The effect of adipose-derived mesenchymal stem cell transplantation on ovarian mitochondrial dysfunction in letrozole-induced polycystic ovary syndrome in rats: the role of PI3K-AKT signaling pathway.

OBJECTIVE: The present study aimed to elucidate how mesenchymal stem cells (MSCs) application could efficiently attenuate pathological changes of letrozole-induced poly cystic ovary syndrome (PCOS) by modulating mitochondrial dynamic via PI3K-AKT pathway. METHODS: Thirty-two female rats were randomly divided into four experimental groups: Sham, PCOS, PCOS + MSCs, and PCOS + MSCs + LY294002. The Sham group received 0.5% w/v carboxymethyl cellulose (CMC); the PCOS group received letrozole (1 mg/kg, daily) in 0.5% CMC for 21 days. Animals in the PCOS + MSCs group received 1 × 106 MSCs/rat (i.p,) on the 22th day of the study. In the PCOS + MSCs + LY294002 group, rats received LY294002 (PI3K-AKT inhibitor) 40 min before MSC transplantation. Mitochondrial dynamic gene expression, mitochondrial membrane potential (MMP), citrate synthase (CS) activity, oxidative stress, inflammation, ovarian histological parameters, serum hormone levels, homeostatic model assessment for insulin resistance (HOMA-IR), insulin and glucose concentrations, p-PI3K and p-AKT protein levels were evaluated at the end of the experiment. RESULTS: PCOS rats showed a significant disruption of mitochondrial dynamics and histological changes, lower MMP, CS, ovary super oxide dismutase (SOD) and estrogen level. They also had a notable rise in insulin and glucose concentrations, HOMA-IR, testosterone level, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, ovarian malondialdehyde (MDA) content as well as a notable decrease in p-PI3K and p-AKT protein levels compared to the Sham group. In the PCOS + MSCs group, the transplantation of MSCs could improve the above parameters. Administration of LY294002 (PI3K-AKT pathway inhibitor) deteriorated mitochondrial dynamic markers, oxidative stress status, inflammation markers, hormonal levels, glucose, and insulin levels and follicular development compared to the PCOS + MSCs group. CONCLUSIONS: This study demonstrated that the protective effects of MSC transplantation in regulating mitochondrial dynamics, promoting mitochondrial biogenesis, competing with redox status and inflammation response were mainly mediated through the PI3K-AKT pathway in the PCOS model.

Astaxanthin supplementation impact on insulin resistance, lipid profile, blood pressure, and oxidative stress in polycystic ovary syndrome patients: A triple-blind randomized clinical trial.

Astaxanthin (ASX) is a natural carotenoid compound found in several of microorganisms and seafood. It may have numerous therapeutic benefits for polycystic ovarian syndrome (PCOS) patients. The aim of this study was to investigate the effect of ASX on lipid profile, insulin resistance (IR), blood pressure (BP), and oxidative stress (OS) levels in infertile PCOS patients. Overall, 58 infertile women with diagnosed PCOS participated in this triple-blind randomized clinical trial. They were randomly assigned to two groups, taking either a placebo or ASX (2 × 6 mg/day) for 8 weeks. Blood serum samples were collected from patients before and after the intervention. Fasting Insulin (FI), fasting blood glucose (FBS), OS markers (malondialdehyde [MDA], superoxide dismutase [SOD], and total antioxidant capacity [TAC]), and lipid profiles were evaluated in serum. Moreover, based on the relevant formula, several indices associated with IR were calculated. BP was also assessed at the start and end of the study. After 8 weeks of ASX consumption, a significant reduction was observed in fasting blood sugar, HOMA-IR, FI, MDA, low-density lipoprotein-cholesterol, and TC/HDL-C. Conversely, ASX significantly increased TAC, HDL-C, and QUICKI. After adjusting the analysis for the baseline values of age, body mass index, and biochemical parameters, non-significant values were obtained for QUICKI and FI, along with no changes in other findings. Overall, ASX appears to be an effective and safe supplement that alleviates insulin metabolism, lipid profile parameters, and OS in infertile PCOS patients.

High Anti-Müllerian Hormone Strongly Correlates with Reproductive Outcomes in Women Undergoing Assisted Reproduction.

