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The Impact of the Using Culture Media Containing with Granulocyte-Macrophage Colony-Stimulating Factor on Live Birth Rates in Patients with a History of Embryonic Developmental Arrest in Previous In Vitro Fertilization (IVF) Cycles. Sipahi M et al. (2021) To investigate the effect of using granulocyte-macrophage colony-stimulating factor (GM-CSF) containing culture media on embryological data and reproductive outcomes in patients with early embryonic developmental arrest. Retrospective case-control study. A total of 39 patients, whose embryos were incubated with GM-CSF containing culture media due to embryonic developmental arrest in two previous IVF cycles in-between January 2016 and November 2017 at … University IVF center, were enrolled. Control group was generated among patients with first IVF attempts due to tubal factor in the same time period. All embryos in the control group were incubated with single step culture medium (without GM-CSF). For the control group selection, matching was done 1:2 ratio considering female age, body mass index, number of M-II oocyte retrieved, and number of embryo transferred (n=80). Demographic features and embryological data were comparable between two groups. Number of fertilized oocytes (2-pronuclear) was 3.7±2.0 in GM-CSF group and 3.9±2.5 in the control (p= 0.576). Overall, number of embryos transferred (1.3±0.5 vs. 1.3±0.5, respectively) and blastocyst transfer rate (67.6% vs. 59.2%, respectively; p=0.401) were similar. For the reproductive outcomes, implantation rate (32.3% vs. 33.1%, respectively; p= 0.937), clinical pregnancy rate (33.3% vs. 32.5%, respectively; p= 0.770), and live birth rate (LBR) (25.2% vs. 26.2%, respectively; p= 0.943) were similar. Using GM-CSF containing culture media in patients with previous two failed IVF attempts due to embryonic developmental arrest might rectify embryological data and reproductive outcomes. To make solid conclusion further randomized controlled trial is warrant.//////////////////Molecular mechanism and functional role of macrophage colony‑stimulating factor in follicular granulosa cells. Wei Z et al. (2017) Our previous demonstrated that macrophage colony‑stimulating factor (M‑CSF) stimulated the production of estradiol (E2) and progesterone (P) in luteinized granulosa cells (GCs), and that its secretion may be regulated by follicle‑stimulating hormone (FSH). The present study aimed to examine the effect of M‑CSF alone or with Letrozole treatment on the function of non‑luteinizing granulosa cells, using the COV434 cell line, and its interaction with FSH. Human luteinized granulosa cells (LGC) were isolated from the follicular fluid of superovulated infertile patients (average age, 30.8±2.1 years) undergoing an intracytoplasmic sperm injection. The LGC were cultured with various concentrations of recombinant human macrophage colony stimulating factor (rhM‑CSF; 0, 10, 25, 50 or 100 ng/ml), rhM‑CSF+Letrozole (10‑6 mol/l), rhFSH (0, 10, 25, 50 or 100 IU/ml), or rhFSH+Letrozole (10‑6 mol/l). E2 concentrations in the media were measured using ELISA. The expression levels of the FSH receptor and the M‑CSF receptor were detected via reverse transcription‑quantitative polymerase chain reaction. Following COV434 cell treatment with M‑CSF, cell proliferation was quantified using the MTS assay and protein expression was detected by western blotting. It was demonstrated that M‑CSF and FSH stimulated the production of E2. The production of FSH receptors was enhanced by rhM‑CSF or rhM‑CSF+Letrozole in vitro in a dose‑dependent manner. It was observed that rhFSH promoted the expression of the M‑CSF receptor, at a certain concentration. Proliferation of COV434 cells increased in a dose‑dependent manner following treatment with RhM‑CSF. Furthermore, M‑CSF induced the phosphorylation of c‑Jun N‑terminal kinase (JNK) and p38; however, the level of E2 in the medium was not altered when the cells were pretreated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580. The present study suggested that M‑CSF may be important in regulating the response of GCs to gonadotropin and may have a promotive effect in the early phase of follicular development. The biological effects of M‑CSF may partially be attributed to activation of the JNK and p38 signaling pathways. M‑CSF may represent a novel follicular development regulator agent in the future.//////////////////
The protective effect of G-CSF on experimental ischemia/reperfusion injury in rat ovary. Bostancı MS et al. (2015) This experimental study was designed to evaluate the effect of granulocyte colony-stimulating factor (G-CSF) in ovarian ischemia and ischemia/reperfusion (I/R) injury. Forty-eight female adult Sprague-Dawley albino rats were divided into six groups as Group 1: sham, Group 2: torsion, Group 3: detorsion, Group 4: sham + G-CSF, Group 5: torsion + G-CSF, and Group 6: detorsion + G-CSF. Except for Groups 1 and 4, all groups underwent a dnexal torsion bilaterally for 3 h. Adnexal detorsion was applied to Groups 3 and 6 for 3 h after a 3-h torsion period. The intraperitoneal injection of G-CSF (100 IU/kg) was administered 30 min previously in Group 4, 5 and 6. At the end of the study process the animals were euthanized and their ovaries were removed for histopathological and biochemical analysis. Total oxidant status (TOS), total antioxidative status and oxidative stress index (OSI) concentrations were determined and compared. Histopathological examination of ovaries was performed for the presence of interstitial edema, congestion, hemorrhage and loss of cohesion to determine tissue damage. In Group 3, 4, 5 and 6, TOS, OSI and total histopathological scores of ovarian tissue were higher than in the sham group (p < 0.05). G-CSF administration decreased mean TOS and OSI levels significantly when compared with the controls (p < 0.001, p < 0.001, respectively). There was a strong correlation between the total histopathological scores of I/R injury and OSI (r = 0.862, p < 0.001). The total histopathological scores for the rats conservatively treated with G-CSF were lower than those of the control groups. G-CSF is effective for the prevention of ischemia and ischemia/reperfusion-induced damage in rat ovary.//////////////////
The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development. Cai L et al. (2015) Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus-oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus-oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF-treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF-treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF-treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF-treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF-treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.//////////////////
Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes. Peralta OA et al. (2014) The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at -50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose-response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days. Immunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2). GM-CSF enhanced cumulus cell expansion of in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development.//////////////////
303 human recombination granulocyte-colony stimulating factor (hrg-csf) have beneficial effects on porcine oocytes quality during in vitro maturation and subsequent viability of embryonic development. Cai L et al. (2014) Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3), is required for the proliferation, differentiation, and survival of cells. In humans, G-CSF is a biomarker of human oocyte developmental competence for embryo implantation. Furthermore, G-CSF concentration increases during the menstrual cycle and levels were significantly higher during ovulatory phase than the other phases. In this study, we examined G-CSF and its receptor gene expression in the porcine granulosa cells, corpus luteum, cumulus cells, and oocytes. The cumulus-oocyte complexes (COC) were aspirated from antral follicles 1 to 3mm (small follicle) and 3 to 6mm (medium follicle). The COC from 2 kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of hrG-CSF (0, 10, and 100ngmL(-1), respectively). Statistical analyses were done by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. After real time-PCR was performed, the CSF3 and its receptor (CSF3R) were observed all of granulosa cells, corpus luteum, cumulus cells, and oocytes. Interestingly, the CSF3 transcript levels were significantly lower in oocytes compared with other cell types, but the CSF3R transcript levels in oocytes were almost similar with granulosa cells. After 44h of IVM, the rates of nuclear maturation had no difference, and the intracellular ROS levels of oocytes from both kind of follicle groups matured with 10ngmL(-1) were significantly decreased compared to other groups (P<0.05). After PA, the cleavage and blastocyst formation rates were significantly (P<0.05) increased for the 100ngmL(-1) of small follicle (SF; 63.29 and 31.18%) group compared to control and 10ngmL(-1) of SF (38.64, 10.4, and 49.0, 15.6%, respectively) group, and significantly (P<0.05) increased in the 10ngmL(-1) of medium follicle (MF; 76.32 and 45.61%) group compared with control and 100ngmL(-1) of MF (52.1, 32.8 and 61.3, 33.9%, respectively). The total cell numbers of blastocyst from SF and MF groups were significantly increased in the 10ngmL(-1) (73.67 and 106.52) groups. At IVF, the blastocysts formation rates were significantly increased in the 10ngmL(-1) of MF group compared to control, 100ngmL(-1) of SF, and control of MF (21.1, 22.8, and 27.8%, respectively). We also examined the Bcl2 and ERK2 transcript levels, which were significantly increased at 100ngmL(-1) of SF and 10ngmL(-1) of MF. These results suggest that hrG-CSF improved the quality of porcine oocyte and embryonic viability.//////////////////
Granulocyte colony-stimulating factor: A relation between serum and follicular fluid levels and in-vitro fertilization outcome in patients with polycystic ovary syndrome. Kahyaoglu I 2014 et al.
