To utilize lobular composition to address questions of extent, duration and individual variation in postpartum breast involution, it is necessary to investigate the influence of clinical and technical parameters which may confound lobule-type quantitation. Here, we address patient age, use of a single tissue section per case, cancer diagnosis, biologic cancer type and potential impact of menstrual cycle on lobular type composition. In the nulliparous group (n = 23 cases), patient age was not found to significantly impact lobular type composition (
P ≥0.19) (Figure
2B), nor was age found to impact lobule type composition in an extended cohort (n = 82 cases) consisting of combined nulliparous and parous cases >2 years postpartum (
P ≥0.18) (Figure
2C). We next addressed the use of a single tissue specimen per case as a potential methodological limitation by performing quadrant analyses on three parous cases >5 years postpartum. As expected, variation in lobular composition between individuals was observed (Additional file
4: Figure S2a). Further, within the same subject, the largest mean difference in lobule type composition was found between the lower inner and upper outer quadrants (Additional file
4: Figure S2a). However, a mixed effect model, which takes the correlation among the tissue samples from the same case into account, indicates that quadrant location is not significantly associated with the presence of lobular types (lower outer (LO) versus lower inner (LI) (
P = 0.33); upper inner (UI) versus lower inner (LI) (
P = 0.40); upper outer (UO) versus lower inner (LI) (
P = 0.25)]. Further, an aggregate analysis revealed no significant morphological differences between quadrants in these cases (
P ≥0.25) (Figure
2D). To address the potential confounder of malignancy, we compared adjacent normal lobular composition of women diagnosed with benign breast lesions to those with cancer. We found the groups indistinguishable (
P = 0.59), indicating that in normal adjacent tissue at least 5 mm distant from the breast cancer, the presence of cancer does not significantly alter lobule type composition (Figure
2E). Next, we examined whether ER expressivity of the breast tumor might influence lobule composition, as variation in lobule types has been reported between patients with luminal A and basal breast cancers [
38]. In our cohort, differences in lobule type composition were not observed between ER positive (n = 47) and ER negative (n = 24) cases (
P = 0.15) (Figure
2F). Finally, to address the potential effect of menstrual cycling, lobule composition for types 1, 2, 3 and 4 lobules was analyzed in initial and subsequent biopsy tissues collected two to three weeks apart in a subset of premenopausal women (n = 12). Although there were small variations in lobular composition between biopsy times, no significant differences between type 1 (
P = 0.38), type 2 (
P = 0.32) or type 3 (
P = 0.46) lobules were observed (Figure
2G). To summarize, in this premenopausal cohort, lobule type composition varies more between women of the same reproductive category than between categories defined by patient age, biopsied quadrant, ER expression of the tumor, presence of breast cancer and stage of menstrual cycle. Although, overall, the power of statistical tests is low due to low sample size, the similarities in mean values indicate that these specific parameters have no strong influence on lobule type analysis in the postpartum involution window, which is the primary objective of this study.