All-trans retinoic acid and fibroblast growth factor-2 enhance the fertility rate and embryo development in polycystic ovary syndrome mouse model.

Objectives: Polycystic ovary syndrome (PCOS) causes a developmental arrest of antral follicles and disrupts oocyte maturation. Retinoic acid (RA) and Fibroblast Growth Factor-2 (FGF2) are effective in follicle growth, thus their effects on histopathology and in vitro fertility of oocytes were investigated in PCOS-induced mice. Materials and Methods: Eighty female NMRI mice were randomly divided into 8 groups including 1-Normal mice, 2-PCOS mice without any treatment, 3-Normal mice treated with RA, 4-Normal mice treated with FGF2, 5-PCOS mice treated with RA, 6- PCOS mice treated with FGF2, 7- PCOS mice treated with RA and FGF2, and 8- Normal mice treated with RA and FGF2. Following PCOS induction, the mice were treated with intraperitoneal RA and FGF2 as a treatment. Then ovarian stimulation, for preparing the oocyte and embryo microscopic examinations was performed. After oocyte morphometry, through in vitro fertilization, the embryo formation was assessed. Data was analyzed by one-way ANOVA and Tukey tests. Results: The results showed simultaneous injection of RA and FGF2 into PCOS-induced mice increases antral follicles and corpus luteum, but decreases cystic follicles. Simultaneous injection of these two substances into healthy mice increases the pre-antral follicles and corpus luteum. Simultaneous injection of RA and FGF2 increases the number of embryos in both control and intervention groups. Conclusion: It can be concluded that RA and FGF2 increase the maturity of ovarian follicles, the number of two-celled embryos, and the number of grade-A embryos in mice with PCOS, which is more effective when these two substances are injected simultaneously.

The relationship between microbial contaminations of embryo transfer catheters with pregnancy outcome after embryo transfer.

Background: Different stages of assisted reproductive technologies are susceptible to contamination by various microorganisms. Objective: The aim of the study was to investigate the relationship between microbial contamination of embryo transfer catheters and the pregnancy outcome after embryo transfer. Methods: This cohort study was conducted on 60 patients candied for in vitro fertilization and embryo transfer cycles from 2021 to 2022. All embryos were transferred using a sterile syringe. The catheter contamination was checked by the microbial culture method, and in the case of microbial culture that were negative, polymerase chain reaction was done to confirm the result. The data analyzed using STATA 17 to determine the impact of catheter contamination on the clinical pregnancy rate. Results: The average age of peoples whose microbial culture was positive was lower than that of people whose microbial culture was negative (P<0.05). Also the results showed that people who live in villages have more positive microbial cultures than people who live in cities (P<0.05). Also there is no difference between the number of successful implantations and the pregnancy outcome between people whose microbial culture results were positive or negative. Conclusion: The results of the current study showed that the contamination of the embryo transfer catheter with microorganisms under our investigation did not affect the pregnancy outcome.

Effect of preconditioning of human umbilical cord mesenchymal stem cells with hydrogen peroxide on the therapeutic potential of MSCs in the cyclophosphamide -induced premature ovarian failure mice model.