BACKGROUND: In a retrospective cohort of 881 women with gynecologic and unexplained infertility, we aimed to study the relationship between serum AMH levels and ART outcomes. This retrospective cohort includes 881 infertile women aged 20 - 45 who underwent their first fresh autologous non-preimplantation genetic diagnosis ART cycles between 2012 and 2020. METHODS: We assessed the correlation between AMH levels and reproductive outcomes among infertile women with different causes of infertility (including endometriosis, polycystic ovary syndrome (PCOS), and unexplained infertility). RESULTS: We found a strong correlation between high AMH levels and reproductive outcomes independent of age and the cause of infertility in women undergoing ART. In all patients with gynecologic and unexplained infertility, higher AMH correlated with the improved number of oocytes (p < 0.001), MII oocytes (p < 0.001), good-quality embryos (p < 0.001), chemical pregnancy rate (p < 0.001 in women < 37; and p = 0.002 in women over 37), clinical pregnancy rate (p < 0.05), and live birth rate (p = 0.05). CONCLUSIONS: Serum AMH concentrations can be invaluable for predicting ovarian reserve and reproductive outcomes in young and advanced-age infertile patients undergoing ART. However, it should not be used as the sole predictive marker for disqualifying infertile women from ART treatment. Further large cohort studies are warranted to determine an exact cutoff point for AMH to provide an accurate ART success prediction.

Association of dietary total antioxidant capacity, alternative healthy eating index, and dietary inflammatory index with semen quality in men seeking infertility treatment.

Background: Since the association between dietary quality scores and semen quality remains unclear, we carried out a hospital-based cross-sectional study to investigate the association of Dietary Total Antioxidant Capacity (dTAC), Dietary Inflammatory Index (DII), and Alternative Healthy Eating Index (AHEI) scores with semen quality in men seeking infertility treatment. Methods: This study enrolled 210 men with unexplained or idiopathic infertility. Semen samples were collected and analyzed according to the WHO 2010 criteria. Dietary data was collected using a 168-item semi-quantitative food frequency questionnaire (FFQ) developed for Tehran Lipid and Glucose Study. Multivariable logistic regression models were used to estimate the relationship between dTAC, AHEI, and DII scores with abnormal semen in crude and adjusted models. Results: There were no significant differences across quartile categories of the dTAC, AHEI, and DII scores regarding semen parameters. There was a trend toward a significant direct association between DII and abnormal semen risk (p = 0.01). Infertile men in the highest quartile of DII had a 2.84 times higher risk of abnormal semen in the crude model (OR: 3.84; 95% CI: 1.64-8.95); such that remained after adjusting for several potential confounders. There was no significant association between dTAC or AHEI and the risk of abnormal semen in infertile men, either before or after adjusting for potential confounders. Total energy (p = 0.05), fat (p = 0.02), saturated fat (p = 0.02), mono-saturated fat (p = 0.009), Thiamine (Vitamin B1) (p = 0.02), Niacin (Vitamin B3) (p = 0.03), Calcium (p = 0.01), and Selenium (p = 0.01) were inversely associated with semen normality. Discussion: The study suggests that certain dietary factors may affect semen quality, and the mechanisms underlying the observed associations are likely multifactorial, involving complex interactions between diet, oxidative stress, inflammation, and hormone levels. Further research is required to confirm the results, fully elucidate the mechanisms underlying the associations, and identify specific dietary interventions that may improve male fertility outcomes.

Astaxanthin ameliorates inflammation, oxidative stress, and reproductive outcomes in endometriosis patients undergoing assisted reproduction: A randomized, triple-blind placebo-controlled clinical trial.

