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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2597-2599
Copyright © 2003 by The Endocrine Society

Serum Androgen Bioactivity in Cryptorchid and Noncryptorchid Boys during the Postnatal Reproductive Hormone Surge

Taneli Raivio, Jorma Toppari, Marko Kaleva, Helena Virtanen, Anne-Maarit Haavisto, Leo Dunkel and Olli A. Jänne

Biomedicum Helsinki, Institute of Biomedicine/Physiology (T.R., O.A.J.), and Department of Clinical Chemistry (O.A.J.), University of Helsinki, and Helsinki University Central Hospital, FIN-00014 Helsinki, Finland; Departments of Pediatrics and Physiology (M.K., H.V., A.-M.H., J.T.), University of Turku, FIN-20520 Turku, Finland; and Hospital for Children and Adolescents (L.D.), Helsinki University Central Hospital, FIN-00029 Helsinki, Finland

Address all correspondence and requests for reprints to: Olli A. Jänne, M.D., Ph.D., Biomedicum Helsinki, Institute of Biomedicine (Physiology), University of Helsinki, P.O. Box 63, Haartmaninkatu 8, FIN-00014 Helsinki, Finland. E-mail: olli.janne{at}helsinki.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The first postnatal months of life in boys are characterized by activation of the hypothalamic-pituitary-testicular axis that results in the well depicted surge of reproductive hormones. Serum testosterone levels at that time are high, but infants do not display signs of virilization, and subsequently there is only indirect evidence that circulating androgens during the surge are biologically active. We used a recombinant cell bioassay to determine serum androgen bioactivity in 80 3-month-old boys born after full-term pregnancies (37–42 wk) in whom localization of the testes was determined by palpation after birth and at a mean age of 3 months. At that age, serum androgen bioactivity ranged from less than 0.8 to 1.9 nM testosterone equivalents and correlated with serum testosterone concentration (r = 0.71; P < 0.0001; n = 34), free androgen index (r = 0.80; P < 0.0001; n = 34), age (r = -0.29; P < 0.01; n = 80), and localization of the testes (r = -0.24; P < 0.05; n = 80). Moreover, all boys in this study with detectable androgen bioactivity (n = 26) had testes located in scrotal or high scrotal position (n = 64), whereas all boys (n = 16) with at least 1 suprascrotal, inguinal, or nonpalpable testis had nonmeasurable androgen bioactivity in serum (P < 0.01). We conclude that 3-month-old boys are exposed to biological effects of androgens during the postnatal activation of the hypothalamic-pituitary-testicular axis, and that this exposure may be reduced in boys with at least 1 testis located superior to the scrotum.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
DURING THE POSTNATAL activation of the hypothalamic-pituitary-testicular axis, boys display high serum gonadotropin, inhibin B, and androgen (testosterone and 5{alpha}-dihydrotestosterone) concentrations (1, 2, 3, 4). However, owing to the concomitant excess of circulating SHBG (3), it is not known whether the androgens are biologically active during this postnatal hormone surge. For example, a proposed indicator of bioactive testosterone, testosterone in saliva, does not increase after birth (5), whereas the involution of the scrotum and the lack of postnatal penile growth without the hormone surge suggest a significant bioactivity of postnatal androgens (6). Likewise, boys with cryptorchidism may display problems with the last androgen-dependent phase (7, 8) of testicular descent during the course of postnatal reproductive hormone secretion. In this work we used a recently developed recombinant cell bioassay (9) to investigate androgen bioactivity in the sera of 80 3-month-old boys with normally or incompletely descended testes.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The mothers of 80 infant boys volunteered to participate in a prospective cryptorchidism study conducted at Turku University Central Hospital. The boys, born after full-term pregnancies (37–42 wk), were clinically examined after birth and at a mean age of 3 months (range, 2.7–3.8 months). On both occasions, localization of the testes was determined by palpation. Testes were localized in 1 of the 5 places: scrotal, high scrotal, suprascrotal, inguinal, and nonpalpable. A scrotal testis located at or below the border of the upper and lower parts of the scrotum. A high scrotal testis could be brought to the upper part of the scrotum. A suprascrotal testis could be pulled out from the inguinal canal, but not below the border of the scrotum (the border of scrotal skin). An inguinal testis was felt within the inguinal canal; a nonpalpable testis was not found during the examination. After birth, 35 boys had normally located testes (2 boys had unilateral retractile testes that were considered normally located) and 45 boys displayed cryptorchidism: 31 boys had unilateral cryptorchidism (5 high scrotal, 5 suprascrotal, 15 inguinal, and 6 unpalpable testes) and 14 boys had bilateral cryptorchidism (18 high scrotal, 2 suprascrotal, 4 inguinal, and 4 unpalpable testes). By the age of 3 months, the testes had descended in 20 boys, whereas 25 boys still displayed cryptorchidism (23 boys with unilaterally undescended testes had 9 high scrotal, 5 suprascrotal, 6 inguinal, and 3 unpalpable testes, and 2 boys with bilaterally undescended testes had 1 high scrotal, 2 suprascrotal, and 1 unpalpable testes). Of these 25 boys, 5 boys with 3 high scrotal and 2 inguinal testes at the age of 3 months displayed spontaneous testicular descent by the age of 18 months. One boy with suprascrotal testis was clearly older (3.8 months) than the other 79 boys (range, 2.7–3.3 months). To investigate the relationship between testicular localization and serum androgen bioactivity, the following scoring system was employed: nonpalpable testis = score 5; inguinal = 4; suprascrotal = 3; high scrotal = 2; and scrotal = 1 (the higher of the 2 scores for each subject was used in the subsequent analyses). At the mean age of 3 months, a single venous blood sample was drawn by an open needle system between 1200 and 1800 h. The blood was allowed to clot, then was centrifuged, and serum was stored at -20 C until analyzed. The study protocol was accepted by the joint ethical committee of Turku University and Turku University Central Hospital.

