Background: Benign prostate hyperplasia (BPH) is the most common benign disease of human prostate. Currently BPH is associated with unregulated proliferation of connective tissue and glandular epithelium within the prostatic transition zone, and it has been described as relevant characteristic of BPH—the increase of the total number of cells, and not only an increase in cell size. To date, there are few studies on the quantitative morphology of glandular tree of BPH compared with normal prostate. The scarce investigations about this particular suggest that the glandular tree branches and expands as the hyperplastic transformation occurs in the prostate. Methods: To verify if this gland expansion and branching was similar to that occurs in the normal prostate, this study deals with the estimation of several stereological parameters as: labeling index for the proliferating cell nuclear antigen to quantify the rate of proliferation of prostate epithelium, average thickness of glandular epithelium, fraction of the volume occupied by the epithelium relative to the total prostate volume, connectivity density of prostate glands, to quantify the branching of prostate glands, and the average volume and the volume-weighted mean glandular volume of prostate acini to assess the mean size of the prostate acini and its variability. Results: All these estimates have been performed in prostate specific antigen immunostained sections from prostates of young men (controls) and in adenomectomy specimens from the adenofibromiomatous variety of BPH. Conclusion: We conclude that the epithelial proliferation is not the only factor intervening in the development of BPH. In addition, a more prolonged survival of epithelial population, together with some degree of hypertrophy of acini expressed by the increase of volume fraction and thickness of acinar epithelium, is relevant in order to the growth and expansion of the BPH glandular tree that shows more abundant and heterogeneous acinar sprouts than in normal prostate.
Benign prostate hyperplasia (BPH) is the most common benign disease of human prostate [
BPH has two phases: pathologic and clinical. The first phase has in turn two stages: microscopic, with the presence of small foci of prostatic hyperplasia, and macroscopic, with evidence of adenomas. Approximately 80% of men over 40 years developed a microscopic BPH, but only half of these evolved to a macroscopic BPH in an average time of five years [
The prostate grows from birth to puberty to reach between 20 and 30 years of age reaching an average weight of 20 g. From here to the 90 years its growth rate decreases. From the fifth decade of life, with the development of histologically identifiable BPH, increased prostate weight occurs reaching an average weight of 33 g [
Prostatic stromal cells play an important role in the pathogenesis of BPH. The first histological changes include microscopic nodules in the stroma surrounding the periurethral glands. The acinar hyperplasia begins around these small nodules and will increase over the years, to produce nodules up to several centimeters in diameter whose histology may show a predominance of epithelial elements, stromal or a mixed pattern. Depending on what elements predominate, BPH can be classified in five varieties: stromal, fibromuscular, muscular, fibroadenomatous and adenofibromiomatous (the most common) [
Although the pathogenesis of BPH is unclear, it is known to be multifactorial, being necessary the presence of two factors for prostate growth occurs: the androgen stimulus and age [
Currently BPH is associated with unregulated proliferation of connective tissue, smooth muscle and glandular epithelium within the prostatic transition zone, and it has been described as relevant characteristic of BPH―the increase of the total number of cells, and not only an increase in cell size [
The cytological findings in BPH are generally of little relevance, and rather unspecific, as alterations including basal cell hyperplasia, increased stromal mass (particularly the amount of smooth muscle cells), enhanced extracellular matrix deposition, reduced elastic tissue, more infiltrating lymphocytes around ducts, acinar hypertrophy and more luminal corpora amylacea and calcifications in the form of prostatic calculi [
Although some increase of cell proliferation has been detected in the glandular epithelium of BPH in comparison with normal prostate [
To date, there have been no studies on the quantitative morphology of glandular tree of BPH compared with normal prostate. The only mention of this particular has been noted by [
To verify if this gland expansion and branching was similar to that occurs in the growth of normal prostate, the present work deals with the estimation of several stereological parameters which can shed some light on the structure and distribution of prostatic glandular tree and its possible changes in BPH.
Briefly, the parameters investigated were:
1) Labeling index for the proliferating cell nuclear antigen (PCNA) to quantify the rate of proliferation of prostate epithelium.
2) Average thickness of glandular epithelium.
3) Fraction of the volume occupied by the epithelium relative to the total prostate volume.
4) Connectivity density of prostate glands, to quantify the branching of prostate glands.
5) The average volume and the volume-weighted mean glandular volume of prostate acini to assess the mean size of the prostate acini and its variability.