OBJECTIVE: Exposure of stem cells to sublethal levels of hydrogen peroxide (H2O2) can prevent oxidative stress-induced apoptosis. In the present study, the effects of H2O2 preconditioning on the therapeutic potential of human umbilical vein cord mesenchymal stem cells (hUCV-MSCs) were evaluated in a murine model of premature ovarian failure. MATERIALS AND METHODS: Mature mice were divided into 4 groups, and 10 mice were incorporated into each group. The control (Ctrl) group received phosphate buffered saline (PBS) intraperitoneal (IP), and the CTX group was injected IP with cyclophosphamide (CTX). The CTX + MSC group after receiving CTX was injected with a single dose of hUCV-MSCs labeled with CM-DiI intravenously (IV), whereas the CTX + preMSCs group after CTX injection received preconditioned MSCs with H2O2 IV. Seven days later, the mice were euthanized, and their ovaries were removed for histological studies such as H&E staining and the TUNEL assay. Furthermore, the numbers of CM-DiI-labeled hUCV-MSCs in the different regions of the ovary were calculated. FSH and estradiol values in the serum were measured. RESULTS: Our studies showed that CTX caused degenerative changes and follicular loss in the ovary. The number of follicles in the CTX + MSCs and CTX + PreMSCs groups was significantly higher compared to the CTX group. In addition, in the CTX + PreMSCs group, the numbers of different types of follicles were higher than in the CTX-MSC group. Immunohistochemical studies in the CTX + MSCs and CTX + PreMSCs groups showed little evidence of TUNEL positivity compared with the CTX group. Moreover, the apoptotic index decreased in the CTX + PreMSCs group compared to the CTX + MSCs group. Moreover, CM-DiI-labeled MSCs in the ovary in the CTX + pre-MSCs group were higher than in the CTX + MSCs group. CONCLUSION: Our experiment offers preconditioning as an effective strategy in stem cell therapy to potentiate MSCs' therapeutic efficacy in ovarian function failure.

Therapeutic potentials of the caffeine in polycystic ovary syndrome in a rat model: Via modulation of proinflammatory cytokines and antioxidant activity.

Recent studies have shown that polycystic ovary syndrome (PCOS) affects about 6% of women worldwide. It is associated with reproductive and metabolic dysfunction. Caffeine is naturally found in tea, cocoa, and coffee. It has been shown that caffeine can change hormonal profiles, stimulate ovulation, and enhance fertility. Therefore, in this study, the effects of caffeine on rats with PCOS were investigated. For this purpose, 40 female rats were divided into five groups: (1) control group (without any intervention), (2) sham group (administration of olive oil as a caffeine solvent), (3) PCOS group (injection of 2 mg of estradiol valerate for each rat), (4) caffeine group (administration of 37.5 mg/kg caffeine for each rat), and (5) PCOS + caffeine group. After 21 days of treatment, the ovaries of rats were removed and prepared for further evaluations, including hematoxylin and eosin staining, TUNEL assay, real-time PCR, and biochemical analysis. Administration of caffeine in PCOS mice considerably reduced both the volume of the ovary (P < 0.05) and follicular clusters (P < 0.01). However, superoxide dismutase and glutathione peroxidase were dramatically active in the PCOS + caffeine group compared to others (P < 0.05). Besides, caffeine treatment in PCOS mice led to Bax reduction and increased Bcl-2 expression. On the other hand, the expression of IL-6 and TNF-α in PCOS + caffeine group was high compared to other groups. We found that caffeine can reduce apoptosis and inflammation in PCOS ovaries and enhance the unpleasant symptoms of PCOS.

Fresh or Frozen Embryo Transfer in The Antagonist In Vitro Fertilization Cycles: A Retrospective Cohort Study.

BACKGROUND: Gonadotropin-releasing hormone antagonist (GnRH-ant), widely adopted protocol, is more in line with the physiological processes, and induces a shorter and more cost-effective ovarian stimulation. In order to assess the success rate of embryo transferring (ET) in the antagonist in vitro fertilization (IVF) cycles, we compared the fresh ET with the frozen ET outcomes. MATERIALS AND METHODS: In this retrospective cohort study, one hundred five cases of ET of the infertility clinic of the Besat hospital (Kurdistan, Iran) between March 2014 to March 2020 that were treated with antagonist cycle (both fresh and frozen) were analyzed. The difference between the two groups in baseline data and reproductive outcomes were evaluated using Independent sample t test, Mann-Whitney U test, Chi-squared test, and Fisher's exact test in SPSS software (version 22). RESULTS: Out of 105 cases, 48 and 57 were in the fresh and frozen ET groups, respectively. The participants age was 35.75 ± 4.9 Y. In the fresh ET group, and 33.98 ± 5.1 Y in the frozen ET group. The percentage of chemical pregnancy was 12 (25%) in the fresh ET group and 15 (26.3%) in the frozen ET group (P=0.8); Clinical pregnancy rate was 11 (22.9%) in the fresh ET group and 11 (19.3%) in the frozen ET group (P=0.6); the rate of abortion in the fresh ET group was 3 (6.3%, P=0.2), and in the frozen ET group was 8 (14%, P=0.2); and the live birth rate was 9 (18.8%) in the fresh ET group, in comparison with 7 (12.3%) in the frozen ET group (P=0.3). CONCLUSION: Not statistically significant, the percentage of chemical pregnancy and abortion were higher in the frozen ET group. The percentage of clinical pregnancy and live birth were higher in the fresh ET group.

