Hui Long

Protective Effect of Quercetin on the Development of Preimplantation Mouse Embryos against Hydrogen Peroxide-Induced Oxidative Injury

Quercetin, a plant-derived flavonoid in Chinese herbs, fruits and wine, displays antioxidant properties in many pathological processes associated with oxidative stress. However, the effect of quercetin on the development of preimplantation embryos under oxidative stress is unclear. The present study sought to determine the protective effect and underlying mechanism of action of quercetin against hydrogen peroxide (H2O2)-induced oxidative injury in mouse zygotes. H2O2 treatment impaired the development of mouse zygotes in vitro, decreasing the rates of blastocyst formation and hatched, and increasing the fragmentation, apoptosis and retardation in blastocysts. Quercetin strongly protected zygotes from H2O2-induced oxidative injury by decreasing the reactive oxygen species level, maintaining mitochondrial function and modulating total antioxidant capability, the activity of the enzymatic antioxidants, including glutathione peroxidase and catalase activity to keep the cellular redox environment. Additionally, quercetin had no effect on the level of glutathione, the main non-enzymatic antioxidant in embryos.

The pregnancy outcome of progestin‐primed ovarian stimulation using 4 versus 10 mg of medroxyprogesterone acetate per day in infertile women undergoing in vitro fertilisation: a randomised controlled trial

To investigate the clinical outcome and endocrinological characteristics of progestin‐primed ovarian stimulation (PPOS) using 4 versus 10 mg of medroxyprogesterone acetate (MPA) per day in infertile women with normal ovary reserve.

New application of dydrogesterone as a part of a progestin-primed ovarian stimulation protocol for IVF: a randomized controlled trial including 516 first IVF/ICSI cycles

STUDY QUESTION Can dydrogesterone (DYG) be used as an alternative progestin in a progesterone primed ovarian stimulation (PPOS) protocol? SUMMARY ANSWER DYG can be used as an appropriate alternative progestin in a PPOS protocol. WHAT IS KNOWN ALREADY PPOS is a new ovarian stimulation regimen based on a freeze-all strategy that uses progestin as an alternative to a GnRH analog for suppressing a premature LH surge during the follicular phase. Medroxyprogesterone acetate (MPA) has been successfully used as an adjuvant to gonadotrophin in the PPOS protocol. However, the use of MPA may lead to stronger pituitary suppression and thus may require a higher dosage of hMG and a longer duration of ovarian stimulation than that of conventional ovarian stimulation protocol. STUDY DESIGN SIZE, DURATION A prospective RCT including 516 patients was performed between November 2015 and November 2016. Computerized randomization was conducted to assign participants at a 1:1 ratio into two treatment groups: an hMG + DYG group (260 patients) or an hMG + MPA group (256 patients) followed by IVF or ICSI with the freeze-all strategy. One cycle per patient was included. The primary outcome of the trial was the number of oocytes retrieved. The sample size was chosen to detect a difference of two oocytes with a power of 90%. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients under 36 years of age with normal ovarian reserve who were undergoing their first IVF/ICSI procedure due to tubal factor infertility were randomized into two groups based on the oral progestin protocol used: hMG co-treatment with DYG (hMG + DYG) or hMG co-treatment with MPA (hMG + MPA). The different progestin was simultaneously administered at the beginning of menstrual cycle 3 (MC3). Oocyte maturation was co-triggered by administration of a GnRH agonist and hCG. All viable embryos from both protocols were cryopreserved for later transfer. Only the first frozen embryo transfer (FET) cycle was included in our study. The embryological and clinical outcomes were measured. MAIN RESULTS AND THE ROLE OF CHANCE Basic characteristics, such as age, BMI and infertility duration, in both groups were comparable. There was no significant difference in the number (mean ± SD) of oocytes retrieved [10.8 ± 6.3 for the hMG + DYG group versus 11.1 ± 5.8 for the hMG + MPA group, P = 0.33] or the oocyte retrieval rate [74.3 ± 19.6% for the hMG + DYG group versus 75.0 ± 19.5% for the hMG + MPA group, P = 0.69] between the groups. The viable embryo rate per oocyte retrieved did not differ between the two groups [odds ratio (OR): 1.08, 95% CI: 0.97-1.21, P = 0.16]: 37.4% (1052/2815) for the hMG + DYG group versus 35.6% (1009/2837) for the hMG + MPA group. During the whole process of ovarian stimulation, the mean LH level in the hMG + DYG group was always higher than that in the hMG + MPA group (P < 0.001); however, no patient from either group experienced a premature LH surge. In addition, no patients experienced moderate or severe ovarian hyperstimulation syndrome during the ovarian stimulation. No significant difference was found in the clinical pregnancy rate of the first FET cycle between the two groups (OR: 0.82, 95% CI: 0.56-1.21, P = 0.33): 57.6% for the hMG + DYG group (125/217) versus 62.3% for the hMG + MPA group (132/212). LIMITATIONS REASONS FOR CAUTION The patients and physician were not blinded to the study. Further, a large proportion of patients were still pregnant at the end of the clinical trial, therefore live birth rates were not observed in the follow-up period. The dose-effectiveness of DYG administration was not addressed in the trial design. WIDER IMPLICATIONS OF THE FINDINGS DYG, which exhibits no or only weak inhibition of ovulation in normal dosage, can serve as an hMG adjuvant during ovarian stimulation. This finding suggests the possibility of a new application of DYG: as an appropriate alternative progestin for a PPOS protocol in IVF. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by The National Nature Science Foundation of China (Grant no. 81503603), Shanghai Three-year Plan on Promoting TCM Development (Grant no. ZY3-LCPT-2-2006) and the Natural Science Foundation of Shanghai (Grant nos. 15401932700 and 15ZR1424900). None of the authors declare any conflict of interest. TRIAL REGISTRATION NUMBER Chictr.org.cn: ChiCTR-IPR-15007251. TRIAL REGISTRATION DATE Chictr.org.cn: 22 October 2015. DATE OF FIRST PATIENT’S ENROLLMENT 1 November 2015.

Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human ‘8’-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii–iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that ‘8’-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01). Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of ‘8’-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in ‘8’-shaped hatching and hatched blastocysts than in ‘U’-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01). Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of ‘8’-shaped hatching, and ‘8’-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

Hypothalamic effects of progesterone on regulation of the pulsatile and surge release of luteinising hormone in female rats

Progesterone can block the oestradiol-induced GnRH/LH surge and inhibit LH pulse frequency. Recent studies reported that progesterone prevented premature LH surges during ovarian hyperstimulation in women. As the most potent stimulator of GnRH/LH release, kisspeptin is believed to mediate the positive and negative feedback effects of oestradiol in the hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nuclei, while the region-specific role of progesterone receptors in these nuclei remains unknown. This study examined the hypothesis that progesterone inhibits LH surge and pulsatile secretion via its receptor in the ARC and/or AVPV nuclei. Adult female rats received a single injection of pregnant mare serum gonadotropin followed by progesterone or vehicle. Progesterone administration resulted in a significant prolongation of the oestrous cycle and blockade of LH surge. However, microinjection of the progesterone receptor antagonist, RU486, into the AVPV reversed the prolonged cycle length and rescued the progesterone blockade LH surge, while RU486 into the ARC shortened LH pulse interval in the progesterone treated rats. These results demonstrated that progesterone’s inhibitory effect on the GnRH/LH surge and pulsatile secretion is mediated by its receptor in the kisspeptin enriched hypothalamic AVPV and ARC respectively, which are essential for progesterone regulation of oestrous cyclicity in rats.

Progestin-primed ovarian stimulation with or without clomiphene citrate supplementation in normal ovulatory women undergoing in vitro fertilization/intracytoplasmic sperm injection: A prospective randomized controlled trial

To compare the endocrinological profiles, cycle characteristics and pregnancy outcomes of progestin‐primed ovarian stimulation (PPOS) with or without clomiphene citrate (CC) supplementation in normal ovulatory women undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI).

