The effects of melatonin and metformin on histological characteristics of the ovary and uterus in letrozole-induced polycystic ovarian syndrome mice: A stereological study

Background: Polycystic ovarian syndrome (PCOS) with anovulation, hyperandrogenism, ovarian and uterine histological changes, menstrual irregularities, etc. signs is an infertility type. It seems that melatonin and metformin can improve these abnormalities. Objective: To evaluate the effects of melatonin and metformin on the ovary and uterus in PCOS-induced mice using stereological methods. Materials and Methods: Seventy-two adult female BALB/c mice (8-wk-old, 20-30 gr) were randomly divided into control (distilled water, gavage), PCOS (90 μg/kg letrozole, gavage), PCOS+metformin (500 mg/kg, gavage), PCOS+melatonin (10 mg/kg, intraperitoneal injection), and PCOS+melatonin control (0.5% ethanol saline) groups (n = 12/each). Another PCOS group was kept for a month to ensure that PCOS features remained. Finally, a stereological evaluation of the uterus and ovary was carried out, and vaginal cytology and serum testosterone levels were assessed. Results: PCOS mice treated with metformin and melatonin had lower testosterone levels, body weight, and more regular estrus cycles than the PCOS group (p ≤ 0.001). A significant decrease in conglomerate and daughter gland numbers, and primary, secondary, atretic, and cystic follicles numbers with a significant increase in primordial and Graafian follicles, and corpus luteum numbers (p ≤ 0.001) was seen in these treated mice. Also, endometrial vessels’ volume and length significantly increased, but ovarian, endometrial, myometrial, stromal, and glands volume, and endometrial and myometrial thickness dramatically declined (p ≤ 0.001). Conclusion: It appears that metformin and melatonin could restore the PCOS phenotype including estrus cycle irregularity, high testosterone level, and ovarian and uterine micromorphology to the control levels. However, the 2 treatments had similar effects on the examined parameters. Key words: Polycystic ovarian syndrome, Melatonin, Metformin, Ovary, Uterus, Mice, Stereology

Protective effect of loboob (a Persian traditional remedy) on sexual hormones, antioxidant activities and stereological changes of testis tissue on busulfan induced oligospermia in rats

Loboob as a traditional drug in Iranis known for its beneficial effects on busulfan-induced oligospermia. In this experimental study, protective effects of loboob (a Persian traditional remedy) on sexual hormones, antioxidant levels and stereological changes of testis tissue were evaluated in an oligospermia rat model induced by busulfan. Fifty male rats were randomly divided into five different groups: control, received no treatments; and the other groups administrated with a single dose of busulfan (10 mg/kg body weight). After 30 days, these groups were treated with 0, 35, 70 or 140 mg/kg/day of loboob for 60 days. Blood samples were collected for hormone and antioxidant enzyme assays. Unbiased stereology was performed on testis tissues to evaluate the volume of different parts of the testis and the number of various testis cells. Data indicated that FSH, LH and MDA were increased, and testosterone, catalase, SOD were decreased in the busulfan group, while treatment with loboob at 70 and 140 mg/kg significantly improved these parameters (P <0.05). Treatment with 70 and 140 mg/kg of loboob ameliorated the germinal epithelium volume, types A and B spermatogonia, spermatocytes, elongated and round spermatids, and Sertoli cells in the seminiferous tubules (P <0.05). High concentration of loboob also improved testis weight and volume, and leydig cell number (P <0.05). Thus, loboob is more effective for the recovery of seminiferous tubules and their cells than for the interstitial tissue. Loboob with various antioxidants, minerals and vitamins could overcome the side effects of busulfan

The Effects of Metal Nanoparticles on the Mammalian Reproductive System

Due to the increasing use of nanoparticles in medical and industrial fields, concerns are growing about the toxicity of them to the body organs especially the reproductive system. In this review, the effect of metal/metal oxide nanoparticles on the mammalian reproductive system was discussed. Nanoparticles are typically toxic to both males and females, depending on their types, administration method, exposure duration, and surface modification. Regarding the embryo, it was also found that the effect of nanoparticles depends on the embryonic stage exposure during development. However, some nanoparticles, depending on the dose and time of administration, not only did not have toxic effects, but also strengthened the reproductive system and increased its efficiency. As the mode of interaction, penetration, and mechanism of nanoparticles action in the reproductive system is unclear, this review highlights the importance of additional tests in these cases

Inhibition of chondrogenic differentiation in chick limb-bud mesenchyme microcultures treated with cyclosporine