Silk fibroin and Nettle extract promote wound healing in a rat model: A histological and morphometrical study.

INTRODUCTION: Considering the anti-inflammatory, antimicrobial ability, and antioxidant effects besides stimulating ability of silk fibroin (SF) in cell migration and proliferation of Nettle, the current study aimed to investigate the effect of Nettle leaf extract (NLE) and SF on histology, morphometrical parameters and apoptosis on the wound in the rat model. MATERIALS AND METHODS: Wistar rats are divided into 5 groups, including 1-control (rats with healthy skin and no treatment); 2-wound (without any treatment); 3-SF (administration of silk fibroin solution for 14 consecutive days); 4- Nettle (administration of Nettle ointment for 14 consecutive days), and 5- Eucerin group (administration of Eucerin substance for 14 consecutive days) and then assessed wound area by photography, angiogenesis, inflammation, and thickness of epidermis using hematoxylin and eosin (H&E) staining, collagen deposition, and structure of dermis layers evaluated by Masson's trichrome staining and the apoptosis index determined by tunnel assay on days 7, 14 and 21. RESULTS: photographic illustrations showed that the wound surface environment on the seventh day in group 4 was significantly different from group 2 (p < 0.002). The rate of wound healing on the fourteenth day was higher in groups 3 and 4 than in group 2 (p < 0.001). Also, at this time, group 4 was significantly different from group 3 and group 5 (p = 0.003 and p = 0.000, respectively). There was a significant difference in epidermal thickness between the wound group and other experimental groups (p < 0.05). The number of apoptotic cells at the wound edges on the seventh day in both group 3 and group 4 had a significant decrease compared to other groups of wounds (p = 0.000), but there was a significant increase on the fourteenth day. Also, on the 21st day, a significant decrease in apoptotic cells was observed in both group 3 and group 4 compared to other wound groups (p = 0.000). DISCUSSION AND CONCLUSION: Nettle and SF maintain cell homeostasis and accelerate wound closure by reducing cell apoptosis and enhancing cell proliferation on the seventh day, but by increasing the apoptosis of fibroblast cells on the fourteenth day, they lead to remodeling and keratinocytes migration to epidermis formation. Increased apoptosis also seems to be one of the pathophysiological mechanisms to prevent the formation of keloid and hypertrophic scar tissue. SF and Nettle extract, by increasing cell proliferation and migration of different cell types to the site of injury, control the remodeling process by inducing and regulating apoptosis in the first two weeks of wound healing and accelerating the process of collagen deposition and epithelialization.

Caffeine modulates apoptosis, oxidative stress, and inflammation damage induced by tramadol in cerebellum of male rats.

