Joy F. Stallings

Hormone Measures in Finger-Prick Blood Spot Samples: New Field Methods for Reproductive Endocrinology

Comparative endocrine studies have notably advanced understanding of ecological factors that contribute to variation in human reproductive function. Such research has relied on methodological advances that permit hormone determinations in samples that are easily and safely collected, stored, and transported, most recently on measurement of steroids in saliva. This report seeks to further expand the scope of endocrine research by demonstrating the value of blood spot samples collected by finger prick. As a sampling strategy, finger-prick blood spot collection offers the advantages of short collection time, low invasiveness, repeatability, absence of postcollection processing, low biohazard risk, and ease of sample storage and transport. We document good sample stability and present sensitive assay methods for a range of steroids and proteins (FSH, LH, PRL, T, E2, DHEAS, androstenedione, cortisol, SHGB) in blood spots that require sample volumes of 3-12 microliters and display good reliability, specificity, precision, accuracy, and convertibility of results to plasma/serum equivalent concentrations. Laboratory evaluation was augmented by a feasibility study at a remote site in Papua New Guinea that confirmed validity and stability of blood spot collections under field conditions. Research applications of blood spot sampling are illustrated with a series of studies, including cross-sectional surveys for developmental and life span endocrinology, a longitudinal, population-based developmental epidemiologic study of puberty, and serial sampling in a dynamic study of neuroendocrine response to suckling. We conclude that the sampling features and wide range of measurable biomolecules of blood spots do constitute a methodological advance for endocrine research.

Epstein-barr virus antibodies in whole blood spots: A minimally invasive method for assessing an aspect of cell-mediated immunity

Objective Study 1: Introduce and validate a method for measuring EBV p18-VCA antibodies in whole blood spots to provide a minimally invasive marker of cell-mediated immune function. Study 2: Apply this method to a large community-based study of psychopathology in children and adolescents. Methods The EBV antibody method was evaluated through analysis of precision, reliability, stability, and comparisons with plasma and indirect immunofluorescence methods. The effects of life events on p18-VCA antibody level were considered in a subsample of 9, 11, and 13 year-old children participating in the Great Smoky Mountains Study in North Carolina. The subsample was stratified by age, sex, and degree of overall life strain. Results Dried blood spots provided a convenient, sensitive, precise, and reliable method for measuring EBV p18-VCA antibody titer. Life events were positively associated with p18-VCA antibodies in girls but not in boys. Conclusions The validity of the blood spot EBV p18-VCA antibody assay, as well as the ease of sample collection, storage, and transportation, may provide an opportunity for psychoneuroimmunology to explore a wider range of stress models in larger, community-based studies.

Germ cell loss in the XXY male mouse: Altered X-chromosome dosage affects prenatal development

Male mammals with two X chromosomes are sterile due to the demise of virtually all germ cells; however, the underlying reasons for the germ cell loss remain unclear. The use of a breeding scheme for the production of XXY male mice has allowed us to experimentally address the question of when and why germ cells die in the XXY testis and whether the defect is due to the presence of an additional X chromosome in the soma, the germ cells themselves, or both. Our studies demonstrate that altered X‐chromosome dosage acts to impair germ cell development in the testis at a much earlier stage than suggested by previous studies of XX sex‐reversed males or XX/XY chimeras. Specifically, we noted significantly reduced germ cell numbers in the XXY testis during the period of germ cell proliferation in the early stages of testis differentiation. Although the somatic development of the XXY testis is morphologically and temporally normal, our studies indicate that germ cell demise reflects a defect in somatic/germ cell communication, since, in an in vitro system, the proliferative potential of fetal germ cells from XXY males is indistinguishable from that of normal males. Mol. Reprod. Dev. 49:101–111, 1998.

Culture Change and Stress in Western Samoan Youth: Methodological Issues in the Cross-Cultural Study of Stress and Immune Function †

This study was designed to pursue three objectives: 1) investigate the impact of culture change on children and adolescents in Western Samoa; 2) introduce a cross‐cultural perspective to studies of psychosocial stress and immune function; and 3) evaluate the utility of minimally invasive methods for assessing immune function. Seven hundred sixty individuals between the ages of 4 and 20 years were recruited from three distinct geographic regions within Western Samoa that differ in degree of westernization. Finger prick samples of whole blood were collected from each individual and analyzed for antibodies against the Epstein‐Barr virus (EBV; an indirect marker of cell‐mediated immune function) and C‐reactive protein (a nonspecific marker of current infection). After controlling for age, sex, and current infection, EBV antibody levels were significantly elevated in urban Apia and rural Upolu, indicating lower levels of cell‐mediated immune function. The results suggest a higher degree of psychosocial stress in these regions, possibly due to exposure to westernizing influences. Am. J. Hum. Biol. 12:792–802, 2000.

Attenuation of nursing-related ovarian suppression and high fertility in well-nourished, intensively breast-feeding Amele women of lowland Papua New Guinea

Intense, sustained nursing lengthens inter-birth intervals and is causally linked with low natural fertility. However, in traditional settings, the effects of such nursing on fertility are difficult to disentangle from those of nutrition. Results from a prospective, direct observational study of reproductive function in well-nourished Amele women who nurse intensively and persistently but who also have high fertility are here presented. Endocrine measures show that ovarian activity resumes by median 11.0 months postpartum. Median duration of postpartum amenorrhoea is 11.3 months, time to next conception is 19.0 months, and the inter-birth interval is 28.0 months. Average life time fertility is 6.8. High fertility in Amele women is due both to refractoriness of reproductive function to suckling stimuli, and to maintenance of equivalent age-specific fertility rates across the reproductive life span.

Ratios of plasma and salivary testosterone throughout puberty: Production versus bioavailability

Because diffusion of testosterone (T) into the salivary gland is thought to be largely limited to the free, biologically active fraction, salivary testosterone is expected to provide a better measure of testosterone bioavailability in the body than is plasma testosterone. Matched saliva and blood spot samples were collected from 218 Zimbabwean males (age 11-23) who were at different stages of puberty, as assessed by self-reported Tanner genital stage ratings. Testosterone concentrations in these matched samples were highly correlated (r = 0.83). Both salivary and plasma testosterone (converted from blood spot value) showed expected significant increases across puberty. However, plasma testosterone distinguished among subjects at different stages of genital development more effectively than did salivary testosterone, suggesting the former to be a better marker of testosterone bioavailability. Sex hormone-binding globulin (SHBG) levels were also measured in a subgroup of 93 of these subjects. After controlling for plasma T concentrations, we found a small but significant inverse correlation between blood spot SHBG levels and the proportion of plasma testosterone recovered in salvia, supporting the hypothesis that SHBG-related changes in T bioavailability are detectable in saliva. We conclude that salivary testosterone accurately reflects testicular production of testosterone, but that neither salivary testosterone nor plasma testosterone is clearly superior to the other as a measure of testosterone bioavailability.

Prolactin response to suckling and maintenance of postpartum amenorrhea among intensively breastfeeding nepali women

The aim of the study was to determine the association between PRL responses to suckling and maintenance of postpartum amenorrhea among breastfeeding mothers. Three blood spot samples (5, 30, and 50 min following a timed nursing bout) were collected from 71 intensively breastfeeding Nepali women for PRL determination. Maternal age, BMI (weight/height2), menstrual status, caste, infant age, nursing bout length, and duration of supplementation were recorded at time of sample collection. Independent and paired t tests, linear regression analyses, and general linear models were used to evaluate differences between cycling (n = 36) and amenorrheic (n = 35) women and associations among variables. Logistic regression analyses were used to relate PRL measures to the odds of maintaining lactational amenorrhea. Amenorrheic breastfeeding mothers had higher (P < .001) PRL levels at all 3 collection times than cycling breastfeeding mothers, and PRL levels declined with time since birth (P < 0.05). The odds (OR) of having ceased lactational amenorrhea was significantly higher (OR = 5.0, 95% Cl = 1.3-19.9) among mothers with lower PRL levels (< or = 10 ng/mL) at 50 min post-sucking, and PRL at 50 min showed a significant dose response relationship with menstrual status. The association between 50 min PRL levels and lactational amenorrhea appears to be independent of time postpartum, maternal age, BMI, nursing bout length, and duration of supplementation. Among intensively nursing women, maintenance of elevated PRL levels across the interbout interval increases the odds of maintaining lactational amenorrhea.

A simple ultrafiltration method for determining unbound estradiol in serum

The Amicon MPS-1 centrifugal ultrafiltration device and the YMT membrane were used to measure free estradiol in serum. Two independent assays were required. The first measured total estradiol-17 beta and the second ultrafiltrable fraction. The unbound fraction was determined by counting [3H]estradiol-17 beta in 0.15-0.3 mL ultrafiltrates of 0.5 to 1 mL of serum that had been equilibrated with [3H]estradiol-17 beta at 37 degrees C. The assay is rapid (1 h), practicable (requires 0.6 mL serum) and reproducible (CV 4.6% within assay and 6.5% between assays). Samples from apparently healthy men and women and from pregnant women gave results that agreed with those obtained by equilibrium dialysis. The percentages of free serum estradiol-17 beta were similar during both early and late follicular phases of the menstrual cycle. Samples from women with hirsutism gave values for total and free estradiol-17 beta which were not significantly different from those of normal females when no consideration was given to the phase of the menstrual cycle. The means for both of these female groups were significantly different from those of males.

Biological and behavioral factors influence group differences in prolactin levels among breastfeeding Nepali women

This study of two natural fertility Nepali groups, the Tamang and Kami, identifies biological and behavioral factors associated with population differences in fertility. Previous research had established that Tamang experience longer interbirth intervals than Kami despite similarly intense nursing practices, and bear considerably higher energy expenditure due to workload. This cross‐sectional study of 71 breastfeeding women includes prolactin determinations on three blood spot samples collected 5, 30, and 50 minutes following a timed nursing bout, and data on maternal age, BMI, menstrual status, previous birth interval, parity, infant age, nursing bout length, and durations of supplementation and postpartum amenorrhea. The findings show that Tamang breastfeeding mothers have higher average prolactin levels than Kami for as long as 22 months postpartum. Tamang mothers sustain average prolactin levels above those of nonpregnant, nonlactating women for nearly 2 years postpartum, whereas prolactin levels among Kami breastfeeding mothers are the same as this latter group after 1 year postpartum. Furthermore, the findings indicate that Tamang mothers have higher average prolactin levels regardless of maternal age, physical status (BMI, weight, or height), or infant age, and the rate of decline in prolactin from 5 to 50 minutes after suckling is significantly greater for Kami than Tamang. Since factors associated with prolactin levels differ by group, the findings also emphasize that populations vary not only in the strength of effects that proximate determinants have on fertility regulation, but also in the pathways by which they exert their effects (direct physiological versus indirect behavioral). Am. J. Hum. Biol. 10:191–210, 1998.