Autophagy, a critical element in the aging male reproductive disorders and prostate cancer: a therapeutic point of view.

Autophagy is a highly conserved, lysosome-dependent biological mechanism involved in the degradation and recycling of cellular components. There is growing evidence that autophagy is related to male reproductive biology, particularly spermatogenic and endocrinologic processes closely associated with male sexual and reproductive health. In recent decades, problems such as decreasing sperm count, erectile dysfunction, and infertility have worsened. In addition, reproductive health is closely related to overall health and comorbidity in aging men. In this review, we will outline the role of autophagy as a new player in aging male reproductive dysfunction and prostate cancer. We first provide an overview of the mechanisms of autophagy and its role in regulating male reproductive cells. We then focus on the link between autophagy and aging-related diseases. This is followed by a discussion of therapeutic strategies targeting autophagy before we end with limitations of current studies and suggestions for future developments in the field.

MicroRNAs, small regulatory elements with significant effects on human implantation: a review.

Embryo implantation is a critical process for achieving a successful pregnancy and live birth. The proper implantation must have a synchronized interaction between blastocyst and a receptive endometrium. Many genes are involved in the modulation of precise molecular events during implantation. MicroRNAs (miRNAs) have been extensively reported as gene regulatory molecules on post-transcriptional levels involved in various biological processes such as gametogenesis, embryogenesis, and the quality of sperm, oocyte, and embryos. A plethora of evidence has demonstrated critical roles for miRNAs in regulating genes involved in the implantation process; hence, dysregulation of miRNAs could be associated with significant impairments in implantation, such as recurrent implantation failure. In addition to the indispensable role of miRNAs in the intracellular control of gene expression, they can also be secreted into extracellular fluid and circulation. Therefore, miRNAs in body fluids and blood may be exploited as non-invasive diagnostic biomarkers for different pathological and physiological conditions. Recently, several studies have focused on the discovery of miRNAs function in the implantation process by appraising miRNAs and their target genes in human embryos, endometrial tissue, and cell culture models. Moreover, it was revealed that there could be a significant association between endometrial receptivity or implantation status and the expression of miRNAs in human body fluids, reinforcing their role as non-invasive biomarkers. In the current work, we reviewed the studies concerning the role of intracellular and extracellular miRNAs in human implantation and the influence of their dysregulation on implantation disorders.

The Efficacy of Vitamin D Supplement in the Expression and Protein Levels of Endometrial Decidualization Factors in Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a challenging situation for infertility specialists, and its treatment is introduced as a difficult case in the field of assisted reproductive technology (ART). Vitamin D (VD) is one of the supplements that have been suggested to improve the implantation process. In the present study, the effect of VD on the expression and protein levels of VD receptor (VDR), progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1), and homeobox protein A10 (HOXA10) in the endometrial cells of unknown RIF women with and without VD deficiency were assessed by qRT-PCR and immunohistochemistry. Twelve women with unknown RIF and VD deficiency (≤ 20 ng/ml) and twelve women with unknown RIF without VD deficiency (≥ 30 ng/ml) from 2021 to 2022 were identified. Endometrial specimens were collected in the mid-luteal stage before treatment or pregnancy. In the group with VD deficiency, oral medication of VD 50,000 units was prescribed for 2 to 3 months and their serum levels of VD were re-measured, then an endometrial biopsy at the same stage of the menstrual cycle was performed. The expression and protein levels of VDR, PR, PRL, IGFBP1, and HOXA10 in RIF patients with VD deficiency were lower than the RIF patients without VD deficiency (P value < 0.05). Our findings suggest that VD can play a key role in the pregnancy process, especially during embryo implantation and decidualization of the endometrial cells.IRCT registration number: IRCT20220528055006N1, Registration date: 2022-10-15, Registration timing: retrospective.

Nanowarming improves survival of vitrified ovarian tissue and follicular development in a sheep model.

