Song Quan

Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes

BACKGROUND Insulin resistance (IR) and hyperinsulinemia compromise fertility in females and are well-recognized characteristics of anovulatory women with polycystic ovary syndrome. Patients with IR and hyperinsulinemia undergoing ovarian stimulation for IVF are at increased risks of impaired oocyte developmental competence, implantation failure and pregnancy loss. However, the precise underlying mechanism remains unknown. METHODS We investigated how IR impairs oocyte quality and early embryonic development by an insulin-resistant mouse model. Oocyte quality, fertilization and embryonic development were analyzed. Furthermore, oxidant stress products and mitochondrial function were evaluated by quantitative real-time PCR and immunofluorescence. RESULTS An imbalance between oxidants and antioxidants revealed by increased concentrations of reactive oxygen species, and a decreased concentration of glutathione (GSH) and a decreased GSH/GSSG ratio resulted in oxidative stress (OS) and impaired mitochondrial function in germinal vesicle (GV) and metaphase II (MII) oocytes of insulin-resistant mice. MII oocytes displayed a decrease in the ATP content and the mitochondrial DNA (mtDNA) copy number. In contrast, GV oocytes were characterized by a high ATP content concomitant with increased clustering of mitochondria and a high inner mitochondrial membrane potential. GV oocytes from insulin-resistant mice showed early stage apoptosis, and fewer MII oocytes could be retrieved from these mice and were of poor quality associated with decreased fertilization and an arrest of embryo development with increased fragmentation. Abnormal spindles and misaligned chromosomes of MII oocyte were significantly increased in IR and hyperinsulinemia mice compared with the control mice. CONCLUSIONS IR contributes to OS and disrupts mitochondrial function in mouse oocytes. This may impair the accurate transmission of mtDNA from one generation to the next. Therefore, our results suggest that OS and mitochondrial dysfunction are responsible for poor oocyte quality of insulin-resistant mice, and may provide novel targets to improve low fertility in females with IR.

GM130, a cis-Golgi protein, regulates meiotic spindle assembly and asymmetric division in mouse oocyte

GM130, a cis-Golgi protein, plays key roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that GM130 was localized to the spindle poles at both metaphase I and metaphase II stages and associated with the midbody at telophase I stage. The association of GM130 with spindle poles was further confirmed by its colocalization with the centrosome-associated proteins, MEK1/2. By nocodazole treatment, we clarified that GM130 localization was consistently dependent on spindle assembly. Then we investigated the possible function of GM130 by specific morpholino microinjection. This treatment caused abnormal spindle formation, and decreased first polar body extrusion. Our results showed that knockdown of GM130 impaired the localization of MTOCs proteins γ-tubulin and Plk1. Using live cell imaging we observed that depletion of GM130 affected spindle migration and resulted in elongated spindle and large polar body extrusion. We further found that depletion of GM130 blocked p-MEK1/2 accumulation at the spindle poles. And, it was shown that GM130 detached from the spindle poles in oocytes treated with MEK specific inhibitor U0126. Taken together, our results suggested that GM130 regulates microtubule organization and might cooperate with the MAPK pathway to play roles in spindle organization, migration and asymmetric division during mouse oocyte maturation.

p38α MAPK Is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation

P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation, and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.

A randomized double blind comparison of atosiban in patients undergoing IVF treatment

