Extracellular microRNAs: key players to explore the outcomes of in vitro fertilization.

BACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that modulate post-transcriptional gene regulation. They are often used as promising non-invasive biomarkers for the early diagnosis of cancer. However, their roles in assisted reproduction are still unknown. METHODS: This prospective study was designed to evaluate the expression profiles of seven extracellular miRNAs (miR-7-5p, miR-202-5p, miR-378-3p, miR-224, miR-320a, miR-212-3p, and miR-21-5p) in human follicular fluid (FF) to explore the outcomes of in vitro fertilization (IVF). Of 255 women, 145 were without polycystic ovary syndrome (PCOS), and their ovarian assets were normal (NOR), while 110 were with normo-androgenic PCOS. RESULTS: The combination of six FF miRNAs expression profile discriminated between PCOS and NOR women with a sensitivity of 79.2% and a specificity of 87.32% (AUC = 0.881 [0.61; 0.92], p = 0.001). MiR-202-5p significantly had a lower abundance level, and miR-378-3p had a high abundance level in pooled FF samples from patients treated with human menopausal gonadotropin (hMG) than those treated with recombinant follicle-stimulating hormone (rFSH) (p < 0.001). Our results showed that miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 in NOR patients with a sensitivity of 80% and specificity of 71%, (AUC = [0.753 (0.651; 0.855)], p = 0.001). For clinical pregnancy outcome prediction, FF miRNA-21 exhibited high sensitivity (74.8%) and specificity (83.7%) with the AUC value of 0.774 (0.682; 0.865). CONCLUSION: Conclusively, our results provide evidence that miR-7-5p, miR-378-3p, miR-224, miR-212-3p were a differentially high expression in normo-androgenic PCOS patients than NOR patients. While miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 (p = 0.001). The expression level of FF miR-212-3p was significantly related to the probability of embryos to develop into a high-quality blastocyst in patients with normal ovarian reserve.

Melatonin levels and microRNA (miRNA) relative expression profile in the follicular ambient microenvironment in patients undergoing in vitro fertilization process.

PURPOSE: Intrafollicular fluid (IFF) melatonin plays a decisive role in maintaining granulosa cells' DNA integrity and protects them against apoptosis. It reduces oxidative stress and improves the oocyte quality with a higher fertilization rate. METHOD: This prospective study investigated the antioxidant property of IFF melatonin and its impact on IVF outcome parameters. We also explored the relative expression of five microRNAs (miR-663b, miR-320a, miR-766-3p, miR-132-3p, miR-16-5p) and levels of cell-free DNA (cfDNA) by real-time PCR in unexplained infertile patients. We collected 425 follicular fluid (FF) samples containing mature oocytes from 295 patients undergoing IVF. RESULTS: Patients were subgrouped based on IFF melatonin concentration (group A ≤ 30 pg/mL, group B > 70 to ≤ 110 pg/mL, group C > 111 to ≤ 385 pg/mL). Our results showed that patients with ≤ 30 pg/mL IFF melatonin levels have significantly higher oxidative stress markers, cfDNA levels, and lower relative expression of miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p compared to other subgroups (p < 0.001). Similarly, they have a low fertilization rate and a reduced number of high-quality day 3 embryos. CONCLUSION: Findings suggest that the therapeutic use of melatonin produces a considerable rise in the number of mature oocytes retrieved, fertilization rate, and good-quality embryo selection. Furthermore, miRNA signature enhances the quality of embryo selection, thus, may allow us to classify them as non-invasive biomarkers to identify good-quality embryos.

The regulation of trophoblast invasion and decidual reaction by matrix metalloproteinase-2, metalloproteinase-7, and metalloproteinase-9 expressions in the rat endometrium.

PURPOSE: We aimed to evaluate how matrix metalloproteinases (MMPs) regulate the trophoblast invasion and placentation. METHODS: Female rats were divided into the estrous cycle and early pregnancy day groups. Obtained uterine tissues and implantation sites were processed for immunofluorescence and real-time PCR examinations. RESULTS: The mRNA expression of MMP-7 was higher than MMP-2 and MMP-9. Immunofluorescence findings confirmed that MMP-2, MMP-7, and MMP-9 were localized in the endometrial stroma, while MMP-7 was high in glandular and lining epithelial cells throughout the entire estrous cycle. However, their immunolocalizations and mRNA expressions were dramatically changed with the early pregnancy days. The MMP-7 reached very strong immunostaining in the giant trophoblast cells (GTCs), and the cytoplasm of mature and differentiating decidual cells, whereas MMP-2 and MMP-9 were mostly seen in the primary decidual zone (PDZ), GTCs, and the endothelium of blood vessels. CONCLUSIONS: All three MMPs seemed likely to be a key mediator of trophoblast invasion into the decidual region as well as angiogenesis during the placentation process. Due to the strong and wide expression of MMP-7 in the mature decidua, it could be suggested that MMP-7 is important for decidual ECM remodeling and it might be used as a new marker of decidual reaction.