Evidence is accumulating in the literature about the potential role of serum and follicular fluid (FF) granulocyte colony-stimulating factor (G-CSF) as a non-invasive biomarker of oocyte competence and embryo selection in in-vitro fertilization (IVF) cycles. In this study, we aimed to evaluate the effect of serum and FF G-CSF levels on IVF outcome in non-hyperandrogenic, non-obese patients with polycystic ovary syndrome (PCOS). Twenty-two patients with PCOS (Group I), and 22 patients with the etiology of male factor infertility (Group II) undergoing IVF treatment were included. Demographic features, controlled ovarian stimulation parameters, neutrophil count (NC), neutrophil/leukocyte (N/L) ratio, serum and FF G-CSF levels of the two groups were compared. Serum E2 level on the day of hCG (2982.5171.4 vs. 2279.0207.2pg/mL), total number of retrieved oocytes (14.70.9 vs. 11.51.3) and mature oocytes (11.60.8 vs. 9.11.1) were significantly higher in group I when compared to group II (p<0.05). On the day of oocyte retrieval, both the mean serum (54.81.7 vs. 48.10.9pg/mL) and FF G-CSF levels (48.81.4 vs. 44.10.5pg/mL), NC (4.40.210(3) vs. 3.60.310(3)/L) and N/L ratio (63.61.4 vs. 56.11.7) in group I were found to be significantly higher than group II ((p<0.05). Despite the increased levels of G-CSF both in the serum and follicular microenvironment in patients with PCOS, a relation between G-CSF and good ovarian response or clinical pregnancy rates could not be demonstrated in this study.
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Evaluation of cytokines in follicular fluid and their effect on fertilization and pregnancy outcome. Gaafar TM 2014 et al.
Cytokines in follicular fluid (FF) are important for reproduction as they modulate oocyte maturation and ovulation which influence subsequent fertilization, development of early embryo and potential for implantation. We evaluated FF cytokines in women who underwent intracytoplasmic sperm injection (ICSI) and their association with fertilized oocytes, embryo quality and pregnancy outcome. FF belonging to 38 patients including 18 polycystic ovary (PCO) and 20 male/unexplained infertility patients were investigated for granulocyte colony stimulating factor (G-CSF), regulated upon activation normal T cell expressed and presumably secreted (RANTES), tumour necrosis factor (TNFa), interferon gamma (IFN?) and interleukins (IL-4 and IL-2) by bead-based sandwich immunoassay. Our findings revealed that on the day of oocyte retrieval, G-CSF was positively correlated with the number of fertilized oocytes, while TNFa detection was associated with reduced number of fertilized oocytes. Only G-CSF showed significant positive effect to the pregnancy outcome although the cytokines studied were not associated with embryo quality. PCO as the cause of infertility did not show an association with cytokines in FF. The functions of cytokines in reproduction are likely to be complex, and cytokine evaluation may offer insight to the understanding of the mechanisms leading to success or failure of assisted reproduction.
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Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes. Peralta OA et al. The objectives of the study were to characterize the expression of the alpha- and beta-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development.
Tamura K, et al. reported that granulocyte-macrophage colony-stimulating factor enhances interleukin-1beta stimulated histamine
release in the preovulatory rat ovary. Histamine release from preovulatory ovarian tissues was stimulated
in a dose-dependent manner at 3-30 ng/ml of GM-CSF in the presence of interleukin-1beta (10 ng/ml).
However, treatment with GM-CSF and interleukin-1beta did not cause any significant change in the
levels of ovarian steroids. These
results indicate that GM-CSF may be involved in the regulation of ovarian histamine secretion in mast
cells partially by enhancing interleukin-1beta-induced histamine release in the process of ovulation.
Exogenous granulocyte-macrophage colony-stimulating factor promotes follicular development in the newborn rat in vivo Wang H, et al .
