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Published-Ahead-of-Print September 6, 2006, DOI:10.2164/jandrol.106.000190
Journal of Andrology, Vol. 28, No. 1, January/February 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.106.000190

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Urinary Prostate-Specific Antigen Is a Noninvasive Indicator of Sexual Development in Male Children

ITARU SATO*,{dagger}, ATSUKO YOSHIKAWA{ddagger},§, MASATOSHI FUGIMOTO||, KEIKO SHIMIZU, ATSUYA ISHIWARI*, TOSHIJI MUKAI{dagger} AND TERUAKI IWAMOTO#

From the * Scientific Crime Laboratory, Kanagawa Prefectural Police, Yokohama, Japan; the {dagger} Department of Legal Medicine, St Marianna University School of Medicine, Kawasaki, Japan; the {ddagger} Department of Microbiology and Immunology, The Institute of Medical Science, Tokyo University, Tokyo, Japan; the || Department of Pediatrics, St Marianna University School of Medicine, Kawasaki, Japan; the Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Japan; and the # Department of Urology, St Marianna University School of Medicine, Kawasaki, Japan.

Correspondence to: Dr Itaru Sato, Forensic Biology Unit, Scientific Crime Laboratory, Kanagawa Prefectural Police, 155-1, Naka-ku, Yamashita-cho, Yokohama, 231-0023, Japan (e-mail: itaru-s{at}m2.ocv.ne.jp).
Received for publication April 7, 2006; accepted for publication August 28, 2006.

   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Testicular androgen induces the synthesis of prostate specific antigen (PSA) in acinar epithelial cells of the prostate. We examined PSA activity in urine from 136 male children from birth up to 17 years of age. We detected PSA at various intervals in early infant urine over a period of 1–4 months. During this period, urinary secretion of testosterone (T) gradually declined, accompanied by 1 or more surges of T prior to a transient increase in PSA in urine from full- and preterm infants (67%, n = 6). Although mean urinary T concentrations during elevations of PSA in preterm infants were 3.1 and 5.6 times greater than in full-term infants and adults, the overall mean urinary PSA concentration of full and preterm infants was just 45% and 18% that of adults, respectively. PSA was not detected in children aged 0.3 to 9 years, after which a gradual increase in urinary PSA activity was observed after 10 years of age. Urinary PSA activity was markedly persistent after Tanner stage III pubertal development. To our knowledge, this is the first study to demonstrate an induction of PSA during early infancy by bioactive T in normally developing human males. We conclude that urinary PSA is a non-invasive, useful indicator for developmental studies from neonatal and adolescent males, which can be measured with a confirmatory semiquantitative PSA assay.

     Key words: Urine, infant, puberty


Androgen is required for the development of secondary sexual characteristics as well as development of the reproductive organs, and also influences the development of social sexual behavior (Mann and Fraser, 1996). Transitory testosterone (T) secretion occurs during early infancy following the appearance of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Work by several investigators has shown effects on testicular development, as well as on development of the external genitalia and sexual behavior, following administration of gonadotropin-releasing hormone (GnRH) agonists and antagonists in nonhuman primates (Mann et al, 1989; Mann et al, 1993; Wallen et al, 1995; Mann and Fraser, 1996; Brown et al, 1999). Although T surges during the first 3 months of life may influence the later onset of puberty in human males, direct evidence regarding the biological function of T during early infancy has yet to be established (Forester et al, 1974).

Prostate-specific antigen (PSA; hK3 for a chymotrypsin-like serine protease) is a member of the human kallikrein (hK) family and might be a useful androgen indicator of pubertal development in boys. Because testicular androgen induces the synthesis of PSA in acinar epithelial cells of the prostate (Stamey et al, 1989), PSA can be detected in both semen and adult male urine (Sato et al, 2002). In addition, T elevation at the onset of puberty might induce permanent synthesis of PSA in the prostate (Kim et al, 1999). Urine can usually be obtained noninvasively; therefore, measurement of T and PSA in urine (Irani et al, 1996, 1997; Barret et al, 2002; Irani et al, 2005) may prove a useful indicator by which to follow sexual development in male children.

