A new method for the sampling and preservation of placental specimens in low-resource settings for the identification of P. falciparum and analysis of nucleic acids.

Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope® hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater™ and RNA-seq.

Risk factors for placental malaria, sulfadoxine-pyrimethamine doses, and birth outcomes in a rural to urban prospective cohort study on the Bandiagara Escarpment and Bamako, Mali.

BACKGROUND: Malaria in Mali remains a primary cause of morbidity and mortality, with women at high risk during pregnancy for placental malaria (PM). Risk for PM and its association with birth outcomes was evaluated in a rural to urban longitudinal cohort on the Bandiagara Escarpment and the District of Bamako. METHODS: Placental samples (N = 317) were collected from 249 mothers who were participants in a prospective cohort study directed by BIS in the years 2011 to 2019. A placental pathologist and research assistant evaluated the samples by histology in blinded fashion to assess PM infection stage and parasite density. Generalized estimating equations (GEE) were used to model the odds of PM infection. RESULTS: In a multivariable model, pregnancies in Bamako, beyond secondary education, births in the rainy season (instead of the hot dry season), and births to women who had ≥ 3 doses of sulfadoxine-pyrimethamine (SP) instead of no doses were associated with reduced odds of experiencing PM (active and past infections combined). Births in later years of the study were strongly associated with reduced odds of PM. Maternal age, which was positively associated with offspring year of birth, was significant as a predictor of PM only if offspring year of birth was omitted from the model. Gravidity was positively associated with both maternal age and offspring year of birth such that if either variable was included in the model, then gravidity was no longer significant. However, if maternal age or year of offspring birth were not adjusted for, then the odds of PM were nearly two-fold higher in primigravida compared to multigravida. Birth outcomes improved (+ 285 g birth weight, + 2 cm birth length, + 75 g placental weight) for women who had ≥ 3 doses of SP compared to no doses, but no difference was detected in birth weight or length for women who had 2 instead of ≥ 3 SP doses. However, at 2 instead of ≥ 3 doses placentas were 36 g lighter and the odds of low birth weight (< 2500 g) were 14% higher. Severe parasite densities (> 10% erythrocytes infected) were significantly associated with decreases in birth weight, birth length, and placental weight, as were chronic PM infections. The women who received no SP during pregnancy (7% of the study total) were younger and lacked primary school education. The women who received ≥ 3 doses of SP came from more affluent families. CONCLUSIONS: Women who received no doses of SP during pregnancy experienced the most disadvantageous birth outcomes in both Bamako and on the Bandiagara Escarpment. Such women tended to be younger and to have had no primary school education. Targeting such women for antenatal care, which is the setting in which SP is most commonly administered in Mali, will have a more positive impact on public health than focusing on the increment from two to three doses of SP, although that increment is also desirable.

Targeted RNA-seq improves efficiency, resolution, and accuracy of allele specific expression for human term placentas.

Genomic imprinting is an epigenetic mechanism that results in allele-specific expression (ASE) based on the parent of origin. It is known to play a role in the prenatal and postnatal allocation of maternal resources in mammals. ASE detected by whole transcriptome RNA-seq (wht-RNAseq) has been widely used to analyze imprinted genes using reciprocal crosses in mice to generate large numbers of informative SNPs. Studies in humans are more challenging due to the paucity of SNPs and the poor preservation of RNA in term placentas and other tissues. Targeted RNA-seq (tar-RNAseq) can potentially mitigate these challenges by focusing sequencing resources on the regions of interest in the transcriptome. Here, we compared tar-RNAseq and wht-RNAseq in a study of ASE in known imprinted genes in placental tissue collected from a healthy human cohort in Mali, West Africa. As expected, tar-RNAseq substantially improved the coverage of SNPs. Compared to wht-RNAseq, tar-RNAseq produced on average four times more SNPs in twice as many genes per sample and read depth at the SNPs increased fourfold. In previous research on humans, discordant ASE values for SNPs of the same gene have limited the ability to accurately quantify ASE. We show that tar-RNAseq reduces this limitation as it unexpectedly increased the concordance of ASE between SNPs of the same gene, even in cases of degraded RNA. Studies aimed at discovering associations between individual variation in ASE and phenotypes in mammals and flowering plants will benefit from the improved power and accuracy of tar-RNAseq.

Loss of Imprinting in Human Placentas Is Widespread, Coordinated, and Predicts Birth Phenotypes.

