Dr. Vladimir Zaichick, Medical Radiological Research Centre, Koroleva Str. 4, Obninsk 249036, Kaluga Region, Russia. E-mail: firstname.lastname@example.org
To clarify age-related histological and zinc content changes in paediatric and nonhyperplastic young adult prostate glands, a quantitative morphometric and energy-dispersive X-ray fluorescence analysis (EDXRF) was performed. The prostates were obtained at autopsy from 50 subjects (European-Caucasian aged 0–30 years) who died from sudden infant death syndrome, acute pulmonary aetiologies and trauma. None of the subjects had any symptoms of prostatic disease and all prostates were classified as histologically normal. Each prostate was divided into two portions. One tissue portion was reviewed by an anatomical pathologist whereas another was intended for a Zn mass fraction measurement using EDXRF. The mean per cent volume of the stroma (S), glandular epithelium (E), glandular lumen (L) and glandular component (GC = E + L) were determined and the mean ratios of per cent volumes (S/E, S/GC and E/L) were calculated for each prostate specimen. It was found that normal prostate tissue undergoes substantial changes from birth through early adulthood. These changes are reflected in an increment of the per cent volume of the glandular epithelium and lumen and in a diminution of the per cent volume of the stroma. The S/E ratio of the prostate falls by a factor of almost 4, between ages <1 and 30 years from 5 : 1 to 1.3 : 1. The Zn mass fraction is nearly 30–35 mg/kg (wet weight basis) and remains steady for first decade of life. Then it begins to increase rapidly during and after puberty. The level of the Zn content in prostate tissue is most closely associated with the volume of the glandular lumen, which reflects the volume of prostatic fluid.
There exists a paucity of literature on the histology of the normal prostate of the neonate, infant, growing child, peripubertal male and adolescent male. Andrews (1951) reported on the gross examination and the histology of the prepubertal prostate. He observed that the most notable change with age was related to the size of the prostate. The total increase in prostate size was attributed to an increase in fibromuscular component (stroma). These observations were based entirely on qualitative examination of stained prostatic tissue sections. Weibel & Gomez (1962) demonstrated that it is possible to quantify morphological data using a stereological approach. According to Bartsch et al. (1979), the prostate can be histologically divided into two major components: the stromal component and the glandular component (GC; epithelium plus lumen). The first quantitative evaluation of the histological changes in the human nonhyperplastic prostate gland occurring from birth through early adulthood was presented by Shapiro et al. (1997). Shapiro et al. observed that the per cent area density of the stromal component (approximately 80%) was not age-dependent. Also, their study provided evidence that the stromal-to-epithelial ratio of the prostate remained constant in 45 male patients between ages 0–30 years and equals 5 : 1. These findings did not agree with morphometric studies of young adult prostate glands (Bartsch et al., 1979). Bartsch et al. reported that in six men ages 20–29 years, with a nonhyperplastic prostate, the stromal compartment comprised only 45% of the gland and that the stromal-to-epithelial ratio was 2 : 1 (Rohr & Bartsch, 1980). Presumably because it was single study with a very small sample size, Shapiro et al. (1997) ignored these findings.
During the last decade, a few morphometric studies of young adult prostate glands were published (Arenas et al., 2001; Chagas et al., 2002; Matsuda et al., 2006). Results of these studies agree well with data obtained by Bartsch et al. (1979) and do not confirm the findings of Shapiro et al. (1997). This disagreement casts a shadow on the data about quantitative evaluation of the histological changes in the human nonhyperplastic prostate gland occurring from birth through early adulthood, presented only in the study of Shapiro et al. (1997).
It is well known that Zn levels in the human prostate are almost 10 times higher than in other soft tissues (Zaichick et al., 1997). The high content of Zn in the prostate suggests that Zn may play a role in prostate function and health. Zinc is the second most abundant metal in the human body, serving as a cofactor for more than 300 enzymes with various physiological functions (Coleman, 1992). Despite a long-term study of Zn metabolism, its specific role in prostate function remains undefined and reasons for the concentration of the element in the gland are unknown. There is only the hypothesis that the normal prostate tissue accumulates Zn, because Zn acts as an inhibitor of an enzyme (m-aconitase), which is part of the Krebs cycle (Costello & Franklin, 1998). Besides that, specialized Zn uptake transporters in prostate epithelial cells were found (Beck et al., 2004; Franklin et al., 2005; Desouki et al., 2007). However, Zn was found not only in glandular epithelium but also in the stromal component (Ide-Ektessabi et al., 2002). Thus, the longstanding question about the main pool and the local distribution of Zn in prostate tissue still remains open up (Mawson & Fischer, 1952; Hoare et al., 1956; Siegal et al., 1961; Kar & Chowdhury, 1968; Dhar et al., 1973; Morita, 1981; Leake et al., 1983; Tvedt et al., 1989; Bataineh et al., 2002; Franklin et al., 2005).