All these estimates have been performed in prostates from young men (controls) and in adenomectomy specimens from the adenofibromiomatous variety of BPH.
Twenty prostate specimens were collected at La Princesa Hospital (Madrid, Spain), 10 were from adults, (CTR group), age (mean ± SD): 45 ± 7; range: 30 - 47 years, all these specimens were of healthy subjects, without endocrine or reproductive pathology, deceased in traffic accidents, and eligible as donors for transplant, the age of the subjects of CTR group was in the range indicated to avoid any histological changes of subclinical BPH, relatively frequents in subjects older than 50 years. The other 10 were surgical specimens (adenomectomies) from patients diagnosed of the adenofibromiomatous type of benign prostatic hyperplasia, (BPH group), age (mean ± SD): 75 ± 10, range: 65 - 85 years. All the ethical requirements were accomplished in order to obtain the prostatic tissue either at the moment of the multiorganic extraction for transplant (CTR group) or at the surgery (BPH group). Immediately after extraction, the specimens were fixed during a week in 10% paraformaldehyde in PBS, pH 7.4 (Probus, Barcelona, Spain).
After fixation, the specimens from the two groups were thoroughly sectioned into 2-mm-thick slices, performed by isotropic uniform random sampling (IUR sections) in order to preserve the isotropy of the tissue [
All the specimens were processed for paraffin embedding. The paraffin blocks were exhaustively sectioned. A total of 20 sections were performed on each block: Ten 5-mm-thick sections for immunohistochemistry and routine haematoxylin-eosine, and other ten 15-µm-thick sections for quantitative studies.
Two immunostainings were performed: i) for visualization of proliferating cell nuclear antigen (PCNA) in order to evidence the epithelial proliferating cells [
At least five randomly selected slides per specimen were immunostained for each antigen in CTR and BPH groups. Deparaffinized and rehydrated tissue sections were treated for 30 min with hydrogen peroxide 0.3% in phosphate-buffered saline (PBS) pH 7.4, to block endogenous peroxidase. To detect PCNA immunoreactivity, sections were incubated with a monoclonal anti-PCNA antibody (Biomeda, Foster City, Ca, USA) diluted at 1:400. To show PSA immunoreactivity, sections were incubated with a monoclonal anti-PSA antibody (Santa Cruz Biotechnology, Santa Cruz, Ca, USA) diluted at 1:50. Pretreatment of sections by heat in citrate buffer pH 6.0 (using a pressure cooker) [
The primary antisera were diluted in PBS pH 7.4 containing 1% bovine serum albumin (BSA) (Sigma, St Louis, USA) plus 0.1% sodium azide (Merck, Darmstad, Germany). The incubation with primary antisera was overnight at 4˚C. The second antibodies employed were a biotin-caproyl-anti-rabbit immunoglobulin (Biomeda, Foster City, CA, USA). The second antibodies were diluted at 1/400 in PBS containing 1% BSA without sodium azide, and incubated for 30 min at room temperature. Thereafter, sections were incubated with a streptavidin-biotin-peroxidase complex (Biomeda). The immunostaining reaction product was developed using 0.1 g diaminobenzidine (DAB) (Sigma) in 200 mL of PBS, plus 40 mL hydrogen peroxide. After immunoreaction nuclear counter-staining with haematoxylin was performed in all the sections immunostained for PCNA, and in some sections immunostained for PSA. No nuclear counter-staining was performed on the 15-µm-thick sections PSA immunostained that were then employed for quantitative purposes. All slides were dehydrated in ethanol, and mounted in a synthetic resin (Depex, Serva, Heidelberg, Germany). The specificity of the immunohistochemical procedures was checked by incubation of sections with nonimmune serum instead of the primary antibody.
Quantitative Methodsi) Estimation of the proliferative index of epithelium
In the two groups, the numerical density (NV) of epithelial cells (cell number per unit of volume of reference space) was estimated for both PCNA immunoreactive (NV PCNA positive) and PCNA negative nuclei (NV PCNA negative), employing the optical disector, an unbiased stereological method [
ii) Quantitative morphology of prostate acini
Three parameters have been employed to quantitate the acinar pattern: The volume fraction of epithelium (VV ep), the average thickness of epithelial lining (Th ep), and the connectivity density of the acinar tree (ConV gl).
