Effects of high-altitude hypoxia on embryonic developmental potential in women undergoing IVF/ICSI procedures.

PURPOSE: In this study we examined the effects of long-term adaptation to hypoxia on embryonic developmental potential of oocytes collected from women who underwent IVF/ICSI procedures. METHODS: We selected young infertile women who lived in a low-altitude normoxic environment (n = 80, altitude < 500 m) or high-altitude hypoxic environment (n = 100, altitude > 2500 m) for a lengthy period of time and who planned to undergo IVF/ICSI procedures. We then determined the baseline reproductive hormone levels, gonadotropin (Gn) dose and Gn treatment duration during controlled ovarian hyperstimulation (COH), number of oocytes retrieved, number of mature oocytes, oocyte maturation rate, fertilization rate, normal fertilization rate, day (D3) embryo-formation rate, blastocyst formation rate, good-quality formation rate, D5 blastocyst formation rate, and D6 blastocyst formation rate between the two groups. RESULTS: Compared with the low-altitude normoxic group, the various reproductive hormone markers of women in the high-altitude hypoxia group were lower, with LH and T levels significantly reduced (P < 0.05) at 72.29 and 72.44% of the normoxic group, respectively (normoxic group vs. hypoxic group, 5.24 ± 1.61 vs. 3.79 ± 1.21; 0.61 ± 0.18 vs. 0.42 ± 0.15; P < 0.05). During ovarian hyperstimulation, a greater Gn dose and longer Gn treatment duration were required for the hypoxic group to complete COH (normoxic group vs. hypoxic group, 2152.08 IU ± 52.76 vs. 2622.09 IU ± 123.28; 9.96 days ± 1.27 vs. 11.54 days ± 1.34, respectively; P < 0.05). The fertilization, cleavage, and D3 embryo-formation rates tended to be higher in the normoxic group than in the hypoxic group (P > 0.05); while the normal fertilization rate tended to lower than in the hypoxic group (P > 0.05). When we conducted an analysis of blastocyst formation rates at different timepoints, we ascertained that the blastocyst formation rate, usable blastocyst rate, and good-quality blastocyst rate of the hypoxic group were all lower than in the normoxic group, with the difference in usable blastocyst rate the most highly significant (normoxic group vs. hypoxic group, 75.31 ± 5.53 vs. 56.04 ± 6.10%, respectively; P < 0.05). In addition, the D5 and D6 blastocyst-formation rates in the normoxic group were slightly higher than in the hypoxic group, revealing that not only were fewer blastocysts formed in the hypoxic group but that there was also a delay in blastocyst formation. CONCLUSION: In young women undergoing IVF/ICSI treatment, long-term hypoxic adaptation required augmented Gn dose and Gn treatment duration during COH, and blastocyst developmental potential was also attenuated.

Effect of BMP-Wnt-Nodal signal on stem cell differentiation.

The generation of germ cells from embryonic stem cells in vitro has current historical significance. Western blot, qPCR, immunofluorescence and flow cytometry assays were used to investigate the differences in expression levels of totipotency and specific markers for Wnt regulation and the related signalling pathways during primordial germ cell-like cell (PGCLC) induction and differentiation. During PGCLC induction, activation of WNT3a increased the expression of NANOG, SOX2 and OCT4, but Mvh, DAZL, Blimp1, TFAP2C, Gata4, SOX17, EOMES, Brachyury and PRDM1 expression levels were significantly reduced. Inhibition of the WNT signal demonstrated the opposite effect. Similarly, inhibitors of BMP and the Nodal/Activin signal were used to determine the effect of signal pathways on differentiation. CER1 affected the Wnt signal and differentiation, but the inhibitor SB only regulated differentiation. BMP-WNT-NODAL were mainly responsible for regulating differentiation. Our results provide a reliable theoretical basis and feasibility for further clinical medical research.

MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells.

BACKGROUND: We elucidated the role of specific MicroRNAs (miRNAs) in the development of polycystic ovary syndrome (PCOS) and explained the changes in the proliferation of granulosa cells. Excised ovarian cortex specimens were collected for miRNA profiling analysis (n = 20 PCOS females and 5 non-PCOS females). Insulin-treated ovarian granulosa cells isolated from mice were used for mechanical studies. RESULTS: High miR-140 expression was observed in PCOS samples and insulin-treated granulosa cells compared to that in non-PCOS and unstimulated cells, respectively. However, the Ras-related protein Rap-2a precursor (RAP2A) was downregulated in in PCOS. MTT assay and EdU staining showed that an miR-140 inhibitor attenuated viability in insulin-treated granulosa cells; cell viability increased with miR-140 overexpression. Reduced expression of miR-140 and the expression of the miR-140 mimic resulted in marked cell apoptosis, as evidenced by the results of PI flow cytometry and Annexin V-FITC; miR-140 overexpression results in downregulated RAP2A expression, and the miR-140 mimic directly bound to the RAP2A 3'-UTR, causing increase in RAP2A levels in insulin-treated granulosa cells; RNA-mediated silencing of RAP2A in insulin-treated granulosa cells restored cell proliferation and apoptosis to normal levels. Phosphorylated AKT was found to be negatively regulated through cross-talk between miR-140 and RAP2A. CONCLUSIONS: In conclusion, PCOS ovarian cortex specimens and insulin-treated granulosa cells showed elevated expression of miR-140, which could lead to increased proliferation and reduced apoptosis of cells by targeting RAP2A. This study may pave the way for future research on the properties of granulosa cells in PCOS.

Establishment of a feeder and serum-free culture system for human embryonic stem cells.

Stem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

AQP8 and AQP9 expression in patients with polycystic ovary syndrome and its association with in vitro fertilization-embryo transfer outcomes.

The aim of the present study was to investigate aquaporin (AQP)8 and AQP9 expression in patients with polycystic ovary syndrome (PCOS) and its association with in vitro fertilization-embryo transfer (IVF-ET) outcomes. A total of 45 patients with PCOS undergoing IVF-ET (test group) and 50 patients with oviduct obstruction or ovarian cyst (control group) were assessed for the mRNA expression of AQP8 and AQP9 in ovarian tissues by reverse transcription-quantitative (RT-q)PCR. The levels of luteinizing hormone, anti-mullerian hormone and testosterone were determined, which were revealed to be significantly different between the two groups (P<0.05). The RT-qPCR results indicated that AQP8 expression in the control group was lower than that in the test group (t=37.75, P<0.01), whereas AQP9 expression in the control group was higher than that in the test group (t=19.59, P<0.01). The number of eggs obtained in the group with high AQP8 expression was significantly lower than that in the group with low AQP8 expression (t=2.64, P<0.01). The number of high-quality embryos in the high AQP8 expression group was not significantly different from that in the low AQP8 expression group (t=1.02, P>0.05). The pregnancy rate in patients with high AQP9 expression was higher than that in the low AQP9 expression group (P<0.05) and the abortion rate in the former was lower than that in the latter (P<0.05). In conclusion, AQP8 and AQP9 are differentially expressed in ovarian tissues of patients with PCOS vs. normal control subjects. The expression of AQP8 is closely associated with the occurrence and development of oocytes, whereas the expression of AQP9 is associated with the success rate of pregnancy in patients with PCOS.

[Influence of basic thyroid-stimulating hormone levels on outcomes of IVF/ICSI in Qinghai].