This work had four aims. The first was to gain precise quantitative information (relative volume in per cent or per cent volume) on the age-related changes in the stroma, glandular epithelium, glandular lumen and GC of the nonhyperplastic prostate of the infant, growing child, peripubertal male, adolescent male and young adult male. The second aim was to calculate the per cent volume ratios of ‘stroma to epithelium’, ‘stroma to GC’ and ‘epithelium to glandular lumen’ for each prostate specimen and to determine mean values of these parameters. The third aim was to investigate the age-related changes of Zn mass fraction in prostate tissue by non-destructive energy-dispersive X-ray fluorescent analysis (EDXRF). The final aim was to estimate the correlations between the Zn content and the per cent volume of prostatic tissue components and between the Zn content and the different per cent volume ratios of prostatic tissue components. All studies were approved by the Ethical Committee of the Medical Radiological Research Center, Obninsk.
Materials and methods
Samples of the human prostate were obtained from randomly selected autopsy specimens of 50 male patients (European-Caucasian) aged <1 day to 30 years. Age ranges for subjects were divided into five groups, with group I, <1 year (0.19 ± 0.06 year, M ± SEM); group II, 1–8 years (3.7 ± 0.5 years, M ± SEM); group III, 9–14 years (13.3 ± 0.7 years, M ± SEM); group IV, 15–20 years (18.2 ± 0.6 years, M ± SEM); and group V, 21–30 years (26.4 ± 0.7 years, M ± SEM). These age groups were selected to reflect the infant (group I, n = 16), childhood (group II, n = 8), peripubertal (group III, n = 5), adolescent (group IV, n = 5) and young adult periods (group V, n = 16). The available clinical data were reviewed for each subject. None of the subjects had a history of an intersex condition, endocrine disorder, neoplasm or other chronic disease that would affect the normal development of the prostate. None of the subjects was receiving medications known to affect prostate morphology and prostatic Zn content. The typical causes of death in most of these patients included sudden infant death syndrome, acute pulmonary aetiologies and trauma. All prostate glands were divided (with an anterior-posterior cross section) into two portions using a titanium scalpel. One tissue portion was reviewed by an anatomical pathologist whereas the other was used for the Zn content determination. Only posterior part of the prostate, including the transitional, central and peripheral zones, was investigated.
Morphometric study of each prostate
The prostate specimens intended for the morphometric study were transversely cut into consecutive slides, which were fixed in buffered formalin (pH 7.4) and embedded in paraffin wax. The paraffin-embedded specimens were sectioned at 5 μm thickness and processed using routine histological methods. All samples were conventionally stained with haematoxylin and eosin (H&E), and then all histological slides were examined by an anatomical pathologist to detect any focus of benign prostatic hyperplasia, carcinoma, or intraepithelial neoplasia, to exclude samples with artefacts and to select appropriate slides for further morphometric evaluation. Morphometric evaluations were then performed quantitatively using stereological methods (Avtandilov, 1973). The stained tissue sections were viewed using microscopy at 120× magnification. To obtain information about changes in prostatic components (glands and stroma), the surfaces occupied by the glands (epithelium plus lumen), the epithelium alone and the stroma were also measured in 10 randomly selected microscopic fields in each histological section. The number of microscopic fields per section studied was determined by successive approaches to obtain the minimum number of microscopic fields required to reach the lowest SD. A greater number of microscopic fields did not decrease the SD significantly. The mean per cent volumes of the stroma (S), glandular epithelium (E), glandular lumen (L) and GC (GC = E + L) were determined and the mean ratios of per cent volumes (S/E, S/GC and E/L) were calculated for each prostate specimen.