Tramadol, an opioid used as analgesic, can induce neurotoxic effects associated to cognitive dysfunction. Moreover, caffeine has been reported to have neuroprotective effects. In this regard, we hypothesized that administration of caffeine can modulate tramadol-induced damages in cerebellum. For this study, forty male Wistar rats were divided into four groups: the control group, the tramadol group (50 mg/kg), the caffeine group (37.5 mg/kg), and the tramadol+caffeine group (50 mg/kg tramadol+37.5 mg/kg caffeine). At the end of study (day 21), after performing rotarod behavioral test, cerebellum tissue samples were removed and prepared for further evaluations including biochemical profile markers (MDA, GPx, and SOD), immunohistochemistry for Caspase-3, as well as the expression of genes involved in cellular processes such as inflammation markers (IL-1ß, HMGB1, IL-6, and TNF), apoptosis markers (Caspase-3, Caspase-8, Bax, and P21), and autophagy markers (LAMP2, ATG5, BECN1, and ATG12). Stereological evaluations were performed to determine the total volume of granular and molecular layers and white matter of cerebellum tissue and numerical density of the Purkinje cells. Our results showed that the stereological parameters, biochemical profiles (except MDA) and behavioral function were significantly higher in the tramadol+caffeine group compared to the tramadol group. Autophagy-related genes were significantly upregulated in tramadol+caffeine group compared to the tramadol group. While the expression of inflammatory and apoptosis genes, MDA level, as well as density of apoptosis cells were significantly lower in the tramadol+caffeine group compared to the tramadol group. Briefly, it can be concluded that administration of caffeine has neuroprotective effects in cerebellar damages induced by tramadol.

In vitro effects of pentoxifylline and coenzyme Q10 on the sperm of oligoasthenoteratozoospermia patients.

The effect of in-vitro sperm incubation with Pentoxifylline (PTX) and Coenzyme Q10 (CoQ10) in Oligoasthenoteratozoospermia (OAT) patients was evaluated. Semen samples were obtained from men with Normozoospermia and men with OAT. Motile sperm from the two groups were subdivided into four subgroups: (i) without incubation with PTX + CoQ10; (ii) incubation with PTX; (iii) Incubation with CoQ10; and (iv) incubation with a combination of PTX + CoQ10. Then, sperm parameters, chromatin, DNA and membrane integrity, protamine deficiency, apoptosis, mitochondrial activity, sperm chromatin dispersion test (SCD), hypo-osmotic swelling test (HOS), chromomycin A3 (CMA3), Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), and diaminobenzidine (DAB) assays were evaluated, respectively. Sperm incubated with CoQ10 and a combination of CoQ10 and PTX resulted in a significant increase in the sperm parameters. Also, a significant decrease was noted with a combination of PTX and CoQ10 in normal men. There was a significant difference between CoQ10 treated and CoQ10 + PTX treated groups in comparison with the OAT group in the percentage of the DNA fragmentation, sperm apoptosis, AB+, HOS test + and sperm mitochondrial activity. Incubated sperm with CoQ10, PTX, and in combination with each other can improve sperm parameters in OAT patients.

The Effect of L-Carnitine and Coenzyme Q10 on the Sperm Motility, DNA Fragmentation, Chromatin Structure and Oxygen Free Radicals During, before and after Freezing in Oligospermia Men.

PURPOSE: The aim of the present study is to assess the effect of L-carnitine and Coenzyme Q10 (CoQ10) on human sperm motility, DNA fragmentation, chromatin structure, and reactive oxygen species (ROS) during, before and after freezing in oligospermia men. MATERIALS AND METHODS: Semen was collected from 30 oligospermic men, who referred to infertility clinic of Beasat Hospital in Sanandaj, Iran. The samples of each individual were divided into 8 equal parts: 1. control group before freezing; 2. incubated with L-carnitine; 3. incubated with coenzyme Q10; 4. incubated with the combination of L-carnitine + CoQ10; 5. control freezing group; 6. the experimental freezing group with L-carnitine; 7. the experimental freezing group with coenzyme Q10 and 8. the experimental freezing with the combination of L-c + CoQ10. Sperm motility was assessed by WET MOUNT method. DNA fragmentation was evaluated by SCD (Sperm Chromatin Desperation), ROS, was evaluated by quantitative fluorescence reaction, and chromatin deficiency was determined by chromatin staining (CMA3). RESULTS: Antioxidant treatments, significantly reduced the number of ROS + in the pre and post freezing groups. Significant improvement was seen in the sperm motility of class B in the pre freezing groups with L-carnitine. Antioxidants also reduced the percentage of DNA fragmentation and protamine deficiency in pre-and post-freezing. CONCLUSION: Addition of Coq10 and L-carnitine to human sperm medium significantly reduced the number of ROS. This reduction in ROS reduced sperm damage during cryopreservation.