Genomic imprinting leads to mono-allelic expression of genes based on parent of origin. Therian mammals and angiosperms evolved this mechanism in nutritive tissues, the placenta, and endosperm, where maternal and paternal genomes are in conflict with respect to resource allocation. We used RNA-seq to analyze allelic bias in the expression of 91 known imprinted genes in term human placentas from a prospective cohort study in Mali. A large fraction of the imprinted exons (39%) deviated from mono-allelic expression. Loss of imprinting (LOI) occurred in genes with either maternal or paternal expression bias, albeit more frequently in the former. We characterized LOI using binomial generalized linear mixed models. Variation in LOI was predominantly at the gene as opposed to the exon level, consistent with a single promoter driving the expression of most exons in a gene. Some genes were less prone to LOI than others, particularly lncRNA genes were rarely expressed from the repressed allele. Further, some individuals had more LOI than others and, within a person, the expression bias of maternally and paternally imprinted genes was correlated. We hypothesize that trans-acting maternal effect genes mediate correlated LOI and provide the mother with an additional lever to control fetal growth by extending her influence to LOI of the paternally imprinted genes. Limited evidence exists to support associations between LOI and offspring phenotypes. We show that birth length and placental weight were associated with allelic bias, making this the first comprehensive report of an association between LOI and a birth phenotype.

Oral contraceptives cause evolutionarily novel increases in hormone exposure: A risk factor for breast cancer.

Background and objectives: In the evolutionary past, women spent most of their reproductive lives either pregnant or in lactational amenorrhea, and rarely menstruated. The current pattern of frequent menses, and the associated increase in endogenous hormonal exposure, has been implicated in the current breast cancer epidemic. It is not known, however, whether oral contraceptives further increase, or actually decrease, hormonal exposure over one menstrual cycle. Here, we examined variation in hormonal exposure across seven oral contraceptive (OC) formulations, and produced the first quantitative comparison of exogenous versus endogenous hormone exposure. Methodology: Data from 12 studies of serum estradiol (E2) and progesterone (P4) were aggregated to create a composite graph of endogenous hormone levels over one menstrual cycle in European or American women (age 19-40 years). Pharmacokinetic package insert data, also from Western women, were used to calculate exposures for hormones in seven different OC formulations. Endogenous and exogenous hormone levels were compared after adjusting for the relative binding affinity (RBA) of progestin to the progesterone receptor and ethinyl estradiol (EE) to the estrogen receptor. Results: After adjusting for RBA, median ethinyl estradiol exposure across 28 days in the OCs was 11.4 nmol/l, similar to median E2 exposure. One formulation, however, was 40% higher in ethinyl estradiol exposure relative to median endogenous estradiol. Median exposure from progestins in OCs (1496 nmol/l) was 4-fold higher than the median endogenous exposure from P4 (364 nmol/l). Exposure from OC progestins ranged from one sixtieth to 8-fold median endogenous P4 over 28 days. Conclusions and implications: Given that breast cancer risk increases with hormonal exposure, our finding that four widely prescribed formulations more than quadruple progestin exposure relative to endogenous progesterone exposure is cause for concern. As not all formulations produce the same exposures, these findings are pertinent to contraceptive choice. We also identify critical gaps in the provision of relevant data on pharmacokinetics and carcinogenicity by drug manufacturers.

The blemishes of modern society? Acne prevalence in the Dogon of Mali.

BACKGROUND AND OBJECTIVES: Non-communicable diseases may reflect an evolutionary mismatch between our human ancestry and modern environments. To explore the mismatch hypothesis for Acne vulgaris, we studied the prevalence and severity of acne in Dogon adolescents in Mali, West Africa. METHODOLOGY: We graded the prevalence and severity of acne in 1182 Dogon adolescents aged 11-18 years from nine villages using facial photos taken as part of a prospective cohort study. Eighty-nine (89%) of the individuals in the cohort migrated to the city during adolescence, enabling us to assess the effect of urban migration. Using multivariable logistic regression, we estimated the effect of predictor variables on the presence of acne. RESULTS: The prevalence of acne in the cohort was 28%, with 90% of cases being mild or very mild. Thus, the prevalence and severity of acne was much lower than for adolescents in high-income countries. Controlling for age, puberty, and body mass index (BMI), the odds of boys developing acne was 85% lower in the city than in the villages (P = 0.002). CONCLUSION AND IMPLICATIONS: Acne is similar to the 'diseases of civilization' in being promoted by the pro-inflammatory properties of modern diets. The low prevalence and severity of acne in the Dogon supports the mismatch hypothesis and suggests that acne should join the list of diseases of modern lifestyles. However, we also observed an unexpected decrease in acne in urban boys. Future research is needed for a deeper mechanistic understanding of the interplay between diet, inflammation, immune function and other environmental exposures that differ between urban and rural environments.