The sampling protocol for the estimate of these parameters is described in detail: For all the specimens in each group of study, three sections immunostained to PSA were randomly sampled. Every section was scanned at a final magnification of ×100, to select an average of 5 microscopical fields per section that were chosen by systematic random sampling [
Subsequently, the images were processed using the public domain Java image processing program, Image J (version 1.48), developed at the US National Institutes of Health and available on the Internet at http://imagej.nih.gov/ij/index.html [
The quantitative parameters studied were estimated onto these stacks of binarised images, employing a plugin that runs in the Image J software [
a) Epithelial volume fraction (VV ep) is the volume of PSA immunoreactive epithelium per unit of volume of the reference space (acini plus stroma), VV ep is simply the number of foreground (epithelium) voxels divided by the total number of voxels in the image.
b) Average thickness of epithelial lining (Th ep). The plugin employed defines the thickness at a point as the diameter of the greatest sphere that fits within the structure (epithelial lining) and which contains the point [
c) Connectivity density of the acinar tree (ConV gland). The number of connected structures in a network can be determined by calculating the Euler characteristic [
iii) Estimations of size of the prostate acini
Two methods have been performed in order to measure the mean volume of the acini, first was estimated the average volume of glands that were sampled independently of their size [
The procedures were as follows:
a) The nucleator, an unbiased stereological estimator [
b) The average volume-weighted mean glandular volume (VV gl) was performed using the point sampled intercept method [
The program used to evaluate VV gl enables the generation of random test-lines directions that were superimposed onto the microscope images. The gland intercepts can be measured along these test lines. The length of gland intercepts (l0) was processed to obtain
All the measurements from VN gl and VV gl were carried out in each group of study onto three sections per specimen stained with haematoxylin-eosine and randomly sampled. The fields selected were visualized using an Olympus microscope at a final magnification of ×40. The software employed by measurements was the Cast-Grid.
c) Estimation of the second moment of the VN gl distribution: As estimates of VN gl and VV gl were made on the same population of glands, the coefficient of variation of the acinar volumes in the VN gl distribution [
where CVN (V) gl is the coefficient of variation of the acinar volume in the VN gl distribution that is a measure of the variability in size of acini.
iv) Statistical analysis
The parameters measured were expressed as mean ± CI (confidence intervals at 95%). Comparisons between the means from CTR and BPH groups were performed by a Student t test (p < 0.05).
Immunoreactivity for PCNA has been detected in some nuclei from basal cells of acinar epithelium in both control and BPH specimens. Not relevant differences were observed in intensity and distribution of PCNA immunoreactive nuclei in both groups of study (
The LI pcna did not experiment significant changes when comparing CTR and BPH groups (
The average volume of the prostate acini (VN gl) does not change significantly in BPH in comparison with CTR groups (
In the present study, it has been noted that in BPH there is no significant increase of cell proliferation measured
by PCNA labeling index. This seems in contrast to that described by other authors [
The end point of this shifting towards the cell proliferation is the increase of epithelium that was reflected in the enlargement of the volume fraction of prostate gland occupied by the epithelium as was described in this study. However, other authors [
The increase of thickness of epithelial lining might be in relation to some hypertrophy at the cellular level that is also present [
The connectivity density from acini in BPH was significantly higher than in control prostate.
This parameter is related with the number of patterns interconnected in a branching structure [
The average volume-weighted mean volume is a simple and efficient method of estimating the size of particles, and has been revealed to be an important prognostic factor in a number of histopathological studies of melanoma [
The VV gl of the acini was significantly increased in BPH cases in comparison with CTR group. This finding is interesting because estimates of VV gl combine information about the three-dimensional glandular size with knowledge of variability of glandular size [
This study sheds some new light on the pathogenesis of BPH, from the point of view of the growing pattern of the hyperplastic glandular tree. We can conclude that the epithelial cell proliferation is not the only factor intervening in the development of BPH, at least, in the stage, or for the type of hyperplasia studied in this work. In addition, a more prolonged survival of epithelial population, together with some degree of hypertrophy of acini expressed by the increase of volume fraction and thickness of acinar epithelium, is relevant in order to the growth and expansion of the BPH glandular tree that shows more abundant and heterogeneous acinar sprouts than in normal prostate.
Luis Santamaría,Ildefonso Ingelmo,Fernando Teba,Almudena Coloma,Laura Martínez, (2016) Quantitative Stereological Estimations of Structural Patterns of the Glandular Tree in Benign Hyperplasia of Prostate. Open Journal of Pathology,06,122-133. doi: 10.4236/ojpathology.2016.63015