Cryptorchidism is one of the main causes of infertility and can result in testicular cancer. This study aimed to present quantitative data on the damage caused by cryptorchidism using stereological analysis.
Thirty newborn rats were randomly divided into control and experimental groups. The experimental group underwent surgery to induce unilateral cryptorchidism in the left testis, whereas the control group underwent a sham surgical procedure 18 days after birth. The testes were removed at designated time points (40, 63, and 90 days after birth) for stereological evaluation and sperm analysis. Total testicular volume, interstitial tissue volume, seminiferous tubule volume and length, and seminiferous epithelium volume and surface area were measured. Other parameters, such as sperm count, sperm morphology, and sperm tail length, were also examined.
Statistically significant differences (
Given the substantial effect of cryptorchidism on different testicular parameters, as well as the irreversible damage it causes in the testes, it is important to take this abnormality seriously to prevent these consequences.
The inhibition of testicular descent into the scrotum of one testis (unilateral undescended testis) or both testes (bilateral undescended testes) [
In most species, including humans, the complete descent of the testes usually occurs prenatally [
Due to the importance of this phenomenon, many studies have been conducted to find effective solutions for cryptorchidism. As of this study, the effects of cryptorchidism on the testis have been evaluated using histology [
In addition, in most cases, cryptorchidism occurs after the testes have naturally descended (acquired cryptorchidism); in this study, however, cryptorchidism was induced surgically in infant rats. We hope that this study can provide further insights into this issue and encourage the further development of treatment strategies.
Adult male and female Wistar rats were purchased from the Pasteur Institute, kept in an adequate environment with a 12-hour light-dark cycle and temperature range of 20 to 26 °C, and provided with
Surgery was performed on the 18th postnatal day before the natural descent of the testes (rat testes descend 21 to 28 days after birth) [
The testes of five rats in each group were removed at designated time points (40, 63, and 90 days after birth) [
Immediately after euthanasia, the testes were excised for stereological analysis (at three time points: 40, 63, and 90 days after birth), and the epididymis (in mature rats only, at 63 and 90 days after birth) was cut into segments, placed in phosphate-buffered saline, and incubated at 37 °C until the sperm was released from the tissue, indicating its readiness for sperm analysis.
In order to count the sperm, a 1:20 sperm solution was prepared, and 10 µL of the dilution was pipetted onto a Neubauer slide with a glass cover. The slide was placed under a light microscope with 100× magnification. The spermatozoa were then counted and recorded in millions [
To evaluate the sperm morphology, 20 µL of the supernatant containing the sperm was smeared. After the smear had dried, it was immersed in acetone-ethanol (1:1) and subsequently placed in aniline blue stain for 7 minutes. It was then observed under a light microscope at ×100 magnification, and 200 sperm cells were studied per testis [
Systematic uniform random fields of view were chosen by maintaining equal distances on the X and Y axes, and 200 sperm cells were studied per testis. A Merz grid with defined measurements was superimposed onto the computer monitor, and a calculation was made using the following formula [
∑L=(π/2)×(a/l)×(1/asf)×∑I
L=∑L/∑N
where ‘‘a/l’’ is the Merz grid constant, which was acquired as follows. The area of each basic tile (smaller frame) was calculated by multiplying the length and width of said tile (xy). Within this tile, there were two semicircles with lengths of pd (circumference of the circle), where ‘‘d’’ was the diameter of 1 semicircle. Therefore, the Merz grid constant ‘‘a/l’’ was calculated as (XY)/pd. ‘‘asf’’ refers to the area of the basic tile divided by the area of the counting frame (larger frame). ‘‘∑I’’ was the total number of intersections between the sperm tails and the semicircles. ‘‘∑N’’ was the total number of the counted sperm cells in the unbiased counting frame.
In order for sperm to be counted, the sperm head had to be located inside the counting frame or on the inclusion lines (green lines) (
On the designated date for each group and following euthanasia, both testes were removed, weighed, and immersed in a 10% formalin solution for fixation. Following this procedure, each testis was embedded in an agar block to obtain isotropic uniform random slabs. The agar block containing the testis was randomly placed at the center of a circle with 90 equidistant divisions along its perimeter, a random number between 0 and 90 was generated and the block was cut along a line parallel to the direction of the selected number. The block was placed on its cut surface at the center of a second equiangular circle, with 96 non-equidistant divisions along its perimeter. The agar block was then divided into different slabs by using a tissue slicer to cut it along a line that was parallel to the direction of a random number ranging from 0 to 96 (
The total volume of the testis was calculated based on its weight. The images were analyzed using a dedicated software program (ImageJ; https://imagej.nih.gov) and specific plugins. In order to calculate the testicular volume fractions of seminiferous tubules and interstitial tissue and the epithelial volume, a test point system was implemented by applying the below formula (
V_{v} (structure)= ∑P (structure)/∑P (testis)
where “V_{v}” is the volume fraction of testicular structures, “ΣP (structure)” is the total number of test points overlying the specific testicular structure, and “ΣP (testis)” is the total number of points overlying the entire testis. The total volume of each structure was calculated by multiplying the volume fraction by the total volume of the testis.