Objectives: To explore the effects of cyclosporine (CsA) on skeletal development (chondrogenesis). Materials and Methods: Mesenchymal cells obtained from stage-23 to stage-24 chick-embryo limb buds were grown in 96-well plates using chemically defined tissue-culture medium. Cultures were treated with CsA (0.01-5.0 μg/ml) and incubated (37°C, 5% CO2) with daily medium changes for 4 days. After incubation of the cells in multiwell plate, cartilage differentiation (chondrogenesis) was assessed by selectively staining sulfated glycosaminoglycans (GAGs) in the cartilage matrix with Alcian blue, extracting the GAGs with 4 M guanidinium HCl, and spectrophotometric analysis of the extracts. Results: CsA treatment had concentration-dependent effects on chick limb-bud mesenchymal cell cultures. At 5 μg/ml, CsA caused cell loss, as judged microscopically by the paucity of cells remaining at the end of the culture period. CsA concentrations between 0.1 and 1 μg/ml caused a marked, dose-dependent decrease in chondrogenesis. At 0.01 μg/ml, CsA had no significant effect on chondrogenesis. At concentrations above 0.01 μg/ml, normalized data showed significant chondrogenic inhibition at 0.5 and 1.0 μg/ml CsA. Conclusions: The findings suggest a possible biological basis for CsA-associated effects on mesenchyme-derived tissues and provide a model system for further studies

Effects of cryopreservation on plasma membrane glycoconjugates of human spermatozoa

Background: Cryopreservation has some detrimental impacts on sperms surface molecules. Modification of the sperm surface molecules can affect on fertility rate. One of the important surface molecules are glycoconjugates. Objective: The objective of this study was to evaluate the changes of content of the glycocalyx after standard cryopreservation procedure. Materials and Methods: Forty five healthy semen samples were frozen in 0.5ml plastic straws and kept in liquid nitrogen and thaw after 48 hours. Sperm smears were prepared before and after freezing and thawing. The smears were stained with the lectins and also with acridin orange. The smears were studied by fluorescents microscopy and the intensities of the reactions to lectins were measured by image analyses software. Results: The reactions of the sperm samples to Peanut agglutinin (PNA), Wheat germ agglutinin (WGA) and Dolichos biflorus (DBA) changed after cryopreservation and the percentage of samples that showed modifications were 46.67%, 34.09% and 73.34%, respectively. The crypreservation led to both increase and decrease the intensities of the reactions. It means that there are various mechanisms that impact on the carbohydrate contents of the sperm surface. There is no correlation between DNA denaturation of sperms and their lectin binding patterns. Conclusion: Cryopreservation affected the surface glycoconjugates at least in a subset of spermatozoa. These results might cause to modify the future application of sperm banking techniques

Effects of Platelet-Rich Plasma & Platelet-Rich Fibrin with and without Stromal Cell-Derived Factor-1 on Repairing Full-Thickness Cartilage Defects in Knees of Rabbits

Background: The purpose of this study was to create biomaterial scaffolds like platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) containing stromal cell-derived factor-1 (SDF1) as a chemokine to induce hyaline cartilage regeneration of rabbit knee in a full thickness defect. Methods: We created a full thickness defect in the trochlear groove of thirty-six bilateral knees of eighteen mature male rabbits. The knees were randomly divided into six groups (group I: untreated control, group II: PRP, group III: PRF, group IV: Gelatin+SDF1, group V: PRP+SDF1, and group VI: PRF+SDF1). After four weeks, the tissue specimens were evaluated by macroscopic examination and histological grading, immunofluorescent staining for collagen type II, and analyzed for cartilage marker genes by real-time PCR. The data were compared using statistical methods (SPSS 20, Kruskal-Wallis test, Bonferroni post hoc test and P<0.05). Results: Macroscopic evaluations revealed that international cartilage repair society (ICRS) scores of the PRF+SDF1 group were higher than other groups. Microscopic analysis showed that the ICRS score of the PRP group was significantly lower than other groups. Immunofluorescent staining for collagen II demonstrated a remarkable distribution of type II collagen in the Gel+SDF1, PRP+SDF1 and PRF+SDF1 groups compared with other groups. Real-time PCR analysis revealed that mRNA expression of SOX9 and aggrecan were significantly greater in the PRF+SDF1, PRP+SDF1, Gel+SDF1 and PRF groups than the control group (P<0.05). Conclusion: Our results indicate that implantation of PRF scaffold containing SDF1 led to the greatest evaluation scores of full-thickness lesions in rabbits