Purpose: In a randomized, triple-blind, placebo-controlled clinical trial (RCT) including 50 infertile women with endometriosis candidate for assisted reproductive techniques (ART), we studied the effect of Astaxanthin (AST) on pro-inflammatory cytokines, oxidative stress (OS) markers, and early pregnancy outcomes. Methods: Before and after 12 weeks of AST treatment (6 mg per day), blood serum and follicular fluid (FF) samples were collected from 50 infertile women with endometriosis stage III/IV undergoing ART. Pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and OS markers (malondialdehyde [MDA], superoxide dismutase [SOD], catalase [CAT], and total antioxidant capacity [TAC]) were measured in the serum and FF. ART outcomes were also compared between the groups. Results: Increased serum levels of TAC (398.661 ± 57.686 vs. 364.746 ± 51.569; P = 0.004) and SOD (13.458 ± 7.276 vs. 9.040 ± 5.155; P = 0.010) were observed after AST therapy in the treatment group. Furthermore, serum MDA (14.619 ± 2.505 vs. 15.939 ± 1.512; P = 0.031) decreased significantly following antioxidant treatment. In addition, significantly lower serum levels of IL-1ß (4.515 ± 0.907 vs. 6.8760 ± 0.8478; P = 0.000), IL-6 (5.516 ± 0.646 vs. 5.0543 ± 0.709; P = 0.024) and TNF-α (2.520 ± 0.525 vs. 2.968 ± 0.548; P = 0.038) were observed after AST treatment. In addition, AST supplementation led to an improved number of oocytes retrieved (14.60 ± 7.79 vs. 9.84 ± 6.44; P = 0.043), number of mature (MII) oocytes (10.48 ± 6.665 vs. 6.72 ± 4.3; P = 0.041), and high-quality embryos (4.52 ± 2.41 vs. 2.72 ± 2.40; P = 0.024). Conclusion: AST pretreatment can modulate inflammation and OS in endometriosis-induced infertile patients. ART outcomes also improved after 12 weeks of AST therapy. Our results suggest that AST can be a potential therapeutic target for infertile patients with endometriosis undergoing ART.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue.

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 µM astaxanthin or 100 µM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 µM AST group more than vitamin E (p < 0.05). Also, 10 µM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin.

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three-week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.

Astaxanthin treatment ameliorates ER stress in polycystic ovary syndrome patients: a randomized clinical trial.

Astaxanthin (ASX), as a natural carotenoid compound, exists in various types of seafood and microorganisms. It has several possible beneficial therapeutic effects for patients with polycystic ovary syndrome (PCOS). Patients with PCOS also suffer from endoplasmic reticulum (ER) stress. In the present work, it was hypothesized that ER stress could be improved by ASX in PCOS patients. Granulosa cells (GCs) were obtained from 58 PCOS patients. The patients were classified into ASX treatment (receiving 12 mg/day for 60 days) and placebo groups. The expression levels of ER stress pathway genes and proteins were explored using Western blotting and quantitative polymerase chain reaction. To assess oxidative stress markers, follicular fluid (FF) was gained from all patients. The Student's t test was used to perform statistical analysis. After the intervention, ASX led to a considerable reduction in the expression levels of 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and X-box-binding protein 1 compared to the placebo group, though the reduction in the messenger RNA (mRNA) expression level of activating transcription factor 6 was not statistically significant. However, ASX significantly increased the ATF4 expression level. GRP78 and CHOP protein levels represented a considerable decrease in the treatment group after the intervention. In addition, a statistically significant increase was found in the FF level of total antioxidant capacity in the treatment group. Based on clinical outcomes, no significant differences were found between the groups in terms of the oocyte number, fertilization rate, and fertility rate, but the ASX group had higher rates of high-quality oocytes, high-quality embryo, and oocyte maturity compared to the placebo group. Our findings demonstrated that ER stress in the GCs of PCOS patients could be modulated by ASX by changing the expression of genes and proteins included in the unfolding protein response.Trial registration This study was retrospectively registered on the Iranian Registry of Clinical Trials website ( www.irct.ir ; IRCT-ID: IRCT20201029049183N, 2020-11-27).

Quercetin and polycystic ovary syndrome; inflammation, hormonal parameters and pregnancy outcome: A randomized clinical trial.

PROBLEM: Women with PCOS have a reduced total antioxidant level in addition to higher oxidative stress. Quercetin is a flavonol-type antioxidant that may be found in many foods. Does quercetin affect inflammatory and hormonal factors and clinical outcomes in PCOS patients? METHOD OF STUDY: Seventy-two women with PCOS were randomly allocated to one of two intervention groups, and each received a daily dosage of 500 mg of Quercetin for the intervention group or a placebo for the control group for a period of 40 days from the start of the menstrual cycle until the day of ovulation. Serum levels of IL-6, TNF-alpha, LH, FSH, and AMH were measured using ELISA. In addition, oocyte and embryo grade before IVF and pregnancy rate have been examined. RESULTS: LH levels reduce significantly in the quercetin group (4.351.62 at baseline to 3.061.43 after 3 months) (p = .029). The results indicated that Quercetin significantly decreased TNF alpha levels in comparison to the pretest (p = .008). Following capsule administration, IL-6 levels significantly decreased in the quercetin group (p = .001). Except for Δ LH, ΔIL6, and ΔFSH, there was no significant difference in any of the hormones and inflammations parameter changes. CONCLUSION: Quercetin consumption causes improvement in oocyte and embryo grade and the pregnancy rate in PCOS patients. As a result, regular consumption of Quercetin has been shown to decrease inflammatory and LH parameters, making it beneficial for the management of PCOS and related diseases.

