Introduction/Background: Some women produce antenatal colostrum during pregnancy and feed it to their baby after birth. However, the composition of antenatal colostrum and how it compares to postnatal colostrum and mature milk are not well described. In fact, there are currently no data on the composition of antenatal colostrum when it comes to human milk oligosaccharides (HMOs), the third most abundant solid human milk component after lactose and lipids. Case Presentation: We report a case of a single healthy donor who collected antenatal colostrum and urine from 19 weeks of gestation all the way to mature milk at 3 months postpartum. We analyzed all samples for HMO composition using high-performance liquid chromatography and for lactose concentrations using an enzymatic assay. Results: The entire spectrum of HMOs typical of a nonsecretor was already present in antenatal colostrum at 19 weeks gestation with a total concentration of 7.5 mg/mL. The HMO concentration further increased to over 12.5 mg/mL at 30 weeks gestation and then declined throughout the remainder of pregnancy and continued to decline in the postpartum period with concentrations of less than 5 mg/mL at 12 weeks postpartum. Concentrations of some of the individual HMOs as well as lactose changed significantly at the time of birth. HMO composition in antenatal colostrum was different in time-matched urine samples. Conclusion: Measuring HMOs in maternal urine does not fully capture the composition of HMOs in antenatal colostrum. Feeding antenatal colostrum to the newborn baby provides the entire set of different HMOs at high concentrations.

Microenvironmental mechanical signals are fundamental regulators of cell behavior both in physiological and in pathological context, particularly in the induction and maintenance of tumorigenic properties. It is thus of utmost importance to experimentally recreate conditions that mimic the physical attributes of real tissues to study their impact on cell behavior and in particular on the induction of cancer stem cell (CSC) properties. Here we present protocols to investigate the role of mechanical stiffness on reprogramming of primary mammary gland cells into CSCs, including the synthesis of hydrogel substrates of the desired stiffness, the isolation and culture of primary differentiated normal cells derived from the human mammary gland, and the assessment of their CSC attributes after oncogene-mediated transformation.

The mammary gland is a vital exocrine organ that has evolved in mammals to secrete milk and provide nutrition to ensure the growth and survival of the neonate The mouse mammary gland displays extraordinary plasticity each time the female undergoes pregnancy and lactation, including a sophisticated process of tertiary branching and alveologenesis to form a branched epithelial tree and subsequently milk-producing alveoli. Upon the cessation of lactation, the gland remodels back to a simple ductal architecture via highly regulated involution processes. At the cellular level, the plasticity is characterised by proliferation of mammary cell populations, differentiation and apoptosis, accompanied by major changes in cell function and morphology. The mammary epithelium requires a specific stromal environment to grow, known as the mammary fat pad. Mammary adipocytes are one of the most prominent cell types in the fat pad, but despite their vast proportion in the tissue and their crucial interaction with epithelial cells, their physiology remains largely unknown. Over the past decade, the need to understand the properties and contribution of mammary adipocytes has become more recognised. However, the development of adequate methods and protocols to study this cellular niche is still lagging, partially due to their fragile nature, the difficulty of isolating them, the lack of reliable cell surface markers and the heterogenous environment in this tissue, which differs from other adipocyte depots. Here, we describe a new rapid and simple flow cytometry protocol specifically designed for the analysis and isolation of mouse mammary adipocytes across mammary gland developmental stages.

The study of the structure and function of the animals\' mammary glands is of key importance, as it reveals pathological processes at their onset, thus contributing to their immediate treatment. The most frequently studied mammary diseases are mastitis in cows and ewes and mammary tumours in dogs and cats. Various imaging techniques such as computed tomography, positron emission tomography, magnetic resonance imaging, and ultrasonographic techniques (Doppler, contrast-enchanced, three-dimensional and elastography) are available and can be applied in research or clinical practice in order to evaluate possible abnormalities in mammary glands, as well as to assist in the differential diagnosis. In this review, the above imaging technologies are described, and the perspectives of each method are highlighted. It is inferred that ultrasonographic modalities are the most frequently used imaging techniques for the diagnosis of clinical or subclinical mastitis and treatment guidance on a farm. In companion animals, a combination of imaging techniques should be applied for a more accurate diagnosis of mammary tumours. In any case, the confirmation of the diagnosis is provided by laboratory techniques.

In this short commentary I recall a long-term experiment that was sketched out to determine if the low incidence of mammary cancer in dairy animals reflects a low incidence in these species generally or is the result of a protective effect of early pregnancy and long lactations. Although that experiment was never done, I discuss these questions in the light of developing knowledge on the incidence of cancer in ruminants generally and in the mammary gland in particular.

Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15-20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland.

Epithelial-stromal interactions play an essential role in regulation of mammary gland development, homeostasis, and tumorigenesis. Fibroblasts constitute a substantial proportion of mammary gland stromal cells in human breast and have been recognized for their paracrine signaling and extracellular matrix production and remodeling roles during normal breast development as well as in breast cancer. However, our current knowledge on human breast fibroblast functions is incomplete. Here we provide a detailed protocol for an organotypic human breast assay to facilitate research in the roles of human breast fibroblasts in mammary epithelial morphogenesis and early tumorigenesis.

Objective: Primary Sjögren\'s syndrome (pSS) is characterized by exocrine glandular inflammation; however, the association between preceding mammary-gland-inflammation-related diseases and newly diagnosed pSS remains unexplored. Methods: We used the 2003-2013 data retrieved from Taiwan\'s National Health Insurance Research Database (NHIRD) to conduct the present population-based study. We identified newly diagnosed pSS female patients during the 2001-2013 period, as well as age-matched (1:20) and propensity-score-matched (1:2) non-SS individuals (as controls). We explored the associations between pSS and a history of mastitis and fibrocystic breast disease by determining adjusted odds ratios (aORs) with 95% confidence intervals (CIs) using a conditional logistical regression analysis after controlling for potential confounders. Results: We identified 9,665 patients with pSS and 193,300 age-matched non-SS controls, as well as 9,155 SS cases and 18,310 propensity-score-matched non-SS controls. We found that fibrocystic breast disease (aOR, 1.75; 95% CI, 1.63-1.88) were independently associated with incident SS, whereas mastitis and childbirth-associated breast infections were not associated with incident SS. We also found positive associations between SS and previously reported SS-associated diseases, including cardiovascular diseases, thyroid diseases, pancreatitis, bronchiectasis, infectious diseases, osteoporosis, and ankylosing spondylitis. In the propensity-score-matched populations, the associations between pSS and fibrocystic breast disease (aOR, 1.74; 95% CI, 1.58-1.91) remained consistent. Conclusion: The present population-based study revealed a previously unexplored association between pSS and history of fibrocystic breast disease, and the finding highlights the need to survey pSS in patients with mammary-gland-inflammation-associated diseases.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription-polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.