. vaginalis, in comparison to the control group, on the standard semen parameters which includes; volume, pH, viscosity, leukocyte count and polymorphonunclear elastase concentration, and the spermatozoa parameters including; motility, concentration, morphology, viability and acrosome reaction.
Figure 3: Flow chart of the simplified experimental procedure for objective 2 CASA
21 3.5 Semen sample analysis
The parameters were assessed in terms of the guidelines outlined by the WHO (WHO, 2010) and the protocols shall be discussed in detail below.
3.5.1 Volume
Following the incubation period (37°C, 5% CO2, 30 min) to allow for the liquefaction of the ejaculate, semen samples were directly decanted from the plastic collection container into a graduated 15 ml plastic Falcon tube. The volume was recorded in millilitres.
3.5.2 pH
pH was assessed by immersing litmus paper in the semen samples and graded according to the pH scale.
3.5.3 Viscosity
An Eppendorf micropipette was used to place 2 µl of semen into the filling area of a single chamber in a Leja® disposable 4 chamber slide (depth, 20 μm; length, 21 mm; width, 6 mm) (Leja® Products B.V., Nieuw-Vennep, The Netherlands). The filling time of each semen sample was measured twice, and the average of these times was recorded as the result. The result was then quantified according to the regression line equation below, according to Rijnders et al. (2007) and expressed in the unit Centipoise (cP):
y = 0.34(x) + 1.34
(x = filling time in seconds)
3.5.4 Leukocyte count
For the identification and quantification of leukocytes in the semen samples, a leukocyte peroxidase test was performed using Leukoscreen (FertiPro; Belgium). A wet mount was initially prepared with 20 µl of mixed semen on a covered slide in order to examine the presence of non-sperm cells under a phase-contrast light
22
microscope (x10 objective). Prior to performing the test, a working solution was prepared with 30 µl of reagent 2 (30% H202) mixed with 1 ml of reagent 1 (benzidine, cyanosine and methanol), which is the stain. Subsequent to the preparation of the working solution, 100 µl of the neat semen sample was mixed with 100 µl of the prepared working solution in an aliquot, mixed thoroughly with a vortex and left for five minutes at room temperature on the laboratory bench.
Following the five minute reaction period, 10 µl was placed on a slide and covered immediately after mixing to avoid the formation of air bubbles that can interfere with the interpretation of the results. A count of the peroxidase positive cells was performed in a similar manner as the standard spermatozoa count, with the aid of a Neubauer haemocytometer (depth 100µm) (Marienfeld, Germany). Upon examination of the slides, read under the bright-field objective (magnification 400x), the following was considered: yellow to brown stained cells can be regarded as peroxidase positive cells, thus neutrophilic polymorphous leukocytes, while the pink and unstained cells can be regarded as peroxidase negative cells. For statistical purposes, the number of leukocytes present in each ejaculate can be quantified and expressed as the number of peroxidase positive cells (x106/ml).
3.5.5 PMN elastase
An ELISA kit (Merck, Darmstadt, Germany) was used for the quantitative detection of extracellular PMN- elastase. The frozen seminal plasma was thawed at room temperature. Once the seminal plasma has completely thawed, it was diluted (1:100) with the sample diluent and 100 µl of sample diluent mixture of seminal plasma and reagents were added in duplicate to each of the blank wells, which are coated with the PMN elastase antibody. The wells were then covered with a plate cover and incubated at room temperature for 1 hour on a rotator and then washed with 300 µl of wash-buffer according to manufacturer’s instructions. Following the wash, 150 µl of Horseradish Peroxidase conjugate was added to each of the wells and incubated at room temperature for 1 hour.
23
Once the incubation period was completed, the wells were washed 4 times as described above. Next, 200 µl of the Tetramethyl-benzidine substrate solution was added to all the wells, including the blanks, and the plate was incubated at room temperature for 20 minutes on a rotator, avoiding direct exposure to intense light. After the incubation period, the enzyme reaction was suspended by pipetting 50 µl of the stop solution into each well. After the reader had been blanked with the previously prepared blank wells, the absorbance of each microwell was read at 450 nm using a Microplate reader (FLUOstar™ Omega, BMG Labtech, Germany). The results were quantified in ng/ml (Figure 4).
Figure 4: ELISA assay microtitre plate for the photometric quantification of the enzymatic activity of elastase in seminal plasma (450 nm)
3.6 Microscopic spermatozoa parameters