Human umbilical cord mesenchymal stem cells improve morphometric and histopathologic changes of cyclophosphamide-injured ovarian follicles in mouse model of premature ovarian failure.

BACKGROUND: Mesenchymal stem cells have restorative effects on premature ovarian failure (POF). The aim of this study was to investigate the effects of human umbilical cord vein MSCs (hUCV-MSCs) on follicular quantitative parameters and histological changes of ovaries in the cyclophosphamide (CTX)-induced POF in mice. MATERIALS AND METHODS: C57BL/6 mice were divided into three groups (10 mice in each group). In the control group, phosphate-buffered saline (PBS) was injected via tail vein following 15 days injection of PBS intraperitoneally (IP). In the CTX group, CTX was administered IP for 15 days and then PBS was injected via tail vein. In the CTX + hUCV-MSCs group, following CTX administration, single dose of the 1 × 106 of hUCV-MSCs were injected into tail vein. H&E, trichrome and PAS staining as well as TUNEL assay were performed on the ovaries tissue sections. The number of follicles, follicular quantitative parameters and apoptotic index were obtained. The serum levels of estradiol and FSH were measured in the mice. RESULTS: In the CTX + hUCV-MSCs group, degenerative changes were decreased and follicular quantitative parameters increased in the ovarian follicles compared to the CTX group. In this group number of follicles was increased, apoptotic index was decreased, estradiol and FSH levels were decreased and increased, respectively, all of them improved compared to the CTX group. The mean percentage areas of collagen fibers content were decreased compared to the CTX group. CONCLUSION: Results showed that, hUCV-MSCs administration increases follicular quantitative parameters and improve degenerative changes in the follicles following CTX injury.

Retinoic acid and fibroblast growth factor-2 play a key role on modulation of sex hormones and apoptosis in a mouse model of polycystic ovary syndrome induced by estradiol valerate.

OBJECTIVE: The main goal of the present study is to investigate the effects of retinoic acid and fibroblast growth factor-2 on serum levels of FSH and LH, histology, and apoptosis in the mouse model of Poly Cystic Ovary Syndrome (PCOS). MATERIALS AND METHODS: 80 female NMRI mice have been randomly divided into eight groups. Group 1 received normal saline as a control, and Group 2 received estradiol valerate (EV) at 4 mg/100 g of body weight. Moreover, Groups 3-4 were administered with RA (a dose of 0.05 µg/µl) and FGF2 (a dose of 0.01 µg/kg), respectively. Groups 5 and 6 respectively received the EV plus the RA (0.05 µg/µl) and FGF2 (0.01 µg/kg). Group 7 received the RA and FGF2 at doses corresponding to healthy mice, and Group 8 received the EV plus the RA + FGF2 (similar to previous doses). RA and FGF2 were injected three times per week for four weeks. Finally, histological and immunohistological parameters of the ovary were evaluated. RESULTS: The study revealed that both single and combined injection of fibroblast growth factor-2 (FGF2) and retinoic acid (RA) in groups 5, 6, and 8 significantly reduced follicular diameters compared to group 2. Measurements confirmed that simultaneous injection of RA and FGF2 into polycystic mice significantly increased antral follicles, corpus luteum (CL), epithelial thickness, and oocyte diameter as well as decreased cystic follicles. Positive TUNEL cells that were considerably increased in the antral follicle of group 2 significantly decreased in the RA and FGF2 recipient groups, either alone or in combination. Besides, the injection of FGF2 increased preantral follicles and CL. CONCLUSION: The findings of the present investigation reveal that injection of RA and FGF2 has both protective and ameliorative effects that can promise new therapies for women with PCOS.