Leukemia inhibitory factor's effect on the growth and survival of sheep's follicles of ovarian tissue during vitrification.

One of the experimental programs for fertility protection in women includes protective cryopreservation. Vitroficasion of ovarian tissue is one of the protective cryopreservation methods that use high concentrations of antifreeze and faster cooling. To reduce its complications, LIF (Leukemia inhibitory factor) was used as a pretreatment in this study. In this study, the ovaries were randomly divided into 8 groups. In NCN (without pretreatment and LIF in culture media), NCP (without pretreatment and with LIF in culture media), PCP (with pretreatment and LIF in culture media), and PCN (with pretreatment and without LIF in culture media) groups, vitrification and reversal were not performed. In the groups NVN (without pretreatment and LIF in culture media), NVP (without pretreatment and with LIF in culture media) PV, PVP (with pretreatment and LIF in culture media), and PVN (with pretreatment and without LIF in culture medium) groups, vitrification and tissue reversal were performed. All groups were cultured and histological, cellular, and molecular evaluations were performed. The results of the present study showed that LIF in the culture medium reduced the number of abnormal, primordial, primary, and secondary follicles, and DNA breakage compared to the group without LIF (P < 0.05) and increases the growth of follicles and expression of GDF9, BMP, AMH, KITLG genes (P < 0.05). The use of LIF pretreatment before vitrification and melting of sheep ovary tissue in its culture medium reduces the damage caused by it and increases the growth and development of ovarian follicles while maintaining their function.

Evaluation of Nrf2/ARE Signaling Pathway in the Presence of Pentoxifylline as a Cryoprotectant in Mouse Spermatogonial Stem Cells.

The process of spermatogonial stem cell cryopreservation (SSCs) in young male cancer survivors is associated with increased reactive oxygen species (ROS), DNA fragmentation, apoptosis, decreased cell activity, and finally reduced fertility of SSCs. Therefore, it is necessary to add cryoprotectants to the freezing medium to minimize the injuries associated with cryopreservation. In addition, the Nrf2/ARE pathway is a main cellular pathway that regulates the antioxidant defense system. The purpose of this study was to evaluate the cryoprotective effect of pentoxifylline (PTX) on SSCs after freezing-thawing through the Nrf2/ARE pathway. SSCs extracted from neonatal mice testes were isolated and their purity was measured by flow cytometry with GDNF family receptor alpha-1 (GFRα1) and inhibitor of differentiation 4 (ID4). After culturing, the cells were frozen in different groups for 1 month. After freezing-thawing, cell viability, colonization rate, and intracellular ROS, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were evaluated. Quantitative real-time polymerase chain reaction and western blotting were done to assess the expression levels of Nrf2, Keap-1, PI3K, and AKT genes and proteins. The survival and colonization rates of SSCs, SOD, and CAT levels, and Nrf2, PI3K, and AKT expression levels were significantly higher in the PTX group compared with the other cryopreservation groups. The Keap-1 expression level and the ROS and MDA production levels also decreased significantly in the PTX group (p-value <0.05). According to our findings, PTX can activate the antioxidant defense through the Nrf2/ARE signaling pathway; therefore, it could be a suitable cryoprotectant candidate for freezing and long-term storage of SSCs in the clinical setting.

Bridging potential of Taurine-loading PCL conduits transplanted with hEnSCs on resected sciatic nerves.

