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Objective: Scrotal hyperthermia poses a significant threat to spermatogenesis and fertility in mammalian species. This study investigated the effects of vitamin B12 and vitamin C on spermatogenesis in adult male mice subjected to long-term scrotal hyperthermia. The rationale is based on the sensitivity of germ cells and epididymal sperm to increased scrotal temperatures. While various factors, both internal and external, can raise the testicular temperature, this study focused on the potential therapeutic roles of vitamins B12 and C. Methods: After inducing scrotal hyperthermia in mice, vitamin B12 and vitamin C were administered for 35 days. We assessed sperm parameters, serum testosterone levels, stereological parameters, the percentage of apoptotic cells, reactive oxygen species (ROS) levels, and glutathione (GSH) levels. Additionally, real-time polymerase chain reaction was used to analyze the expression of the c-kit, stimulated by retinoic acid gene 8 (Stra8), and proliferating cell nuclear antigen (Pcna) genes. Results: Vitamin C was more effective than vitamin B12 in improving sperm parameters and enhancing stereological parameters. The study showed a significant decrease in apoptotic cells and a beneficial modulation of ROS and GSH levels following vitamin administration. Moreover, both vitamins positively affected the expression levels of the c-kit, Stra8, and Pcna genes. Conclusion: This research deepens our understanding of the combined impact of vitamins B12 and C in mitigating the effects of scrotal hyperthermia, providing insights into potential therapeutic strategies for heat stress-related infertility. The findings highlight the importance of considering vitamin supplementation as a practical approach to counter the detrimental effects of elevated scrotal temperatures on male reproductive health. [ABSTRACT FROM AUTHOR]

Introduction: 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. Methods: 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. Results: 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. Conclusion: Based on the present findings, laser therapy can reduce complications caused by scrotal hyperthermia during prepuberty and ameliorate spermatogenesis during puberty. [ABSTRACT FROM AUTHOR]

Introduction: Unhealthy lifestyle choices such as alcohol, chemicals, and heat stress can worsen male infertility. Heat stress can cause damage to the essential structure known as the blood-testis barrier (BTB). Photobiomodulation therapy (PBMT) has been employed in various studies to enhance sperm quality in individuals with genital inflammatory conditions in recent times. The current research sought to study how laser therapy affects spermatogenesis and the structure of the BTB in a mouse model of scrotal heat exposure. Methods: Thirty adult male NMRI mice, 8 weeks old, were divided into three groups: Control, Hyperthermia, and Hyperthermia + Laser 0.03 J/cm2. The animals in the hyperthermia group had their testicles exposed to water at 43 °C for 20 minutes five times every other day. Then, the testicles were exposed to laser radiation every other day for 35 days, lasting 3 minutes each time, with an energy density of 0.03 J/cm2. Animals were sacrificed, and sperm parameters, reactive oxygen species (ROS) and glutathione (GSH) levels, stereological parameters, and gene expression were assessed in the end. Results: The study showed that PBMT can significantly enhance sperm quality, quantity of spermatogenic cells, testicular volume, levels of ROS and GSH, and gene expression related to the blood-testis barrier. Conclusion: Currently, PBMT is a novel approach for addressing male infertility by preserving the integrity of the BTB in Sertoli cells, which in turn supports the growth and specialization of germ cells. [ABSTRACT FROM AUTHOR]

Background: Insulin is an essential factor that controls female reproductive system. Insulin signaling via Foxo1 and Akt1 can improve steroidogenesis, cell proliferation, and protein synthesis. We aimed to determine the effect of insulin on possible changes in gene expression, hormonal status, and histological aspects of the ovary following the induction of the animal model of polycystic ovary syndrome (PCOS). Materials and Methods: In this experimental study, 24 adult female NMRI mice weighing 25-30 g were randomly placed in three groups: control, PCOS (60 mg/kg dehydroepiandrosterone (DHEA) for 20 days, and PCOS+insulin (60 mg/kg DHEA for 20 days+100 µL insulin diluted in water twice a week for 30 consecutive days). Blood specimens were obtained from the heart and the serum levels of testosterone, progesterone, and estradiol were measured. Right, and left ovaries were removed for real-time polymerase chain reaction (PCR) and stereological study. Results: DHEA injection significantly amplified the concentration of testosterone, progesterone, and estradiol. While insulin treatment amended the level of reproductive hormones. DHEA injection significantly reduced the expression levels of Irs1-4, Pdk1, Pi3k, and Akt1-3 and raised the expression level of Caspase-3. However, insulin administration amplified expression levels of Irs1-4, Pdk1, Pi3k, and Akt1-3, and reduced Caspase-3. The total volume of ovarian tissue in mice receiving DHEA significantly declined compared to the control group. Besides, a substantial decrease was detected in the number of ovarian antral, Graafian, and primordial follicles and also in the total number of corpus luteum following DHEA administration. Comparison of structural alterations in ovarian tissue between the PCOS+insulin and the PCOS groups displayed that insulin administration improved the total number of Graafian, primordial, and antral follicles and also corpus luteum. Conclusion: In general, short-term insulin treatment showed improvement in hormonal balance, folliculogenesis, and insulin resistance in the ovaries of the PCOS mice model. [ABSTRACT FROM AUTHOR]