Comparison of Cell Viability and Embryoid Body Size of Two Embryonic Stem Cell Lines After Different Exposure Times to Bone Morphogenetic Protein 4

Background: Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB), size, and cavitation of ES cells. Methods: Embryonic stem cells (R1 and B1 lines) were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4) or after 48 h (+BMP4) of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P<0.05 was considered as significant. Results: In EB culture protocol, cell number significantly decreased in +BMP4 culture condition with greater cavity size compared to the ++BMP4 condition at day 5 (P=0.009). In contrast, in monolayer culture system, there was no significant difference in the cell number between all groups (P=0.91). Conclusion: The results suggest that short-term exposure of BMP4 is required to promote cavitation in EBs according to lower cell number in +BMP4 condition. Different cell lines showed different behavior in cavitation formation

The effects of activated-omental extract on nuclear and cytoplasmic in vitro maturation of rat oocytes

Objective: The role of growth factors, including vascular endothelial growth factor of activated omentum on mitosis is clearly known, though not on all the aspects of in vitro oocyte maturation. This study was designed to assess the effect of activated-omental extract (AOE) on in vitro maturation (IVM) of rat cumulus-oocyte complexes (COCs). Materials and Methods: In this experimental study, the COCs were incubated in Ham’s F-10 supplemented with either 20% AOE, 20% fetal bovine serum (FBS) or serum-free media. Post-culture COCs were studied according to the cumulus cells (CCs) expansion, nuclear maturation and cytoplasmic maturation. Cumuli expansion was evaluated by inverted microscope without staining; nuclear maturation was assessed by aceto-orcein staining (light microscope) and cytoplasmic maturation was also observed by TEM. Results: Expansion of CCs and nuclear maturation of the oocytes in in vitro for 24 hr was significantly higher in AOE- and FBS-supplemented groups (P=0.000 and 0.013) and (P=0.004 and 0.014), respectively, compared to serum-free group. At ultra-structural level, after 24 hr, both FBS and AOE-supplemented media showed uniformly wide perivitelline space (PVS). After 12 hr, the cortical granules were found in the oocytes cultured in FBS and AOE-supplemented media. Within 24 hr, both granules and mitochondria were large without any detectable topographic tendency across the ooplasm. In AOE and FBS- supplemented oocytes, the number and size of microvilli were more than those in serum-free one. Conclusion: Although AOE supplementation induced a higher rate of the CCs expansion, and resuming meiosis, it was not as potent as FBS to provide cytoplasmic maturation of rat oocytes

Effect of Follicular Fluid and Platelet-Activating Factor on Lactate Dehydrogenase C Expression in Human Asthenozoospermic Samples

Background: Application of follicular fluid (FF) and platelet-activating factor (PAF) in artificial insemination improves sperm motility. Lactate dehydrogenase C (LDH-C) is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples. Methods: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction (q-RT PCR) and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples. Results: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms. Conclusion: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples (P=0.0001), although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients

Effects of sera taken from women with recurrent spontaneous abortion on sperm motility and apoptosis

Background: Recurrent spontaneous abortion impacts almost 1% of couples. The sera from women with unexplained recurrent spontaneous abortion (URSA) have toxic effects on embryos that grow in the uterus. Therefore, the abnormal condition of the uterus may also affect sperm qualities. Objective: The objectives of this study were to search if these sera could induce DNA denaturation in sperm nuclei and also it could reduce sperm motility. Materials and Methods: Sera of 20 women with URSA history and sera from 20 women with at least two healthy children were added to the sperms samples from 20 healthy men for 2 hours. The sperm motility was assessed after incubation with sera. The samples were stained with Tdt mediated dUTP nick end labeling (TUNEL) assay for DNA fragmentation. The samples were analyzed with flow cytometry and the percentage of the TUNEL positive sperms were calculated. The data were analyzed by t-test. Results: The incubation of the sperm samples in sera with URSA lead to a decrease in the percentage of the motile sperm from 55% in control to 41% in the treated group, significantly (p=0.038). The percentage of the sperm with abnormal fragmented DNA increased after incubation with URSA (26.6%) compare to the control (21.2%); however, it was not significant. Conclusion: It seems that sera from URSA patients could not induce a significant increase in the percentage of the sperms with nuclei contain DNA fragmentation. However, the sera of women with URSA could affect the fertility rate by reduction of the sperm motilit