Reconstruction of nerve conduits is a promising method for functional improvement in peripheral nerve repair. Besides choosing of a suitable polymer for conduit construction, adding factors such as Taurine improve a more advantageous microenvironment for defect nerve regeneration. Showing several major biological properties of Taurine, for example, regulation of the osmotic pressure, modulation of neurogenesis, and calcium hemostasis, makes it an appropriate option for repairing of defected nerves. To this, we examined repairing effects of Taurine-loading PCL conduits cultured with human endothelial stem cells (hEnSCs) on resected sciatic nerves. PCL/Taurine/Cell conduits transplanted to a 10-mm sciatic nerve gap. Forty-two wistar rats were randomly divided to seven groups: (1) Normal group, (2) Negative control (NC), (3) Positive control (nerve Autograft group), (4) PCL conduits group (PCL), (5) Taurine loaded PCL conduits group (PCL/Taurine), (6) hEnSCs cultured on the PCL conduits (PCL/Cell), (7) hEnSCs cultured on the PCL/Taurine conduits (PCL/Taurine/Cell). Functional recovery of motor and sensory nerves, the action potential of exciting muscle and motor distal latency has seen in PCL/Taurine/Cell conduits. Histological studies showed also remarkable nerve regeneration and obvious bridging has seen in this group. In conclusion, PCL/Taurine/Cell conduits showing suitable mechanical properties and biocompatibility may improve sciatic nerve regeneration.

The Effect of Astaxanthin on Motility, Viability, Reactive Oxygen Species, Apoptosis, and Lipid Peroxidation of Human Spermatozoa During the Freezing-Thawing Process.

Cryopreservation of spermatozoa is a general procedure to preserve viable sperm for an indefinite period. Despite the efficiency of sperm cryopreservation, excessive reactive oxygen species (ROS) production during cryopreservation can induce structural and functional changes in spermatozoa. Also, cryopreservation has been shown to decrease the spermatozoa's antioxidant activity inducing them to be more sensitive to damage caused by ROS. Experimental evidence suggests that astaxanthin (AXT) has essential activities such as antioxidant, antibacterial, and antithrombotic properties. Therefore, this study aimed to evaluate the effect of AXT on the sperm quality of healthy men during freezing-thawing. In the first phase, 10 semen samples with different concentrations of AXT (0.0, 0.5, 1, and 2 µM) were cryopreserved to achieve an optimal dose of AXT. Then, motility, viability, and phosphatidylserine (PS) externalization were evaluated. In the second phase, 25 samples were collected and divided into 3 groups: fresh group, control group (untreated frozen-thawed samples), and AXT group (treated frozen-thawed with AXT). Then, samples were cryopreserved in freezing media supplemented with or without the optimal concentration of AXT (1 µM). After thawing, the levels of sperm parameters, including motility (using a computer-assisted sperm analyzer), viability (eosin-nigrosin), early apoptotic change (annexin V/propidium iodide), ROS (flow cytometry), and lipid peroxidation (LPO) (using enzyme-linked immunosorbent assay), were evaluated. Our results showed that the addition of 1 µM AXT to sperm freezing media improved all parameters of sperm motility and viability (p ≤ 0.05). Furthermore, it could reduce the levels of ROS parameters (intracellular hydrogen peroxide and superoxide) compared with the control group (p ≤ 0.05). Also, AXT significantly decreased the level of PS externalization (p ≤ 0.05) and LPO (p ≤ 0.05) after the freezing-thawing process. In conclusion, our findings demonstrated that human semen treatment with 1 µM AXT before the freezing-thawing process has protective effects against oxidative stress and could diminish the destructive effects of this process on sperm quality.

Investigating the effect of Sulforaphane on AMPK/AKT/NRF2 pathway in human granulosa-lutein cells under H2O2-induced oxidative stress.

Excessive production of reactive oxygen species (ROS) in granulosa cells (GCs) plays a role in pathogenesis of polycystic ovarian syndrome (PCOS) by developing oxidative stress (OS). It was shown that Sulforaphane (SFN), with known antioxidant properties, can have protective effects in different diseases through affecting the nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Thus, the purpose of the current work was to examine the protective impact of SFN through the activation of the AMPK/AKT/NRF2 pathway against OS produced by H2O2 in granulosa-lutein cells (GLCs). Individuals' GLCs were obtained during ovum retrieval in intracytoplasmic sperm injection (ICSI) cycles. First, the induced OS model was created in GLCs using H2O2 exposure. To examine the protective effect of SFN against OS, the cells were cultured for 24 h in presence or absence of SFN. Eventually, the levels of intracellular ROS and apoptosis were measured by flow cytometry, and genes and proteins expression levels of AMPK, AKT, and NRF2 were evaluated using qRT-PCR and western blotting. Compared to the control group, the levels of intracellular ROS and apoptosis rose dramatically in GLCs with enhanced OS. SFN therapy decreased ROS and apoptosis levels and increased the overexpression of AMPK, AKT, and NRF2 genes and proteins. This study's results revealed that SFN exposure results in the alleviation of ROS and apoptosis levels possibly through activating the overexpression of genes and proteins of AMPK, AKT, and NRF2, and exerts its protective effects against OS in GLCs.