Tissue cryopreservation has allowed long term banking of biomaterials in medicine. Ovarian tissue cryopreservation in particular helps patients by extending their fertility window. However, protection against tissue injury during the thawing process has proven to be challenging. This is mainly due to the heterogenous and slow distribution of the thermal energy across the vitrified tissue during a conventional warming process. Nanowarming is a technique that utilizes hyperthermia of magnetic nanoparticles to accelerate this process. Herein, hyperthermia of synthesized PEGylated silica-coated iron oxide nanoparticles was used to deter the injury of cryopreserved ovarian tissue in a sheep model. When compared to the conventional technique, our findings suggest that follicular development and gene expression in tissues warmed by the proposed technique have been improved. In addition, Nanowarming prevented cellular apoptosis and oxidative stress. We therefore conclude that Nanowarming is a potential complementary candidate to increase efficiency in the ovarian cryopreservation field.

The effect of kisspeptin on the maturation of human ovarian follicles in culture following vitrification-thawing processes.

OBJECTIVE: Ovarian cryopreservation is one of the effective methods to preserve fertility for cancer patients. Still, this approach has some problems, namely ROS, resulting in adverse effects on oocytes and ovarian follicles. Kisspeptin as an antioxidant to control ovarian function, directly or indirectly. In this study, the effect of kisspeptin on follicle maturation was evaluated in culture following ovarian cryopreservation. METHODS: Ovarian tissue samples of women between 20 and 35 years old (n=12) were laparoscopically collected. The samples were randomly divided into four groups: 1) control, 2) vitrification, 3) vitrified+1µM kisspeptin, and 4) vitrified+10µM kisspeptin. After vitrification and thawing processes, the tissues were cultured in DMEM medium for 7 days. H&E staining for histological evaluation, Real-Time PCR for GDF9 and BMP15 gene expression, and immunohistochemical staining for GDF9 and BMP15 protein expression were performed. RESULTS: In the vitrification group, ovarian tissue morphology was incoherent, and more primordial follicles than other follicle types were found. The expression of GDF9 and BMP15 genes and proteins were significantly decreased in this group compared with other groups (p<0.05). In the vitrification groups with kisspeptin (1 and 10 µM), the number of primary and secondary follicles was more than in the vitrification group. Besides, the expression of these genes and proteins was dramatically elevated in the vitrification groups with kisspeptin compared to the vitrification group alone (p<0.05). CONCLUSIONS: It seems that kisspeptin is an effective substance to improve the quality of the human ovarian cryopreservation medium by improving follicle maturation.

Impact of ejaculatory abstinence period and semen characteristic on the reproductive outcomes after intracytoplasmic sperm injection.

OBJECTIVE: The prognostic of semen characteristics in intracytoplasmic sperm injection (ICSI) outcomes is not clear. Also, there is no evidence-based recommendation for the abstinence period before ICSI. So, we aimed to assess the influence of the abstinence period and semen characteristics on ICSI outcomes. METHODS: A total of 1003 fresh ICSI cycles were divided into six groups; group 1 (1-day abstinence), group 2 (2 days abstinence), group 3 (3 days abstinence), group 4 (4 days abstinence), group 5 (5 days abstinence), and group 6 (6-10 days abstinence). RESULTS: We showed that semen volume (p=0.0001) and total sperm count (p=0.005) were increased in the groups with higher abstinence periods. Other semen parameters did not significantly associate with the abstinence period. The percentage of progressively motile sperm was associated with fertilization rate (p=0.007), and the sperm morphology was associated with cleavage-stage embryo rate (p=0.036). No influence of abstinence or semen parameters on rates of pregnancies was observed. CONCLUSIONS: The abstinence period before ICSI can influence the semen volume and total sperm count, and possibly fertilization. Although the sperm with the highest quality are selected for ICSI, the percentages of progressively motile and morphologically normal sperm in the ejaculated semen have a predictive value for fertilization and cleavage rates after ICSI, respectively.

Sitagliptin/metformin improves the fertilization rate and embryo quality in polycystic ovary syndrome patients through increasing the expression of GDF9 and BMP15: A new alternative to metformin (a randomized trial).

