Pablo A. Nepomnaschy

Evolutionary functions of early social modulation of hypothalamic-pituitary-adrenal axis development in humans.

The hypothalamic-pituitary-adrenal axis (HPAA) is highly responsive to social challenges. Because stress hormones can have negative developmental and health consequences, this presents an evolutionary paradox: Why would natural selection have favored mechanisms that elevate stress hormone levels in response to psychosocial stimuli? Here we review the hypothesis that large brains, an extended childhood and intensive family care in humans are adaptations resulting from selective forces exerted by the increasingly complex and dynamic social and cultural environment that co-evolved with these traits. Variations in the modulation of stress responses mediated by specific HPAA characteristics (e.g., baseline cortisol levels, and changes in cortisol levels in response to challenges) are viewed as phenotypically plastic, ontogenetic responses to specific environmental signals. From this perspective, we discuss relations between physiological stress responses and life history trajectories, particularly the development of social competencies. We present brief summaries of data on hormones, indicators of morbidity and social environments from our long-term, naturalistic studies in both Guatemala and Dominica. Results indicate that difficult family environments and traumatic social events are associated with temporal elevations of cortisol, suppressed reproductive functioning and elevated morbidity. The long-term effects of traumatic early experiences on cortisol profiles are complex and indicate domain-specific effects, with normal recovery from physical stressors, but some heightened response to negative-affect social challenges. We consider these results to be consistent with the hypothesis that developmental programming of the HPAA and other neuroendocrine systems associated with stress responses may facilitate cognitive targeting of salient social challenges in specific environments.

Is Cortisol Excretion Independent of Menstrual Cycle Day? A Longitudinal Evaluation of First Morning Urinary Specimens

Background Cortisol is frequently used as a marker of physiologic stress levels. Using cortisol for that purpose, however, requires a thorough understanding of its normal longitudinal variability. The current understanding of longitudinal variability of basal cortisol secretion in women is very limited. It is often assumed, for example, that basal cortisol profiles do not vary across the menstrual cycle. This is a critical assumption: if cortisol were to follow a time dependent pattern during the menstrual cycle, then ignoring this cyclic variation could lead to erroneous imputation of physiologic stress. Yet, the assumption that basal cortisol levels are stable across the menstrual cycle rests on partial and contradictory evidence. Here we conduct a thorough test of that assumption using data collected for up to a year from 25 women living in rural Guatemala. Methodology We apply a linear mixed model to describe longitudinal first morning urinary cortisol profiles, accounting for differences in both mean and standard deviation of cortisol among women. To that aim we evaluate the fit of two alternative models. The first model assumes that cortisol does not vary with menstrual cycle day. The second assumes that cortisol mean varies across the menstrual cycle. Menstrual cycles are aligned on ovulation day (day 0). Follicular days are assigned negative numbers and luteal days positive numbers. When we compared Models 1 and 2 restricting our analysis to days between −14 (follicular) and day 14 (luteal) then day of the menstrual cycle did not emerge as a predictor of urinary cortisol levels (p-value >0.05). Yet, when we extended our analyses beyond that central 28-day-period then day of the menstrual cycle become a statistically significant predictor of cortisol levels. Significance The observed trend suggests that studies including cycling women should account for day dependent variation in cortisol in cycles with long follicular and luteal phases.

Validation of a New Multiplex Assay Against Individual Immunoassays for the Quantification of Reproductive, Stress, and Energetic Metabolism Biomarkers in Urine Specimens

 Measuring multiple hormones simultaneously in a single assay saves sample volume, labor, time, reagents, money, and consumables. Thus, multiplex arrays represent a faster, more economically and ecologically sound alternative to singleton assays.

Who is stressed? Comparing cortisol levels between individuals

Cortisol is the most commonly used biomarker to compare physiological stress between individuals. Its use, however, is frequently inappropriate. Basal cortisol production varies markedly between individuals. Yet, in naturalistic studies that variation is often ignored, potentially leading to important biases.

Number of Children and Telomere Length in Women: A Prospective, Longitudinal Evaluation.