BACKGROUND: Expression and selective cellular localization of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in ovarian tissue imply an autocrine/paracrine role in ovarian function. Evidence indicating a functional role for GM-CSF in ovarian follicular cell function has been provided by studies with GM-CSF knockout (GM-/-) mice, which suggest that GM-CSF influences events associated with murine follicular maturation. METHODS: Immature female rats were treated with GM-CSF, FSH or saline for 5 or 10 days. Ovaries were collected for histologic examination and immunostaining determination of CYP17, a theca cell marker. In addition, ovarian section slides were evaluated by immunofluorescence for CD45, an ovarian leukocyte marker. To investigate the possible mechanism of GM-CSF action on follicular development, theca-interstitial cells (T-I) were separated and cultured. Cells were treated with increasing concentrations of GM-CSF, then evaluated for CYP17 mRNA and protein expression assays. RESULTS: After 10 days of treatment with GM-CSF, the number of small preantral and large preantral follicles was significantly increased compared with the control group (P < 0.05). Similarly, treatment with FSH increased the number of small preantral and large preantral follicles (P < 0.05). CD45 expression measured by immunofluorescence was not different in the three groups, indicating that the distribution of leukocytes was unchanged. In addition, CYP17 was increased in the T-I cells both in vivo and in vitro after GM-CSF treatment. CONCLUSION: The present results suggest that GM-CSF may play a significant role in follicular development.
Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor. L?e N et al. BACKGROUND The cytokine/chemokine levels of individual follicular fluids (FFs) were measured to determine whether a biomarker could be linked to the developmental potential of the derived embryo. METHODS Fluid was collected from 132 individual FFs that were the source of oocytes subsequently fertilized and transferred. In each, a bead-based multiplex sandwich immunoassay (Luminex) was used to measure 28 cytokines and chemokines simultaneously. RESULTS Significantly higher levels of interleukin (IL-2) and interferon (IFN-gamma) were detected in FF for embryos that underwent early cleavage. IL-12 was significantly higher in FF corresponding to highly fragmented embryos and the chemokine CCL5 was significantly higher in FF related to the best quality (Top) embryos. The level of granulocyte colony-stimulating factor (G-CSF) in individual FF samples was correlated with the implantation potential of the corresponding embryo. The area under the receiver operating characteristics curve, which distinguished the embryos that definitely led to delivery from those that did not, was 0.84 (0.75-0.90) (P = 0.0001) for FF G-CSF. FF G-CSF was significantly lower in patients older than 36 years compared with those <30-year old. When the FF G-CSF was 20 pg/ml or higher, the ratio between Top and non-Top embryos was significantly higher than for the group with FF G-CSF below 20 pg/ml (45 versus 20.45%, P = 0.007). CONCLUSIONS Individual FF composition is related to the development of the corresponding in vitro generated embryo and its potential of implantation. Individual FF G-CSF may provide a non-invasive biomarker of implantation that needs to be evaluated together with in vitro observation to select the oocyte, and hence the embryo, to transfer.
Culture of human oocytes with granulocyte-macrophage colony-stimulating factor has no effect on embryonic chromosomal constitution. Agerholm I et al. The effect on ploidy rate in donated human oocytes after in-vitro culture with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; 2ng/ml) from fertilization until day 3 was examined in a multicentre, prospective placebo-controlled and double-blinded study including 73 women donating 86 oocytes. The primary endpoint was to investigate the chromosomal constitution of human embryos (fluorescence in-situ hybridization analysis for chromosomes 13, 16, 18, 21, 22, X and Y) cultured with or without GM-CSF. The secondary endpoints were number of top-quality embryos (TQE) and number of normally developed embryos evaluated morphologically on day 3. The cytogenetic analyses demonstrated non-inferiority and therefore the chromosomal constitution of human embryos cultured in vitro in the presence of 2ng/ml GM-CSF was no worse than the control group cultured without GM-CSF. In-vitro culture of human embryos in the presence of 2ng/ml GM-CSF resulted in 34.8% (8/23) uniformly normal embryos. Culture without 2ng/ml GM-CSF resulted in 33.3% (9/27) uniformly normal embryos. A trend towards a higher number of TQE in the test group was observed; however, due to lack of TQE in the control group, this was considered a random finding.
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