Protective roles of seminal plasma exosomes and microvesicles during human sperm cryopreservation.

RESEARCH QUESTION: Do seminal plasma microvesicles and exosomes, as two subtypes of extracellular vesicles, exert cryoprotective properties in sperm cryopreservation? DESIGN: Microvesicles and exosomes isolated from normozoospermic semen samples (n = 10) by serial ultracentrifugation were determined using scanning electron microscopy, dynamic light scattering and western blot analysis. The interactions between extracellular vesicles and spermatozoa were detected using Dil labelling. Purified spermatozoa from different normozoospermic samples (n = 25) were then treated individually with exosomes or microvesicles for 1 h and subsequently cryopreserved. The effects of extracellular vesicles during cryopreservation were investigated by determining post-thaw sperm motility, morphology, viability, reactive oxygen species (ROS) generation, lipid peroxidation, total antioxidant capacity (TAC), mitochondrial membrane potential (MMP), DNA integrity, and apoptosis rate. RESULTS: Microvesicles and exosomes displayed a round-shape morphology, with about 70% of exosomes ranging from 43-144 nm, microvesicles ranging from 144.5-486 nm and both expressed tetraspanin markers. Fluorescence microscopy showed that exosomes and microvesicles absorbed mainly in the sperm head and less frequently in the neck and tail. The post-thawing results indicated that the diluent with exosomes or microvesicles had improved sperm motility (P = 0.007), morphology (P < 0.001) and viability (P < 0.001) compared with untreated samples. The ROS levels decreased significantly (P = 0.001), with a consequent decrease in DNA damage (P = 0.001). The TAC activity (P = 0.001) and MMP levels (P = 0.001) were also significantly improved; levels of malondialdehyde (MDA) (P = 0.62) and apoptosis rate (P = 1.000) remained unchanged. CONCLUSION: Seminal plasma microvesicles and exosomes could protect spermatozoa from cryopreservation chilling injuries.

Randomized controlled trial of astaxanthin impacts on antioxidant status and assisted reproductive technology outcomes in women with polycystic ovarian syndrome.

PURPOSE: Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women, is typically accompanied by a defective oxidative defense system. Here, we investigated the effect of astaxanthin (AST) as a powerful antioxidant on the oxidative stress (OS) response and assisted reproductive technology (ART) outcomes in PCOS patients. METHODS: In this double-blind, randomized, placebo-controlled trial, PCOS patients were randomly assigned into two groups. The intervention group received 8 mg AST, and the control group received the placebo daily for 40 days. The primary outcomes were the serum and follicular fluid (FF) levels of the OS biomarkers and the expression levels of the specific genes and proteins in the oxidative stress response pathway. The secondary outcomes were considered ART outcomes. RESULTS: According to our findings, a 40-day course of AST supplementation led to significantly higher levels of serum CAT and TAC in the AST group compared to the placebo group. However, there were no significant intergroup differences in the serum MDA and SOD levels, as well as the FF levels of OS markers. The expression of Nrf2, HO-1, and NQ-1 was significantly increased in the granulosa cells (GCs) of the AST group. Moreover, the MII oocyte and high-quality embryo rate were significantly increased in the AST group compared to the placebo group. We found no significant intergroup difference in the chemical and clinical pregnancy rates. CONCLUSION: AST treatment has been shown to increase both serum TAC levels and activation of the Nrf2 axis in PCOS patients' GCs. TRIAL REGISTRATION: ClincialTrials.gov Identifier: NCT03991286.

The boosting effects of melatonin on the expression of related genes to oocyte maturation and antioxidant pathways: a polycystic ovary syndrome- mouse model.