STUDY QUESTION Does atosiban (oxytocin/vasopressin V1A receptor antagonist), given around embryo transfer improve the live birth rate of women undergoing IVF treatment? SUMMARY ANSWER The use of atosiban around embryo transfer did not improve the live birth rate in a general population of IVF patients. WHAT IS KNOWN ALREADY Uterine contractions in IVF cycles were significantly increased following ovarian stimulation and women with frequent uterine contractions had a lower pregnancy rates. A few observational studies suggested that the use of atosiban around embryo transfer resulted in higher pregnancy rates in women with repeated implantation failure (RIF). A non-randomized trial of IVF patients also reported higher implantation and clinical pregnancy rates after the use of atosiban. STUDY DESIGN, SIZE, DURATION This multi-centre randomized double blind study recruited 800 general subfertile women undergoing IVF treatment between November 2011 and March 2013. Subjects were randomized into the atosiban (n = 400) and placebo (n = 400) groups according to a computer-generated randomization list. PARTICIPANTS/MATERIALS, SETTING, METHODS Subjects were recruited and randomized in the three IVF units in Guangzhou, Hong Kong and Ho Chi Minh City. Women in the atosiban group received i.v. atosiban 30 min before embryo transfer with a bolus dose of 6.75 mg, and the infusion was continued at 18 mg/h for ∼1 h. The dose of atosiban was then reduced to 6 mg/h continued for another 2 h. Those in the placebo group received i.v. normal saline only. The primary outcome measure was the live birth rate. MAIN RESULTS AND THE ROLE OF CHANCE There was no significant difference in the live birth rate between the atosiban and placebo groups (39.8 versus 38.0%, P = 0.612, rate ratio 1.051, 95% confidence interval: 0.884-1.251). No significant differences were found between the two groups in the positive pregnancy test, clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy, ectopic pregnancy rates and implantation rate per woman. Similar results were found between the groups at different IVF centres, with a repeated cycle, presence of uterine fibroids or a serum estradiol level on the day of hCG above the median level. LIMITATIONS, REASONS FOR CAUTION Limitations include the transfer of early cleavage embryos, no measurement of uterine contractions, no documentation of adenomyosis and incomplete tracking of congenital abnormalities in newborns. WIDER IMPLICATIONS OF THE FINDINGS This randomized double blind study demonstrated that the use of atosiban given around embryo transfer did not improve the live birth rate in a general population of IVF patients; therefore atosiban should be given only in the context of clinical research. STUDY FUNDING/COMPETING INTERESTS Centres in Hong Kong and Vietnam received research funding from Ferring, which was not involved in study design, execution, data analysis and manuscript preparation. There are no conflicts of interest. TRIAL REGISTRATION NUMBER ClinicalTrials.gov Identifier: NCT01501214.

Comparison of serum anti-Mullerian hormone levels following hysterectomy and myomectomy for benign gynaecological conditions.

OBJECTIVE To compare serum anti-Mullerian hormone (AMH) levels following hysterectomy and myomectomy. STUDY DESIGN Prospective longitudinal observational study. Serum AMH, follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were measured pre-operatively (T1) and 2 days (T2) and 3 months (T3) following hysterectomy and myomectomy in 70 women aged 36-45 years. Hysterectomy (laparoscopy-assisted vaginal hysterectomy=10; total abdominal hysterectomy=25) with conservation of both ovaries for benign diseases of the uterus was performed in 35 women, and myomectomy (laparoscopy myomectomy=15; open myomectomy=20) was performed in another 35 women. The follow-up period was 3 months following surgery. The results were analysed using the t-test or one-way analysis of variance by repeated-measures ANOVA. RESULTS Serum AMH in the hysterectomy group was 1.08±0.77 ng/ml at T1, 0.78±0.58 ng/ml at T2 and 0.81±0.58 ng/ml at T3; the level was significantly lower at T2 and T3 compared with T1. In the myomectomy group, the corresponding values were 1.54±0.95 ng/ml, 1.18±0.77 ng/ml and 1.50±0.58 ng/ml; serum AMH was significantly lower at T2 compared with T1, but the difference between T3 and T1 was not significant. There were no significant differences in serum FSH and LH in either group between these three time points. CONCLUSION Serum AMH was significantly lower 2 days and 3 months following hysterectomy compared with the pre-operative level. Following myomectomy, serum AMH was significantly lower than the pre-operative level 2 days following the procedure, but was similar to the pre-operative level 3 months after surgery. Therefore, hysterectomy may have a more lasting adverse effect on ovarian reserve than myomectomy. A long-term study of AMH levels is needed.

Decreased endometrial vascularity in patients with antiphospholipid antibodies-associated recurrent miscarriage during midluteal phase.

OBJECTIVE To explore uterine arterial impedance and endometrial-subendometrial vascularity determined by two-dimensional Doppler ultrasonography (2D-DU) and three-dimensional ultrasonography and power Doppler angiography (3D-PDA) between patients with antiphospholipid antibodies-associated recurrent miscarriage (aPL-RM) and normal fertile women, and to further investigate the relationship between these parameters and endometrial microvessel density (MVD). DESIGN Prospective observational study. SETTING Tertiary-care fertility center. PATIENT(S) A total of 109 aPL-RM patients (aPL-RM group) and 49 normal fertile women (control group). INTERVENTION(S) Uterine measurement by transvaginal ultrasonography and endometrial MVD in the midluteal phase was assessed for both groups. MAIN OUTCOME MEASURE(S) Endometrial thickness, volume, and MVD, uterine arterial pulsatility index (PI) and resistance index (RI), and the vascularization index (VI), flow index (FI), and vascularization flow index (VFI) of endometrial and subendometrial regions were measured on day 7 of a natural cycle after ovulation. RESULT(S) Both groups had similar endometrial thickness, volume, and MVD and uterine arterial PI and RI. Endometrial VI, FI, VFI, and subendometrial FI were significantly reduced in the aPL-RM group compared with the control group. None of the uterine arterial 2D-DU and endometrial-subendometrial 3D-PDA parameters correlated with the endometrial MVD in both groups. CONCLUSION(S) Endometrial and subendometrial vascularity was significantly impaired in aPL-RM patients during natural midluteal phase compared with normal fertile women, and endometrial MVD did not correlate with any of the acquired vascularity parameters.

