Poorly-Controlled Type 1 Diabetes Mellitus Impairs LH-LHCGR Signaling in the Ovaries and Decreases Female Fertility in Mice

PURPOSE: The aim of this study was to investigate how type I diabetes mellitus (T1D) affects the folliculogenesis and oocyte development, fertilization, and embryo development. MATERIALS AND METHODS: A comparative animal study was conducted using two different mouse models of T1D, a genetic AKITA model and a streptozotocin-induced diabetes model. Ovarian function was assessed by gross observation, immunoblot, immunohistochemistry, oocyte counting, and ELISA for serum hormones (insulin, anti-Mullerian hormone, estradiol, testosterone, and progesterone). Maturation and developmental competence of metaphase II oocytes from control and T1D animals was evaluated by immunofluorescent and immunohistochemical detection of biomarkers and in vitro fertilization. RESULTS: Animals from both T1D models showed increased blood glucose levels, while only streptozotocin (STZ)-injected mice showed reduced body weight. Folliculogenesis, oogenesis, and preimplantation embryogenesis were impaired in both T1D mouse models. Interestingly, exogenous streptozotocin injection to induce T1D led to marked decreases in ovary size, expression of luteinizing hormone/chorionic gonadotropin receptor in the ovaries, the number of corpora lutea per ovary, oocyte maturation, and serum progesterone levels. Both T1D models exhibited significantly reduced pre-implantation embryo quality compared with controls. There was no significant difference in embryo quality between STZ-injected and AKITA diabetic mice. CONCLUSION: These results suggest that T1D affects folliculogenesis, oogenesis, and embryo development in mice. However, the physiological mechanisms underlying the observed reproductive effects of diabetes need to be further investigated.

The Regulators of VEGF Expression in Mouse Ovaries

The objectives of this study were to explore whether ovarian vascular endothelial growth factor (VEGF) expression in mice can be regulated by IL-6 (interleukin-6), angiotensin II, FSH, and hCG; and to test whether the mouse ovarian VEGF expression can result in angiogenesis. The ICR mice were sacrificed, and their ovaries were recovered. Recovered ovaries were treated with IL-6, angiotensin II, FSH, and hCG separately and incubated for 24 hours in alpha-MEM. Expression of mRNA and protein of VEGF were assessed by RT-PCR and immunohistochemistry. The resulting angiogenesis was evaluated through immunohistochemical analysis for CD34. Treatment of mice ovaries with IL-6, FSH, and hCG resulted in a significant increase of VEGF mRNA, and IL-6 was the most potent inducer of VEGF. IL-6 and FSH resulted in increased neovascularization in the follicular phase of mouse ovaries. In contrast, angiotensin II could not increase VEGF expression or neovascularization. We documented an in vitro increase in VEGF expression by IL-6, FSH, and hCG; and reaffirmed that the proliferative response of murine ovarian endothelial cells paralleled an increase of VEGF expression.

Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model / 대한불임학회지

OBJECTIVES: The aim of this study was to assess toxicities of cryoprotectants. METHODS: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. RESULTS: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH (75.9+/-27.0) or the control (99.0+/-18.3) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO (14.2+/-1.5) and PROH (11.2+/-1.4) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.2+/-0.9, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). CONCLUSIONS: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

Effect of GM-CSF on the Embryonic Development and the Expression of Implantation Related Genes of Mouse Embryos / 대한불임학회지

OBJECTIVE: The purpose of the current series of experiments were to assess the effect of GM-CSF, as a medium supplement, on the development of mouse embryos and the expression of LIF and IL-1beta mRNA. MATERIALS AND METHODS: Mouse 2-cell embryos were collected from the oviducts of 6 weeks old ICR mice at 48 hours after hCG injection. Embryos were cultured in P-1 medium supplemented with mouse GM-CSF(0,1,5,10 ng/ml). The embryo development to blastocysts and hatching blastocysts was assessed and the cell number in blastocyst was also examined. Using RT-PCR, the expressions of LIF and IL-1beta mRNA in blastocyst were evaluated in the GM-CSF supplemented group and control group. RESULTS: In mouse, the addition of GM-CSF increased the percentage of blastocysts(65.5%, 68.6%, 73.0% and 76.1% for control and 1, 5 and 10 ng/ml, respectively), and increased the proportion of hatching blastocysts(35.2%, 36.4%, 43.2% and 53.0% for control and 1, 5 and 10 ng/ml, respectively). The mean cell numbers in blastocyst were significantly increased in GM-CSF supplemented groups compared to control group. LIF and IL-1beta expression in blastocysts were significantly higher in GM-CSF supplemented group than in control group. CONCLUSION: The results of experiment by mouse embryos showed beneficial effects of GM-CSF as a medium supplement. Furthermore, the addition of GM-CSF significantly increased the expression of LIF and IL-1beta in mouse embryos. These results suggest that GM-CSF might be a important molecule in embryo implantation.

