In recent decades there has been an increasing recognition of the need to account for sex and gender in biology and medicine, in order to develop a more comprehensive understanding of biological phenomena and to address gaps in medical knowledge that have arisen due to a generally masculine bias in research. We have noted that as basic experimental biomedical researchers, we face unique challenges to the incorporation of sex and gender in our work, and that these have remained largely unarticulated, misunderstood, and unaddressed in the literature. Here, we describe some of the specific challenges to the incorporation of sex and gender considerations in research involving cell cultures and laboratory animals. In our view, the mainstreaming of sex and gender considerations in basic biomedical research depends on an approach that will allow scientists to address these issues in ways that do not undermine our ability to pursue our fundamental scientific interests. To that end, we suggest a number of strategies that allow basic experimental researchers to feasibly and meaningfully take sex and gender into account in their work.

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Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.

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We investigated the effects of a natural disaster (a sudden flood) as a source of prenatal maternal stress (PNMS) on the placental glucocorticoid system and glucose transporters. Whether the gestational age at the time of the flood moderated these effects was also evaluated. Placental samples were collected from participants in the 2011 Queensland Flood Study (QF2011) who were pregnant in the first or second trimester at the onset of the flood. Detailed questionnaire results for objective hardship and composite subjective distress were obtained to assess stress levels. Subjective distress was significantly associated with a reduction in placental NR3C1-β mRNA levels for males only (β = -0.491, p = 0.005). In female placentas, objective hardship was marginally linked with lower SLC2A1 mRNA levels while subjective distress was a marginally significant predictor of higher placental SLC2A4 mRNA levels. Gestational age at the time of the flood was a significant moderator of the effect of subjective distress on placental mRNA levels for NR3C1-α (p = 0.046) and HSD11B1 (p = 0.049) in male placentas: if the flood occurred in mid-pregnancy, lower subjective distress predicted higher HSD11B1 while higher subjective distress predicted lower NR3C1-α placental mRNA level. While results did not show any PNMS effects on placental HSD11B2 mRNA and protein levels, and activity, we showed a reduction in placental NR3C1-β mRNA level in male placentas. Our results show evidence of distinct placental glucocorticoid and glucose systems adaptations to PNMS as a function of fetal sex and gestational timing of exposure, with high subjective PNMS in mid-pregnancy associated with lower levels of expression of glucocorticoid-promoting gene in males, leaving the fetus less protected against maternal stress. The exact mechanism by which natural disaster-related PNMS acts on the placenta and the impact on fetal programming requires further investigation.

Normalization with proper reference genes is a crucial step in obtaining accurate mRNA expression levels in RT-qPCR experiments. GeNorm and NormFinder are two commonly used software packages that help in selecting the best reference genes, based on their expression stability. However, GeNorm does not take into account a group variable, such as sample sex, in its calculation. We demonstrate a simple calculation step to assess the variability of such parameters by multiplying the GeNorm M value with the difference of Cq values between groups. To test this, we used 28 reference gene candidates, to analyze 20 placental samples (10 of each sex), and by using HPRT1 (lower Cq values in male placentas (P = 0.017)), as a target gene. Our calculation demonstrates that the RPL30 – GAPDH reference gene combination is the better option to assess small placental sex differences in mRNA level, versus the selection obtained from GeNorm or NormFinder. The HPRT1 normalized mRNA expression level is different between placental sexes, using RPL30 and GAPDH as reference genes (P = 0.01), but not when using genes suggested by GeNorm or NormFinder. These results indicate that the proposed calculation is appropriate to assess small variations in mRNA expression between 2 groups.

Respiratory monitoring, using impedance with implanted telemetry in socially housed animals, was not possible until the recent development of digital signal transmission. The objective of this study was to evaluate digital telemetry monitoring of cardiopulmonary parameters (respiratory rate, tidal volume, minute volume, electrocardiography (DII), systemic arterial blood pressure, physical activity, and body temperature) in conscious, single-housed, non-rodent species commonly used in toxicology studies following administration of positive/negative controls (saline, dexmedetomidine, morphine, amphetamine, and doxapram), and also, the effects of various social housing arrangements in untreated female and/or male cynomolgus monkeys, Beagle dogs, and Göttingen minipigs (n = 4 per species). Aggressions were observed in socially housed male minipigs, however, which prevented pair-housed assessments in this species. All tested pharmacological agents significantly altered more than one organ system, highlighting important inter-organ dependencies when analyzing functional endpoints. Stress-related physiological changes were observed with single-housing or pair-housing with a new cage mate in cynomolgus monkeys and Beagle dogs, suggesting that stable social structures are preferable to limit variability, especially around dosing. Concomitant monitoring of cardiovascular and respiratory parameters from the same animals may help reduce the number of animals (3 Rs) needed to fulfill the S7A guidelines and allows for identification of organ system functional correlations. Globally, the data support the use of social housing in non-rodents for safety pharmacology multi-organ system (heart and lungs) monitoring investigations.