Prenatal synthetic glucocorticoid exposure alters hypothalamic-pituitary-adrenal regulation and pregnancy outcomes in mature female guinea pigs

Abstract

Preterm delivery occurs in approximately 10% of all pregnancies. Prenatal exposure to synthetic glucocorticoids (sGCs) reduces the incidence of respiratory distress syndrome (RDS) in these babies. Therefore, administration of multiple courses of sGCs became common practice. Animal and human studies have demonstrated that multiple courses of sGCs can have long-term effects. While the majority of animal studies have been undertaken in male offspring, it is emerging that there are profound sex differences in the consequences of prenatal sGC exposure. To our knowledge, no studies have determined the effects of prenatal sGC exposure on hypothalamic-pituitary-adrenal (HPA) axis function in female offspring while accounting for reproductive cycle status, or determined if there are effects on pregnancy parameters. Pregnant guinea pigs were administered three courses of betamethasone (Beta), dexamethasone (Dex) or vehicle on gestational days 40/41, 50/51 and 60/61. In adulthood (age range: postnatal days 126-165), basal and activated HPA axis function were assessed at various stages of the reproductive cycle. The female offspring were then mated and underwent an undisturbed pregnancy. Females were killed in the luteal phase of the reproductive cycle following litter weaning, and molecular analysis undertaken. In the luteal phase, Beta-exposed females exhibited significantly lower basal salivary cortisol levels (P < 0.05). Dex-exposed females also exhibited significantly lower basal salivary cortisol levels during the luteal phase (P < 0.05), but increased basal salivary cortisol levels during the ostrous phase (P < 0.01). The Beta-exposed females exhibited increased glucocorticoid receptor (GR) mRNA expression in the CA1/2 region of the hippocampus (P < 0.05) and MC2R mRNA in the adrenal cortex (P < 0.05). The Dex-exposed animals exhibited higher hippocampal GR and mineralocorticoid receptor (MR) mRNA levels (P < 0.05). Beta-exposed females showed reduced fecundity (P < 0.05). In Dex-exposed females there was a lower male to female sex ratio. In conclusion, prenatal sGC exposure affects HPA axis activity, in a cycle-dependent manner, and long-term reproductive success. The clinical implications of the findings on endocrine function and pregnancy in females are profound and further follow-up is warranted in human cohorts. Furthermore, we have shown there are considerable difference in phenotypes between the Beta- and Dex-exposed females and the specific endocrine and maternal outcome is contingent on the specific sGCs administered during pregnancy.

Figure 1. Basal salivary cortisol concentrations

Basal salivary cortisol concentrations at 08.00 h, 12.00 h and 16.00 h on day 0 (ostrous phase) (A), day 8 (mid-luteal phase) (B) and day 11 (late-luteal phase) (C) of the reproductive cycle in adult female guinea pig offspring whose mothers had been injected with vehicle (Veh; open circles, continuous line; n= 12) or betamethasone (Beta; 1 mg kg⁻¹, filled circles, dashed line; n= 11) or dexamethasone (Dex; 1 mg kg⁻¹, filled squares, dashed line; n= 14) on days 40/41, 50/51 and 60/61 of gestation. Inset, total area under the curve. Data are presented as means ±s.e.m.^aP < 0.01 vehicle vs. Dex over time, ^bP < 0.05 vehicle vs. Dex over time, ^cP < 0.01 vehicle vs. Beta over time, *P < 0.05 vs. vehicle.

Figure 2. Salivary cortisol response to a strobe light stressor

Salivary cortisol was analysed before (time 0), immediately after exposure to the stressor (30 min) and during the recovery phase (60 and 120 min) on day 1 (A), day 9 (B) and day 12 (C) of the reproductive cycle in adult female guinea pig offspring whose mothers had been injected with vehicle (Veh; open circles, continuous line; n= 12) or betamethasone (Beta; 1 mg kg⁻¹, filled circles, dashed line; n= 11) or dexamethasone (Dex; 1 mg kg⁻¹, filled squares, dashed line; n= 14) on days 40/41, 50/51 and 60/61 of gestation. Net area under the curve inset. Data are presented as means ±s.e.m.

Figure 3. Pregnancy parameters

Pregnancy parameters in adult female guinea pig offspring whose mothers had been injected with vehicle (Veh; n= 12), betamethasone (Beta; 1 mg kg⁻¹; n= 11) or dexamethasone (Dex; 1 mg kg⁻¹; n= 14) on days 40/41, 50/51 and 60/61 of gestation. A, number of reproductive cycles needed for successful conception. B, percentage of female (filled bar) and male (open bar) offspring in litters of first pregnancies of vehicle, Beta and Dex females. C, salivary cortisol levels on gestational days 30, 40, 50 and 60 in first pregnancy of vehicle (open circles, continuous line), Beta (filled circles, dashed line) and Dex (filled squares, dashed line) females. *P < 0.05 vehicle vs. Beta. §Significant difference in the female/male ratio compared to vehicle and Beta.

Figure 4. Hippocampol corticosteroid receptor gene expression

Relative optical density (ROD) (mean ±s.e.m.) of GR mRNA (A) and MR mRNA (B) in CA1/2, CA3, CA4, and dentate gyrus (DG) of the hippocampus following in situ hybridization in adult female guinea pig offspring whose mothers had been injected with vehicle (Veh; white bar; n= 12), betamethasone (Beta; 1 mg kg⁻¹; black bar; n= 11) or dexamethasone (Dex; 1 mg kg⁻¹; grey bar; n= 14) on days 40/41, 50/51 and 60/61 of gestation. *P < 0.05, significant difference between prenatal treatment groups. Exposure time for GR mRNA was 21 days and for MR mRNA was 14 days. *P < 0.05 vehicle vs. Beta or Dex. **P < 0.01 vehicle vs. Beta or Dex.

Figure 5. Hypothalamic gene expression

Relative optical density (ROD) (mean ±s.e.m.) of GR mRNA (A), CRH mRNA (B) and AVP mRNA (C) in hypothalamic paraventricular nucleus following in situ hybridization in adult female guinea pig offspring whose mothers had been injected with vehicle (Veh; white bar; n= 12), betamethasone (Beta; 1 mg kg⁻¹; black bar; n= 11) or dexamethasone (Dex; 1 mg kg⁻¹; grey bar; n= 14) on days 40/41, 50/51 and 60/61 of gestation. *P < 0.05, significant difference between prenatal treatment groups. Exposure time for GR mRNA was 21 days, for CRH mRNA was 35 days and for AVP mRNA was 8 h. *P < 0.05 vehicle vs. sGC. **P < 0.01 vehicle vs. Beta or Dex.

Figure 6. Adrenocortical gene expression

Relative optical density (ROD) (mean ±s.e.m.) of steroidogenic factor 1 (SF-1) mRNA (A) and MC2R mRNA (B) in the adrenal cortex following in situ hybridization in adult female guinea pig offspring whose mothers had been injected with vehicle (Veh; white bar; n= 12), betamethasone (Beta; 1 mg kg⁻¹; black bar; n= 11) or dexamethasone (Dex; 1 mg kg⁻¹; grey bar; n= 14) on days 40/41, 50/51 and 60/61 of gestation. Exposure time for SF-1 and MC2R mRNA was 10 days. *P < 0.05 vehicle vs. Beta or Dex.