The aim of this study was to investigate the effects of sitagliptin/metformin (sitaformin), metformin and sitagliptin in PCOS patients. PCOS is a hormonal disorder and therefore the use of treatments that modulate hormone levels Like AMH, testosterone, insulin, leptin and especially FAI and HOMA-IR can reclaim the symptoms of PCOS. PCOS also increases oxidative stress and lipid peroxidation. Therefore, in clinical and research trials, the level of these factors is usually examined to reduce patients' symptoms. Participants were randomly assigned to receive metformin, sitagliptin, sitaformin or placebo Treatment was carried out 2 months before the start of the ovulation cycle and continued until the day of ovum pick up. The serum levels of HOMA-IR and FAI decreased significantly in the treated groups compared to the placebo. The serum and the FF levels of leptin also decreased significantly in the sitaformin group when compared to the metformin and sitagliptin groups. Moreover, the serum and FF levels of AMH and MDA had a significant decrease in the sitaformin and sitagliptin group compared to the placebo. The mRNA expression and protein levels of GDF9 and BMP15 in the cumulus cells increased significantly in the sitaformin compared to metformin and sitagliptin groups. The expression level of GDF9 and BMP15 mRNA were positively correlated with the fertilization rate and grade I embryos. Sitaformin improves levels of GDF9 and BMP15 in PCOS compared to metformin and sitagliptin, which can increase the rate of fertilization and grade I embryos.

Comparative Effect of Photobiomodulation on Human Semen Samples Pre- and Post-Cryopreservation.

The primary objective of this study is to evaluate and to compare the effects of photobiomodulation (PBM) on sperm parameters both before and after cryopreservation. In this regard, 24 freshly ejaculated semen samples from normozoospermic men were included in this study. Each semen sample was randomly divided into three groups (1 ml aliquot for each group): the control group (group one) underwent conventional sperm cryopreservation (n = 24), group two underwent pre-freezing PBM exposure (810 nm, diode laser, and 0.6 J/cm2) (n = 24), and group three underwent post freezing and thawing PBM exposure (n = 24). Indicators of sperm quality, including total sperm motility (TSM), progressive sperm motility (PSM), DNA fragmentation, lipid peroxidation levels, apoptosis-like changes, and gene expression levels of protamine (PRM) 1, PRM2, and adducin 1 alpha (ADD1), were investigated in a blinded style. Due to the beneficial effect of pre-freezing PBM therapy, group 2 exhibited the highest TSM and PSM levels compared to groups 1 and 3. At the same time, DNA fragmentation and lipid peroxidation were significantly reduced in the group 2 compared to the group 1 (p = 0.024 p = 0.016, respectively). Evaluation of apoptotic/necrotic changes revealed that parameters including early apoptosis, dead, and necrotic cells decreased in the group 2 compared to the either groups 1 (p = 0. 008, p = 0. 032, p = 0. 02, respectively) or group 3 (p = 0.037, p = 0.108, p = 0.083). There were no significant differences in the expression levels of PRM1, PRM2, and ADD1 among the study groups. Based on our results, PBM therapy prior to cryopreservation, even in the normal semen samples, plays a significant protective role against cryo-damage by preserving the functional parameters of spermatozoa.

Exosomal microRNA panels as biomarkers for hematological malignancies.

Hematological malignancies are classified as a heterogeneous category of cancers with various degrees of incidence and prognosis and different etiologies. Due to their aggressive essence they should be diagnosed as early as possible to improve prognosis, treatment outcome and survival. Bases on the limitations of previously identified biomarkers in terms of sensitivity, specificity and predictability, it is necessary to develop new diagnostic tools and biomarkers for the early diagnosis of hematological malignancies. Exosomes are nanovesicles secreted by almost all cell types in both physiological and pathological conditions. They play major roles in intercellular communication and are recently being considered as disease biomarkers. These nanovesicles carry proteins, lipids and nucleic acids like microRNAs (miRNAs). miRNAs are small noncoding RNAs, which act as translational suppressors via regulating protein-coding genes. The aberrant expression of miRNAs has been shown in various conditions including hematological malignancies. Moreover, it is now known that tumor cells secrete higher amounts of exosomes compared to normal cells. The idea of using exosomal miRNAs in serum as biomarkers is based on their surprisingly high stability and specificity. In the present paper, we reviewed and recommended exosomal miRNA panels including (miR-150, miR-155 and miR-1246), (miR-17-5p, miR-20a-5p, miR-16-5p and miR-5a-5p), (miR-18a, Let-7b) and (miR192-5p, miR21-5p, miR320b and Let-7d), for their potential to be used as non-invasive biomarkers in different hematological malignancies such as multiple myeloma, leukemia, and lymphoma.