Zn content measurement in each prostate
After the samples intended for the Zn content determination were weighed, they were transferred to be stored at −20°C, until they were freeze-dried, weighed once again and homogenized. The pounded sample weighing about 8 mg was applied to a piece of adhesive tape serving as a sample backing.
To determine the Zn content, by comparison with a known standard, aliquots of commercial, chemically pure compounds were used. The microlitre standards were placed on discs made of thin, ash-free filter papers fixed on the adhesive tape pieces and dried in a vacuum. Ten subsamples of the Certified Reference Material (CRM) IAEA H-4 (animal muscle) each weighing about 8 mg were analysed to estimate the precision and accuracy of the results. The CRM IAEA H-4 subsamples were prepared in the same way as the samples of dry homogenized prostate tissue. All samples of prostate tissue were prepared in duplicate, and mean values of Zn mass fraction were used in final calculation.
The facility for EDXRF analysis included an annular 109Cd source with an activity of 2.56 GBq, a Si(Li) detector and a PC-based portable multichannel analyser. Its resolution was 270 eV at the 5.9 keV line of a 55Fe-source. The duration of the Zn measurements was 20 min. The intensity of Kα-line of Zn for samples and standards was estimated using a calculation based on the total area of the corresponding photopeak in the spectra. The Zn mass fraction was calculated by a relative method, comparing the intensities of Kα-lines for samples and standards. Details of the sample preparation, the facility for performing EDXRF, the method of analysis and quality control of analytical results were presented in our previous publication concerning the EDXRF of chemical element contents in prostate specimens (Zaichick & Zaichick, 2010, 2011).
Using the Microsoft Office Excel program to provide a summary of statistical results, the arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels were calculated for all the morphometric parameters obtained and the Zn mass fraction. For the construction of ‘age – investigated parameter’ diagrams and the estimation of the Pearson correlation coefficient between the morphometric parameters and Zn mass fraction in prostate tissue the Microsoft Office Excel programs were also used. The reliability of difference in the results between all age groups was evaluated by parametric Student's t-test.
Age-related changes and correlations
Figure 1 shows individual data sets for the per cent volumes (stroma, epithelium, lumen and GC), the per cent volume ratios (stroma/epithelium, stroma/GC and epithelium/lumen) and Zn mass fraction in the nonhyperplastic prostate gland of male patients in the age range 0–30 years and their trend lines.
Table 1 presents basic statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of the quantitative morphometric parameters (S, E, L, GC, S/E, S/GC and E/L) and Zn mass fraction in nonhyperplastic prostate glands for each age group.
Table 1. Certain statistical characteristics of the histological components (per cent volumes), some of their ratios and zinc mass fraction (mg/kg, wet weight basis) in paediatric and young adult nonhyperplastic prostate glands
Age group years
S, stroma, E, epithelium, L, lumen; GC, glandular component; M, arithmetic mean; SEM, standard error of mean; Min, minimum value; Max, maximum value; Med., median value; Per. 0.025, percentile with 0.025 level; Per. 0.975, percentile with 0.975 level.
GC = E + L (%)
S/(E + L)
Zn, mg/kg wet weight basis
To estimate the effect of age on the morphometric parameters, some ratios of morphometric parameters and Zn contents of the prostate (Table 2) we examined the five age groups, described above. The ratios of means and the reliability of difference between mean values of morphometric parameters and Zn contents in all different age groups and the age group I are presented in Table 2.
Table 2. Effect of age on the morphometric parameters and zinc mass fraction in paediatric and young adult nonhyperplastic prostate glands
Ratio of means in different age groups to mean value in group I
S, stroma, E, epithelium, L, lumen; GC, glandular component; M, arithmetic mean; SEM, standard error of mean. Student's t-test, statistically significant difference (*p ≤ 0.05, **p ≤ 0.01).
The comparison of our results with data published by Shapiro et al. (1997) for paediatric and young adult nonhyperplastic prostate glands is shown in Table 4. Because in that study mean area densities of prostate smooth muscle and connective tissue were measured separately, in our calculations, we took the fibromuscular component (stroma) to be the sum of prostate smooth muscle and connective tissue. Then the means of the stromal-to-epithelial ratios were estimated using data calculated for the stroma and the authors’ data for the epithelium.