Assays

The androgen bioassay was performed as previously described (9). In short, COS-1 cells were transiently transfected with plasmids encoding the ligand-binding domain and the amino-terminal region of the androgen receptor. The presence of androgen leads to interaction of the 2 receptor-derived polypeptides; the interaction is amplified by a coactivator, androgen receptor-interacting protein 3, also transfected to COS-1 cells. The reporter gene (luciferase) activity in cell lysates is derived from androgen bioactivity in human serum. The lowest standard employed in the bioassay (0.8 nM testosterone in fetal calf serum) is the lowest testosterone concentration that could be reliably distinguished from the assay noise (mean + 2 SD of multiple measurements with no testosterone) (9). Serum androgen bioactivity levels were considered unnmeasurable when the relative reporter gene activity brought about by 1 or both replicate measurements of subject’s serum sample did not exceed the sensitivity of the assay. The serum SHBG concentration (n = 34) was measured using a time-resolved fluoroimmunoassay (Perkin-Elmer Corp., Turku, Finland) with intra- and interassay coefficients of variation less than 6%. The serum testosterone concentration was measured in 34 boys born with testis scores of 2–5; at the age of 3 months, 23 boys had testis scores of 1–2, and 11 boys displayed scores of 3–5. Serum testosterone was determined using an RIA kit (Orion Diagnostica, Espoo, Finland) with a sensitivity of 0.1 nM and intra- and interassay coefficients of variation less than 8%. In this work the measurements were carried out after denaturation of serum SHBG by heating (60 C for 30 min) to minimize the possible interference of this carrier protein with the RIA.

Data analyses

The relationships between two related variables were assessed with linear correlation coefficients and respective P values. In the correlation analyses, unmeasurable androgen bioactivity levels were set equal to 0.8 nM testosterone equivalents. The dependence between serum androgen bioactivity (detectable vs. nondetectable) and testicular localization (scores 1–2 vs. scores 3–5) was investigated using a {chi}2 test. Comparisons between two groups were carried out by t test. The differences between ages in three subgroups were tested with ANOVA, followed by Scheffé’s post hoc analysis. Results were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
We used a recombinant cell bioassay to investigate serum androgen bioactivity in 80 3-month-old boys with normally located or incompletely descended testes. Overall, serum androgen bioactivity level ranged from less than 0.8 to 1.9 nM testosterone equivalents and correlated negatively with age (r = -0.29; P < 0.01; n = 80) and positively with serum testosterone concentration (r = 0.71; P < 0.0001; n = 34) and serum free androgen index (serum testosterone/SHBG x 100; r = 0.80; P < 0.0001; n = 34; Fig. 1Go). The mean ± SEM testosterone and SHBG levels in these 34 subjects were 3.6 ± 0.4 and 161 ± 6 nM, respectively. Importantly, the score describing testicular localization correlated negatively with androgen bioactivity (r = -0.24; P < 0.05; n = 80; Fig. 2Go), but not with age (r = 0.13; P = NS; n = 80). Thus, high bioactivity was associated with normal descent of the testes.



