Determination of the Association of Urine Prostate Specific Antigen Levels with Anthropometric Variables in Children Aged 5-14 Years

Purpose: Calculation of PSA is possible in human fluids even if it presents in very low concentrations with the help of hypersensitive immunodiagnostic methods. The periurethral glands represent one of the potential sources of urine prostate specific antigen (uPSA) in both sexes but the purpose of studying PSA levels in children is still unclear in the literature. In this pilot study we studied uPSA in a small cohort of normal, pre and post pubertal children, in relation to standard anthropometric variables. Materials and Methods: The study cohort consisted of 58 children 5-14 years old (42 boys/16 girls). Height, weight, body mass index (BMI) and the respective stature-for-age, weight-for-age and BMI-for-age percentiles of the sample were determined. uPSA levels were measured using a third generation immunodiagnostic method (DPC Immulite®) that has a lower limit of detection of 3 ng/L. When levels of PSA were above the upper limit of detection, uPSA levels were assessed using the ROCHE technique. Results: uPSA levels tend to be higher in male than female children (p = 0.091, linear regression analysis). uPSA was measurable only in 3/16 girls (18.75%). Measurable uPSA was found in 18/42 boys (42.8%). The range of urine PSA in boys was 0-161000 ng/L (mean 10561.9 ± 31830.48 ng/L). Statistical analysis with linear regression showed correlation with height and age in boys. Conclusions: The use of hypersensitive assays allows calculation of uPSA in childhood. The values of this variable are measurable in both sexes and related with gender. In boys, uPSA was correlated with age and height but not with other variables tested. Further studies are required to clarify this field.


INTRODUCTION
Prostate specific antigen (PSA) was discovered initially in seminal fluid as γ-seminoglobulin in the late 1960s (1).Since then there has been a great number of discoveries and developments in the field of this marker.It is now clear that PSA is not tissue or gender specific.Studies have shown its presence in other tissues and especially in female tissues, such as periurethral glands, breast, endometrium and ovarian tissue (2,3).
Advances in clinical biochemistry in the last decade have made possible measurement of PSA using highly sensitive assays in body fluids with extremely low concentrations.One of these methods, the third generation DPC Immulite ® method (Diagnostics Products Corp., Los Angeles, CA) has a detection limit of 3 ng/L and a calibration range up to 20000 ng/L (4).
Previous studies have shown that serum PSA can be calculated not only in boys in the different stages of puberty but also in girls, although in the latter it is encountered in lower concentrations and with lower frequency (5,6).Recently PSA was measured in the urine of children of both sexes (7).The role of uPSA in children has not been investigated yet.Prostate development in childhood is related with testosterone activity, androgen receptor and 5-alpha reductase activity in the target organ (8)(9)(10).Diseases that affect the levels and activity of the above substances such as disorders of sexual differentiation and hypospadias (11) could potentially decrease PSA production in urine.uPSA could be used as a non-invasive marker for these diseases.
In the present study, we collected urine sample from healthy boys and girls aged 5-14 years, calculated uPSA levels and investigated the association of PSA with standard anthropometric variables.

MATERIALS AND METHODS
We studied 58 healthy children (42 boys and 16 girls) aged 5-14 years, not displaying any endocrine, neurological, nephrological or urogenital disorders.Children taking any kind of medication or with a fever at the time of collection of urine specimen were excluded from the study.Samples were collected from children who visited the outpatient department for minor health problems and from normal children of the hospital staff .Subjects were natives of Greece.This research study was approved by the Research Ethics Committee and local school authorities.
Each sample was collected according to the following protocol: Written consent was first ob-tained from the parents of each child who were fully informed about the study design.Then the first 5 mL of urine was collected from the first morning urination between 7.00-9.00hours.The sample was centrifuged at 3000 s/min for 2 minutes and the top 2 mL of supernatant was stored at -30 o C. In addition, height and body weight was recorded in order to calculate the respective stature-for-age, weight-forage and body mass index (BMI)-for-age percentiles of the sample.uPSA was calculated using the DPC Immulite method, which has a lower limit of detection of 0.003 μg/L.Samples that had PSA levels outside of the measurable range of the DPC Immulite were measured using the ROCHE technique.Biostatistical analysis was performed using the Stata 9.0 software suite.We used the Pearson correlation to study correlations between continuous variables, the ANOVA or linear regression analysis to study continuous over categorical variables and Pearson's chi square test was utilized to study categorical variables.For all statistical tests a significance level of 0.05 was used.As far as the age of the subjects was concerned, we examined it not only as a continuous variable, but also as a binomial categorical variable as well in two groups: a childhood group (26 boys, ≤ 12 years old) and adolescent group (16 boys, > 12 years old).