Religion as a means to assure paternity.

The sacred texts of five world religions (Buddhism, Christianity, Hinduism, Islam, and Judaism) use similar belief systems to set limits on sexual behavior. We propose that this similarity is a shared cultural solution to a biological problem: namely male uncertainty over the paternity of offspring. Furthermore, we propose the hypothesis that religious practices that more strongly regulate female sexuality should be more successful at promoting paternity certainty. Using genetic data on 1,706 father-son pairs, we tested this hypothesis in a traditional African population in which multiple religions (Islam, Christianity, and indigenous) coexist in the same families and villages. We show that the indigenous religion enables males to achieve a significantly (P = 0.019) lower probability of cuckoldry (1.3% versus 2.9%) by enforcing the honest signaling of menstruation, but that all three religions share tenets aimed at the avoidance of extrapair copulation. Our findings provide evidence for high paternity certainty in a traditional African population, and they shed light on the reproductive agendas that underlie religious patriarchy.

Cooperation and competition in a cliff-dwelling people.

In animals that breed cooperatively, adult individuals will sometimes delay reproduction to act as helpers at the nest who raise young that are not their genetic offspring. It has been proposed that humans are also a cooperatively breeding species because older daughters, grandmothers, and other kin and nonkin may provide significant childcare. Through a prospective cohort study of children's (n = 1,700) growth and survival in the Dogon of Mali, I show that cooperative breeding theory is a poor fit to the family dynamics of this population. Rather than helping each other, siblings competed for resources, producing a tradeoff between the number of maternal siblings and growth and survival. It did not take a village to raise a child; children fared the same in nuclear as in extended families. Of critical importance was the degree of polygyny, which created conflicts associated with asymmetries in genetic relatedness. The risk of death was higher and the rate of growth was slower in polygynous than monogamous families. The hazard of death for Dogon children was twofold higher if the resident paternal grandmother was alive rather than dead. This finding may reflect the frailty of elderly grandmothers who become net consumers rather than net producers in this resource-poor society. Mothers were of overwhelming importance for child survival and could not be substituted by any category of kin or nonkin. The idea of cooperative breeding taken from animal studies is a poor fit to the complexity and diversity of kin interactions in humans.

Alternatives to the grandmother hypothesis: a meta-analysis of the association between grandparental and grandchild survival in patrilineal populations.

We conducted a meta-analysis of 17 studies that tested for an association between grandparental survival and grandchild survival in patrilineal populations. Using two different methodologies, we found that the survival of the maternal grandmother and grandfather, but not the paternal grandmother and grandfather, was associated with decreased grandoffspring mortality. These results are consistent with the findings of psychological studies in developed countries (Coall and Hertwig Behavioral and Brain Sciences 33:1-59, 2010). When tested against the predictions of five hypotheses (confidence of paternity; grandmothering, kin proximity, grandparental senescence, and local resource competition), our meta-analysis results are most in line with the local resource competition hypothesis. In patrilineal and predominantly patrilocal societies, the grandparents who are most likely to live with the grandchildren have a less beneficial association than those who do not. We consider the extent to which these results may be influenced by the methodological limitations of the source studies, including the use of retrospective designs and inadequate controls for confounding variables such as wealth.

Testing for archaic hominin admixture on the X chromosome: model likelihoods for the modern human RRM2P4 region from summaries of genealogical topology under the structured coalescent.

A 2.4-kb stretch within the RRM2P4 region of the X chromosome, previously sequenced in a sample of 41 globally distributed humans, displayed both an ancient time to the most recent common ancestor (e.g., a TMRCA of approximately 2 million years) and a basal clade composed entirely of Asian sequences. This pattern was interpreted to reflect a history of introgressive hybridization from archaic hominins (most likely Asian Homo erectus) into the anatomically modern human genome. Here, we address this hypothesis by resequencing the 2.4-kb RRM2P4 region in 131 African and 122 non-African individuals and by extending the length of sequence in a window of 16.5 kb encompassing the RRM2P4 pseudogene in a subset of 90 individuals. We find that both the ancient TMRCA and the skew in non-African representation in one of the basal clades are essentially limited to the central 2.4-kb region. We define a new summary statistic called the minimum clade proportion (pmc), which quantifies the proportion of individuals from a specified geographic region in each of the two basal clades of a binary gene tree, and then employ coalescent simulations to assess the likelihood of the observed central RRM2P4 genealogy under two alternative views of human evolutionary history: recent African replacement (RAR) and archaic admixture (AA). A molecular-clock-based TMRCA estimate of 2.33 million years is a statistical outlier under the RAR model; however, the large variance associated with this estimate makes it difficult to distinguish the predictions of the human origins models tested here. The pmc summary statistic, which has improved power with larger samples of chromosomes, yields values that are significantly unlikely under the RAR model and fit expectations better under a range of archaic admixture scenarios.

