Modified Nag Assay Procedure to Diagnose Obstructive Azoospermia
Published Date: December 07, 2017
Modified Nag Assay Procedure to Diagnose Obstructive Azoospermia
Rajasingam S. Jeyendran*, Milica Ivanovic, Krithika Jeyendran, and Seth Levrant
Andrology Laboratory Services Inc, Chicago, Illinois 60611, USA
*Corresponding author: Rajasingam S. Jeyendran, Andrology Laboratory Services Inc, Chicago, Illinois 60611, USA, E-mail: firstname.lastname@example.org
Citation: Jeyendran RS, Ivanovic M, Jeyendran K, Levrant S (2017) Modified Nag Assay Procedure to Diagnose Obstructive Azoospermia. Andr Gyn Rep 1(1): 103.
The objective was to assess the usefulness of modified neutral β- glucosidase (NAG) assay procedure in the diagnosis of obstructive azoospermia. Ejaculates from 30 normozoospermic men, 27 azoospermic men and 24 men before and after vasectomy were analyzed. Relative NAG activity was determined at 30 and 90 minute time points during incubation of NAG assay on all ejaculates. The difference in relative NAG activity was calculated for each ejaculate compared within and between normozoospermic, azoospermic and before and after vasectomy groups respectively. The difference in the relative NAG activity obtained between the two assay time points were similar for all ejaculates after vasectomy and significantly different (p < 0.0001) when compared with normozoospermic and before vasectomy ejaculates. Thirteen of the 27 azoospermic ejaculates had difference in relative NAG activity similar to that of the vasectomized ejaculates. This suggest that the diagnosis of obstructive azoospermia would be confirmed if the difference in the relative NAG activity obtained between the 30 minute and 90 minute assay time points is similar.
Keywords: Obstructive azoospermia; Neutral β- glucosidase; NAG; Azoospermia
Epididymal obstruction is clinically difficult to diagnose and often is diagnosed by exclusion  or by testicular biopsy, an invasive procedure [2,3]. The complete absence of sperm in an ejaculate is essential for the confirmation of either obstructive or non obstructive type of azoospermia. A normal level of follicle stimulating hormone (FSH) may suggest an obstruction of the reproductive tract but cannot rule out maturation arrest during spermatogenesis [4,5]. An elevated level of FSH may suggest a non obstructive azoospermia. However, sperm could be retrieved from the testicles in more than 40% of men diagnosed as having non-obstructive azoospermia [6,7].
Azoospermia cannot be confirmed with absolute certainty even when the centrifuged pellet reveals no sperm, probably because the minuscule volume of ejaculate is examined to assess presence or absence of sperm. Thus the relative chance of finding a sperm is extremely small especially when the sperm in an ejaculate under normal circumstances is not uniformly distributed. Therefore a marker other than sperm is needed to confirm epididymal obstruction.
There are proteins that are exclusively produced in the epididymis that either forms an integral part of sperm or is essential for sperm maturation during their development. These are soluble proteins and therefore are homogenously distributed in an ejaculate. However, no such protein is currently available that is proven to be an effective to conclusively diagnose obstructive azoospermia [8,9]. But the activity of one such protein namely neutral α-glucosidase (NAG), an enzyme exclusively produced by the epididymis [10,11] has been reported to be significantly reduced when there is an occlusion distally to the epididymis [12–17]. Although the normozoospermic NAG concentration has been established , there is no defined minimum below which the NAG level is diagnostically useful. This is perhaps due to other non-specific glucosidase present in low concentration in an ejaculate which may mask the NAG activity or interfere with the interpretation of the results. NAG could be used as a definitive marker if only the activity of the non-specific glucosidases could be eliminated.
To eliminate or minimize non-specific glucosidases activity, it was theorized that the activity of these non specific glucosidase would be insignificant over time. Our objective was to test the hypothesis and to develop a procedure that is reliable to determine the true amount of NAG present in an ejaculate and to assess its usefulness to aid in the diagnosis of obstructive azoospermia due to epididymal occlusion.
Materials and Methods
Ejaculates from 81 men with mean age of 40 +/- 7 years were obtained by self-masturbation. Institutional Review Board of Advocate Health Care (Park Ridge, IL 60068) exempted this study under 45 CFR 46.101(b) . Informed consent was obtained from each participant. These samples were then analyzed for semen quality; the remaining samples were kept frozen at -20°C until needed.
Of the 81 men, 30 were apparently healthy with normozoospermic samples and 24 were presumably fertile who desired to have vasectomy. Ejaculates were obtained from normozoospermic men and from men before and after vasectomy. These ejaculates served as positive and negative controls for the development of the new assay procedure.
The ejaculates of the remaining 27 men were centrifuged at 1000 g for 10 minutes and the resulting pellet was vortexed and a drop placed on a glass slide under a cover glass. The slide was then scanned in more than ten random fields to identify any sperm present. There were no sperm found and the ejaculates were then classified as azoospermic.
These frozen samples were then thawed and analyzed for NAG activity by measuring the amount of p-nitrophenyl glucopyranoside (PNPG) converted to p-nitrophenol as described previously . Briefly, 100 µl of p-nitrophenyl glucopyranoside (PNPG) in phosphate buffer containing 1% sodium dodecyl sulfate (SDS) at pH 6.8 was mixed with 15 µl of seminal plasma from each of the ejaculates and incubated for 2 hours at 37°C. The enzyme reaction was then stopped by adding 1 ml of 0.1 M sodium carbonate 250 µl was transferred to a 96 plate and the absorbance of each sample was recorded at 405 nm. For the present study the assay procedure was modified as follows.