Introduction: Although the effect of adriamycin, a chemical drug used to treat cancer, is known to affect gametogenesis and ovarian function, the drug's effect on endometrial receptivity and expression of integrin genes is unclear. To fill this gap, we aimed to investigate the effect of adriamycin on the expression of integrin genes that play a role in endometrial receptivity. Methods: A total of 32 female rats weighing about 200 g were tested in the present study. The rats were divided into 4 groups: Healthy (sham), control, and experimental, with adriamycin at 4 mg/kg and 8 mg/kg doses for 6 weeks. Two weeks after the injection of adriamycin at an 8 mg/kg dose in the treatment group, all rats were sacrificed, but the treatment group with a 4 mg/kg dose of adriamycin continued the treatment. Then, the estrus phase was determined in the three groups using vaginal smears. The endometrial tissue was removed, and gene expression levels of a1ß1, a1ß4, and ανβ3 genes were evaluated using real-time PCR methods in the three groups. Results: The rats' weight decreased significantly in the treatment group with adriamycin. The process of weight loss was found to be dose-dependent. Integrin gene expression in the adriamycin group in the estrus phase had no significant difference compared with that in the control and sham groups (P>0.05). In the adriamycin group, the expression of integrin a1 (intga1) increased significantly (P<0.05; P=0.013). Conclusion: The expression of integrin genes did not change, except for integrin alpha1, during the estrus phase in the endometrium treated with adriamycin drug. Probably, adriamycin did not have any effect on the expression of integrin genes. [ABSTRACT FROM AUTHOR]

Background: Recurrent implantation failure (RIF) is defined as three or more failed in vitro fertilization attempts and is due to several factors such as oocyte and embryo quality. Methods: Fifty-one RIF patients and 19 controls were selected based on the inclusion criteria. EFNB2, CAMK1D, AREG, and PTGS2 as well as miR-34, miR-145, miR-204-5p, and miR-26-5p were selected since the microRNAs (miRNAs) targeted the genes based on bioinformatic predictions and literature review. Total RNA was extracted from cumulus cells (CCs) and follicular fluid (FF) of the oocytes. We performed real-time polymerase chain reaction to evaluate the expression of the genes and the miRNAs in CC and FF of pregnant and nonpregnant RIF patients. The expression of CAMK1D, AREG, miR-34-5p, and miR-26-5p was higher in CC than FF. Results: The expression of CAMK1D, PTGS2, and miR-26-5p in CC of the pregnant group was higher than FF. The expression of EFNB2, PTGS2, miR-145, and miR-204-5p was lower in the CC, and the expression of EFNB2, AREG, miR-34-5p, mR-145, and miR-204-5p was lower in the FF of the pregnant group. The expression of CAMK1D, AREG, PTGS2, miR-34-5p, and miR-26-5p was higher in the CC and FF of the high quality (HQ) embryos than non-HQ (NHQ) embryos. The expression of EFNB2, miR-145, and miR-204-5p was higher in the CC and FF of the NHQ embryos. The difference was statistically significant for EFNB2 in CC and FF as well as miR-145 in CC. The level of progesterone and prostaglandin E2 in the FF of the pregnant group was higher than their level in the nonpregnant group. Conclusion: CAMK1D expression and overexpression of miR-34-5p and miR-26-5p could be considered as markers of successful pregnancy. In addition, the results show that normal FF treatment of RIF patients may result in the production of high-quality embryos. [ABSTRACT FROM AUTHOR]