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  		Figure 1. The relationship between serum androgen bioactivity and testosterone concentration (left) and serum androgen bioactivity and free androgen indexes (right) in 34 3-month-old boys. Linear regression lines are shown.

 


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  		Figure 2. The relationship between testicular localization (x-axis) and serum androgen bioactivity (y-axis) in 80 3-month-old boys (23 boys had unilateral cryptorchidism, and 2 boys had bilaterally undescended testes; testicular localization refers to the position of the superior of the 2 testes in each subject). The number of study subjects in each group is shown.

 
All boys in this study with detectable androgen bioactivity (n = 26) had testes located in the scrotal or high scrotal position (testis score, 1–2; n = 64), whereas all 16 boys with at least 1 suprascrotal, inguinal, or unpalpable testis (testis score, 3–5) had unmeasurable androgen bioactivity (P < 0.01; Fig. 2Go). Age did not differ between boys with testis scores of 1–2 and detectable (n = 26) or undetectable (n = 38) serum androgen bioactivity or between the latter subgroup and those with testis scores of 3–5 (n = 16; by ANOVA). However, the boys with scores of 3–5 were older than those with detectable androgen bioactivity (3.1 ± 0.06 vs. 2.9 ± 0.03 months; P < 0.05, by ANOVA). This difference was due to the fact that 1 boy with a testis score of 3 was clearly older than the other boys studied; exclusion of this subject abolished the age difference (P = NS, by ANOVA), but did not influence the correlation between testis score and serum androgen bioactivity (r = -0.24; n = 79; P < 0.05) or the relationship between the presence or absence of measurable androgen bioactivity and testicular localization (P < 0.01, by {chi}2 test).

Of the 34 boys in whom serum testosterone was measured, the boys with testis scores of 1–2 (n = 23) had higher serum testosterone concentrations than the 11 boys with testis scores of 3–5 (4.3 ± 0.5 vs. 2.1 ± 0.3 nM; P < 0.01); this difference remained significant after exclusion of the oldest subject (P < 0.05). Twenty boys born with undescended testes still had cryptorchidism at the age of 18 months. However, at the age of 3 months serum androgen bioactivity did not differ significantly between them and the other 60 boys (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The postnatal gonadotropin and sex steroid surge seems to represent the response of the hypothalamus and the pituitary gland to the decline in circulating androgens after birth (10, 11). The proposed roles for the surge involve endocrine alterations, imprinting of sexual orientation and behavior, and priming of target tissues for subsequent growth and maturation later in life (10). These proposals thus suggest that androgens are biologically active during the surge, and the results of the present work provide direct evidence to support this. Assuming that the peak of serum testosterone concentration occurs between 30 and 60 d of age (3), it is likely that the proportion of boys with detectable androgen bioactivity would have been even higher if they had been studied earlier than at the mean age of 3 months.

The etiology of cryptorchidism usually remains unknown; the condition may result from anatomical obstacles, intrinsic testicular causes, or endocrine abnormalities (12, 13, 14). For example, the postnatal rise of serum testosterone may be blunted in some cryptorchid babies (15), whereas other investigators have demonstrated an intact function of the hypothalamic-pituitary-testicular axis during the first year of life regardless of testicular localization (8). In children with unilateral cryptorchidism, even the normally located testis may show cellular changes (16). The fact that undetectably low serum androgen bioactivity and low serum testosterone concentration were both found among the boys with at least one testis superior to the scrotum implies that there is no major discrepancy between serum testosterone concentration and androgen bioactivity in cryptorchidism. The suppressed androgenic milieu in these boys could not be explained by differences in age or serum LH levels (data not shown). Our results suggest that infants with at least one testis located superior to the scrotum are exposed to reduced androgen bioactivity during the postnatal reproductive hormone surge. The possible relationship between this finding and the subsequent development of the genitalia, fertility, and sexual behavior in adulthood requires long-term follow-up of the present cohort and/or investigation of these aspects in men with histories of cryptorchidism.