RESULTS
PSA was detected in the urine of 18 of 42 boys (42.8%) and in three of 16 girls (18.75%).uPSA levels tend to be higher in male than female children (p = 0.091, linear regression analysis).Two girls 6 and 11 years old respectively had a uPSA value of 10 ng/L and another one 13.5 years old had uPSA of 316 ng/L.The range of uPSA in boys presented greater variability.Especially in boys 5-8 years old, uPSA activity was detected in 5 of 14 specimens (35.7 %).In 4 cases the range was 3-10 ng/L and in one case the activity was < 3 ng/L.In the group of boys aged 8-12 years old it was detected in 2/12 (16.6%) specimens with values 20 and 79 ng/L.In boys older than 12   for-age or BMI-for-age percentiles (Table-1).Boys were divided according to their age into two groups: childhood (26 boys, ≤ 12 years old) and adolescent group (16 boys, > 12 years old) in order to study if there was any relation of weight to uPSA.Sub-classification of each group according to BMI-for-age percentiles in normal, overweight and obese was also performed.We also investigated whether BMI-for-age percentiles classification correlates with age group using Pearson's chi square.No relation was found (p = 0.69).Linear regression analysis did not show any relation of uPSA with obesity in neither childhood nor in adolescence groups.

COMMENTS
It is well known that serum PSA levels rise sharply with increasing stage after males reach puberty and this rise has been shown to correlate with testosterone levels (6,12).A recent study from Antoniou et al. showed that serum PSA levels do not differ significantly between the two sexes until the age of 12 years, with median values of 38.41 ng/L (range: 1-2768) and 4.059 (1-287) ng/L for boys and girls respectively.After the age of 12, serum PSA levels in boys are seen to increase sharply (13).Another study by Sato et al. found that there is a gradual increase in uPSA activity from 1-4 month old infant males and boys older than 10 years of age.In boys 0.3-9 years there was no uPSA activity (7).These results reflect the previous study by Goldfarb et al. who showed that PSA levels in prostatic tissue were high at birth and decreased after 6 months, and subsequently reappeared by 10 years of age and increased during puberty (14).
In our study, we showed that uPSA was detectable in 26% of boys aged 5-12 years old within the range of 2-79 ng/L.In boys older that 12 years uPSA values increased up to 161000 ng/L.There was a statistically significant correlation between uPSA levels and height.Taller boys had higher levels of uPSA.This correlation disappeared when it was investigated in relation to stature for age percentiles because this variable is corrected for age.Statistical analysis of uPSA levels in boys with weight, BMI and the respective stature-for-age, weight-for-age and BMI-for-age percentiles of the population did not reveal any correlation.
uPSA was found only in a small percentage of girls in our sample (18.75% of girls).Other studies have shown that PSA is detectable in 11-38% of adult females with mean values of 290 and 3.720 ng/L respectively (15,16).Periurethral glands or the "female prostate" are considered to be homologous to the prostate gland as these organs have the same embryological origin (17,18).Periurethral glands in females are considered to be the primary site of PSA secretion in urine and manifest active secretion in approximately 66.7-80% of women (2,17,18).These glands do not undergo developmental changes from fetal to adult age (17).Another possible source of female adult urine PSA is the reactive metaplastic changes of cystitis cystica and cystitis glandularis that occur in adult women (19).The presence of PSA in females is under regulation of steroid hormones especially androgens and progestin.Our results support the concept that periurethral glands do not (if at all) influence significantly the serum PSA in both sexes (20).
uPSA could be potentially used as a non-invasive marker of sexual development and abnormalities in boys.However, there are major disadvantages for its use.It requires hypersensitive PSA assays that are not always available in the biochemistry department.In addition values are not standardized and may fluctuate in the same subject from one urine sample to another (21).Our study design has certain limitations as it has involved a relatively small cohort of children, a single urine specimen from each child and the Tanner stage of the individuals was not recorded.Better designed studies which consider the above limitations will help to better define uPSA levels and variation in this population.

CONCLUSION
The use of hypersensitive assays allows calculation of uPSA in childhood.The values of this variable are measurable in both sexes and related with gender.In boys, uPSA is correlated with age and height but not with other variables tested.Further studies are required to clarify this field.

Figure 1 -
Figure 1 -Urine PSA in boys in relation to age.There is a sharply increase of uPSA in boys after the age of 12 years, when they come into adolescence.

Figure 2 -
Figure 2 -Urine PSA in boys in relation to height.The slope is proportional to that of Figure-1 reflecting as a major contributor of increase entry of boys into adolescence.

Table 1 -
Results of statistical analysis in boys.For all statistical tests a significance level of 0.05 was used.BMI = mass body weight. *