The exact relationship between varicocele and infertility remains to be explained. Clinical studies showed that unrelieved venous stasis, persistent hypoxia/ischemia, and overproduction of ROS that occur in varicocele lead to decreased testicular volume and poor semen quality. These changes were obvious if varicocele was presented early in puberty. If it was neglected, the testes became atrophic with oligospermia or even with an absence of spermatozoa in seminal fluid (azoospermia) [24–28].
In this study, after comparing two periods of EV, we found that testicular impairment was time dependent as it progressively increased with the period of induction. Examination of sections of the testes of subgroup EV6 of the EV group (group 2) showed that testicular parenchyma was formed of many distorted STs with irregular outlines, disorganized epithelium, and wide lumina. Some areas of interstitium were wide. The disorganized epithelium showed separation and sloughing. The epithelial cells contained small vacuoles. Examination of sections from the EV18 subgroup showed that most of the STs were markedly distorted with very wide lumina and increased reduction in the thickness of their epithelial lining. A few tubules had a relatively narrow lumina. The epithelium was formed of a few spermatogenic cells with darkly stained nuclei. Most of the tubules were mostly lined by sertoli cells that had euchromatic nuclei and mitochondria. Morphometrical and statistical analyses of the epithelial height of the STs confirmed these results as it showed a highly significant decrease in EV18 as compared with others. Earlier studies clarified that the pathophysiological processes of massive loss of germ cells in varicocele was apoptosis-mediated and it increased with time [19,29]. This could lead to germ cell depletion (sertoli-only-syndrome-like feature) that occurred in parts of STs . Although sertoli cells were extremely resistant to cell death, their metabolic and regulatory pathways could be disturbed, resulting in germ cell degeneration. If sertoli cells were permanently affected, the recovery of spermatogenesis might not be possible or at least would be incomplete .
Some STs in the EV6 subgroup showed multinucleated cells in their lumina. Some investigators clarified that most of these cells were spermatocytes or spermatids that failed to separate as a result of failure of breakdown of intercellular bridges in the early phases of spermiogenesis . Other investigators observed that they were formed from fusion of spermatids secondary to retraction of sertoli cell processes .
In this study, examination of the same group showed that acidophilic hyaline material was present in most of the interstitial spaces, and congested blood vessels were observed. The acidophilic hyaline material could be attributed to excess lymphatic exudates oozing from degenerative lymphatic vessels  or to an increase in vascular permeability .
Electron microscope examination of the EV6 subgroup showed that almost all transverse sections in the mid pieces of sperms showed markedly affected disarranged axoneme, fibrous, and mitochondrial sheaths. In most of the STs of the EV18 subgroup, no sperms were observed. It was reported that the ROS might cause lipid peroxidation of sperm cell membranes, hence damaging the midpiece, axonemal structure, or disrupting the capacitation and acrosomal reaction . Subsequently, increased sperm abnormality and decreased sperm count and motility were associated with decreased fertility [35,36].
This ultrastructure examination of the EV6 subgroup showed that the STs were enveloped by a relatively thick, irregular, highly infolded basement membrane. This membrane became extremely thick and irregular in the EV18 subgroup. Myoid cells appeared with irregular heterochromatic nuclei. It was documented by some investigators that altered peritubular myoid cells caused an enlargement of the collagen fibril layer that was normally produced by these cells , whereas others attributed this thickness to an increased production of glycosaminoglycans and proteoglycans in the basement membrane as a defense reaction against the damaging activity of free radicals . Contrarily, other investigators claimed that the initial involvement of basal lamina could represent one of the mechanisms responsible for varicocele-induced histological alterations of the testes .