The surface density of the germinal layer was calculated using test line probes and the following formula (
where “ƩI” is the number of intersections between the test lines and the luminal surface of the germinal layer, “I/p” is the length of each test line associated with each point of the test grid, and “∑P” is the number of points overlying the germinal layer. The total surface area of the germinal layer was ultimately calculated by multiplying the surface density by the volume of the germinal epithelium.
The length of the seminiferous tubules was estimated by superimposing an unbiased counting frame onto each microscopic field of view. The tubule profiles located inside the counting frame or crossing the inclusion lines (green lines) were counted (
Lv (seminiferous tubules)= 2∑Q/(∑P×a/p)
where “∑Q” is the total number of the counted profiles, “∑P” is the total number of counted frames, and “a/p” is the area per frame [
SPSS version 27 (IBM Corp.) was used to conduct the statistical analysis, and the normality of the distribution of the data was analyzed using the Kolmogorov-Smirnov test. The Kruskal-Wallis and Mann-Whitney tests were used for statistical analysis of the sperm count. One-way analysis of variance and the Tukey test were used to analyze the sperm morphology, sperm tail length, and stereological parameters. A
Observations of the left testes at 63 and 90 days revealed that the sham group had a higher sperm count than the experimental group (
In the testes at 63 and 90 days, the normal sperm count was considerably higher in the sham group than in the experimental group (
The sperm tail length in the left testes did not vary between the sham and experimental groups at both 63 and 90 days (
The calculated volumes of the various parameters are summarized in
The values for epithelial surface area and seminiferous tubule length are reported in
This is the first study to report three-dimensional quantitative data regarding testicular morphometry using unbiased design-based stereology in rat testes following experimentally-induced cryptorchidism. Cryptorchidism can damage the germinal epithelium, decrease the concentration of healthy sperm, reduce semen quality, and eventually lead to sperm disorders such as immaturity, necrosis, and apoptosis [
Previous studies have shown that evaluating testicular volume is one of the most efficient indirect ways to predict fertility in men, especially in pediatric patients where semen analysis is not feasible [
Intra-abdominal testes are predisposed to seminiferous tubule alterations. These alterations depend on the thickness of the lamina propria, which may interfere with metabolic exchanges between the seminiferous tubules and the interstitial tissue [
Cryptorchidism leads to increased interstitial tissue volume and increased fibrosis in the testes [
After birth, the seminiferous tubules start to gradually increase in length, become more compact, and ultimately form a dense structure. The length of seminiferous tubules and their structure influences spermatogenic cell growth and the movement of sperm towards the epididymis [
Histologically, the seminiferous tubules of cryptorchid testes tend to show structural abnormalities, such as increased tubule branching and tubular blind ends, smaller tubular diameter, and areas completely free of germ cells, as opposed to normally descended testes, which have evenly distributed spermatogonia and regular tubular morphology. The structural abnormalities seen in cryptorchid testes suggest that injury tends to occur early in testicular development [
Studies have observed damage in the contralateral testis as a result of unilateral cryptorchidism, especially spermatogonia cell depletion, which, once present, is irreversible even after orchiopexy [
In conclusion, although the effects of cryptorchidism are well known, this abnormality is still a major challenge that affects male fertility. In order to devise optimal treatment measures, this condition must be better understood. This study aimed to provide a better understanding of cryptorchidism by precisely measuring the biological features of cryptorchid testes as a whole. The results confirmed that the total volume of the testis and the volume of the seminiferous tubules decrease due to cryptorchidism, while the volume of the interstitial tissue increases. In addition, the length of the seminiferous tubules and the surface area of the germinal epithelium of the seminiferous tubules decrease due to cryptorchidism. These changes were observed at all examined time points but did not affect the opposite testis.
No potential conflict of interest relevant to this article was reported.
Conceptualization: JS, MMD. Data curation: ER, MAA, AS. Formal analysis: JS. Methodology: JS, MMD, SFM. Writing-original draft: JS, FY, HB. Writing-review & editing: JS, MDS.
Calculation of sperm tail length using a Merz grid. The arrowheads indicate counted sperms, whereas the arrows indicate the intersections between the sperm tails and the semicircles (aniline blue staining, ×400 magnification).
Isotropic, uniform random sections of the rat testes were obtained by applying the orientator method. (A) The testis for each animal was embedded in an agar block and placed at the center of a circle with 90 equidistant divisions along the perimeter. A random number between 0 and 90 was chosen. (B) The agar medium was cut along a parallel line according to the direction of the selected number (here, 35). (C) The agar block was placed on its cut surface at the center of a second circle, with 96 non-equidistant divisions along its perimeter. The agar was cut into multiple equidistant pieces along a parallel line according to the direction of a random number from 0 to 96 (here, 50) using a tissue slicer.