Effect of Partial Laser Assisted Hatching on Mouse Embryos / 대한불임학회지

OBJECTIVE: The present study was performed to investigate the efficiency of partial laser assisted hatching (p-LAH; lased 1/2 ZP width from ZP edge) on hatching of mouse blastocysts. METHODS: We used non-contact 1.48 micrometer diode laser (MTM, Switzland) to create a precise hole on zona pellucida. 2-cell embryos were collected from the mouse (ICR) oviduct at 48 hours after hCG administration. Collected 2-cell embryos were cultured in the P-1 medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after 20~22 hours in culture. After conventional (c-LAH) or partial laser assisted hatching, the embryos were further cultured in P-1 medium supplemented with 0.4% BSA for 3 days. To compare efficiency of complete and partial laser assisted hatching, hatching rate, hatching time and blastocyst diameter and zona pellucida thickness at hatching time were investigated. Embryos were examined every 12 hours. Blastocyst diameter and zona pellucida thickness at hatching time were measured with an ocular micrometer. RESULTS: Hatching rates of p-LAH group (84.2%) was significantly higher than that of control group (39.3%), but there was no difference between the p-LAH (84.2%) and c-LAH (91.2%). p-LAH group was hatched 12 hours earlier than control group, but hatched 12 hours later than c-LAH group. The diameter of blastocyst at hatching time of p-LAH group (113.1+/-6.4 micrometer) was smaller than that of control group (122.2+/-5.0 micrometer), but larger than that of c-LAH group (102.2+/-2.7 micrometer). Zona pellucida thickness at hatching time of p-LAH group (6.4+/-0.9 micrometer) was thicker than that of control group (4.5+/-1.5 micrometer), but thinner than that of c-LAH group (10.0+/-0.8 micrometer). CONCLUSION: These results suggest that p-LAH may maintains the cell arrangement of early embryos to ensure successful development and prevent precocious hatching of blastocyst when compare to c-LAH and conventional (acidic tyrode) AH. Thus, p-LAH may provide a valuable and effective AH technique for human ART program.

The Effect of GM-CSF Supplementation in Culture Medium in the Human IVF Programs / 대한불임학회지

OBJECTIVE: Granulocyte-macrophage colony stimulating factors known to be secreted in murine and human reproductive tract. The development of human, bovine and murine embryos could be promoted by addition of GM-CSF in culture medium. However, the pregnancy and implantation rate of embryos cultured in GM-CSF have not been evaluated. The aim of this study was to assess the effect of GM-CSF in embryo development, pregnancy and implantation rate. METHODS: A total of 191 IVF cycles were divided into control and GM-CSF supplement group (control =96, GM-CSF=95). The embryos were cultured for three day with or without 2 ng/ml of recombinant human GM-CSF. The quality of embryo, developmental velocity, pregnancy and implantation rates were compared. RESULTS: There was no difference in age, number of gonadotropin ampules used, number of oocytes and fertilization. The number of ICSI cycle was higher in GM-CSF group. In GM-CSF group, G-1 grade embryos were the highest in proportion (56.4%), while G-2 grade embryos were highest (44.3%) in control group. The developmental velocity of embryos were not different between GM-CSF and control group. The pregnancy and implantation rates were significantly higher in GM-CSF group than control (47.4% vs. 33.3%, 17.0% vs. 11.1% respectively). CONCLUSION: By adding GM-CSF in culture medium, the quality of embryo, pregnancy and implantation rate could be improved.