BACKGROUND: Melatonin, as a free radical scavenger exhibiting genomic actions, regulates the antioxidant genes expression and apoptosis mechanisms. In polycystic ovary syndrome (PCOS) patients, an imbalance between free radicals and antioxidants in follicular fluid leads to oxidative stress, aberrant folliculogenesis, and intrinsic defects in PCOS oocytes. In this experimental mouse model study, oocytes of PCOS and the control groups were cultured in different melatonin concentrations (10- 5, 10- 6, and 10- 7 M) to investigate the expression of oocyte maturation-related genes (Gdf9/Bmp15), antioxidant-related genes (Gpx1/Sod1), apoptotic biomarkers (Bcl2/Bax) and total intracellular ROS levels. RESULTS: Gdf9 and Bmp15, Gpx1 and Sod1 were up-regulated in PCOS and control oocytes cultured in all melatonin concentrations compared to those cultured in IVM basal medium (P < 0.05). A significant decrease in the total ROS level was observed in all groups cultured in the supplemented cultures. Melatonin increased Bcl2 and decreased Bax gene expression in PCOS and control oocytes compared to non-treated oocytes. CONCLUSIONS: Melatonin increased antioxidant gene expression and regulated the apoptosis pathway, effectively reducing the adverse effects of culture conditions on PCOS oocytes. Furthermore, it influenced the expression of oocyte maturation-related genes in PCOS, providing valuable support during the IVM process.

Evaluating the Differential Expression of miR-146a, miR-222, and miR-9 in Matched Serum and Follicular Fluid of Polycystic Ovary Syndrome Patients: Profiling and Predictive Value.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder of women in reproductive age with significant effects on reproductive and metabolic functions. Many molecular players may be involved in PCOS pathology; however, miRNAs possess great ability in gene expression control in normal ovarian function and folliculogenesis. We appraised the relative expression of miR-146a, miR-222, miR-9, and miR-224 in serum and follicular fluid (FF) of PCOS patients compared to control subjects. PCOS (n = 35) and control (n = 30) subjects were recruited in the study during their enrolment in IVF cycles. Serum and FF of human subjects were collected and stored. Total RNA was isolated from samples and cDNA was synthesized using miRNA-specific stem-loop RT primers. Quantitative real-time PCR was used to evaluate the expression of miRNAs relative to U6 expression. The predictive value of miRNAs' expression for discrimination of PCOS patients from control subjects was evaluated by receiver-operating characteristic (ROC) curve analysis. miR-224 was not detected in serum and FF samples. Significantly, higher levels of miR-146a and miR-9 in serum of PCOS group were detected. In contrast, relative expression of miR-146a and miR-9 significantly decreased in FF. In PCOS group, relative expression of all detected miRNAs was elevated in serum in comparison to FF, whereas in control group no change was noticed. Combination of FF miRNAs showed improved predictive value with area under the ROC curve (AUC) of 0.84, 93.8% sensitivity, and 83.3% specificity. Contradicting alternations of miRNAs in serum and FF are indicative of different sources of miRNAs in body fluids. Presumptive target genes of studied miRNAs in signalling pathways may show the potential role of these miRNA in folliculogenesis.

The effect of mitochondria-targeted antioxidant MitoQ10 on redox signaling pathway components in PCOS mouse model.

PURPOSE: Considerable evidence suggests that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of Polycystic ovary syndrome (PCOS). We aimed to evaluate the effectiveness of mitochondria-targeted antioxidant, MitoQ10, on the redox signaling pathway's component in PCOS. METHOD: We assessed TXNIP, TRX, and ASK1 expression in granulosa cells (GCs) of the DHEA-induced PCOS mouse model. Female BALB/c mice in five groups of Control, DHEA, and DHEA + MitoQ10 in three doses of 250, 500, and 750 µmol/L MitoQ10 were treated for 21 days. RESULTS: Histological investigation showed a probable improvement in folliculogenesis; besides, ASK1 and TXNIP expression were significantly increased in GCs of the PCOS mouse F4Fmodel as compared to the control groups and decreased steadily in groups treated by MitoQ10. However, TRX expression showed a drop that was restored by MitoQ10 meaningfully (P ≤ 0.05). CONCLUSION: The work presented herein suggests mitochondria-targeted antioxidant, MitoQ10, have modulating effects on folliculogenesis in the ovary and also on the redox signaling pathway in GCs of PCOS mouse model which may have potential to attenuate oxidative stress and its relative damages.

Astaxanthin Relieves Busulfan-Induced Oxidative Apoptosis in Cultured Human Spermatogonial Stem Cells by Activating the Nrf-2/HO-1 pathway.

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10µM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.