engleski

Blood vessels in testicular biopsies of infertile men

INTRODUCTION The human testis is composed of two major compartments: the seminiferous tubules compartment and the interstitial tissue compartment. Seminiferous tubules are limited by the lamina propria and highly depend upon the normal function of adjacent interstitial tissue (where the majority of small blood vessels are situated). These small blood vessels are arterioles, venules, capillaries and lymph vessels. Occasionally, some capillaries could be found within the lamina propria itself. Testicular capillaries of elderly men have thickened basal lamina. Infertile men were shown to have reduced lumen of the testicular blood vessels. The reduced lumen is mostly due to the process called &#8220; hyalinization&#8221; , pathological accumulation of glycoprotein substance within the blood vessel wall. Hyalinization could be found in the case of cryptorchidism, immune orchitis, Klinefelter&#8217; s syndrome and in the significant number of patients with idiopathic infertility. Microangiographic data on the testicular arterioles in severe cases of male infertility demonstrated irregular blood flow, damage of the blood vessel wall and reduced spermatogeneis within the seminiferous tubules. AIM Despite some microangiographic, morphological and other data on the microvasculature of the testis of infertile patients, at present, there is a lack of data on the most frequently affected blood vessel type/s in the case of male infertility. In addition, there is a lack of electron microscopic and morphometric studies dealing with the structure of testicular blood vessels in infertile men. This especially goes for the patients with a non-obstructive azoospermia. These patients commonly suffer form idiopathic infertility. Therefore, the aim of our study was: a) to identify the most frequent blood vessel type affected in this group of infertile patients ; b) to describe blood vessels changes both at light and electron microscopic level ; c) to perform a morphometric (stereological) analysis of small blood vessels of the testicular biopsies of infertile men. Thus, we wanted to check if there were some differences in the number, volume, length and curvature of these vessels when compared to controls. MATERIALS & METHODS 48 couples that consulted andrologist at the Urology Clinic (University of Zagreb, Medical School) for infertility in the period 1997-2003 were included in the current study. The infertility problem was due to the male factor. In 40 infertile men, a non-obstructive azoospermia could be diagnosed. 8 patients had obstructive azoospermia with completely preserved testicular tissue and served as controls. Whenever possible, a bilateral biopsy was performed. A detailed diagnostic procedure (including determination of the testicular volume by ultrasound and/or orchidometer) preceded testicular biopsy. All patients were subjected to the open biopsy of the testis. Briefly, an incision of 8-10 mm in length in the t. albuginea of the testis has been made. This incision allows 4-5 testicular lobules to be included into the biopsy. The protruding testicular tissue was dissected using surgical microscissors. Several pieces were taken from different parts of the male gonad. After dissection, all pieces were immediately transferred into a transport medium (Sperm-Prep, Medi-Cult) and brought to the Dept. of Histology and Embryology (University of Zagreb, Medical School) for consequent freezing and histological analysis. Thus, each piece of testicular tissue was divided into two parts: one part was immersed in 0.5 ml Sperm Freezing Medium (Medi-Cult) and another fixed in a buffered 5.5% glutaraldehyde. Tissue in the Sperm Freezing Medium was subjected to a programmed freezing using a Nicool LM 10 freezing device (Air Liquid). During first 5 min. the tissue was cooled up to -70&ordm ; ; C, then exponentially to -120&ordm ; ; C in the following 55 min. Finally, the tissue samples were stored in the liquid nitrogen until the in vitro fertilization attempt. Tissue fixed in glutaraldehyde was rinsed several times in 0.05M phosphate buffer (pH=7.1-7.4, 800 mOsm), postfixed with 1%OsO4 and dehydrated. After a routine histological procedure, the testicular tissue was embedded in Durcopan (Agar). Semi-thin sections (section thickness = 0.9 &micro ; ; m) were made by Reichert ultramicrotome and stained with 1% toluidine blue. Each specimen was sectioned extensively, thus resulting in six slides bearing 10 semi-thin sections which were made and stained. In some cases, ultra-thin sections were made (section thickness = 70 nm), contrasted with lead citrate and uranyl acetate and examined by transmission electron microscope Zeiss 902A (Centre for Electron Microscopy, Medical School Univ. of Zagreb). The histological evaluation of the testicular biopsy was performed using a modified Johnsen score. Morphometric (stereological) analysis was made using Weibel&#8217; s test system with 42 points. The sections were analysed by Nikon binocular microscope (Eclipse E200) applying a physical dissector principle. Magnification used was x100, where the test surface area of the Weibel&#8217; s grid was 1.245 mm2, the length of the test line (d) was 0.185 mm and the total length of all test lines (Lt) was 3.88 mm. The following stereological variables were determined: the number, volume, length and curvature of testicular arterioles, venules and capillaries per mm3 of the tissue and per the whole organ. Assessed data were