Age-related nomograms of serum anti-Mullerian hormone levels in female monkeys: Comparison of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys

AMH is regarded as a promising predictor for ovarian reserve in humans and non-human primate, and widely used in human medicine to predict ovarian response to gonadotropin, menopause and premature ovarian failure. However, large data set on the range of AMH levels in nonhuman primates is still scarce, which limited its applications largely. In this study, age-related AMH nomograms of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) were produced and compared. 219 rhesus and 529 cynomolgus monkeys ranging from infancy to adult were included. In total, the mean serum AMH levels in cynomolgus monkeys were higher than that of rhesus monkeys (14.6 ± 5.3 ng/ml vs 9.5 ± 6.0 ng/ml, P < 0.001). AMH was inversely correlated with age (r = -0.371, P < 0.001) in rhesus, while the negative correlation did not reach statistical significance in cynomolgus monkeys (r = -0.044, P = 0.156). The maximum mean AMH levels were attained at the subgroup of 4-11 yr and the lowest AMH levels were obtained at the subgroup of ≧12 yr in both primates, corresponding to their fertility potential. In rhesus monkeys, from 1 to 11 years old, AMH level remained stable (1-3 yr: ß = 2.784, P = 0.340; 4-11 yr: r = 0.100, P = 0.110) whereas from 12 yr onward, an inverse correlation between AMH and age (r = -0.450, P = 0.02) was observed. Similarly, AMH appeared stable from 1 to 3 yr (ß = -2.289, P = 0.429) and showed an inverse correlation with age (r = -0.521, P < 0.001) from 12 yr onward in cynomolgus monkeys, while a positive correlation was observed (r = 0.156, P = 0.001) from 4 to 11 yr. AMH levels were relatively stable across the menstrual cycle in both primates and no seasonal difference for AMH levels was observed in rhesus monkeys. Body mass index did not affect serum AMH levels in both primates. Our nomograms of serum AMH provide a reference guide on AMH longitudinal distribution by age for Macaca monkeys and might facilitate its applications.

Effect of Frozen Embryo Transfer and Progestin-primed Ovary Stimulation on IVF outcomes in women with high body mass index

Among women undergoing IVF, high BMI negatively affects pregnancy outcomes when using the conventional ovary stimulating protocols combined with fresh embryo transfer. Therefore, finding a proper treatment for these high BMI women is more important and urgent when obesity is prevalent. In our study, we reported a retrospective study of 4457 women who were divided into normal BMI group (18.5 kg/m2–24.9 kg/m2) and high BMI group (≥25 kg/m2) undergoing 4611 IVF treatment cycles with frozen embryo transfer (FET). We found the high BMI group originally had the poor oocytes performance, but after FET they got the similar pregnancy outcomes as the normal BMI group. Then under FET we analyzed the IVF outcomes of our new progestin-primed ovary stimulation (PPOS) protocol, indicating that the hMG + MPA (4 or 10 mg/d) groups had the obvious better pregnancy results than the conventional short group in the high BMI group, and binary logistic regression analysis showed the hMG + MPA (10 mg/d) group was associated with better pregnancy outcomes than the hMG + MPA (4 mg/d) group. These results indicate PPOS - hMG + MPA (10 mg/d) combined with FET might be a new potential treatment choice for the high BMI women undergoing IVF treatments.

Andrographolide disrupts meiotic maturation by blocking cytoskeletal reorganisation and decreases the fertilisation potential of mouse oocytes

Andrographolide (AG) is a diterpenoid lactone isolated from the stem and leaves of Andrographis paniculata Nees that is used for the effective treatment of infectious diseases in Asian countries. Previous studies have reported adverse effects of AG on female fertility in rodents; however, the underlying mechanisms are unknown. The aim of the present study was to investigate the effects of AG on the IVM of mouse oocytes and their fertilisation potential. Immature oocytes incubated for 6, 14 or 24h in medium containing 5, 10 or 20μM AG showed time- and dose-dependent decreases in maturation rates compared with the control group. Immunostaining revealed that AG exposure disrupted spindle organisation and migration, as well as actin cap formation and cytokinesis. Furthermore, most oocytes exposed to 20μM AG underwent apoptosis, and the few oocytes exposed to 5 or 10μM AG that reached MII exhibited lower fertilisation rates after intracytoplasmic sperm injection. The findings of the present study suggest that AG may disrupt mouse oocyte meiotic maturation by blocking cytoskeletal reorganisation, and may thus have an adverse effect on female fertility.