Contrasting signatures of population growth for mitochondrial DNA and Y chromosomes among human populations in Africa.

A history of Pleistocene population expansion has been inferred from the frequency spectrum of polymorphism in the mitochondrial DNA (mtDNA) of many human populations. Similar patterns are not typically observed for autosomal and X-linked loci. One explanation for this discrepancy is a recent population bottleneck, with different rates of recovery for haploid and autosomal loci as a result of their different effective population sizes. This hypothesis predicts that mitochondrial and Y chromosomal DNA will show a similar skew in the frequency spectrum in populations that have experienced a recent increase in effective population size. We test this hypothesis by resequencing 6.6 kb of noncoding Y chromosomal DNA and 780 basepairs of the mtDNA cytochrome c oxidase subunit III (COIII) gene in 172 males from 5 African populations. Four tests of population expansion are employed for each locus in each population: Fu's Fs statistic, the R(2) statistic, coalescent simulations, and the mismatch distribution. Consistent with previous results, patterns of mtDNA polymorphism better fit a model of constant population size for food-gathering populations and a model of population expansion for food-producing populations. In contrast, none of the tests reveal evidence of Y chromosome growth for either food-gatherers or food-producers. The distinct mtDNA and Y chromosome polymorphism patterns most likely reflect sex-biased demographic processes in the recent history of African populations. We hypothesize that males experienced smaller effective population sizes and/or lower rates of migration during the Bantu expansion, which occurred over the last 5,000 years.

Cortisol levels and very early pregnancy loss in humans.

Maternal stress is commonly cited as an important risk factor for spontaneous abortion. For humans, however, there is little physiological evidence linking miscarriage to stress. This lack of evidence may be attributable to a paucity of research on maternal stress during the earliest gestational stages. Most human studies have focused on "clinical" pregnancy (>6 weeks after the last menstrual period). The majority of miscarriages, however, occur earlier, within the first 3 weeks after conception (approximately 5 weeks after the last menstrual period). Studies focused on clinical pregnancy thus miss the most critical period for pregnancy continuance. We examined the association between miscarriage and levels of maternal urinary cortisol during the first 3 weeks after conception. Pregnancies characterized by increased maternal cortisol during this period (within participant analyses) were more likely to result in spontaneous abortion (P < 0.05). This evidence links increased levels in this stress marker with a higher risk of early pregnancy loss in humans.

Contrasting patterns of Y chromosome and mtDNA variation in Africa: evidence for sex-biased demographic processes.

To investigate associations between genetic, linguistic, and geographic variation in Africa, we type 50 Y chromosome SNPs in 1122 individuals from 40 populations representing African geographic and linguistic diversity. We compare these patterns of variation with those that emerge from a similar analysis of published mtDNA HVS1 sequences from 1918 individuals from 39 African populations. For the Y chromosome, Mantel tests reveal a strong partial correlation between genetic and linguistic distances (r=0.33, P=0.001) and no correlation between genetic and geographic distances (r=-0.08, P>0.10). In contrast, mtDNA variation is weakly correlated with both language (r=0.16, P=0.046) and geography (r=0.17, P=0.035). AMOVA indicates that the amount of paternal among-group variation is much higher when populations are grouped by linguistics (Phi(CT)=0.21) than by geography (Phi(CT)=0.06). Levels of maternal genetic among-group variation are low for both linguistics and geography (Phi(CT)=0.03 and 0.04, respectively). When Bantu speakers are removed from these analyses, the correlation with linguistic variation disappears for the Y chromosome and strengthens for mtDNA. These data suggest that patterns of differentiation and gene flow in Africa have differed for men and women in the recent evolutionary past. We infer that sex-biased rates of admixture and/or language borrowing between expanding Bantu farmers and local hunter-gatherers played an important role in influencing patterns of genetic variation during the spread of African agriculture in the last 4000 years.

Stress and female reproductive function: a study of daily variations in cortisol, gonadotrophins, and gonadal steroids in a rural Mayan population.