analyzed using biostatistical programme SAS (version 6.12). RESULTS Histological analysis demonstrated that the control group had normal structure of seminiferous tubules as well as the interstitial compartment of the testis. Seminiferous tubules had diameter ranging from 170 to 210 &micro ; ; m. Lamina propria consisted of 5-7 layers of peritubular (myoid) cells. Tubules were lined with seminiferous epithelium, resting on the basement membrane of a normal thickness. Within the seminiferous epithelium, Sertoli cells had indented nucleus with a prominent nucleolar complex. The cytoplasm of these cells contained few lipid droplets and vacuoles. Spermatogenic cells consisted of spermatogonia, primary and secondary spermatocytes, round and elongated spermatids. Occasionally, sperm could be noted released into the lumen of tubules. Leydig cells in the interstitial tissue had oval or round nucleus, sometimes with a prominent nucleolus. Abundant cytoplasm contained few lipid droplets. Within the cytoplasm of some cells, Reinke&#8217; s crystal/s could be noted. Blood vessels had normal thickness of t. intima, media and externa. Infertile group of patients presented various degrees of testicular damage. In most cases, a &#8220; mixed atrophy&#8221; of seminiferous tubules has been observed. This testicular damage involves seminiferous tubules with different morphology: some tubules had normal morphology with full spermatogenesis going on ; in some tubules seminiferous epithelium was lacking and the tubules were transformed into the fibrous tissue (so-called &#8220; tubular shadows&#8221; ) ; a number of seminiferous tubules were lined with Sertoli cells or spermatogonia exclusively ; finally, some tubules had seminiferous epithelium composed of primary and secondary spermatocytes and, rarely, round and elongated spermatids. Interstitial blood vessels in the control group displayed regular architecture and thickness of the t. intima, media and externa (Fig. 1). Capillaries were composed of 1-3 endothelial cells with a regular basement membrane and accompanying pericytes. In infertile group, there was a change in the morphology of some arterioles and venules. In the subendothelial layer of these vessels, an accumulation of a hyaline could be observed (Figs. 2, 3). In the arterioles, hyalinization could be noted also within the t. media, affecting the smooth muscle cells (Fig. 3). Observed by the transmission electron microscope, the thickened subendothelial layer had an accumulated material of a moderate electron density, located under the basement membrane of endothelial cells. In the case of tubular fibrosis, an increased number of capillaries with the thickened basement membrane could be observed. Stereological analysis pointed out a significantly increased number, volume, length and curvature of capillary network (in mm3 of the tissue) in the infertile group of patients (p<0.05). DISCUSSION The observed changes in the structure of testicular arterioles and venules were accompanied with the progressive hyalinization within the lamina propria of seminiferous tubules. Within the affected blood vessels, the subendothelial layer of the t. intima was mostly changed, thus suggesting the accumulation of glycoproteins from the blood. It seems that endothelial cells are actively involved in the process of hyaline deposition to the subendothelial layer. The mechanism of the deposition is still unclear, but could involve an active transport of glycoproteins via membrane carriers, pinocytosis, and channels passing through the endothelial cells. Increased capillary network in infertile group of patients (particularly in the case of tubular fibrosis) could be the result of factors that induce angiogenesis as a result of hypoxia. Experiments on animals that were subjected to hypoxia pointed out that there was a significant increase in the production of Vascular Endothelial Growth Factor (VEGF) in all tissues. However, the highest production of VEGF was in the brain and in the testis. Therefore, it has been postulated that each organ has its specific VEGF factor. As for the testis, it could be speculated that the Endocrine Gland-Derived Vascular Endothelial Growth Factor (EG-VEGF) is involved. EG-VEGF could induce proliferation, migration and fenestration of endothelial cells of capillaries within the interstitium of the testis. This factor is produced by Leydig cells in response to hypoxia. Recently, another factor that induces the proliferation of endothelial cells has been described. It is called Bv8 or prokinectin-2. It seems that the major source of Bv8 are primary spermatocytes. CONCLUSION Herein, we describe the structure of blood vessels in infertile men with non-obstructive azoospermia. Histological analysis on semi-thin sections pointed out that the subendothelial layer of testicular arterioles and venules is affected. Within this layer, a hyalinization of the blood vessel takes place and could be extended to the t. media (smooth muscle layer), especially in arterioles. The stereological analysis confirmed the morphological observations that the number (as well as volume, length and curvature) of capillaries in infertile group of patients is significantly increased. The mechanisms that cause the above mentioned pathological change of the subendothelial layer in testicular arterioles and venules as well as the proliferation of capillaries are discussed.

male infertility; blood vessels; testicular biopsies

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