Sex-Specific Survival, Growth, Immunity and Organ Development in Preterm Pigs as Models for Immature Newborns

Abstract

Background: After very preterm birth, male infants show higher mortality than females, with higher incidence of lung immaturity, neurological deficits, infections, and growth failure. In modern pig production, piglets dying in the perinatal period (up to 20%) often show signs of immature organs, but sex-specific effects are not clear. Using preterm pigs as model for immature infants and piglets, we hypothesized that neonatal survival and initial growth and immune development depend on sex. Methods: Using data from a series of previous intervention trials with similar delivery and rearing procedures, we established three cohorts of preterm pigs (90% gestation), reared for 5, 9, or 19 days before sample collection (total n = 1,938 piglets from 109 litters). Partly overlapping endpoints among experiments allowed for multiple comparisons between males and females for data on mortality, body and organ growth, gut, immunity, and brain function. Results: Within the first 2 days, males showed higher mortality than females (18 vs. 8%, P < 0.001), but less severe immune response to gram-positive infection. No effect of sex was observed for thermoregulation or plasma cortisol. Later, infection resistance did not differ between sexes, but growth rate was reduced for body (up to -40%) and kidneys (-6%) in males, with higher leucocyte counts (+15%) and lower CD4 T cell fraction (-5%) on day 9 and lower monocyte counts (-18%, day 19, all P < 0.05). Gut structure, function and necrotizing enterocolitis (NEC) incidence were similar between groups, but intestinal weight (-3%) and brush-border enzyme activities were reduced at day 5 (lactase, DPP IV, -8%) in males. Remaining values for blood biochemistry, hematology, bone density, regional brain weights, and visual memory (tested in a T maze) were similar. Conclusion: Following preterm birth, male pigs show higher mortality and slower growth than females, despite limited differences in organ growth, gut, immune, and brain functions. Neonatal intensive care procedures may be particularly important for compromised newborns of the male sex. Preterm pigs can serve as good models to study the interactions of sex- and maturation-specific survival and physiological adaptation in mammals.

Keywords: animal model; cohort; gender; immune; preterm; sex.

Figure 1

Illustration of clinical care procedures (green text) and possible morbidities (red text) for cesarean-delivered 90% gestation preterm pigs, reared as models for preterm infants. Preterm pigs show clinical and physiological characteristics reflecting very preterm infants (<32 weeks gestation) but comparisons to infants are both age- and organ-specific (37). Based on the reports of gender-specific morbidities in preterm infants, we investigate if sex-specific effects are present in preterm pigs at different stages after preterm birth.

Figure 2

Hematological and blood gas parameters in female and male preterm pigs (open and filled circles, respectively) inoculated with live S. epidermidis bacteria shortly after birth. Neutrophil fractions (A, n = 38). Platelet counts (B, n = 38). Lymphocyte fractions (C, n = 38). Blood acidity (D, n = 38). Blood oxygen pressure (E, n = 38). Blood lactate levels (F, n = 38). All shown as means with corresponding SE. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Incidence of bone marrow bacterial infection (A, n = 74 and 130) with corresponding bacterial densities (B, n = 74 and 130), as well as fractions of T cells (C, n = 52–148), CD4 positive T cells (D, n = 52–148) and expressions of IL2, IFNG, TLR2, and MPO (E–H, n = 38–81) in male and female preterm pigs. Shown as fractions (A), means with corresponding error (B–D) and fold- changes in relation to housekeeping gene, before and after stimulation with lipopolysaccharide (LPS, E–H). (*)Tendency to an effect, P < 0.1, *P < 0.05. ^§Effect of LPS, P < 0.1, ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001.

Figure 4

Growth rates for 5, 9, and 19 day cohorts (A, g/kg/day, n = 1,135, 319, and 221), incidence of necrotizing enterocolitis (NEC score ≥3) at day 5 and 9 (B, n = 1,152 for day 5 and n = 319 for day 9), and brush border enzyme activities at day 5 (C, n = 1,028) for male and female preterm piglets. Shown as means with corresponding SE (A,C) or as proportion of animals (%) in the cohort (B). (*)Tendency to effect, P < 0.1, *P < 0.05, **P < 0.01.