We therefore investigated whether male sexual development from birth to adolescence can be evaluated using urinary PSA levels.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
We obtained morning (0930–1200 hours) spot urine samples from 92 boys between the ages of 2 months and 14 years with nonendocrinological conditions attending the Pediatrics Unit at St Marianna University School of Medicine, Kawasaki, Japan, as well as from 44 healthy male volunteers between 6 and 18 years of age. Clinical data was obtained for these boys by their pediatricians, including height, body weight, testicular mass, and development of pubic hair, after which they were classified into 5 groups, spanning Tanner stages I to V (Table 1). We obtained morning urine samples from 6 male infants from birth up to 18 weeks of age at weekly intervals. We obtained the history of each individual, including his date of birth, expected date of confinement, birth weight, and feeding manner, as well as the parity of the mother. We also obtained spot urine samples from 6 female infants (aged 0.3–1.2 years) and 6 pubertal girls (aged 8–12) as negative controls in this study. A local research ethics committee approved the study, and the parents or guardians of the children provided written informed consent to all procedures associated with the study. We measured PSA activity in spot urine samples using a SMITEST PSA card (Seratec Diagnostica, Gottingen, Germany) and used an immunochromatographic membrane method for semiquantitative screening. Although the SMITEST PSA card has a limit of detection of 4 ng/mL PSA, a faint immunoreaction still appears in the range of 1–3 ng/mL according to the manufacturer. Therefore, all responses were compared with the reference sample (4 ng/mL) and quantified as follows: PSA 4+ (4 ng/mL), PSA 3+ (3 ng/mL), PSA 2+ (2 ng/mL), PSA + (1 ng/mL), or PSA negative (–) (Sato et al, 2002). In addition, we measured PSA activity in spot urine samples using an IMMULITE third-generation PSA enzyme-amplified luminescence system (Diagnostic Products Corp, Los Angeles, Calif) according to the method of Randell et al (1996) as an ultrasensitive quantitative assay for monitoring changes in total PSA concentrations in 6 male infants. The analytical sensitivity of this system is 0.003 ng/mL. Total urinary T levels were measured using a commercially available solid-phase radioimmunoassay (Diagnostic Products Corp) according to the manufacturer's instructions, with slight modifications according to the method of Huhtaniemi et al (1986). Prior to analysis, urine samples were treated with 12 N NaOH to ensure hydrolysis and then boiled for 15 minutes. PSA and T levels were expressed as micrograms (µg) per millimole (mmol) of urinary creatinine (Irani et al, 1997).


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  		Table 1. Urinary PSA in relation to pubertal stage in 59 healthy boys*
 


Figure 1
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  		Figure 1. Urinary PSA activity from birth to adolescence was screened using the immunochromatographic membrane test for PSA on the SMITEST PSA card. The line is a polynominal regression fit to the data points (•).

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
We measured urinary PSA activity in normal urine samples from birth (age: 0 days) up to adolescence (age: 17 years) using an immunochromatographic membrane PSA test, which can visually identify the presence of PSA (negative: 0; positive range: from 1+ to 4+), as shown in Figure 1. Within 4 months of birth, faint PSA activity appeared in 20 (25%) of 80 urine samples (n = 14, PSA range: 1–3 ng/mL). Although undetectable in urine from children between the ages of 4 months and 9 years (n = 64, 73 urine samples), urinary PSA activity was identified in 11 (32%) of 34 boys (34 urine samples) aged between 10 and 12 years (PSA range: 1–3 ng/mL). Thereafter, urinary PSA was typically detected in individuals over 13 years of age (n = 24, 24 urine samples, PSA range: greater than 4 ng/mL). When the relationship between urinary PSA activity and pubertal stage was examined (Table 1), PSA activity was detected in pubertal boys meeting the criteria for Tanner stages II and III (aged 12–13 years), as well as boys meeting the criteria for Tanner stage I development (aged less than 1 year). PSA activity was not detected in urine from 6 female infants and 6 pubertal girls (data not shown).