To determine whether SDS which selectively inhibits acidic alpha glucosidase is essential for determining NAG activity at pH 6.8 as published (WHO 2010), NAG activity from 28 ejaculates was determined in PNPG either no or containing 1% SDS. The relative NAG activity with and without 1% SDS on 28 different ejaculates incubated for 90 minutes revealed statistically no significant difference (0.75 +/- 0.40 vs. 0.77 +/- 0.39; p > 0.05) respectively and was excluded from further analysis (see discussion).
To test the hypothesis whether the relative activity of NAG over time would eliminate the activity of the other non-specific glucosidases NAG activity, ejaculates from 24 apparently fertile men before and after vasectomy was determined as follows: To 300 µl of PNPG in phosphate buffer 15 µl of semen from each of the ejaculates was added and incubated for 90 minutes at 37°C. At 30 and 90 minutes of incubation, 150 µl of the mixture respectively was transferred into a vial containing 1 ml of 0.1 M sodium carbonate to stop the reaction. The amount increase in the NAG activity over time was determined.
To test whether the relative increase in the NAG activity over time activity of NAG could identify the obstructive azoospermia, ejaculates from 27 azoospermic men were analyzed as described above.
All data are presented as the mean +/- standard deviation (SD) of the absorbance obtained in lieu of the true NAG activity. To identify differences in the mean, one-way analysis of variance was employed. A p value of < 0.05 was considered statistically significant.
The relative NAG activity at 30 and 90 minutes of incubation for all 30 men with normozoospermic ejaculates and 24 men with ejaculates before and after vasectomy is listed in table 1.
The mean relative NAG activity at 90 minutes incubation was significantly higher (p < 0.0001) for normozoospermic ejaculates and for ejaculates before vasectomy as compared to ejaculates incubated for 30 minutes or for ejaculates after vasectomy for both time periods (Table 1). Statistically there was no difference (p > 0.05) between the relative NAG activity observed at 30 minutes for normozoospermic ejaculates, ejaculates before vasectomy and for ejaculates after vasectomy both time periods. Similarly when the mean differences in the relative NAG was compared a significant difference (p < 0.0001) was observed (Table 1).
Table 1: Mean +/- SD (range) of relative NAG activity at 30 and 90 minutes in 30 men with normozoospermic ejaculates and 24 men with ejaculates before and after vasectomy. (*Mean with a common superscript are not significantly different from each other but significantly different (p < 0.0001) from the corresponding mean value).
Figure 1 details the individual relative difference in NAG activity of azoospermic ejaculates obtained between 30 and 90 minute incubation time points during the assay. Using a mean cut off of 0.09 (95 percentile of mean difference in NAG activity of post vasectomy ejaculates), individual differences among azoospermic ejaculates could be clearly identified and separated into two distinct groups.
Ejaculates from 13 of the 27 azoospermic men had their difference in relative NAG (0.05 +/- 0.04) statistically similar to those obtained for vasectomized ejaculates (0.03 +/- 0.03). The remaining 14 azoospermic men had their difference in relative NAG (0.20 +/- 0.06) statistically similar to those obtained for pre vasectomy ejaculates (0.24 +/- 0.20) (Figure 1).
Figure 1: Relative difference in NAG activity obtained between 30 and 90 minute time points during incubation of the azoospermic ejaculates in relation to the 95 percentile of the vasectomized ejaculate value.
The use of SDS to inhibit the acidic alpha glucosidase has been reported since the introduction of NAG as an epididymal marker . However, NAG is the most prevalent in ejaculate as compared to acidic alpha glucosidase  which is active at pH 6.0. Therefore its activity should not significantly influence the NAG activity especially at pH 6.8. The data presented clearly shows that the SDS has no significant effect (p > 0.05) in inhibiting the activity of acidic alpha glucosidase at pH 6.8. Since SDS had no influence during the NAG assay at pH 6.8, ejaculates were assayed in the absence of SDS.
A statistically insignificant difference in the relative NAG activity obtained between 30 and 90 minute incubation time points (0.10 +/- 0.03 VS 0.13 +/- 0.03) during the assay for the post vasectomized ejaculates was observed (Table 1). This confirms our hypothesis that the relative activity of the non specific glucosidases would be insignificant over time and thus the difference in relative activity between the two incubation time points during the NAG assay could be effectively employed to determine the true activity of NAG.
The eight fold difference in the relative activity between 30 and 90 minute incubation time points during the NAG assay before (0.24 +/- 0.20) and after (0.03 +/- 0.03) vasectomy ejaculates also validates our hypothesis (Table 1). Thus the assay procedure as described above could be effectively used to confirm occlusion of the vas deferens.
Using the 95 percentile of mean difference in NAG activity (0.09) between the two time points of incubation of the assay of post vasectomy ejaculates as the cut off value, the results of the azoospermic ejaculates were identified as either being above or below this level (Figure 1).
Thirteen (48.1%) azoospermic ejaculates were identified as having their relative mean difference of NAG activity below the cut off value and the individual values were similar to those obtained for after vasectomy ejaculates. This would suggest that these ejaculates represent men with obstructive azoospermia. Thus the modified NAG assay procedure may effectively and non invasively diagnose obstructive azoospermia and perhaps sperm may be retrieved from these patients, provided no other clinical entity is present that may affect sperm production.
The remaining 14 (51.9%) azoospermic ejaculates were identified as having their relative mean difference of NAG activity above the cut off value and the individual values were similar to those obtained before vasectomy ejaculates and therefore may suggest these ejaculates represent men with non obstructive azoospermia. Inability of patient follow up due to HIPPA regulations precluded from further involvement to clinically confirm the diagnosis.
It is proposed, that the diagnosis of obstructive azoospermia would be confirmed if the difference in the relative NAG activity obtained between the 30 minute and 90 minute assay time points is similar.
Conflict of Interest
No conflict of interests.
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Copyright: © 2017 Jeyendran RS, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.