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin.

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

The expressions of NLRP1, NLRP3, and AIM2 inflammasome complexes in the contusive spinal cord injury rat model and their responses to hormonal therapy.

Spinal cord injury (SCI) is a devastating condition with a growing incidence in developing countries. The activity of inflammasome complexes initiates neuroinflammation, which is a key player in SCI pathogenesis. Here, NLRP1, NLRP3, and absent in melanoma 2 (AIM2) inflammasome complexes were assessed in the contusive (T6) SCI rats for their expression profiles and their response to hormonal therapy (10 mg/kg melatonin or 25 µg/kg 17ß-estradiol [E2] every 12 h until 72 h). Two phases was considered in this study: the dominant time of inflammasome activation, which was 72 h post-SCI and the response from each complex to hormonal therapy at this time. Gene and protein expressions of NLRP1, NLRP3, AIM2, ASC, and caspase-1 were evaluated by real-time PCR (for gene analysis), western blot, and immunohistochemistry (IHC), and biochemical presence of IL-18 and IL-1ß in spinal cord tissue homogenates was analyzed by enzyme-linked immunosorbent assay (ELISA). The whole inflammasome complexes showed high expressions in the SCI group, while after hormonal therapy, these alterations were counteracted, which were more conspicuous for the NLRP1 and NLRP3. Melatonin had no predilection over E2 for such effect. Finally, the expression profile of signaling related to the synthesis (TLR4/NF-κB) and activation (NADPH oxidase 2 [NOX2]/TXNIP) of inflammasome complexes was surveyed, and there were low activities for the two pathways in SCI rats that underwent hormone therapy. From the findings, it is concluded that both melatonin and E2 are efficient to target inflammasome activation in the SCI rats.

Hydrogen Peroxide Preconditioning Promotes Protective Effects of Umbilical Cord Vein Mesenchymal Stem Cells in Experimental Pulmonary Fibrosis.

Purpose: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder with few available treatments. Mesenchymal stem cell therapy (MSCT), an innovative approach, has high therapeutic potential when used to treat IPF. According to recent data, preconditioning of MSCs can improve their therapeutic effects. Our research focuses on investigating the anti-inflammatory and antifibrotic effects of H2 O2 -preconditioned MSCs (p-MSCs) on mice with bleomycin-induced pulmonary fibrosis (PF). Methods: Eight-week-old male C57BL/6 mice were induced with PF by intratracheal (IT) instillation of bleomycin (4 U/kg). Human umbilical cord vein-derived MSCs (hUCV-MSCs) were isolated and exposed to a sub-lethal concentration (15 µM for 24 h) of H2 O2 in vitro. One week following the injection of bleomycin, 2×105 MSCs or p-MSCs were injected (IT) into the experimental PF. The survival rate and weight of mice were recorded, and 14 days after MSCs injection, all mice were sacrificed. Lung tissue was removed from these mice to examine the myeloperoxidase (MPO) activity, histopathological changes (hematoxylin-eosin and Masson's trichrome) and expression of transforming growth factor beta 1 (TGF-ß1) and alpha-smooth muscle actin (α-SMA) through immunohistochemistry (IHC) staining. Results: Compared to the PF+MSC group, p-MSCs transplantation results in significantly decreased connective tissue (P<0.05) and collagen deposition. Additionally, it is determined that lung tissue in the PF+pMSC group has increased alveolar space (P<0.05) and diminished expression of TGF-ß1 and α-SMA. Conclusion: The results demonstrate that MSCT using p-MSCs decreases inflammatory and fibrotic factors in bleomycin-induced PF, while also able to increase the therapeutic potency of MSCT in IPF.

Biological Effects of Magnetic Resonance Imaging on Testis Histology and Seminiferous Tubules Morphometry.