FoxM1 Directs STAT3 Expression Essential for Human Endometrial Stromal Decidualization

Human endometrium decidualization, which involves endometrial stromal proliferation and differentiation, is a prerequisite for embryo implantation, thus successful pregnancy. The Forkhead Box M1 (FoxM1), previously known as HNF-3, HFH-11, MPP2, Win, and Trident, is a transcriptional factor that plays crucial roles in cell proliferation and cell cycle progression. However, the molecular mechanism of FoxM1 during human endometrial decidualization remains unexplored. In this study, we first found FoxM1 is dynamically expressed in human endometrium during menstrual cycle. Employing a human endometrial stromal cell (HESC) line, we then demonstrated that FoxM1 inhibition downregulates cyclin B1 expression, delaying G2/M phase transition during HESC proliferation. Additionally, loss of FoxM1 expression blocks the differentiation of HESCs in response to estrogen, progesterone, and dbcAMP. Applying chromatin immunoprecipitation (ChIP) technique and luciferase assay, we further approved that FoxM1 can transcriptionally active signal transducer and activator of transcription 3 (STAT3), ensuring normal HESC differentiation. Besides enriching our knowledge on molecular basis underlying stromal decidualization, these findings help to shed light on the potential molecular causes for the endometrial disorders in humans.

Sphingosine-1-phosphate inhibits ceramide-induced apoptosis during murine preimplantation embryonic development

Sphingolipids are a complex family of naturally occurring molecules enriched with lipid rafts that contribute to their unique biochemical properties. Sphingolipid metabolites, including ceramide (Cer) and sphingosine-1-phosphate (S1P), are bioactive signaling molecules that regulate cell movement, differentiation, survival, and apoptosis, but their effects on preimplantation development of murine embryos are not well-characterized. In this study, murine zygotes were collected, cultured in vitro, and treated with 50 μM C2-Cer plus various concentrations of S1P. The blastocyst formation rate was decreased in the C2-Cer-treated group, compared with that in the control group and the group treated with 50 μM C2-Cer plus 25, 50, or 100 nM S1P (P < 0.05), respectively. The total cell number of the blastocysts from various treatment groups was similar at 110 hours post-hCG treatment, but that from the group treated with 50 μM C2-Cer was significantly decreased at 120 hours post-hCG treatment, compared with the control group and the group treated with 50 μM C2-Cer plus 50 nM S1P. However, the apoptotic cell number of blastocysts from the group treated with 50 μM C2-Cer was significantly increased at 110 and 120 hours post-hCG treatment, compared with the control group and the group treated with 50 μM C2-Cer plus 50 nM S1P. Moreover, expression of p53 in the group treated with 50 μM C2-Cer was higher than that in the control group and the group treated with 50 μM C2-Cer plus 50 nM S1P (P < 0.05). In conclusion, Cer decreases the blastocyst formation rate and induces embryonic cell apoptosis, but S1P partly inhibits the effects of Cer during preimplantation development of murine embryos.

NEDD8-mediated neddylation is required for human endometrial stromal proliferation and decidualization