Protective Effects of Fisetin in the Mice Induced by Long-Term Scrotal Hyperthermia.

Exposure to heat in the male reproductive system can lead to transient periods of partial or complete infertility. The current study aimed to examine the beneficial effects of  Fisetin against spermatogenic disorders in mice affected by long-term scrotal hyperthermia. For this purpose, hyperthermia was induced daily by exposure to the temperature of 43 °C for 20 min for 5 weeks. Except for the Healthy group, six other groups were exposed to heat stress: two treated groups including Preventive and Curative which received oral administration of fisetin (10 mg/kg/day) starting immediately before heat exposure and 15 consecutive days after the end of the heat exposure, respectively. And for each treated group, two groups including Positive Control (Pre/Cur+PC group) and vehicle (Pre/Cur+DMSO group) were considered. Our results showed that the testicular volume; the density of spermatogonia, primary spermatocyte, round spermatid, and Sertoli and Leydig cells; and sperm parameters, as well biochemical properties of the testis tissue, were remarkably higher in both Preventive and Curative groups compared to the other hyperthermia-induced groups and were highest in Preventive ones. Unlike the c-kit gene transcript which was significantly increased in the  Fisetin treatment groups (specially the Preventive group), the expression of HSP72 and NF-kß genes, Caspase3 protein, and DFI in sperm cells were significantly more decreased in Preventive and Curative groups compared to other hyperthermia-induced groups and were lowest in Preventive ones. Overall,  Fisetin exerts preventive and curative effects against spermatogenic disorders induced by long-term scrotal hyperthermia.

Evaluation of Expression and Phosphorylation of Progesterone Receptor in Endometrial Stromal Cells of Patients with Recurrent Implantation Failure Compared to Healthy Fertile Women.

Recurrent implantation failure (RIF) is the repeated failure of good-quality embryos in implantation process following several assisted reproduction cycles. Disruption of the endometrial receptivity is one of the main causes of RIF. Progesterone plays a pivotal role in the endometrial receptivity through the regulation of gene expression pattern by binding to its receptors in the endometrial cells. The aim of this study was to evaluate the expression level of progesterone receptor (PR) and its phosphorylated form in the endometrial stromal cells (eSC) of RIF patients and compare it to the eSC of healthy fertile women as control group. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, expression levels of PR mRNA, PR protein, and phospho-Ser294 PR protein were evaluated by quantitative real-time PCR and immunofluorescence staining, respectively. The results demonstrated a significant reduction in PR mRNA expression (P < 0.01.) and phospho-Ser294 PR protein (P < 0.05) level in RIF patients compared to the control group. These data for the first time suggest that the expression of PR and its phosphorylated form are impaired in RIF patients. Therefore, designing therapeutic methods for improving PR expression status and its regulation in the endometrium of RIF patients may help in improving the final reproductive outcomes of these cases.

Effect of 1,25(OH)2-vitamin D3 on expression and phosphorylation of progesterone receptor in cultured endometrial stromal cells of patients with repeated implantation failure.

Repeated implantation failure (RIF) occurs in a condition when good quality embryos fail to implant in the endometrium following several in vitro fertilization (IVF) cycles. Suboptimal endometrial receptivity is one of the main underlying factors that causes this failure. Progesterone is the key regulator of endometrial receptivity which regulates gene expression through binding to its receptors in the endometrial stromal cells (eSC). The aim of this study was to evaluate the effect of 1,25(OH)2-vitamin D3 on progesterone receptor (PR) expression level and its phosphorylation on Ser294 residues in eSC of RIF patients and healthy fertile women. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, the cells were incubated with vitamin D3 and the expression level of PR mRNA, PR protein and phospho-Ser294 PR protein were evaluated after treatment. The results showed that vitamin D3 treatment increases PR mRNA and protein level and phospho-Ser294 PR protein level in the isolated eSC of both RIF patients and the control group. These results suggest that vitamin D3 may possibly play a key role during the embryo implantation process by affecting the expression pattern and regulatory modifications of the PR in the eSC.