Figure 2
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  		Figure 2. Trends in urinary PSA and testosterone concentrations in 6 neonates. Urinary PSA and T levels were measured using an enzyme-amplified luminescence method known as IMMULITE Third Generation (Randell et al, 1996), and a solid-phase radioimmunoassay, respectively. PSA and T levels were expressed as micrograms per Nmillimole of urinary creatinine. Symbols indicate testosterone (•), PSA ({circ}), and expected date of confinement ({blacktriangleup}). Values within parentheses indicate birth weight. Arrows indicate T surge.

 
Following this, we quantitatively monitored urinary T and PSA levels in 6 male infants from birth to 18 weeks at weekly intervals (Figure 2). Although T levels in full-term infants (Figure 2, numbers 1, 2, and 3) remained low throughout early infancy, high levels were observed in preterm infants (Figure 2, numbers 4, 5, and 6), after which levels were noted to decline with age. In all infants, a peak in PSA occurred once (Figure 2, numbers 1 and 5), 2–4 times (Figure 2, numbers 2 and 6), or continuously (Figure 2, number 4) in each individual prior to age 9 weeks.

We compared urinary T and PSA levels during early infancy among full-term and preterm infants (Table 2). Urinary T levels in preterm infants were significantly higher during early infancy than in full-term infants. Although mean urinary T concentrations during each PSA peak in preterm infants were 3.1- and 4.2-fold those in full-term infants and adults, respectively, mean PSA concentrations were only 45% and 18% those of adults.


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  		Table 2. Comparison of reproductive parameters between full-term and preterm delivery during early infancy*
 


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Urinary PSA activity was examined during infancy and adolescence, as shown in Figure 1. Our results support those of an immunohistochemical study demonstrating PSA activity in the lumina of primary prostatic ducts, as well as the transitional epithelium of infants aged between 0 and 6 months (Goldfarb et al, 1986), with detectable changes in PSA activity in neonatal serum (Randell et al, 1996). This prior study also suggests a lack of PSA expression in prostate tissue between 6 months and 10 years of age, after which increased levels are observed until puberty (Goldfarb et al, 1986). Furthermore, a polynomial regression line of PSA production parallels T production from birth until puberty (Mann and Fraser, 1996).

PSA reappears at the onset of development of secondary sexual characteristics in boys aged between 10 and 12 years (Randell et al, 1996; Sato et al, 2002), a period corresponding to testicular growth and the development of pubic hair (Rosenfield, 1990). Although increased serum PSA activity corresponds to increased serum T levels in healthy pubertal boys that have reached Tanner stages III (T-III) development, PSA activity in boys with precocious puberty exceeds that of healthy boys (T-II), even in subjects under 10 years of age (Viera et al, 1994; Juul et al, 1997; Kim et al, 1999). The present data of boys aged 12 indicates greater urinary PSA activity in taller boys with increased testicular mass and body weight, compared to shorter boys of similar (Table 1, Tanner stage I–III). In shorter boys of lesser weight, weak or absent urinary PSA activity was detected (Table 1, Tanner stage I at 13 years of age). The onset of puberty is well correlated with bone density, and T plays a role in skeletal development (Rosenfield, 1990; Mann et al, 1993). Therefore, abnormal androgen production in boys with a delayed mean age of onset of puberty might influence PSA production as well as sexual behavior (Rosenfield, 1990; Mann et al, 1993; Wallen et al, 1995). Measuring urinary PSA activity and urinary T with respect to pubertal development might be useful in examining their relationship with the various Tanner stages. Lack of detection of PSA in the urine of boys aged 4 months to 9 years might reflect a decreased production of sex steroid hormones during this period. As of yet, the physiological significance of PSA production between 1 and 4 months of age remains unclear. The present findings suggest that synthesis of PSA in acinar epithelial cells of the prostate might be promoted and enhanced by peaks in T (Goldfarb et al, 1986).