A triplet spiral channel coupled with cross-flow filtration has been designed and fabricated in an effort to separate sperm cells from either semen or simulated testicular sperm extraction (TESE) samples. This device separates a fraction of cells from the sample by taking advantage of inertial focusing combined with hydrodynamic filtration in multiple micro-slits. Compared to the conventional swim-up technique, the proposed microfluidic device is capable of efficiently separating sperm cells without any tedious semen sample processing and centrifugation steps with a lower level of reactive oxygen species and DNA fragmentation. The device processing capability on the simulated TESE samples confirmed its proficiency in retrieving sperm cells from the samples with an approximate yield of 76%. Conclusively, the introduced microfluidic device can pave the path to proficiently separate sperm cells in assisted reproductive treatment cycles. [ABSTRACT FROM AUTHOR]

There are a number of risk factors, especially viral diseases, which can lead to infertility. Among the various viral infections, much attention has been given to the role of the Papillomaviridae and Herpesviridae. After collecting 82 semen samples (37 teratospermia, 2 asthenozoospermia, 2 oligoasthenospermia, 1 oligospermia, 6 asthenoteratospermia and 34 normal semen samples), and washing them, the DNA from both freshly ejaculated spermatozoon and washed spermatozoa was extracted. Subsequently, the prevalence of EBV, CMV, HSV‐1, HSV‐2, VZV and HPV was evaluated using Multiplex PCR and Nested PCR. In this study, 1 normal and 5 abnormal semen samples were infected with HSV‐1 (1 normal, 4 teratospermia and 1 oligoasthenospermia). In addition, there were 2 VZV‐positive samples (both were teratozoospermia). Nested PCR indicated that 1 asthenozoospermia, 1 asthenoteratospermia, 3 teratospermia and 4 normal samples were HPV positive (including 8 HPV‐18 and 1 HPV‐33). Among 9 HPV‐positive subjects, 3 samples were negative after washing the infected samples. The prevalence of EBV, CMV, VZV, HSV‐1 and HSV‐2 remained unchanged prior to and after washing. Maybe sperm washing can be useful to eliminate HPV infection from semen samples, but further investigation is required because of the small number of samples. [ABSTRACT FROM AUTHOR]

Endometriosis, a chronic inflammatory disease, is identified by the presence of endometrial tissue outside the uterus. The prevalence of this disease among reproductive-age women is almost 10–15%. High levels of IL-6 and IL-8 have been found in the peritoneal fluid (PF) of women with endometriosis and are involved in its pathogenesis. Isolated stromal cells from 12 ectopic and eutopic endometrial biopsies of women with ovarian endometrioma and also 12 endometrial biopsies of nonendometriotic controls were treated with 1.1 µM pyrvinium pamoate, a Wnt/β-catenin signaling pathway inhibitor, for 72 hrs. Before treatment, mRNA gene expression and secretion of IL-6 and IL-8 were significantly higher in ectopic (EESCs) than eutopic (EuESCs) and control (CESCs) endometrial stromal cells. After treatment, mRNA gene expression and also secretion of IL-6 and IL-8 were significantly reduced. Our Findings showed that pyrvinium pamoate suppresses the mRNA gene expression and secretion of IL-6 and IL-8 in human endometriotic stromal cells. Additional investigations on this compound are required before clinical application. [ABSTRACT FROM AUTHOR]

The imbalance of Th17/Treg cells has been recently suggested as a new risk factors for recurrent implantation failure (RIF). Furthermore Th17/Treg cells are involved in immune regulation in peripheral blood and endometrial tissue of patients with RIF. In this research, we investigated the effects of Hydroxychloroquine (HCQ) on the level and function of Th17 and Treg cells in women with RIF. It may be possible to improve pregnancy outcomes by modulating high cytokine levels. Women with RIF received oral HCQ (n = 60) on day 4 of the menstrual cycle and continued until day 20 of the menstrual cycle and 2 days before embryo transfer and continued until the day of the pregnancy test, for a total of 16 days in another cycle. The serum levels of IL-17 and IL-10, the expression of transcription factors related to Th17 and Treg cells and the immune-reactivity of IL-17, IL-21 as Th17 related cytokines and IL-10, TGF- β as Treg related cytokines in endometrial tissues were evaluated by ELISA, real-time PCR, and fluorescent immunohistochemistry respectively. Results: Treatment with HCQ down-regulated Th17 related cytokines and function and up-regulated Treg related cytokines and function significantly (p <.001). RORγt, the Th17 transcription factor, expression was down-regulated and FOXP-3, the T-reg transcription factor, expression was up-regulated. The biochemical pregnancy rate was not significantly different in RIF patients before and after treatment. Our results demonstrated that the administration of HCQ in RIF women with immune cell disorders during pregnancy could affect the Th17/Treg ratio and enhance Treg and diminish Th17 responses which may be associated with successful pregnancy outcomes. However, significant difference in pregnancy outcomes was not observed in the present study. [ABSTRACT FROM AUTHOR]

Objectives: Choriocarcinoma spheroids, as acceptable three-dimensional (3D) models of the embryo, vary in the number of incorporated cells based on the procedures of production. Since the secretion of emitted signals from spheroids can modulate endometrial confrontation responses, improving a high throughput standard assay is precisely demanding for normalizing the characteristics of spheroids based on cellular enumeration. Materials and Methods: Spheroids derived from the suspension culture of the BeWo cell line were dimensionally categorized into three groups with sizes of 50, 100, and 200 µm in diameters. The volume of each spheroid was determined according to 4/3pr3 formula and MTT and neutral red uptake (NRU) assays were applied as conventional and modified procedures. Furthermore, the categorized groups of spheroids were enzymatically dissociated and stained by the Trypan blue dye to precisely enumerate the number of incorporated cells based on volume alterations. Results: The absorbance of spheroids in modified MTT and NRU procedures with respect to the conventional ones resulted in higher correlation coefficients with volume alterations. Similarly, modified MTT and NRU procedures with respect to the conventional ones resulted in higher correlation coefficients between the cells numeration and volume alterations. Finally, the sensitivity of modified MTT assay was higher than modified NRU assay regarding accurately estimating the number of incorporated cells per spheroid. Conclusion: In general, the modification of MTT and NRU procedures can amplify the potential of both assays to enumerate the cell number of 3D aggregates. Exclusively, the modification of MTT assay brought it as an ideal high throughput assay with high sensitivity to screen the cell number of spheroids with a diameter of 200 µm and less. [ABSTRACT FROM AUTHOR]

Reproductive senescence is accompanied by a reduced number and quality of ovarian follicles in response to the accumulation of free radicals and the process of apoptosis. Having selected mice as models, we examined the hypothesis that curcumin as an antioxidant and anti-inflammatory agent might prevent or retard ovarian aging. Female NMRI 21-day-old mice were divided into control, vehicle and curcumin groups. In the treatment group the mice received curcumin at 100 mg kg–1 day–1 intraperitoneally. After 6, 12 and 33 weeks several parameters were examined including ovarian reserve, oocyte quality, oxidative status, in vitro fertilisation and expression of ovulation-related (growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15)) and anti-aging-related (sirtuin 1 (SIRT-1) and SIRT-3) genes. Curcumin treatment up to 12 and 33 weeks resulted in increased ovarian volume and number of follicles and was associated with elevated anti-Müllerian hormone and oestrogen and diminished FSH serum levels. Furthermore, enhanced oocyte maturation, fertilisation and embryo development plus reduced oxidative stress were seen in the curcumin group. Also, the expression of GDF - 9 , BMP - 15 , SIRT - 1 and SIRT - 3 genes was increased in the curcumin group. Concerning gestational age, the findings of the study suggested that administration of curcumin could delay the process of oocyte aging in a mouse model. Ovarian aging as the earliest-aging organ is characterised by reduction of ovarian follicle quantity and quality, which is associated with impairment of oocyte developmental competence. This study was designed to prevent ovarian reserve loss in female mice by natural aging using curcumin as a well known antioxidant. Our data offer new insights into antioxidant therapy for postponing oocyte aging. [ABSTRACT FROM AUTHOR]

Since proliferation and differentiation of spermatogonial stem cells (SSCs) in culture system provide successful transplantation in this study, culture of human SSCs was compared to SACS (soft agar culture system), gelatin and control groups. The cells were isolated from seminiferous tubules of non-azoospermia patients (NOA) and cultured in DMEM for 3 weeks. The presence of SSCs in culture system was confirmed by immunocytochemistry of GFR-α1 and ITGα6 antibodies. The proliferated cells were cultured in three mentioned groups in the presence of retinoic acid and Sertoli cells conditioned medium for another 2 weeks. The number of colonies in the SACS group was significantly higher than two other groups. Before 2 weeks of culture, only Oct4 expression was observed in testicular cells (2.32 ± 0.25). After 2 weeks, the expression of Oct4 in the gelatin group was higher than that of the SACS group on day 7. The maximum expression of Stra8 was observed in SACS and gelatin groups after 7 days, but its expression was significantly decreased after 14 days of culture (p <.05). The expression of Scp3 and Acrosin genes were higher after 14 days in the SACS group compared to other groups. SACS has positive effects on proliferation and differentiation of hSSCs. [ABSTRACT FROM AUTHOR]