We report here on a longitudinal study of stress and women's reproduction in a small Kaqchikel Mayan community in rural Guatemala. Current understanding of the effects of stress on the reproductive axis in women is mostly derived from clinical studies of individual stressors. Little is known, however, about the cumulative effects of "real life" stress. Cortisol increases in response to a broad variety of individual stressors (Tilbrook et al., 2002). In this article, we evaluate the association between daily fluctuations in women's urinary cortisol and reproductive hormones: estrone conjugates (E(1)C), pregnandiol glucuronide (PdG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). To assess the association between daily changes in cortisol levels and changes in the profiles of the reproductive hormones, we used a random coefficients model based on polynomial regression. The sample includes 92 menstrual cycles provided by 24 participants over a year-long prospective study. Increases in urinary cortisol levels were associated with significant increases in gonadotrophin and progestin levels during the follicular phase. Also, in a time window between days 4 and 10 after ovulation, increased cortisol levels were associated with significantly lower progestin levels. These results are significant because untimely increases in gonadotrophins and low midluteal progesterone levels have previously been reported to impinge on the ovulatory and luteinization processes and to reduce the chances of successful implantation (Ferin, 1999; Baird et al., 1999). Future research should consider the possibility that stress may affect fecundability and implantation without necessarily causing amenorrhoea or oligomenorrhoea.

Water sources are associated with childhood diarrhoea prevalence in rural east-central Mali.

BACKGROUND: Water supply improvements generally reduce the incidence of diarrhoea. However, populations with limited access to a safe water supply may continue to draw water from unimproved sources, thereby increasing their risk of diarrhoea. Furthermore, young children who are not breastfed may be even more susceptible to water-borne diarrhoeal pathogens. Our study explored the interactive protective effects against diarrhoea of exclusively using improved water sources and breastfeeding among children in rural Mali. METHODS: Interviews were conducted with parents or guardians of children under 7 years of age in seven villages with access to a variety of water supplies. Water sources used, breastfeeding status, demographics and recent diarrhoea symptoms were determined for 1117 children. The cross-sectional findings were used to compare diarrhoea prevalence among exclusive and non-exclusive users of improved water sources. Variation in prevalence by age and exclusive breastfeeding status was evaluated using chi-square and multivariate analyses. RESULTS: Children whose water was drawn exclusively from wells had a significantly lower prevalence of diarrhoea as compared with children whose water was drawn from a spring or stream (5.9% vs. 8.7%; P=0.04). The exclusive use of improved water sources had no impact on diarrhoea prevalence among children who were exclusively breastfed. Similarly, the strongest protective effect was observed among children who were not exclusively breastfed. CONCLUSIONS: Our results indicate that using surface water as a primary or secondary water source exposes children to greater risk of diarrhoeal disease than using only improved sources such as wells. It is particularly beneficial for young children who are not exclusively breastfed to be supplied with water drawn from improved sources.

How to measure reproductive success?

To date there have been few empirical comparisons between alternative methods for measuring reproductive success (RS). We consider the pros and cons of alternative measures of RS to provide guidance for the design of field studies in human behavioral ecology. We compare cross-sectional measures that count offspring alive at the time of the interview and retrospective measures that require data on offspring age at death or censoring. We consider analyses that include adult women (yielding age-specific estimates of RS) as well as analyses restricted to postreproductive women (yielding data on lifetime RS). These methods are applied to reproductive data for the Dogon of Mali, West Africa.

Life-history theory, fertility and reproductive success in humans.

According to life-history theory, any organism that maximizes fitness will face a trade-off between female fertility and offspring survivorship. This trade-off has been demonstrated in a variety of species, but explicit tests in humans have found a positive linear relationship between fitness and fertility. The failure to demonstrate a maximum beyond which additional births cease to enhance fitness is potentially at odds with the view that human fertility behaviour is currently adaptive. Here we report, to our knowledge, the first clear evidence for the predicted nonlinear relationship between female fertility and reproductive success in a human population, the Dogon of Mali, West Africa. The predicted maximum reproductive success of 4.1+/-0.3 surviving offspring was attained at a fertility of 10.5 births. Eighty-three per cent of the women achieved a lifetime fertility level (7-13 births) for which the predicted mean reproductive success was within the confidence limits (3.4 to 4.8) for reproductive success at the optimal fertility level. Child mortality, rather than fertility, was the primary determinant of fitness. Since the Dogon people are farmers, our results do not support the assumptions that: (i) contemporary foragers behave more adaptively than agriculturalists, and (ii) that adaptive fertility behaviour ceased with the Neolithic revolution some 9000 years ago. We also present a new method that avoids common biases in measures of reproductive success.