testis

Cryopreservation of immature testicular tissue is a suitable method for spermatogonial stem cell (SSC) preservation in prepubertal boys, who are at risk of infertility due to cancer treatments. Viable spermatozoa can be obtained by transplantation or in vitro culture of cryopreserved testicular tissue. Optimizing the culture conditions is essential for reducing tissue damage caused by oxidative stress produced during cryopreservation and culture. Our ob jective was to improve the culture conditions of vitrified immature mouse testicular tissue by using N-acetylcysteine (NAC) antioxidant. In this experimental study, testicular tissues of 6-day-old immature NMRI mice were iso lated, vitrified, and distributed into three groups: control, culture I (cultured without NAC), and culture II (cultured in the presence of 125 mM NAC). After seven days of culture, histological analysis, cell viability, apoptotic-related gene expression, promyelocytic leukaemia zinc finger (Plzf) gene expression, and Caspase-3 protein expression were assessed. Moreover, the malondialdehyde (MDA) level was measured in the culture media. Tissue integrity and higher viability level were observed in the culture II group compared to the other two groups. Furthermore, the Bax/Bcl-2 ratio and MDA level were decreased significantly in the culture ӀӀ group, whereas Caspase-3 and Plzf gene expression were significantly increased. Our data revealed that the presence of 125 mM NAC improves the developmental process of vitrified-warmed immature mouse testicular fragments during in vitro culture, thus it may have potential implications for in vitro culturing of human prepubertal testicular tissues.

Cyclophosphamide (Cy) as an alkylating chemotherapeutic agent with broad-spectrum efficacy in cancer treatment. Despite its wide spectrum of clinical usage, off-target multiple organ toxicity such as sperm and testicular injury is one of its toxic side effects. Since the Nasturtium officinale L. hydroalcoholic extract (NOE) contains a wide range of phytochemicals with various biological functions, the current study was designed to explore the protective potential of NOE on testicular toxicity caused by Cy in rats.

Forty-eight adult male Wistar rats were randomly allocated into eight groups (n=6): control, Cy [received a single dose of 75 mg/kg, intraperitoneal (i.p)], NOE+Cy (Prevention): received NOE 500 and 1000 mg/kg/day, orally for 21 consecutive days and on the last day received Cy, Cy+NOE (Treatment): received NOE 500 and 1000 mg/kg/day, orally for 7 days after Cy administration for 21 consecutive days, and NOE (500 and 1000 mg/kg/day). After experiments, the testicular weight and volume, testosterone level, and sperm parameters as well as histologic and histomorphometric changes of testis were examined.

Base on the results, Cy caused significant decreases in testicular weight and volume, decreased testosterone level and reduced sperm count, and motility whereas increased sperm abnormality (p<0.05). Cy significantly reduced seminiferous tubules diameter, and height of the seminiferous epithelium (p<0.05). Furthermore, disorganization of seminiferous tubules diameter was increased in Cy group (p<0.05). Interestingly, pre and post-treatment with NOE could effectively improve testicular weight and volume, and testosterone level as well as sperm parameters. Furthermore, NOE administration ameliorated seminiferous tubules diameter diameter, seminiferous epithelium height (p<0.05).

It is concluded that NOE may provide a potential protective effect for Cy-induced testicular damage.

Some natural compounds studied in the field of infertility, despite having positive effects, may also have negative effects, including amygdalin, which is abundant in the seeds and kernels of plants of the rose family. The aim of this study was to investigate the effect of amygdalin on some sperm characteristics and testicular structure in mice.

In this experimental study, 40 male NMRI mice were used in five groups. Four groups received concentrations of 6.25 mg/kg, 12.5 mg/kg, 25 mg/kg, and 50 mg/kg of amygdalin, and one group received saline intraperitoneally for 28 days. Blood samples were taken to measure testosterone. In addition, epididymis and testes were used to assess sperm motility and tissue structure, respectively. The data were analyzed using a one-way analysis of variance in GraphPad software. P<0.05 was considered a significant level.

The dose of 50 mg/kg body weight of amygdalin significantly increased serum testosterone levels compared to the control group and the dose of 6.25 mg/kg. Further, all doses of amygdalin (6.25 mg/kg, 12.5 mg/kg, 25 mg/kg, and 50 mg/kg body weight) could significantly increase sperm count compared to the control group (P<0.01 and P<0.05). Other characteristics, including body weight, testicular weight, gonadosomatic index, sperm motility, and seminiferous tubule structure, were not affected by amygdalin.