Life history theory (LHT) predicts a trade-off between reproductive effort and the pace of biological aging. Energy invested in reproduction is not available for tissue maintenance, thus having more offspring is expected to lead to accelerated senescence. Studies conducted in a variety of non-human species are consistent with this LHT prediction. Here we investigate the relationship between the number of surviving children born to a woman and telomere length (TL, a marker of cellular aging) over 13 years in a group of 75 Kaqchikel Mayan women. Contrary to LHT’s prediction, women who had fewer children exhibited shorter TLs than those who had more children (p = 0.045) after controlling for TL at the onset of the 13-year study period. An “ultimate” explanation for this apparently protective effect of having more children may lay with human’s cooperative-breeding strategy. In a number of socio-economic and cultural contexts, having more chilren appears to be linked to an increase in social support for mothers (e.g., allomaternal care). Higher social support, has been argued to reduce the costs of further reproduction. Lower reproductive costs may make more metabolic energy available for tissue maintenance, resulting in a slower pace of cellular aging. At a “proximate” level, mechanisms involved may include the actions of the gonadal steroid estradiol, which increases dramatically during pregnancy. Estradiol is known to protect TL from the effects of oxidative stress as well as increase telomerase activity, an enzyme that maintains TL. Future research should explore the potential role of social support as well as that of estradiol and other potential biological pathways in the trade-offs between reproductive effort and the pace of cellular aging within and among human as well as in non-human populations.

A longitudinal evaluation of the relationship between first morning urinary and salivary cortisol

Cortisol is one of the most frequently used stress biomarkers in humans. Urine and saliva are the matrices of choice to longitudinally monitor cortisol levels. Salivary and urinary cortisol are often discussed as though they provide similar information. However, the relationship between “free” cortisol levels in urine (nonconjugated) and saliva (non‐protein‐bound) has yet to be properly evaluated using naturalistic designs.

Evolutionary endocrinology: Integrating proximate mechanisms, ontogeny, and evolved function

Natural selection leads to the evolution of strategies that tend to optimize the allocation of resources to the competing somatic and reproductive demands faced by any organism. This theoretical approach, known as life history theory, is useful for understanding unavoidable trade-offs in resource allocation and the resulting variation in phenotypes (e.g., physical attributes, expressed behaviors, the number and size of offspring, etc.) (Charnov 1993; Stearns 1992). In recent years, life history theory has begun to influence research on the proximate mechanisms mediating ontogenetic strategies. The endocrine system plays a central role in coordinating ontogenetic processes and modulating cross-talk among different functional domains (e.g., hypothalamic–pituitary–gonadal and hypothalamic–pituitary–adrenal axes). These processes are integral to the evolution of phenotypic plasticity (a key feature of adaptive responses) in developmental schedules and other life-history traits. From conception to death, hormones orchestrate growth, differentiation, maintenance, reproduction, and senescence. Pheromones may help parents-to-be to attract each other (Kohl et al., 2001; Hays, 2003). Androgen steroids increase libido encouraging them to mate (Hutchinson, 1995; Stern and McClintock, 1998). Gonadotrophins and gonadal steroids mediate gamete maturation and release. Endometrial steroids intervene in the capacitation of spermatozoa and chemotaxis, luring them towards the oocyte. Recognition, binding, and fusion between sperm and oocyte, and the consequent activation of the egg are mediated by a multitude of hormones (Sun et al., 2005). After fertilization, but before development of the endocrine system, chemical signals help guide cleavage, cell differentiation, migration, and growth. Once developed, the endocrine system transmits information between cells regarding internal and external conditions, coordinating a intricate dance between the epigenome and the genome, steering the organism through its ontogenetic road map. All organisms of a given species follow a similar ontogenetic road map. Yet, as environmental opportunities, restrictions, and challenges are heterogeneously distributed between and within populations, each individual passes life-history mileposts at its own speed and according to its own strategy. The endocrine system is an integral part of the mechanisms that provide organisms with the plasticity and dynamism that are crucial for adapting to their individual contexts. The challenges to be met are broad in type and temporal scales. Some challenges involve almost all biologic systems and take place over long periods of time, such as coordinating proportionate growth or undergoing sexual maturation. Others involve fewer tissues but need to be achieved within minutes or hours, such as adjusting oxygen or blood glucose levels. Some challenges are predictable, such as the sequences of night and day, or the succession of changes that follow conception. Other challenges, such as a tornado or an aneurysm, may be less predictable. Organisms rely on the endocrine system to regulate responses to these challenges as well as to inform ontogenetic strategies based on them. When and how much to grow at each life stage, when to begin sexual maturation, when to stop growing, the timing of the first reproductive venture, the length of inter-birth intervals, and when to stop reproducing are just a few examples of critical life history ‘‘decisions’’ mediated by hormonal pathways. Ontogenetic strategies are modified through neuroendocrine cues that trigger changes in gene expression. Phenotypic plasticity is ultimately achieved by changes in DNA methylation patterns and chromatin remodeling. These epigenetic changes provide great dynamism to genotypic expression. Recent research developments suggest that while some epigenetic DNA methylation patterns are preserved through multiple generations, others are not as stable as originally believed and can change in response to postnatal environmental stimuli at different times within an individual’s life span (McGowan et al., 2008; Szyf et al., 2008). The epigenome, informed by neuro-endocrine cues, is critical for fine tuning the expression of the more static genotype in response to the demands that the environment places on the phenotype. Given the dynamic and complex nature of the endocrine system, a holistic approach to its study should consider not only the longitudinal variation of hormones across days, seasons, and life stages, but also its interactions with the nervous system and the epigenome. Anthropologists are in the privileged position necessary for conducting the multidisciplinary research that will help to realize this admittedly daunting research agenda. The collection of high quality longitudinal data achieved through an intimate knowledge of study participants allows for the description of normal ranges of endocrine function and its variability in response to environmental challenges in nonclinical populations exposed to a broad range of social, economic, and physical contexts. The 2008 Plenary Session of the Human Biology Association explored recent advances in the study of endocrine pathways approached from this holistic perspective. The