OBJECTIVE: To study basic thyroid stimulating hormone (bTSH) levels impact on outcomes of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in Qinghai. METHODS: Totally 282 cases with IVF cycles and 93 cases with ICSI cycles were studied prospectively, according to bTSH level, patients were divided into four groups. Reproduction rate, clinical pregnancy rate, miscarriage rate and live birth rate were studied among four groups. RESULTS: (1) In 375 cases with IVF/ICSI cycles, bTSH was positively correlated with abortion rate (r = 0.42, P = 0.04), but live birth rate and growing rate showed negative correlations with bTSH (r = -0.42, -0.28; P = 0.04, 0.03). bTSH and the number of eggs, the number of fertilized eggs, the number of embryos, biochemical pregnancy rate, and clinical pregnancy rate were no significant correlation (all P > 0.05). (2) Among women at group of ≤ 1.7, >1.7 and ≤ 2.5, >2.5 and ≤ 3.5, >3.5 mU/L, the implantation rates were 28.7%, 27.3%, 37.7% and 19.2%, live birth rates were 80.9%, 75.0%, 82.7%, and 59.8%, abortion rates were 19.0%, 15.0%, 16.7%, 40.1%; they all showed significant difference (all P < 0.05). Abortion rate in women with high bTSH level was higher than that of women with lower bTSH level, however implantation rate, live birth rate in women with high bTSH level were lower. CONCLUSION: When bTSH level is >3.5 mU/L, the abortion rate were increased, but live birth rate, rate of implantation were decreased.

Maternal-fetal fluid balance and aquaporins: from molecule to physiology.

Maternal-fetal fluid balance is critical during pregnancy, and amniotic fluid is essential for fetal growth and development. The placenta plays a key role in a successful pregnancy as the interface between the mother and her fetus. Aquaporins (AQPs) form specific water channels that allow the rapid transcellular movement of water in response to osmotic/hydrostatic pressure gradients. AQPs expression in the placenta and fetal membranes may play important roles in the maternal-fetal fluid balance.

Pregnant phenotype in aquaporin 8-deficient mice.

AIM: Aquaporin 8 (AQP8) is expressed within the female reproductive system but its physiological function reminds to be elucidated. This study investigates the role of AQP8 during pregnancy using AQP8-knockout (AQP8-KO) mice. METHODS: Homozygous AQP8-KO mice were mated, and the conception rate was recorded. AQP8-KO pregnant mice or their offspring were divided into 5 subgroups according to fetal gestational day (7, 13, 16, 18 GD) and newborn. Wild type C57 pregnant mice served as the control group. The number of pregnant mice, total embryos and atrophic embryos, as well as fetal weight, placental weight and placental area were recorded for each subgroup. The amount of amniotic fluid in each sac at 13, 16, and 18 GD was calculated. Statistical significance was determined by analysis of variance of factorial design and chi-square tests. RESULTS: Conception rates did not differ significantly between AQP8-KO and wild type mice. AQP8-KO pregnant mice had a significantly higher number of embryos compared to wild type controls. Fetal/neonatal weight was also significantly greater in the AQP8-KO group compared to age-matched wild type controls. The amount of amniotic fluid was greater in AQP8-KO pregnant mice than wild type controls, although the FM/AFA (fetal weight/amniotic fluid amount) did not differ. While AQP8-KO placental weight was significantly larger than wild type controls, there was no evidence of placental pathology in either group. CONCLUSION: The results suggest that AQP8 deficiency plays an important role in pregnancy outcome.

[Expression of aquaporin-1 in human oligohydramnios placenta and fetal membranes].

OBJECTIVE: To detect aquaporin-1 mRNA (AQP1) expression in human oligohydramnios placenta and fetal membranes. METHODS: Placenta and fetal membranes samples were obtained from 5 women with oligohydramnios and 5 with normal amniotic fluid volume. AQP-1 mRNA expression in the tissue samples was detected by semi-quantitative RT-PCR. RESULTS: The expression of AQP1 mRNA was significantly lower in oligohydramnios placenta than in normal pregnancy placenta at term (P<0.05), and also significantly lower in oligohydramnios fetal membranes than in normal fetal membranes at term (P<0.05). CONCLUSION: Alterations in AQP1 mRNA expressions in human placenta and fetal membranes may play an important role in the disorder of maternal-fetal fluid exchange and amniotic fluid volume.