OBJECTIVES: Spermatogenesis is a regular and lengthy process in which the function of testicular cells may potentially be influenced by several extrinsic and intrinsic stressors, including environmental factors such as magnetic resonance imaging (MRI) waves and radiation. Our study aimed to investigate the effects of MRI waves and fields on the testicular histology and morphometry of seminiferous tubules in mice. METHODS: The experiment was conducted on 40 adult Naval Medical Research Institute mice. The control group was located in the center of the MRI bore while it was turned off, while the exposed group was exposed to the active scanner for 36 minutes once a week for three weeks. Our study included four groups: group I (control group at one hour after last exposure), group II (experimental group at one hour after last exposure), group III (control group at 35 days after last virtual exposure), and group IV (experimental group at 35 days after last exposure). We then assessed the tube and lumen diameters, as well as epithelium thickness of the seminiferous tubules. RESULTS: Our data showed that MRI waves partially reduced testicular weight one hour after the last exposure (group II) compared to group I (p = 0.240). On the other hand, in group II the Johnson's score (score 10, complete spermatogenesis and perfect tubules) was 87.5% which was slightly less than recorded in groups I, III, and IV (91.4%, 92.2%, and 90.5%, respectively). Furthermore, the MRI in group II revealed induces vacuolization in the epithelium, arrest in primary spermatocytes in the pachytene stage as well as disruption in the testicular parenchyma. CONCLUSIONS: Long-term exposure to MRI waves has deleterious effects on the male reproductive system, fertility parameters, and the quantity of germ cells in the seminiferous tubules with the exception of the number of round spermatid cells and epithelial thickness. All these effects were reversible after a new period of spermatogenesis.

Proliferation and differentiation of mouse spermatogonial stem cells on a three-dimensional surface composed of PCL/gel nanofibers / Proliferación y diferenciación de células madre espermatogónicas de ratón en una superficie tridimensional compuesta de nanofibras PCL / gel

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.

Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.

Distribution of the CM-Dil-Labeled Human Umbilical Cord Vein Mesenchymal Stem Cells Migrated to the Cyclophosphamide-Injured Ovaries in C57BL/6 Mice

Background: Mesenchymal stem cells (MSCs) can be used to treat premature ovarian failure (POF). Different methods have already been applied to detect MSCs in tissues. This study aimed to investigate the quantitative distribution of CM-DiI-labeled human umbilical cord vein MSCs (hUCV-MSCs) in different regions of the ovarian tissue of the cyclophosphamide (CTX)-induced POF in mice. Methods: Adult female C57BL/6 mice (n = 40) were divided into four groups: (1) Mice receiving PBS as control (Ctrl) group; (2) mice receiving hUCV-MSCs intravenously as Ctrl + hUCV-MSCs group; (3) mice receiving CTX intraperitoneally (i.p.) as CTX group; (4) mice receiving CM-DiI-labeled hUCV-MSCs after CTX injection as CTX + hUCV-MSCs group. Histological changes and CM-DiI-labeled hUCV-MSCs distribution were analyzed in the ovarian tissues. Quantitative real-time PCR was performed to detect human mitochondrial cytochrome b (MTCYB) gene in the ovarian tissues of the mice. Results: The mean number of the fluorescent hUCV-MSCs was 20 ± 2.5 (57.1%) in the medulla, 11.3 ± 2.8 (32.2%) in the cortex, and 5.5 ± 1 (15%) in the germinal epithelium of the ovarian tissue (p < 0.05). Moreover, MTCYB gene was detected in the mice ovaries of the CTX + hUCV-MSCs group, but not in other groups. Conclusion: Our findings suggest that the distribution of the transplanted hUCV-MSCs in different regions of the ovarian tissue is not equal, and it is greater in the medulla than the cortex and germinal epithelium. This is the first report of quantitative distribution of MSCs in different regions of ovarian tissue in the POF model.

Colonization of Mouse Spermatogonial Cells in Modified Soft Agar Culture System Utilizing Nanofibrous Scaffold: A New Approach.