The ex-pat effect: presence of recent Western immigrants is associated with changes in age at first birth and birth rate in a Maya population from rural Guatemala

Abstract Background: Economic transitions expose indigenous populations to a variety of ecological and cultural challenges, especially regarding diet and stress. These kinds of challenges are predicted by evolutionary ecological theory to have fitness consequences (differential reproduction) and, indeed, are often associated with changes in fertility dynamics. It is currently unclear whether international immigration might impact the nature of such an economic transition or its consequences for fertility. Aim: To examine measures of fertility, diet and stress in two economically transitioning Maya villages in Guatemala that have been differentially exposed to immigration by Westerners. Subjects and methods: This study compared Maya women’s ages at first birth and birth rates between villages and investigated whether these fertility indicators changed through time. It also explored whether the villages differed in relation to diet and/or a proxy of stress. Results: It was found that, in the village directly impacted by immigration, first births occurred earlier, but birth rate was slower. In both villages, over the sampled time window, age at first birth increased, while birth rate decreased. The villages do not differ significantly in dietary indicators, but the immigration-affected village scored higher on the stress proxy. Conclusion: Immigration can affect fertility in host communities. This relationship between immigration and fertility dynamics may be partly attributable to stress, but this possibility should be evaluated prospectively in future research.

Child mortality, hypothalamic-pituitary-adrenal axis activity and cellular aging in mothers

Psychological challenges, including traumatic events, have been hypothesized to increase the age-related pace of biological aging. Here we test the hypothesis that psychological challenges can affect the pace of telomere attrition, a marker of cellular aging, using data from an ongoing longitudinal-cohort study of Kaqchikel Mayan women living in a population with a high frequency of child mortality, a traumatic life event. Specifically, we evaluate the associations between child mortality, maternal telomere length and the mothers’ hypothalamic-pituitary-adrenal axis (HPAA), or stress axis, activity. Child mortality data were collected in 2000 and 2013. HPAA activity was assessed by quantifying cortisol levels in first morning urinary specimens collected every other day for seven weeks in 2013. Telomere length (TL) was quantified using qPCR in 55 women from buccal specimens collected in 2013. Results: Shorter TL with increasing age was only observed in women who experienced child mortality (p = 0.015). Women with higher average basal cortisol (p = 0.007) and greater within-individual variation (standard deviation) in basal cortisol (p = 0.053) presented shorter TL. Non-parametric bootstrapping to estimate mediation effects suggests that HPAA activity mediates the effect of child mortality on TL. Our results are, thus, consistent with the hypothesis that traumatic events can influence cellular aging and that HPAA activity may play a mediatory role. Future large-scale longitudinal studies are necessary to confirm our results and further explore the role of the HPAA in cellular aging, as well as to advance our understanding of the underlying mechanisms involved.

High sensitivity fluorescence detection using smart phone cameras

A low cost and highly sensitive fluorescence detection system using smart phone cameras was developed for biological and biochemical detection experiments. The system was designed for standard 96-well plates, which are typically used in life-sciences laboratories. Traditional fluorescence detection is done using specialized optics, filters and ultra-sensitive detectors such as photo-multipliers, which makes the system expensive. In our method, the 96-well plate is imaged using a smart phone camera inside a light tight enclosure. No specialized filters are used for the imaging. The image is then analysed using an algorithm, developed by us, that separates out the Red-Green-Blue (RGB) component. The Green component is then further processed to extract the fluorescence intensity. The developed hardware system and the algorithm were tested using two types of samples, fluorescein and Green Fluorescent Protein (GFP) incorporated yeast cells, prepared in varied concentrations. The performance of the developed system was compared with measurements taken using a PerkinElmer VICTOR™ X5, 2030 Multilabel Reader. Our system is capable of reliably detecting 1 nM concentrations of fluorescein. We believe the system can be improved further to detect even lower concentrations.