Estimation of testicular volume, testicular surface area, and the length of the seminiferous tubules. (A) Estimation of testicular volume by employing the point-counting system. The volume of testicular structures was estimated by randomly superimposing a point-counting probe onto each section. The upper-right corner of each point (arrow) was used as a reference to count the number of points hitting the targeted region of interest (hematoxylin-eosin stain, scale bar: 200 µm). (B) Calculation of the surface area of seminiferous tubules using test line probes randomly superimposed onto each section. The test points hitting the seminiferous tubule epithelium (arrowhead) and the points where test lines and the seminiferous tubule lumen intersect (arrows) were counted (hematoxylin-eosin staining, scale bar: 200 µm). (C) The unbiased counting frame principle was applied to estimate the length of the seminiferous tubules. The profiles located inside the counting frame or crossing the inclusion lines (green lines) were counted (hematoxylin-eosin staining, scale bar: 200 µm).
Comparison of (A, B) sperm count, (C, D) normal sperm morphology, and (E, F) sperm tail length at 63 and 90 days in the sham and experimental (cryptorchid) groups. ^{a),b)}Significant differences between the groups are indicated with different letters (
Total testicular volume and volume fractions of testicular structures in rat testes at 40, 63, and 90 days
Parameter | Age (day) | Sham group | Experimental group | ||
---|---|---|---|---|---|
Right | Left | Right (scrotal) | Left (abdominal) | ||
Total testicular volume (cm^{3}) | 40 | 0.58±0.07^{a)} | 0.60±0.04^{a)} | 0.53±0.04^{a)} | 0.29±0.02^{b)} |
63 | 1.39±0.09^{a)} | 1.41±0.08^{a)} | 1.32±0.15^{a)} | 0.41±0.07^{b)} | |
90 | 1.28±0.17^{a)} | 1.23±0.12^{a)} | 1.39±0.16^{a)} | 0.45±0.07^{b)} | |
Interstitial volume (%) | 40 | 25.98±2.19^{a)} | 24.08±3.75^{a)} | 25.35±2.98^{a)} | 36.18±3.32^{b)} |
63 | 26.48±1.05^{a)} | 27.04±1.29^{a)} | 25.22±1.67^{a)} | 33.18±1.68^{b)} | |
90 | 25.29±3.55^{a)} | 27.06±3.13^{a)} | 27.58±2.24^{a)} | 38.70±6.53^{b)} | |
Epithelium volume (%) | 40 | 56.78±2.51^{a)} | 58.46±3.54^{a)} | 55.08±2.19^{a)} | 42.43±2.95^{b)} |
63 | 53.15±2.66^{a)} | 53.98±2.14^{a)} | 53.55±1.44^{a)} | 50.62±4.77^{a)} | |
90 | 54.69±2.80^{a)} | 55.50±4.03^{a)} | 54.17±4.27^{a)} | 52.41±4.86^{a)} | |
Seminiferous tubule volume (%) | 40 | 74±2.19^{a)} | 75.89±3.74^{a)} | 74.63±2.98^{a)} | 63.80±3.32^{b)} |
63 | 74.51±2.19^{a)} | 72.95±1.29^{a)} | 75.92±2.01^{a)} | 66.81±1.68^{b)} | |
90 | 74.69±3.55^{a)} | 72.92±3.13^{a)} | 72.40±2.24^{a)} | 61.28±6.53^{b)} |
Values are presented as mean±standard deviation.
^{a),b)}Different letters in each row indicate significant differences between the groups (
Epithelial surface area and tubular length in rat testes at 40, 63, and 90 days
Parameter | Age (day) | Sham group | Experimental group | ||
---|---|---|---|---|---|
Right | Left | Right (scrotal) | Left (abdominal) | ||
Epithelial surface area (cm²) | 40 | 27.08±3.76^{a)} | 30.40±6.41^{a)} | 23.71±4.50^{a)} | 11.13±2.58^{b)} |
63 | 62.68±5.74^{a)} | 60.25±2.01^{a)} | 65.00±11.81^{a)} | 21.83±8.71^{b)} | |
90 | 59.98±12.87^{a)} | 61.26±16.34^{a)} | 68.47±3.03^{a)} | 27.04±13.19^{b)} | |
Seminiferous tubule length (m) | 40 | 13.58±0.73^{a)} | 15.56±0.87^{a)} | 13.54±1.25^{a)} | 10.65±2.03^{b)} |
63 | 20.91±5.29^{a)} | 22.27±2.00^{a)} | 24.34±3.90^{a)} | 12.87±3.07^{b)} | |
90 | 19.67±2.63^{a)} | 22.15±3.62^{a)} | 23.40±2.00^{a)} | 13.13±1.39^{b)} |
Values are presented as mean±standard deviation.
^{a),b)}Different letters in each row indicate significant differences between the groups (