In conclusion, we have employed a mammalian cell bioassay to investigate circulating androgen bioactivity in 3-month-old boys. Our results suggest that infant boys are exposed to biological effects of androgens during the postnatal activation of the hypothalamic-pituitary-testicular axis, and that the degree of this exposure may be reduced in boys with a testis located superior to the scrotum.


   Acknowledgments
 
We thank Ms. Johanna Iso-Oja for excellent technical assistance.


   Footnotes
 
This work was supported in part by grants from Medical Research Council (Academy of Finland), National Technology Agency, Sigrid Jusélius Foundation, Helsinki University Central Hospital, Foundation for Pediatric Research, European Union Contract QLK4-1999-01422, and Turku University Central Hospital.

Received October 25, 2002.

Accepted March 3, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Forest MG, Sizonenko PC, Cathiard AM, Bertrand J 1974 Hypophyso- gonadal function in humans during the first year of life. I. Evidence for testicular activity in early infancy. J Clin Invest 53:819–828
  2. Andersson A, Toppari J, Haavisto AM, Petersen JH, Simell T, Simell O, Skakkebaek NE 1998 Longitudinal reproductive hormone profiles in infants: peak of inhibin B levels in infant boys exceeds levels in adult men. J Clin Endocrinol Metab 83:675–681[Abstract/Free Full Text]
  3. Bolton NJ, Tapanainen J, Koivisto M, Vihko R 1989 Circulating sex hormone-binding globulin and testosterone in newborns and infants. Clin Endocrinol (Oxf) 31:201–207[Medline]
  4. Pang S, Levine LS, Chow D, Sagiani F, Saenger P, New MI 1979 Dihydrotestosterone and its relationship to testosterone in infancy and childhood. J Clin Endocrinol Metab 48:821–826[Medline]
  5. Huhtaniemi I, Dunkel L, Perheentupa J 1986 Transient increase in postnatal testicular activity is not revealed by longitudinal measurements of salivary testosterone. Pediatr Res 20:1324–1327[Medline]
  6. Main KM, Schmidt IM, Skakkebaek NE 2000 A possible role for reproductive hormones in newborn boys: progressive hypogonadism without the postnatal testosterone peak. J Clin Endocrinol Metab 85:4905–4907[Abstract/Free Full Text]
  7. Hutson JM, Donahoe PK 1986 The hormonal control of testicular descent. Endocr Rev 7:270–283[Medline]
  8. De Muinck Keizer-Schrama SMPF, Hazebroek FWJ, Drop SLS, Degenhart HJ, Molenaar JC, Visser HKA 1988 Hormonal evaluation of boys born with undescended testes during their first year of life. J Clin Endocrinol Metab 66:159–164[Abstract]
  9. Raivio T, Palvimo JJ, Dunkel L, Wickman S, Jänne OA 2001 Novel assay for determination of androgen bioactivity in human serum. J Clin Endocrinol Metab 86:1539–1544[Abstract/Free Full Text]
  10. Quigley CA 2002 Editorial: the postnatal gonadotropin and sex steroid surge: insights from the androgen insensitivity syndrome. J Clin Endocrinol Metab 87:24–28[Abstract/Free Full Text]
  11. Bouvattier C, Carel J-C, Lecointre C, David A, Sultan C, Bertrand A-M, Morel Y, Chaussain J-L 2002 Postnatal changes of T, LH, and FSH in 46,XY infants with mutations in the AR gene. J Clin Endocrinol Metab 87:29–32[Abstract/Free Full Text]
  12. Toppari J, Kaleva M, Virtanen H 2001 Trends in the incidence of cryptorchidism and hypospadias, and methodological limitations of registry-based data. Hum Reprod Update 7:282–286[Abstract/Free Full Text]
  13. Toppari J, Kaleva M 1999 Maldescendus testis. Horm Res 51:261–269[CrossRef][Medline]
  14. Sizonenko PC 1993 Disorders of the testis. In: Bertrand J, Rappaport R, Sizonenko PC, eds. Pediatric endocrinology: physiology, pathophysiology, clinical aspects, 2nd ed. Baltimore: Williams & Wilkins; 430–446
  15. Gendrel D, Roger M, Job JC 1980 Plasma gonadotropin and testosterone values in infants with cryptorchidism. J Pediatr 97:217–220[CrossRef][Medline]
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