Table 4. Some morphometric parameters of paediatric and young adult nonhyperplastic prostate glands according to data from the literature in comparison with the results of this work
Some morphometric parameters of young adult nonhyperplastic prostate glands according to data from the literature (Bartsch et al., 1989; Arenas et al., 2001; Chagas et al., 2002; Matsuda et al., 2006) are compared with our results in Table 5. In the study of Arenas et al. (2001) each prostate was divided into three regions (periurethral, central and peripheral) and the volume of each region, as well as the average volume occupied by stroma and epithelium in each region were quantified in mm3. For comparison of these results with other published data and data of our study, the per cent volumes for stroma and epithelium were calculated by us for each region and the medians of values are presented in Table 5. The results for glandular lumen (L) were found as L (%) = 100 – S (%) – E (%).
Table 5. Some morphometric parameters of young adult nonhyperplastic prostate glands according to data from the literature in comparison with our results
E + L (%)
S/(E + L)
S, stroma, E, epithelium, L, lumen; –, no data available.
Calculation from this work using means (medians) of reference data.
Calculation from this work using median of means for S, E and L is expressed in bold.
(E) calculated as (E) = (E + L) − L for keep a balance S+ E+ L=100%.
Table 6 depicts the comparison of our results with published data for Zn mass fraction in paediatric and young adult nonhyperplastic prostate glands (Hienzsch et al., 1970; Leissner et al., 1980; Feustel et al., 1982; Tisell et al., 1982; Picurelli et al., 1991; Oldereid et al., 1993). Because a number of values for Zn mass fraction were not expressed on a wet weight basis in the above works, we calculated these values using published data for water content 85 and 83% in paediatric and young adult prostate respectively (Leissner et al., 1980; Woodard & White, 1986).
Table 6. Means (M ± SD) of Zn mass fraction (mg/kg, wet weight basis) in paediatric and young adult nonhyperplastic prostate glands according to data from the literature in comparison with our results
Group number (age range in years)
Group I <1
Groups I + II <1–8
Group II 1–8
Group III 9–14
Groups III + IV + V 9–30
Group V 21–30
M, arithmetic mean; n, number of prostatic specimens; –, no data available; Dor, Lat and Med, dorsal, lateral and medial prostatic lobes respectively; AAS, atomic absorption spectrometry; ICP-AES, inductively coupled plasma-atomic emission spectrometry; EDXRF, energy-dispersive X-ray fluorescence analysis.
Using the technique of morphometric and EDXRF analysis, we quantified the histological components and Zn mass fraction of the prostates of male patients ranging in age from <1 day to 30 years (Fig. 1). The mean values and all selected statistical parameters were calculated for per cent volumes of the stroma, glandular epithelium, glandular lumen and GC, for per cent volume ratios of stroma/glandular epithelium, stroma/GC, and glandular epithelium/glandular lumen and for Zn mass fraction (Table 1). The similarity of arithmetic means and median values for all the parameters investigated (Table 1), testifies to the normal distribution of individual results.
In the histologically normal prostates, we have observed an increase in per cent volume of the glandular epithelium and lumen with age up to 30 years, accompanied by a decrease in per cent volume of the stroma (Fig. 1). The per cent volume of prostatic stroma (81%) was greatest and the per cent volume of prostatic glandular epithelium (17%) and lumen (~2%) were lowest in the infant group (Table 1). A decrease of almost 50% in per cent volume of the stroma with age, from birth up to the age range 15–30 years (groups IV and V), was found (Table 2). Men, 15–30 years of age, had a higher per cent volume of glandular epithelium and lumen in their prostate than had infant subjects, by ca two and five times respectively (Table 2). The per cent volume ratios of stroma/glandular epithelium, stroma/GC and glandular epithelium/glandular lumen decreased with age up to 15–30 years, by almost four, five and three times respectively (Table 2). A strongly pronounced tendency of age-related increase in Zn mass fraction was observed in the prostate (Tables 1 and 2). For example, in the prostate of 21–30 year old men, the mean Zn mass fraction was 3.5 times greater than that in prostate of infants.