Wingless ligands and beta-catenin expression in the rat endometrium: The role of Wnt3 and Wnt7a/beta-catenin pathway at the embryo-uterine interface.

Wnt/beta-catenin signaling may play an essential role in endometrial decidualization, placentation, and the establishment of pregnancy. We investigate here the possible roles, immunolocalizations, and synthesis of the Wnt3, Wnt7a, and beta-catenin proteins in the rat endometrium during the estrous cycle and early postimplantation period. Wnt3 and Wnt7a had a similar localization and dynamic expression relative to the endometrial stages. Wnt7a immunostaining was not limited only to the luminal epithelial cells, but also to strong stainings in the stromal and endothelial cells. Wnt3, Wnt7a, and beta-catenin were highly synthesized and colocalized at the trophoblast-decidual interface; and were more obvious in the primary decidual zone, the GTCs, and the ectoplacental cone. Beta-catenin was strongly localized at the borders of the mature decidual cells; however, Wnt3 and Wnt7a immunolocalizations were decreased in those cells. As such, the immunolocalization of Wnt3, Wnt7a, and beta-catenin shifted with decidualization and placentation. The expression level of Wnt3, Wnt7a, and beta-catenin messenger RNAs increased in early pregnancy, and especially between Days 8.5 and 9.5. The dramatic changes in the expression of Wnt3, Wnt7a, and beta-catenin observed during the early days of pregnancy and the estrous cycle may indicate their roles in decidualization, stromal cell proliferation, and trophoblast invasion.

Effects of vitrification and transplantation on follicular development and expression of EphrinB1 and PDGFA in mouse ovaries.

The aim of this study was to assess the follicular development and the patterns of EphrinB1 and PDGFA immunostaining in vitrified mouse ovarian tissue (OT) with and without transplantation. Histological evaluation was performed on fresh and vitrified OTs, whether transplanted or not. RT-PCR was performed on fresh and vitrified ovarian samples (OSs) and vitrified OS graft. Vitrification alone did not significantly reduce the normal primordial, primary, and secondary follicles except antral ones (p > 0.05). However, transplantation decreased all the follicle types. The EphrinB1 immunoexpression showed high intensity in all follicular types in vitrified OT and the significant increased was detected in secondary and antral follicles (p < 0.05). PDGFA protein immunoexpression of primordial and primary follicles was decreased in vitrified OT (p < 0.05). However, the lowest immunoexpression of EphrinB1 and PDGFA was detected after transplantation (p < 0.05). The levels of ephrinb1 and pdgfa mRNA expressions in vitrified OS and vitrified OS grafts were found as comparable to the fresh OS. In conclusion, vitrification has no detrimental effect on the follicles at the different developmental stages, majority of ovarian follicular loss takes place after transplantation rather than vitrification. Overall, vitrification and grafting do not change the ephrinb1 and pdgfa gene expressions. In addition, EphrinB1 and PDGFA are expressed during different stages of folliculogenesis in a different manner in fresh, vitrified, or grafted OTs. Vitrification and/or grafting appear to affect the follicular expression of EphrinB1 and PDGFA. These findings suggest that these proteins could have several functions related to the development of follicles and angiogenesis after transplantation.

The long-term effects of extracorporeal shock waves on the epiphysis of the adolescent rat.

BACKGROUND: Extracorporeal shock waves (ESW) have been successfully used to treat musculoskeletal injuries, tendinopathy, and plantar fasciitis. Different side effects of high-energy ESWT on bones, tendons, nerves, epiphysis, and cartilage have been discussed. Although the effects of ESW on the epiphysis in animal models have been described, no studies have investigated the long-term effect of ESW on the immature epiphysis in an animal model. The purpose of this study was to investigate the long-term effects of ESW on the immature epiphysis in rats. METHODS: Sixteen 4-week old Wistar albino rats, average weight 116.3 grams (109.6-120.2 g) were used for these experiments. The rats were randomly divided into two groups, the ESW 1500 pulses (ESW1500p) group (n = 8) and the ESW 3000 pulses (ESW3000p) group (n = 8). In the ESW1500p group, 1500 pulses, at 4 bar, of 1-Hz shockwaves were applied, once, to the left knees of the rats. In the ESW3000p group, 3000 pulses, at 4 bar, of 1-Hz shockwaves were applied, once, to the left knees of the rats. The right knees (n = 16) of the rats in the two groups were used as the controls. The animals were sacrificed after an 8-month follow-up period. Femoral epiphyses were assessed by use of histology. The femoral length (FL), tibial length (TL), and femoral supracondylar medio-lateral width (MLW) were measured. RESULTS: There was no statistically significant difference between FL, TL, and MLW for the three groups (P > 0.05). The average histological scores were 8.8 (7-10), 17.8 (15-22), and 2.7 (0-4) in the ESW1500p, the ESW3000p groups, and the controls, respectively. The average histological score for the ESW3000p group was significantly higher than those for the ESW1500p group and the controls (P < 0.001). The average histological score for the ESW1500p group was significantly higher than that for the controls (P < 0.001). CONCLUSIONS: The histological findings of this study indicate that ESW increased the cellularity and basophilia of the extracellular matrix (ECM) in the adolescent rat epiphysis and there were no negative effects on extremity measurements in the long term.

