Immunohistochemistry

Endometrial biopsies were conducted at baseline and 16 weeks. Paraffin-embedded endometrial biopsy tissue sections were used for immunohistochemistry. Antigen retrieval using citrate buffer (pH 6.0) was performed. Sections were incubated in primary antibody against Ki67 (BD Pharmigen, CA) or P27 (R & D System, Minneapolis, MN) at 4°C overnight, followed by incubation with biotinylated anti rabbit and anti-mouse IgG, and streptavidin-HRP (Dako Incorporation). Diaminobenzidine solution (Dako Incorporation) was applied to visualize the complex and sections were counterstained with Mayer’s hematoxylin. The total number of Ki67 staining nuclei was counted and reported as the percentage of positively nuclear stained endometrial cells; procedures consistent with international guidelines for Ki67 assessment were followed (36). P27 loss has been suggested to be a marker of endometrial risk related to obesity and p27 expression was scored as intact, slightly reduced, moderately reduced, or severely reduced, as previously described (37).