The intraperitoneal injection of amygdalin can affect some sperm parameters. This effect is probably due to the improvement of the antioxidant system. However, the effects of amygdalin on the reproductive system probably depend on the injection method, amygdalin dose, and exposure time.

Restoring fertility in male cancer individuals through testicular tissue transplantation faces challenges due to hypoxia-induced loss of spermatogonial stem cells (SSCs). Hydrogel encapsulation was explored to minimize hypoxic damage in testicular tissue transplantation. For this purpose, human amnion membrane (hAM)-derived hydrogel could be an alternative.

The potential of hAM-derived hydrogel to support testis tissue grafts was evaluated.

In this experimental study, testicular tissue samples (1-3 mm3) were obtained from 16 male NMRI mice (4-5 wk, 22 ± 2 gr). These tissue fragments were either encapsulated within a hydrogel derived from a hAM or left unencapsulated (control) prior to being autologously transplanted beneath the dorsal skin of mice subjected to hemilateral or bilateral orchiectomy. The grafted testicular tissues were histologically evaluated for key parameters, including the integrity of seminiferous tubules, survival of SSCs, Sertoli cell functionality, as well as hypoxia and apoptosis on day 21.

No significant differences were observed between groups regarding ST integrity, number of SSCs, Sertoli cell functionality, or the rate of hypoxia-inducible factor 1-alpha and apoptosis (p ≤ 0.05).

In conclusion, this study demonstrated no effect of hAM hydrogel encapsulation on the outcomes of testicular tissue transplantation.

Small extracellular vesicles (sEVs) have been recognized as a promising therapeutic modality due to their low immunogenicity, and the ability to penetrate biological barriers. They contain significant amounts of lipids, proteins, and microRNAs, effectively participating in intra- and inter-cellular communications. sEVs derived from mesenchymal stem cells (MSCs) are being explored as a potential therapeutic option due to their immunomodulatory, anti-inflammatory, antioxidant, and regenerative properties, offering advantages over stem cell transplantationbased treatments. Chemotherapy induces side effects on various organs, particularly those with high proliferative capacity, such as testicular tissue. Exposure to some groups of chemotherapeutic agents, such as cyclophosphamide, cisplatin, and doxorubicin can cause DNA damage and induce apoptosis in spermatogonia and primary spermatocytes. Chemotherapy has been shown to induce cellular stress in testicles, leading to testicular dysfunction and the activation of apoptotic pathways in response to external and internal stress. The current research aims to review the potential therapeutic advantages of sEVs derived from MSCs in addressing sperm abnormalities and male infertility resulting from chemotherapy. Several lines of evidence indicate that treatment with sEVs can reduce testicular tissue damage caused by chemotherapy by decreasing oxidative stress and inflammatory responses. sEVs boost the growth and motility of spermatogenic cells and protect them from apoptosis by activating internal pathways. Therefore, as a non-invasive approach, they have shown promising results in regenerating damaged spermatozoa and restoring spermatogenesis.

Testicular torsion is a critical urological emergency that can lead to testicular ischemia and significant tissue damage. Citrulline, a supplement known for enhancing cellular metabolism and mitigating oxidative stress and inflammation, has been explored for its protective effects against testicular injury resulting from torsion and detorsion in rat models.

This study involved 42 Wistar rats, divided into six groups: Sham, torsion/detorsion (T/D), and four groups receiving varying doses of Citrulline (300, 600, 900 mg/kg) and vitamin E (20 mg/kg). A surgical procedure was performed to induce torsion by rotating the left testicle for 4 hr, followed by reperfusion. Daily oral administration of the supplements continued for one week post-surgery. Assessments included oxidative stress markers, apoptosis, inflammation, pathology, and sperm parameters. Statistical analysis was conducted using GraphPad Prism.

Citrulline administration at doses of 600 and 900 mg/kg significantly reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels. Additionally, it increased glutathione (GSH) levels and decreased protein carbonyl levels at the 900 mg/kg dose. The expression of interleukin-6 (IL-6) decreased at 900 mg/ kg, tumor necrosis factor-alpha (TNF-α) levels dropped at 600 and 900 mg/kg, and the pro-apoptotic factor Bax was reduced at all doses. Sperm analysis showed improved sperm count and motility at the 900 mg/kg dose. Histological examination revealed significant positive effects of Citrulline on testicular tissue.

Citrulline effectively lowers oxidative stress, inflammation, while enhancing sperm quality and pathological outcomes. These results indicate that Citrulline has potential as a therapeutic agent for testicular torsion.

Diabetes mellitus (DM), one of the most pervasive and enduring metabolic diseases, has been demonstratedto adversely impact male fertility. Conversely, both exercise training and Chrysin have been identified aspotential interventions capable of mitigating the deleterious effects of diabetes on spermatogenesis. Thus, the currentstudy aims to explore the individual and combined influences of Chrysin supplementation and running exerciseon oxidative stress and germ cell apoptosis in the testicular tissue of diabetic adult rats. In this experimental study, the DM was induced by streptozotocin (STZ,50 mg/kg). Ratswere divided into control (received STZ solvent), DM-sole, Chrysin-sole (50 mg/kg, daily), moderate-intensityrunning exercise training (MIRET-sole, warm-up, 5 minutes at 30% of Smax1 (Maximum speed); Moderate intensityexercise, 60 minutes at 60% of Smax1, and recovery, 5 minutes to 30% of Smax1), DM+Chrysin, DM+MIRET,and DM+MIRET+Chrysin. Following 8 weeks, the histopathological changes (Johnson’s score, epithelial height,and tubular diameter), testicular malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase(GPX), and the mRNA levels of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax was analyzed. Chrysin solely and simultaneous with MIRET could remarkably (P=0.001) improve the DM-induced histopathologicaldamages, increase the testicular SOD and GPx levels, and decline the DM-increased MDA content.Moreover, our results showed that Chrysin solely and more simultaneously with MIRET could significantly (P=0.001)decrease the mRNA expression of Bax and improve the Bcl-2 expression and rebalance the Bax/Bcl-2 balance. Our findings showed that co-administration of Chrysin along with MIRET can significantly amelioratethe DM-induced histopathological, and biochemical impairments and reduce the pro-apoptotic impact of DMon testicular tissue.

Diazinon is an organophosphorus compound widely used as a pesticide in agriculture. It causes oxidative stress and has toxic effects on the reproductive system. Quercetin is a carotenoid with antioxidant activity.

This research aimed to evaluate quercetin's protective effects on oxidative stress and reduce testicular tissue damage in rats treated with diazinon.

In the present study, 40 adult Wistar rats were divided into five groups. Each group had eight rat, including control-sham (0.2 mg/kg/B.W of corn oil), diazinon (300 mg/kg/B.W), experimental 1; diazinon (300 mg/kg/B.W) and quercetin (25 mg/kgB.W), experimental 2; diazinon (300 mg/kg/B.W) and quercetin (50 mg/kgB.W), experimental 3; diazinon (300 mg/kg/B.W) and quercetin (100 mg/kgB.W). The rats were given orally daily for two months.

According to the results, diazinon decreased repopulation index (RI), FRAP testosterone levels, and carbohydrate reserves and increased malondialdehyde (MDA), sperm index (SI), negative tubule differentiation index (TDI), and lipid reserves in the testicular tissue of rats compared to the control sham. All the above parameters in the diazinon group with quercetin were returned to near normal.

Based on the results, diazinon negatively affected the histochemical and histology of the testis in the Wistar rat. The rat's reproductive system is protected against diazinon if combined with quercetin.

 Heat stress is one of the environmental causes of damage to the testis, whose effects are less known before puberty. The aim of the present study was to investigate the impact of photobiomodulation (PBM) on the testis of prepubertal mice subjected to hyperthermia.

 Twenty-four three-week-old prepubertal male mice were allocated to the following groups: I) control, II) scrotal hyperthermia (Hyp), and III) Hyp+PBM (n=8/each group). In order to induce hyperthermia, the scrotum was placed in water at 43 °C for 20 minutes every other day for a total duration of 10 days. In the Hyp+PBM group, after hyperthermia induction, the testis of the mice was subjected to laser irradiation at a wavelength of 890 nm (0.03 J/cm2 for 30 seconds)
for 35 days. After the mice were sacrificed, the testis and epididymis were removed for testing.

 Compared with those of the Hyp group, the sperm parameters of the laser irradiation group improved notably. In addition, histological examinations revealed that the final number of testis cells and the volume of tissue in the Hyp+PBM group were dramatically greater than those in the Hyp group. The analysis of molecular data revealed an increase in the expression of mitotic genes and testosterone levels and a decrease in the formation of reactive oxygen species (ROS) and the expression of the apoptotic gene in the testis subjected to PBM.

 Based on the present findings, laser therapy can reduce complications caused by scrotal hyperthermia during prepuberty and ameliorate spermatogenesis during puberty.

