Vitamin D supplementation prior to in vitro fertilisation in women with polycystic ovary syndrome: a protocol of a multicentre randomised, double-blind, placebo-controlled clinical trial.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is one of the leading causes of female infertility, affecting around 5% of women of childbearing age in China. Vitamin D insufficiency is common in women with PCOS and is associated with lower live birth rates. However, evidence regarding the effectiveness of vitamin D supplementation in women with PCOS is inconclusive. This multicentre randomised, double-blinded, placebo-controlled trial aims to evaluate the effectiveness of vitamin D supplementation prior to in vitro fertilisation (IVF) on the live birth rate in women with PCOS. METHODS AND ANALYSIS: We plan to enrol women with PCOS scheduled for IVF. After informed consent, eligible participants will be randomised in a 1:1 ratio to receive oral capsules of 4000 IU vitamin D per day or placebo for around 12 weeks until the day of triggering. All IVF procedures will be carried out routinely in each centre. The primary outcome is live birth after the first embryo transfer. The primary analysis will be by intention-to-treat analysis. To demonstrate or refute that treatment with vitamin D results in a 10% higher live birth rate than treatment with placebo, we need to recruit 860 women (48% vs 38% difference, anticipating 10% loss to follow-up and non-compliance, significance level 0.05 and power 80%). ETHICS AND DISSEMINATION: This study has been approved by the Ethics Committee in Women's Hospital of Zhejiang University on 2 March 2020 (reference number: IRB-20200035-R). All participants will provide written informed consent before randomisation. The results of the study will be submitted to scientific conferences and a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04082650.

Isolation and characterization of a flowering plant male gametic cell-specific promoter.

Flowering plant male gametic cell-specific gene expression has been reported recently but the regulatory elements controlling specificity of such genes expressed in generative cell and sperm cells have not been identified and studied. Here, we report the 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell-specific gene, LGC1, sufficient to regulate the expression of reporter genes in a cell-specific manner. In addition, the diphtheria toxin A-chain- (DT-A)-coding region under the control of the LGC1 promoter sequence confirmed unequivocally the lack of LGC1 expression in vegetative tissues. Transgenic tobacco plants carrying the LGC1-DT/A construct showed normal phenotype except for anthers of these plants that contained sterile and aborted pollen. Truncation and internal deletion analysis of the LGC1 promoter identified -242 bp as the minimal sequence necessary for male gametic cell-specific expression. In addition, a regulatory sequence required for determining generative cell-specific expression of LGC1 was identified. Deletion of this regulatory sequence led to loss of the generative cell specificity resulting in activation of this promoter in other tissues where it is normally repressed. Therefore, male gametic cell specificity of the LGC1 gene seems to be regulated by factors that suppress its activation in other plant cells. This is the first report of a male gametic cell-specific promoter, hence can be used as a novel tool in molecular analyses and experimental manipulation of flowering plant spermatogenesis and fertilization.

Caspase proteolysis of the cohesin component RAD21 promotes apoptosis.

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.