4.1. Sampling

Breast milk samples were collected in winter, at two times: the first between August and September 2018, and the second in August 2019. In total, 37 samples were obtained from lactating mothers living in the municipality of Belize in the province of Cabinda, an enclave in the Congo north of the rest of Angolan territory. Samples were obtained at the Health Center, and milk collection was performed by manual extraction, with the help of a nurse, in sterile storage bags. After this collection, the samples were immediately frozen at −20 °C and preserved at a constant temperature and protected from direct light throughout the transport. These conditions were maintained until their analysis in the Laboratory of Cellular and Molecular Biology of the Escola Universitária Vasco da Gama, in Coimbra.

The participants included in this study met the following criteria: breastfeeding without any record of systemic or breast disease, a signed informed consent form, and a completed questionnaire. The defined exclusion criteria were: under 18 years of age and delivery within the last month (to avoid the analysis of colostrum or transitional milk).

The procedures of this work were developed in accordance with the Declaration of Helsinki of the World Medical Association (WMA) [55], the Declaration of Taipei of the WMA on ethical considerations related to health databases and biobanks [56], and also the Oviedo Convention for the Protection of Human Rights and Human Dignity in the Face of Biology and Medicine [57]. This study was approved by the Scientific Council of the Faculty of Pharmacy of the University of Coimbra and by the Ethics and Animal Welfare Committee (CEBEA) of the Vasco da Gama University School (opinion n° 2019/001). The collection of samples was also authorized by the Directorate of the Hospital of Belize, the Municipal Health Department of Belize, and the Provincial Health Department in Cabinda. Participants voluntarily entered this study after signing the informed consent form, in which the objectives and procedures of the study were clarified, and the confidentiality and anonymity of the collected data were guaranteed.