Acute appendicitis commonly presents with right iliac fossa pain. However, it may present with right scrotal pain and swelling, which may be confused with other scrotal pathologies such as orchitis, scrotal abscess, and testicular torsion. Acute appendicitis in children and adolescents may present with scrotal pain due to inflammatory intraperitoneal fluid tracking down into the scrotal sac via a patent processus vaginalis. In rare cases, acute appendicitis may present concurrently with testicular torsion, making the diagnosis much more challenging. Herein, we present an even rarer case of acute appendicitis with right testicular torsion and concurrent right inguinoscrotal hernia.

Chromosome 7 open reading frame 61 (C7orf 61) was a testis-specific gene, and may be involved in the process of spermatogenesis. This study aimed to investigate the expression of C7orf61 in the testis and determine its role in spermatogenesis.

Reverse transcription–quantitative polymerase chain reaction, Western blot and immunofluorescence were performed to evaluate the expression characteristics of C7orf61 in mice and humans. In vitro fertilization assay was used to determine the role of the C7ORF61 protein in sperm-egg fusion.

The results demonstrated that C7orf61 was a testis-specific gene; the C7ofr61 mRNA expression level sharply increased in the fourth postnatal week and gradually increased until the adult stage. The C7ORF61 protein was located throughout the subacrosomal area and close to the nucleus in both mouse and human sperm. The incubation with the C7ORF61 antibody significantly decreased the fertilization rate of mouse eggs.

The present findings suggested that the C7ORF61 protein might be involved in sperm–egg fusion, and could serve as a useful target for contraceptives. However, further research is still needed to know the detailed molecularmechanismofitsrole.

Cisplatin (CPN) is widely used for the management of various malignant tumors.

This study investigated the effects of Thymoquinone (TQN) on the expression of the p62 gene in CPN-induced testicular damage in mice.

Histomorphometry, testis injury scores, expression of p62, and protein levels of LC3-II were assessed.

Cisplatin induced histological changes, increased p62 expression (P < 0.01), and reduced LC3-II levels (P < 0.001). Thymoquinone pretreatment decreased p62 expression while increasing LC3-II protein levels. Thymoquinone significantly reversed the testicular injury scores and improved histomorphometric parameters.

The results indicate that TQN enhances autophagy and improves testicular tissue in CPN-intoxicated mice.

In this study, we aimed to investigate the protective effect of Thymoquinone (THQ) against testicular damage caused by Methotrexate (MTX).

This study consists of 5 groups: Control, Olive oil, THQ, MTX, and MTX+THQ. At the end of the experiment, spermiogram analysis was performed on the rats. In addition, testicular tissues were taken and histopathology, immunohistochemistry, and biochemistry analysis were performed. Biochemical analyses were performed on the serums.

According to the results obtained, spermiogram values, Johnson’s testicular biopsy score, SOD, CAT, GPx, FSH, LH, and testosterone values were statistically significantly decreased in the MTX group compared to the control group. In the MTX+THQ group, spermiogram values, Johnson’s testicular biopsy score, SOD, CAT, GPx, FSH, LH, and testosterone values increased statistically significantly compared to the MTX group. NRF2 and HO-1 immunoreactivity were statistically significantly decreased in the MTX group compared to the control group. In the MTX+THQ group, NRF2 and HO-1 immunoreactivity were statistically significantly increased compared to the MTX group. The level of MDA, which is important in lipid damage, and the level of biochemistry results of TNF-α, IL1-β, and IL-6, which are important markers, and the results of p-NF-kB and P38 immunoreactivity were statistically significantly increased in the MTX group compared to the control group. In the MTX+THQ group, these parameters showed a significant decrease compared to the MTX group.

According to these results, it is thought that THQ will play a protective role against infertility caused by chemotherapy-induced testicular damage.

Testicular torsion is a surgical emergency leads to severe acute ischemia injuries, and may eventuallycause male infertility. The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domaincontaining3 (NLRP3) inflammasome is involved in testicular torsion pathophysiology. The aim of this study was toevaluate the effects of nanocurcumin (nCur) on testicular tissue and the NLRP3 inflammasome components. In this experimental study, male Wistar rats (n=36) were randomly divided into six equalgroups: controls, ischemia-reperfusion (I/R), I/R+nCur (50 or 100 mg/kg thirty minutes before reperfusion), and I/R+nCur (50 or 100 mg/kg thirty minutes before reperfusion and continued for seven days). The left testis was rotated720 (2×360) degrees counterclockwise to induce testicular torsion. After two hours of ischemia, detorsion was performed.At the end of treatment, an orchiectomy was carried out. The testis histopathology and the mRNA levels ofNLRP3, apoptosis-associated speck-like protein (ASC), and Caspase-1 were evaluated. Our results revealed that, testicular I/R had a detrimental effect on testis histology such as the number ofspermatogonia (14.5 ± 0.57, P<0.001) and the seminiferous tubules epithelium thickness (28.5 ± 11.7, P=0.007).It also significantly increased the expression of the NLRP3 inflammasome components (P<0.001). Treatment withnCur (in both doses) improved testicular damage and significantly reduced the expression of NLRP3 (P=0.007), ASC(P=0.003), and Caspase-1 (P<0.001). These results imply that nCur might be a useful therapeutic strategy in the field of reproductive medicineto diminish the side effects of testicular I/R via its anti-inflammatory properties and may be employed as adjuvanttherapy to lessen testicular torsion complications.

Studies indicate that phytoestrogens and phytosterols have adverse effects on the male reproductive system. To our knowledge, the effects of Tanacetum parthenium on testicular tissue, spermatozoa chromatin integrity and free radical damage have not yet been investigated. Therefore, this study aimed to evaluate the effect of T. parthenium administration on sperm parameters, testis histology, sperm DNA integrity and oxidative damage in adult male mice. 

Eighteen adult male mice (2-3 months old) were randomly divided into 3 groups: control, TP1 and TP2. TP1 and TP2 groups were separately gavaged with 50 and 100 mg/kg T. parthenium. After harvesting the epididymis, sperm analysis was performed according to the guidelines of the World Health Organization (WHO). The testicular tissue also passaged through the tissue routine process after being placed in a formalin fixative solution. To check the quality of sperm chromatin, a sperm smear was prepared and then stained with acridine orange dye and was examined with a fluorescent microscope. Biochemical parameters, including malondialdehyde (MDA), thiol, catalase enzyme, and superoxide dismutase (SOD), were measured in testicular tissue. Finally, data were analyzed by the analysis of variance in SPSS software, version 16. 

A significant reduction was seen in sperm count and sperm morphology percentage and the germinal epithelium thickness in the TP1 and TP2 groups versus the control group. The spermatozoa with DNA damage in percentage were higher in the TP1 group (21.22±3.70) and TP2 group (42.60±3.73) compared to the control group (2.40±4.3). There were remarkable differences between the three groups in MDA (P≤0.001) and thiol (P≤0.001) levels. Catalase level (P≤0.001) was lower in the TP1 and TP2 groups than in the control group. 

The results of this study showed that T. parthenium caused a significant decrease in sperm chromatin quality, MDA level and germinal epithelium thickness at both doses. A reduction was found in the antioxidant enzyme level in the mice administrated with 50 and 100 mg/kg of T. parthenium.

This study aims to comprehensively assess the therapeutic potential of clove extract in mitigating testicular damage and preserving male reproductive function under hypoxic conditions in male rats.

We randomly divided 24 male rats into three groups: a hypoxia group that just received normal saline, a hypoxia + clove extract (4 mg/kg) group, and a sham operation group without treatment. After eight weeks, the serum level of oxidative stress parameters was examined, and the testicle histopathological examination, such as seminiferous tubule diameters and epithelium thickness, was assessed.

The serum malondialdehyde (MDA) concentration was significantly enhanced in the hypoxic group, and the levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) declined (P<0.05). The diameters and thickness of the seminiferous tubule were diminished notably (P<0.05). Johnson’s score decreased in the hypoxic group. Treatment with clove extract also led to improvements in this parameter.

Our study provides compelling evidence for the therapeutic potential of clove extract in mitigating testicular injury associated with varicocele-induced testicular hypoxia in male rats.

This study aimed to investigate the potential protective impact of anthocyanin against tissue damage and oxidative stress provoked by varicocele within the testes of adult rats.

A total of 32 male rats were divided into four groups as follows: a control group undergoing a sham procedure, a varicocele group without intervention (V), a varicocele group treated with anthocyanin (VA), and a group receiving anthocyanin treatment alone. Following a 56-day treatment period, the indicators of oxidative stress were gauged in the blood plasma, while histological modifications were evaluated utilizing the hematoxylin and eosin staining technique.

In the varicocele group treated with anthocyanin, we observed noteworthy enhancements in Johnsen score, epithelium thickness, and seminiferous tubule diameter compared to the untreated varicocele group. Treatment groups exhibited substantial elevations in testosterone levels and antioxidant enzyme levels. Furthermore, there was a reduction in the levels of malondialdehyde (MDA), an established marker of oxidative damage, and a decline in histological damage in the treatment groups.

The outcomes underscore the potential safeguarding influence of anthocyanin against testicular damage stemming from varicocele induction, suggesting its beneficial role in countering oxidative stress and tissue impairment.