Norrin immunolocalization and its possible functions in rat endometrium during the estrus cycle and early pregnancy.

Mutations in Norrie Disease Pseudoglioma (NDP) gene cause serious sight loss, deafness and mental retardation in Norrie disease patients via the impairment of angiogenesis. Since norrin is a Wnt pathway ligand, it could function in several tissues other than eye and nervous systems. Therefore, the aim of the present study was to determine the possible function of norrin in angiogenesis, cellular differentiation in stroma and in decidua and the survival of those cells using immunofluorescent labeling. While norrin had a uniform distribution in stroma and in blood vessels, it had a strong expression in luminal and glandular epithelia during the estrus cycle. Norrin had strong immunolocalization in the antimesometrial decidual reaction zone on day 7 of gestation, whereas it had a decreased expression in the mesometrial uterine luminal epithelium along with an increased localization in blood vessels and decidual cells of the same region on day 8 of gestation. As from day 9 of gestation, norrin demonstrated rather strong expression in the decidual cells and blood vessels of the mesometrial region in which the chorioallantoic placenta was going to develop. In all periods studied, norrin had rather weak expression in the primary decidual zone surrounding the embryo. Findings of the present study suggested that norrin might regulate the decidual reaction and the placental angiogenesis along with the survival and the differentiation of luminal and glandular epithelial and decidual cells in rats. In addition, it could play indirect important roles in the control of trophoblastic invasion and the programmed cell death.

Extracellular matrix remodelling in rat endometrium during early pregnancy: the role of fibronectin and laminin.

The endometrial extracellular matrix (ECM) remodelling has a crucial role in the establishment of a successful pregnancy. In addition to its basic function such as regulation of cell function, differentiation, migration, proliferation, the substantial alterations in the endometrial ECM may play a specific role in the trophoblast invasion, placentation, cell death and formation of the proper and functional implantation chamber around the embryo. In the present study, immunolocalizations of fibronectin and laminin were determined using avidin-biotin complex-peroxidase in rat implantation sites during 7-10 days of pregnancy. Both proteins were present in the basal membrane of blood vessels and in decidual matrix whereas they were absent or had very weak reactivity in the primary decidual zone on day 7. When placentation has begun, the immunoreactivity of both proteins was increased in the placental bed and in the basal membrane of blood vessels of the mesometrial region. The immunolocalization of both proteins seemed to be decreased in the antimesometrial decidua, however, it was increased in the mesometrial decidual matrix on days 9 and 10. Therefore, it could be suggested laminin and fibronectin demonstrating dynamic expressions in relation with the morphological differentiation of endometrial stroma may play crucial roles in the control of trophoblast adhesion and invasion, in placentation and angiogenesis, in the determination of cell shape and fate thus contributing the endometrial receptivity and a successful pregnancy.

Effect of high-energy extracorporeal shock waves on the immature epiphysis in a rabbit model.

INTRODUCTION: We investigated the effect of high-energy extracorporeal shock wave (ESW) on immature tibial epiphysis in a rabbit model. MATERIALS AND METHODS: Twenty New Zealand white immature rabbit were used in this study. Animals were divided into two groups and they received 14 kW, 0.6 mJ/mm(2), 1,500 or 3,000 shots for three times with an interval of 7 days. After 6 weeks all rabbits were killed. The all tibia epiphysis were assessed by histology. The epiphyseal plaque thickness of right tibiae of the 3,000-shot ESW group was significantly higher than those of the 1,500-shot ESW group and the left tibia epiphyses of the 1,500- and 3,000-shot ESW groups as controls (P < 0.05). RESULTS: The epiphyseal plaque thickness of right tibiae of the 1,500-shot ESW group was significantly higher than that of the left tibia epiphyses of the 1,500- and 3,000-shot ESW groups as controls (P < 0.05). DISCUSSION: We demonstrated that high-energy ESW stimulated the growth of immature rabbit epiphysis. The long-term effect of shock wave on the immature rabbit epiphysis deserves further study.

Vascular endothelial growth factor production by rat granulated metrial gland cells and their morphological features in normal and pathological conditions.

Granulated metrial gland (GMG) cells are pregnancy-specific cells that may have many functions in successful placentation and pregnancy. In the present study, changes in the rat GMG cell structure, distribution and vascular endothelial growth factor (VEGF) expression during early pregnancy were evaluated by light microscopy. Implantation sites taken from females with spontaneous abortion were also investigated. On Day 7 of pregnancy, GMG cells were distributed through the implantation and interimplantation sites. They formed metrial glands in the mesometrial triangle on Day 9, and were observed in the decidua basalis on Day 14 of pregnancy. Avidin-biotin complex immunohistochemistry revealed that GMG cells showed moderate staining for VEGF at the beginning of pregnancy and intense staining on Days 9 and 10 of pregnancy. They were localised mostly near the newly formed blood vessels. The implantation sites from spontaneously aborting females showed numerous leucocytes in the lumen of mesometrial blood vessels. In spontaneously aborting females, GMG cells showed a distinct morphology, increased in number and volume, their granules were denser and degranulation was observed. These results suggest that rat GMG cells might be a guide for placental angiogenesis and they might share a role with leucocytes in pathological conditions.

[The effect of definitive continuous distraction employed with the Ilizarov type external fixation system on fracture healing: an experimental rabbit model]. / Ilizarov tipi eksternal fiksatörle belirli sürekli distraksiyonun kirik iyilesmesi üzerine etkisi: Tavsanda deneysel çalisma.

OBJECTIVES: The objective of this study was to evaluate the effect of definitive continuous distraction (DCD) employed through an Ilizarov type external fixator on fracture healing in a rabbit tibia model. METHODS: Fifteen mature New Zealand white rabbits weighing 2.8 to 4.0 kg were randomly assigned to three groups following osteotomy of the tibia and fibula. The first group (controls) was treated only with the Ilizarov type external fixator while the second and third groups were subjected to DCD amounting to 10% and 30% of the body weight, respectively, being employed through modulated springs on the Ilizarov external fixation system. All the rabbits were evaluated by radiographs on the 12th, 21st, 28th, and 35th days following surgery and were sacrificed on the 45th day. Radiographic evaluations were made according to the Lane and Sandhu's scoring system, and histologic evaluations according to the scoring system proposed by Heiple et al. The results were analyzed using the Kruskal-Wallis test. RESULTS: The highest radiographic and histologic scores were obtained from the second group, being 8.6 and 11.6, respectively (p<0.05). Radiographic and histologic scores of the third group were lower than those of the control group. CONCLUSION: Application of DCD of 10% of body weight through modulated springs on the Ilizarov external fixator improves fracture healing while a greater amount of instability results in delayed healing.

Comparison of effects of cyclooxygenase inhibitors on myometrial contraction and constriction of ductus arteriosus in rats.

The aim of this study was to compare the tocolytic effect of a selective cyclooxygenase-2 inhibitor, DFU (5,5-dimethyl-3(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone), indomethacin and nimesulide on myometrial strips isolated from rats in both lipopolysaccharide-induced preterm labour and term labour. We also compared the constrictor effects of DFU and indomethacin on the fetal ductus arteriosus. Myometrial strips were obtained from preterm and term labour Wistar albino rats and were mounted in organ baths for the recording of isometric tension. DFU, nimesulide and indomethacin significantly inhibited KCl-, oxytocin-, prostaglandin E(2)- and prostaglandin F(2 alpha)-stimulated contractions of myometrial strips isolated from rats in preterm and term labour. The E(max) value of indomethacin was significantly lower than those for DFU and nimesulide (P<0.05), with no change-log (10) EC(50) values. There was no significant difference between in -log (10) EC(50) and E(max) values of DFU and nimesulide for any of the tissues (P>0.05). In addition, there was no significant difference between -log (10) EC(50) and E(max) values for each of these three agents in myometrial tissues isolated from rats in preterm and term labour (P>0.05). Fetal ductus arteriosus was significantly constricted by DFU (10 or 100 mg/kg) in preterm and term rats, although DFU (10 or 100 mg/kg)-induced constriction ratios were significantly lower than those for indomethacin (P<0.05). These data demonstrate that DFU, a specific cyclooxygenase-2 inhibitor, could be considered as a new therapeutic agent for preterm labour. However, careful attention should be given to constriction of the fetal ductus arteriosus.