In this study, the EV6 subgroup showed Leydig cells with lipid droplets, variable-size electron-dense granules, and multiple cytoplasmic processes. Those of subgroup EV18 had variable-size electron-dense granules and extensive cytoplasmic processes interdigitating with the neighboring ones. In this study, we suggested that the observed electron-dense granules could be lipid in nature. One of the theories that attempted to explain the changes induced by varicocele was the venous lesion that led to vasodilatation and interference with the vascular countercurrent heat exchange. Therefore, the blood flow increased and testes became warmer . According to this theory, some studies claimed that infertility with varicocele might be attributable to dysfunction of Leydig cells. Temperature higher than the body temperature might cause damage of Leydig cells. This damage was associated with an unusual accumulation of lipid droplets, which was considered as storage depots of the precursors used in its synthesis of androgens, indicating that less substrate was being utilized. As the degree of stabilization of fatty acids by osmium tetraoxide fixative depended on their extent of unsaturation, the lipid droplets differed in electron densities. Its different sizes and densities meant that Leydig cell activity was reduced and steroid precursors were accumulated [1,41–43]. Leydig cells with cytoplasmic processes of different length and a few secondary branches were observed in testes with reduced spermatogenesis. These cells were present in small or large cell groups and had desmosome-like contacts and gap junctions between these processes and neighboring cells. These junctions carried signals that regulated their secretory activity .
In this study, examination of sections of the testes of group 3 (EV-carnitine) showed that they nearly regained their normal general architecture. Most of their STs were packed together with regular outlines and narrow interstitium. Acidophilic hyaline material was still observed between some tubules. In addition, most of the tubules restored their normal epithelial stratification. A few tubules still had disorganization in their germinal epithelium. These results were confirmed by statistical analysis that clarified a highly significant increase in their epithelial height as compared with the EV18 subgroup. Some investigators tried to evaluate the impact of surgical treatment (varicocelectomy) in varicocele. They found that surgical repair of the short-term experimental varicocele could restore full recovery of bilateral testicular weight, epididymal sperm content, and sperm motility. However, only partial recovery of the semen parameters after repair of the long-term ones was observed. Therefore, they decided that surgical repair alone was not enough in such cases [44,45]. Meanwhile, other investigators tried to evaluate the role of L-carnitine in the reduction or prevention of the testicular damage that occurred as a result of other conditions such as drug toxicity (i.e. etoposide)  or γ irradiation . Carnitines act as anti-ROS drugs as they are capable of returning the ROS concentrations to their physiological level with an improvement in sperm parameters [18,47,48].
Ultrastructural study of the EV-carnitine group examination showed that spermatogonia, primary spermatocytes, spermatids, and sertoli cells showed their normal fine structure. However, intercellular separations were still present between some cells. The basement membrane was thin and regular. Leydig cells contained numerous mitochondria, a few variable-size electron-dense granules, and a few lipid droplets. No cytoplasmic processes were observed. Earlier studies had identified the sertoli cell as a possible testicular target of carnitine activity because the latter had a direct action on sertoli cell metabolism . It influenced fat and carbohydrate metabolism by increasing fatty acid oxidation, glucose uptake, and lactate/pyruvate secretion . Other investigators demonstrated that carnitine could act through carnitine/organic cation transporter in sertoli cell membrane. They added that, if these cells were injured, spermatogenesis was impaired . Other investigators claimed that carnitine could act on stem spermatogonia by minimizing the DNA damage. These cells had extensive DNA repairing mechanisms. Hence, the ability of the testis to recover spermatogenesis depended on the survival of some stem spermatogonia and their ability to repopulate the testis with differentiating cells [21,46,51].
In this work, examination of sections of the testes of the same group showed that sperms were observed in most of the tubules, although they were a few in some of them. Ultrastructurally, most of the transverse sections of the midpieces of sperm had a normal structure, except for a few of them that showed an affected axoneme. Some investigators reported that L-carnitine played a key role in sperm metabolism by providing readily available energy for use by spermatozoa [16,47,52]. Hence, several experimental and clinical studies used carnitine supplementation to treat oligoasthenospermia because carnitine therapy improved the sperm function, spermatozoa kinetics, fertilization capacity, and morphological characteristics [48,53–56].
From the results of this study, we concluded that varicocele led to a deleterious effect on the ipsilateral testis that increased progressively with time. In addition, L-carnitine administration improved the structure of testis of long-duration varicocele. Therefore, L-carnitine could be considered as a therapeutic hope for those with long-duration varicocele and for those who failed to become fertile after varicocelectomy, or who are not suitable for surgery. We recommend further studies using a combination of varicocelectomy and L-carnitine therapies for long-duration varicoceles.
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