A significant positive correlation between the prostatic Zn and per cent volume of glandular epithelium (r =0.56, p ≤0.001), glandular lumen (r =0.68, p ≤0.001) and GC (r =0.63, p ≤0.001) was seen (Table 3, column ‘Σ all age groups’). Strongly pronounced negative correlation between the prostatic Zn and the per cent volume of stroma (r =−0.63, p ≤0.001) and the per cent volume ratios of stroma/glandular epithelium (r =−0.50, p ≤0.001), stroma/GC (r =−0.50, p ≤0.001) and glandular epithelium/glandular lumen (r =−0.37, p ≤0.01) were also observed. This indicates that there is a special relationship of Zn with the GC of the prostate. This relationship starts at the peripubertal period (ages 9–14 years, as shown in Table 3, for the age group III) when the GC of the prostate starts to produce prostatic fluid. Thus, the GC is a main pool of Zn concentration in the normal human prostate, for the age range 9–30 years. Moreover, we can conclude that the Zn more tightly binds within the prostatic fluid than with glandular cells. This conclusion follows from a negative correlation between the Zn content and the per cent volume ratio of glandular epithelium/glandular lumen (Table 3), because the volume of glandular lumen reflects the volume of prostatic fluid.
The means obtained for the per cent volume of the stroma, glandular epithelium, glandular lumen and the stromal-to-epithelial ratio in the infant group as shown in Table 4, agree well with values cited by Shapiro et al. (1997). In a more recent study (Matsuda et al., 2006) very similar results for the stroma (82.6 ± 8.4%) and the stromal-to-epithelial ratio (4.8) in the infant group were also demonstrated. However, morphometric data obtained in our work agree well with values cited by Shapiro et al. (1997) for the infant group only. For all other age groups our morphometric parameters differ from the results of Shapiro et al. (1997) and for age groups IV and V these differences are statistically significant. Shapiro et al. (1997) did not give details on what zones of the prostate was examined. The differences in examined zones may be the cause of the disagreement of their and our findings. The validity of our data for young adult men is confirmed by the good agreement with medians of results cited by other researches (Bartsch et al., 1989; Arenas et al., 2001; Chagas et al., 2002; Matsuda et al., 2006) for the human prostate of men aged 15–30 years (Table 5). The relatively big dispersion of data in Table 5 may be explained by the differences in zones of the prostate, racial distribution of the population and exact morphometric methods that were used.
The means obtained for Zn mass fraction, as shown in Table 6, agree well with values cited by other researches for the human prostate of different age groups. The present study demonstrated age-related increases in the Zn mass fraction of prostate tissue occurring from birth through early adulthood. Zinc levels, being nearly 30–35 mg/kg (wet weight basis) remain steady for the first decade of life and begin to increase at puberty. This level of the Zn mass fraction in human prostate tissue is not higher than the mean values of this element's content in many other soft tissues of the human body, such as skeletal muscle, liver, lung and kidney [International Commission on Radiological Protection (ICRP; 1975); Iyengar, 1998]. Only at the age of puberty, does the value of the Zn mass fraction in the human prostate begin to increase rapidly and after the age about 20 years it exceeds a level of 100 mg/kg (wet weight basis). This level is higher the Zn mass fraction of all other soft and hard tissues of the human body [International Commission on Radiological Protection (ICRP; 1975); Iyengar, 1998]. No published data referring to correlations between Zn mass fraction and morphometric parameters of human prostate have been found.
Quantitative morphometric analysis may provide information on structural changes in the prostate with advancing age. Such methodology provides objective and reproducible values for several morphological structures and thus allows statistical comparison of morphological structures with each other in different age groups and with other quantitative characteristics of prostate tissue, including biochemical parameters. The present study represents the first comprehensive evaluation of the histological and Zn content changes in the human nonhyperplastic prostate gland occurring from birth through early adulthood. It was found that histologically normal prostate tissue undergoes substantial changes from birth through early adulthood. These changes are reflected in an increment of the per cent volume of the glandular epithelium and lumen and in a diminution of the per cent volume of the stroma. The stromal-to-epithelial ratio of the prostate falls almost by a factor of 4 between ages <1 and 30 years, from 5 : 1 to 1.3 : 1. A zinc mass fraction of 30–35 mg/kg (wet weight basis) remains steady for the first decade of life and then begins to increase very rapidly during and at puberty. The level of Zn content in prostate tissue is most closely associated with the volume of the glandular lumen, which in turn reflects the volume of prostatic fluid.
The authors are grateful to the late Prof. A.A. Zhavoronkov, Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow, for supplying prostate specimens and for his help in the morphometric study.