1. Abnormally enhanced cystic fibrosis transmembrane conductance regulator-mediated apoptosis in endometrial cells contributes to impaired embryo implantation in controlled ovarian hyperstimulation 
Jian Zhi Yang, M.D., Xiaohua Jiang, Ph.D., JianDa Dong, M.Phil., JingHui Guo, M.Phil., Hui Chen, M.Phil., Lai Ling Tsang, M.Phil., Yiu Wa Chung, M.B., B.S., XiaoHu Zhang, Ph.D., Hsiao Chang Chan, Ph.D. 
Fertility and Sterility 
Volume 95, Issue 6, Pages e2 (May 2011)
DOI: /j.fertnstert
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Controlled ovarian hyperstimulation (COH) endometrium exhibited more apoptosis during implantation. Either COH or natural endometrium was retrieved at day 4 after mating. (A) Isolated natural endometrial epithelial cells were plated on Matrigel-coated inserts and grown until confluence. Polarized epithelial cell monolayer was stained with 6-diamino-2-phenylindole (DAPI). Bar = 50 μm. (B) After digestion, the natural endometrial cells were smeared on the slides and immunostained for cytokeratin 10, a protein present in mice uterine epithelial cells (red), nuclei were counterstained with DAPI (blue). Negative controls were performed by substituting nonimmune mouse immunoglobulin for the primary antibody. Bar = 50 μm. (C) Cells from either natural or COH endometrium grown on transwell inserts were fixed and immunostained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (green) and nuclei were stained with DAPI (blue) before visualization. In the experiments of blastocyst coculture, the same amount of natural blastocysts were cocultured with polarized epithelial cells from either natural endometrium or COH endometrium in DMEM/F12 media for 3 days. Bar = 50 μm. (D) Quantification of TUNEL positivity in control or COH-treated endometrial cells. ce = COH endometrium with blastocyst; c = COH endometrium only; ne = natural endometrium with blastocyst; n = natural endometrium only. The results were expressed as ratio of TUNEL-positive cells of total cells. Results displayed as mean ±SE of three independent experiments. ∗P<.05, endometrium only versus endometrium with blastocysts; #P<.05, versus ce; ∧P<.05, versus ne.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Cystic fibrosis transmembrane conductance regulator (CFTR) is aberrantly up-regulated in controlled ovarian hyperstimulation (COH) endometrium. (A) Either COH or natural endometrium was retrieved at day 4 after mating from four mice separately in each group. Cell lysates were collected and Western blot performed. Endometrial cells with or without blastocysts were analyzed for CFTR expression using Western blot and immunofluorescence as indicated in Materials and Methods section. (B) Cells grown on transwell insert were fixed and immunostained for CFTR expression and nuclei were stained with 6-diamino-2-phenylindole (DAPI) (blue) before visualization. Bar = 50 μm. (C) Quantification of CFTR positivity. ce = COH endometrium cocultured with blastocyst; c = COH endometrium only; ne = natural endometrium cocultured with blastocyst; n = natural endometrium only. The results were expressed as ratio of CFTR-positive cells versus total cells. Results displayed are mean ±SE of three independent experiments. ∗P<.05; ∗∗P<.01.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Ectopic supraphysiologic concentration of E2 induced cystic fibrosis transmembrane conductance regulator (CFTR)-mediated apoptosis. Primary endometrial epithelial cell culture was established by using premature female imprinting control region mice to exclude the influence of estrous cycle. The cells were treated with 0, 0.1, 1, 10 μg/mL E2 for 24 hours. (A) The CFTR (green) expression was determined by immunofluorescence and nuclei were stained with DAPI (blue). Bar = 50 μm. (B) Cells were treated with 10 μg/mL E2 with or without CFTR inh172 for 24 hours. The cells were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (red) and nuclei were stained with 6-diamino-2-phenylindole (DAPI) (blue). Bar = 50 μm. (C) Quantification of TUNEL-positive cells. Data were presented as mean ±SE from three independent experiments. ∗P<.01; ∗∗P<.05.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

5. Effect of controlled ovarian hyperstimulation (COH) on embryo development, blastocyst adhesion, and hatching. Effect of COH on blastocyst development. Embryos from natural cycle (n = 10) or COH mice (n = 7) were counted for blastocysts and morula numbers 3 days after mating. C-b = the blastocyst number of COH cycle mice; C-m = the morula number of COH cycle mice; N-b = the blastocyst number of natural cycle mice; N-m = the morula number of nature cycle mice. ∗P<.05.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions