The proposed HBM4EU effort to assess human adverse health effects due to environmental chemicals exposure using biomarkers of effect, has continued with the implementation of these indicators, as described in D14.8. In the HBM4EU aligned studies, a broad panel of classical and novel biomarkers of effect (e.g. brain-derived neurotrophic function, kisspeptin, reproductive hormones, thyroid hormones, metabolic markers, oxidative stress markers, among others) has been implemented. However, some proposed biomarkers have not been validated and implemented, such as leptin and adiponectin. These important cytokines play a key role in the proper development of metabolic and reproductive function. Additionally, the relationships between exposure and effect biomarkers data in the HBM4EU aligned studies have also not been reported. Therefore, the aims of this deliverable were to show: i) the quality control and quality analysis of leptin and adiponectin, as well as their measured concentrations in the aligned studies; ii) the physiological validation of kisspeptin (serum protein concentrations, kiss54, and whole blood DNA methylation of the KISS1 gene), in a pilot study within the INMA-Granada cohort, to investigate its suitability as effect biomarker of the reproductive function prior to their application in HBM4EU aligned studies; and iii) the preliminary results of the exposure-effect analyses conducted in the Aligned studies. For this purpose, leptin and adiponectin protein levels were measured using different commercial enzymelinked immunosorbent assays (ELISA). The new quality control procedure for leptin measurements showed that the chosen ELISA kit was a reproducible and confident analytical technique. Adiponectin was then measured in adolescent’s serum samples from the aligned studies, including the FLEHS, PCB, BEA, and SLO CRP studies; as well as in children from the SLO CRP study. Leptin concentrations were also measured in adolescents from the FLEHS studies and in children from the SLO CRP studies. On the other hand, preliminary data with INMA-Granada blood and serum samples demonstrate the physiological feasibility of implementing new biomarkers of reproductive function, such as KISS, at different levels of biological organisation. The results showed that percentages of KISS1 DNA methylation were inversely correlated with the serum concentrations of kiss54 protein, as well as with the serum concentrations of the selected reproductive hormones (FSH, estradiol, testosterone, SHBG). Moreover, levels of kiss54 were positively correlated with all reproductive hormones. Thus, the pilot study with samples from INMA-Granada demonstrated the technical feasibility of implementing kisspeptin at different levels of biological organisation to assess the reproductive function in the HBM4EU aligned studies. Finally, when the kisspeptin effect biomarker (serum kiss54) data were analysed together with serum concentrations of reproductive hormones in the aligned studies, consistent results with the findings described in the pilot study were found, indicating that the novel effect biomarker KISS is a valuable marker of reproductive function useful for assessing possible alterations caused by environmental pollutants.