BACKGROUND: Spermatogonial stem cells (SSCs) are considered in fertility management approaches of prepubertal boys facing cancer therapies. However, in vitro propagation has become an important issue due to a small number of SSCs in testicular tissue. The present study aimed to investigate a modified soft agar culture system by using a nanofibrous scaffold as a new approach to mimic in vivo conditions of SSCs development. MATERIALS AND METHODS: The SSCs were isolated from neonate mouse testes, cultured on polycaprolactone scaffold, and covered by a layer of soft agar for 2 weeks. Then, the number and diameter of colonies formed in experimental groups were measured and spermatogonial markers (i.e., Plzf, Gfrα1, Id4, and c-Kit) in SSCs colonies were evaluated by a real-time polymerase chain reaction and immunostaining. RESULTS: Our results indicated that the colonization rate of SSCs was significantly higher in the present modified soft agar culture system (P<0.05). Only Plzf indicated a significant increased at the levels (P<0.05), the gene expression levels of Id4, Plzf, and Gfrα1 were higher in the present culture system. In addition, the expression of the c-Kit gene as a differentiating spermatogonia marker was higher in presence of scaffold and soft agar compared with the amount of other experimental groups (P<0.05). CONCLUSION: The culture system by using nanofibrous scaffold and soft agar as a new culture method suggests the potential of this approach in SSCs enrichment and differentiation strategies for male infertility treatments, as well as in vitro spermatogenesis.

Combined Effect of Retinoic Acid and Basic Fibroblast Growth Factor on Maturation of Mouse Oocyte and Subsequent Fertilization and Development.

BACKGROUND: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. MATERIALS AND METHODS: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. RESULTS: As compared with the control group, the rate of maturation was significantly increased in the RA (P<0.001) and bFGF+RA (P<0.02) groups with 58 ± 10 and 57 ± 3.46, respectively. The rate of maturation was significant in the RA (P<0.02) and bFGF+RA (P<0.03) groups, in comparison with the bFGF group. The bFGF+RA group had higher rate (83 ± 1.52) of two-cells development, than control (33 ± 1, P<0.001). CONCLUSION: Our findings showed beneficial effects of 2 µM RA and 20 ng/ml bFGF combination on mouse oocyte IVM.

Attenuating Effect of Long-term Culture of Umbilical Cord Vein Mesenchymal Stromal Cells on Pulmonary Fibrosis in C57BL/6 Mice.

In recent studies, mesenchymal stromal cells (MSCs) have been increasingly employed to treat various diseases like pulmonary fibrosis (PF). There are very few MSCs in tissues so in order to obtain their sufficient numbers for therapeutic applications, their in vitro expansion is necessary. The aim of this study was to investigate the effects of long-term culture of the human umbilical cord vein MSCs (hUCV-MSCs) on pulmonary fibrosis in mice. MSCs were first isolated from human umbilical cord vein and cultured up to 18 passages. In C57BL/6 mice, 15 min after belomycin instillation, UCV-MSCs at passages (P) 0, 4, 8, 12, and 18 (long-term culture) were transplanted intratracheally. Mice were weighted every 5 days and were euthanized on day 21. For histopathological examination, the lung sections were stained with hematoxylin-eosin (HE) and Masson's trichrome. The mRNA expression of TGF-ß1, alpha-smooth muscle actin (α-SMA), and collagen type I alpha 1 (COL1A1) in lung tissues were assessed using RT-PCR. For cell tracking, human cytochrome B DNA was detected in mice lung tissues by PCR. The weight of mice receiving long-term culture of UCV-MSCs increased compared to other mice (p=0.056). Also, transplantation of UCV-MSCs at P18 led to increased alveolar space and decreased connective tissue and collagen deposition of the lung tissues. The mRNA expression of TGF-ß1, α-SMA, and COL1A1 also decreased in this group. The results showed that intratracheally transplanted long-term culture of the UCV-MSCs attenuated lung fibrosis in mice.