STUDY QUESTION Does NEDD8-mediated neddylation regulate human endometrial stromal proliferation and decidualization? SUMMARY ANSWER Neddylation inhibition by a selective NEDD8-activating enzyme inhibitor, MLN4924, significantly impairs human endometrial stromal cell (HESC) proliferation and decidualization and facilitates cell senescence, via p21 accumulation. WHAT IS KNOWN ALREADY Neddylation regulates cell proliferation and tissue remodeling during embryogenesis and tumorigenesis, while human endometrial stroma undergoes sequential proliferation, differentiation, as well as dynamic tissue remodeling during each menstrual cycle. STUDY DESIGN, SIZE, DURATION We first analyzed the expression of NEDD8 in human endometrial tissues from 50 subjects, and then explored the consequence of neddylation inhibition by MLN4924 on HESCs proliferation, decidualization and cellular senescence. PARTICIPANTS/MATERIALS, SETTING, METHODS We collected 50 dated human endometrial tissues from early proliferative stage to late secretory phase of the menstrual cycle and analyzed the NEDD8 expression and cellular location in human endometrium by employing quantitative real-time PCR (qRT-PCR) and immunohistochemistry staining. Similar approaches were also used to explore the mRNA and protein expression of NEDD8 in an immortalized human endometrial stromal cell line (HESC) during proliferation and decidualization (N = 6). An MTS assay was performed to evaluate the effects of neddylation inhibition by MLN4924 on HESC proliferation. Flow cytometry and BrdU incorporation assay were conducted to determine the HESC cell cycle progression in response to MLN4924 exposure during proliferation. We also analyzed F-actin distribution by phalloidin staining and decidual marker gene expression by qRT-PCR to accesses the consequence of neddylation inhibition on HESC decidualization. Immunoblotting analysis of cullin1 and p21, and SA-β-Galactosidase staining were performed to reveal the potential molecular basis for the impaired HESC proliferation, decidualization and cellular senescence. The siRNA technique was applied to knockdown p21 expression to test whether a clearance of p21 accumulation would correct the HESC defects from neddylation inhibition. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrated that NEDD8 is ubiquitously expressed in human endometrium including luminal epithelium, glandular epithelium and the stromal cells during the menstrual cycle, as well as in the HESCs during proliferation and differentiation in culture. Employing multiple molecular, cellular and pharmacological approaches, we further observed that neddylation inhibition by MLN4924 significantly attenuates HESC proliferation (P-value < 0.05), impairs decidual transformation (P-value < 0.05), and facilitates cellular senescence. These abnormal HESC activities upon MLN4924 exposure were accompanied with reduced cullin1 neddylation and an aberrant accumulation of p21. While a clearance of p21 accumulation by siRNA knockdown could partially restore HESC proliferation and cellular viability, it failed to correct the decidualization defects. LIMITATIONS, REASONS FOR CAUTION Since NEDD8 was also intensely expressed in the endometrial epithelium, it is interesting to further study its potential role in stroma-epithelial interactions through isolating and culturing epithelial cells. p21 siRNA knockdown experiments revealed that there are differential molecular machineries, other than p21, that are subject to neddylation regulation during HESC proliferation compared with differentiation. This alternative mechanism warrants further investigation in future. WIDER IMPLICATIONS OF THE FINDINGS Our findings add novel evidence showing, for what we believe the first time, that NEDD8-mediated neddylation is required for normal human endometrial functions, which raises the possibility of approaching the neddylation system for diagnosis and treatment of infertility in women. STUDY FUNDING/COMPETING INTERESTS This work was supported in parts by the National Basic Research Program of China (2011CB944400 to H.W.) and the National Natural Science Foundation (81130009, 81330017 to H.W., 81170575 to S.Q. and 31471106 to S.Z.). The author declares that there is no conflict of interest.

ZEB1 promotes epithelial–mesenchymal transition in cervical cancer metastasis

OBJECTIVE To investigate role of Zinc finger E-box binding homeobox 1 (ZEB1) in cervical cancer tissue (squamous cell carcinoma, SCC). DESIGN Exploratory study. SETTING University hospital. PATIENT(S) Sixty patients with SCC, including stage CINIII (n = 10), IB1 (n = 10), IB2 (n = 10), IIA1 (n = 10), IIA2 (n = 10), and IIB (n = 10) were studied. INTERVENTION(S) Caski cells were transfected with recombinant shZEB1 lentivirus or shCtrl lentivirus to generate stable ZEB1-knockdown Caski cells. MAIN OUTCOME MEASURE(S) ZEB1 expression was analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry in cervical cancer tissues. ZEB1 expression in Caski cells was down-regulated by short-hairpin RNA (shRNA) interference, and changes in ZEB1 expression corresponded with changes in the proliferation and migratory ability of Caski cells. RESULT(S) Quantitative real-time polymerase chain reaction and immunohistochemistry results revealed that ZEB1 expression and the ratio of Vimentin to E-cadherin were high in 27 of 50 SCC patients and correlated with advanced International Federation of Gynecology and Obstetrics stage, tumor size >4 cm, and parametrial invasion. However, the expression of ZEB1 in cervical cancer tissue was independent of age and SCC antigen level. Transfection of ZEB1 shRNA in Caski cells significantly decreased the messenger RNA and protein expression of ZEB1, parallel with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker Vimentin. Furthermore, the proliferation and migratory ability of Caski cells were significantly lower in the transfected group than in the nontransfected control group. CONCLUSION(S) Down-regulation of ZEB1 expression may protect the invasive front of the tumors from converting to a mesenchymal phenotype by reducing the proliferation and motility of cervical cancer cells, suggesting that ZEB1 might be a potential therapeutic target for SCC.