Recently, Barrett et al (2002) reported similar changes in urinary and serum T levels in nonhuman primates. To better understand the relationship between T and PSA, we quantitatively monitored urinary T and PSA levels in male infants from birth up to 18 weeks of age (Figure 2). Although a direct comparison between urinary T concentrations (Figure 2) and previous reported salivary T concentrations (Huhtanieme et al, 1986) could not be made to give differences in reporting results, similar trends were observed because urinary T was expressed as micrograms per millimole of urinary creatinine. Transient PSA peaks were observed in all subjects prior to 9 weeks of age, indicating fluctuations in urinary PSA during this period. During the neonatal period, urinary PSA levels reached adult levels (Table 2). The highest (H) and lowest (L) urinary PSA values prior to correction for creatinine were 9.1- and 600-fold greater than serum PSA levels during this period (0.007 µg/L; Randell et al, 1996). Urinary PSA reached a high of 4.25 ng/mL 5 weeks after birth in subject number 1, and a low of 0.064 ng/mL 4 weeks after birth in subject number 4. This indicates that almost all of the PSA synthesized by acinar epithelial cells of the prostate was concentrated in urine. In addition, we did not observe any PSA activity in the urine of females during the neonatal period using a SMITEST PSA card. These results suggest that androgen activity in male neonates might stimulate the prostate to produce PSA, and that PSA might modulate the interaction between male infants and their mothers. During the first year of life in rhesus monkeys, females spend more time close to their mothers than males, and neonatal T-suppressed males show more maternal dependence, while T-augmented males exhibit less maternal interaction and more independence (Wallen et al, 1995).

Although T levels in full-term infants remained low throughout early infancy in the present study, preterm infants showed elevated levels, which declined with age. When T levels in full-term infants were compared with T levels in preterm infants, mean levels were 3.1-fold lower throughout early infancy (Table 2). Significant differences in T levels according to mode of delivery were also observed (t test; P < .01). However, T concentrations did not appear to be related to PSA activity following either mode of delivery (Figure 2 and Table 2). Therefore, T levels may not modulate PSA activity. In addition, all individuals initially exhibited urinary PSA within a few weeks after the first T surge (arrows in Figure 2). This indicates that PSA synthesis by acinar epithelial cells of the prostate may be promoted and/or enhanced by the T surge (Goldfarb et al, 1986).

Although the mature prostate of the adult does not require abundant T to synthesize PSA, increased stimulation by T might be required for synthesis in the immature prostate in early infancy (Table 2). In full-term infants (Table 2, numbers 1, 2, and 3), PSA appeared uniformly in urine from 5 weeks after birth, while detection of urinary PSA was delayed in preterm infants (Table 2, numbers 4, 5, and 6). However, PSA activity appeared in urine at 5 weeks of age, around the normal time of birth.

We first demonstrated that an adult reproductive characteristic is evident in male neonates by measuring urinary T and PSA. Although PSA is also detected in neonatal serum (Sato et al, 2002), it can be difficult to obtain from infants and children; thus, the presence of PSA in urine is of considerable interest. Urine can usually be directly obtained from individuals with minimal damage or stress. The PSA assay used in this study might be used to monitor PSA levels in accordance with various stages of sexual development in the future. However, PSA activity within spot urine samples is difficult to detect and may fluctuate during the neonate period (eg, a full-term infant; Figure 2, number 3). Therefore, functional studies of the immature prostate, specifically hormonal and protein assays, should be used performed on pooled urine rather than spot urine samples.

Further investigation into the responsiveness of the prostate to androgen, as well as the behavioral effects of PSA production, during the neonatal period, is required.


   Acknowledgments
 
This study was performed by the Cooperation Research Program of Primate Research Institute, Kyoto University. The authors wish to thank Miss Mieko Askaham, M Sci, Department of Pediatrics, St Marianna University School of Medicine, for providing excellent secretarial support during the preparation of the manuscript.


   Footnotes
 
Supported by Grants-in-Aid for scientific research of IS (15922076 and 16922185) from the Japan Society for the Promotion Science as well as a 21st Century COE Research grant (Kyoto University, A14) from the Ministry of Education, Science, Sports and Culture, Japan, and in part by a Cooperation Research Program of Primate Research Institute, Kyoto University.

§ Present address: Research Institute, International Medical Center of Japan. Back


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