228 In vitro embryo culture alters fetal neurodevelopment, which leads to brain alterations in adult life

Placental adaptations induced by adverse in utero environments can influence brain development via the placenta-brain axis. Altered availability of serotonin (5HT) and oxidative stress (OS) during pregnancy can lead to abnormal brain development that results in behavioural changes later in life. In vitro embryo culture (IVC) is associated with OS for the developing embryo and increased risk of neurodevelopmental disorders. Our aim was to evaluate whether IVC causes an oxidative state and altered 5HT levels during prenatal development and during adulthood. Levels of 5HT and oxidative status were evaluated in placenta and brain of mouse C57BL6 fetuses collected at 19 days post-coitum (dpc) and in brain (hypothalamus) of adult 4-month-old male C57BL6 offspring. Pregnancies were obtained after transfer of IVC embryos for 72 h. Control group consisted of pregnancies developed after natural mating (CTR). Superoxide dismutase (SOD) activity and levels of reduced (GSH) and oxidised (GSSG) glutathione as well as Malondialdehyde (MDA), were determined by colourimetric assays, and 5HT levels by ELISA kit, in tissue homogenates of placenta, fetal brain, and hypothalamus (n = 8/group/tissue). Statistical significance was assessed by Mann-Whitney test, data are described as median ± IQR, all values are normalised to mg of total protein. Our data shows that altered oxidative status, reflected by low SOD activity (8.88 ± 3.69 vs 11.96 ± 7.49 U/mg; P = 0.02) low GSSG (0.05 ± 0.04 vs 0.1 ± 0.07 μM/mg; P = 0.02), and low 5HT levels (0.09 ± 0.07 vs 0.29 ± 0.08 ng/mg; P = 0.001) in IVC placentas can be due to compensatory mechanisms to ensure resources for brain development. This is reflected by normal levels of GSSG, high activity of SOD (27.11 ± 20 vs 15.63 ± 6.3 U/mg; P = 0.002), and high 5HT levels (0.41 ± 0.2 vs 0.26 ± 0.1 ng/mg; P = 0.01) in the fetal brain of IVC group. An oxidative status (low GSSG = 0.11 ± 0.02 vs 0.43 ± 0.3; P = 0.01 and GSH = 0.62 ± 0.2 vs 1.2 ± 0.7 μM/mg; P = 0.002) and low levels of 5HT (309.9 ± 249 vs 451.2 ± 228 ng/mg; P = 0.03) were also observed in the adult brain of IVC offspring. Alterations initially observed in placenta (GSSG and 5HT levels), although not observed at the level of fetal brain, are present in adult brain. Furthermore, higher MDA levels in fetal brain (391.7 ± 225 vs 193.5 ± 258 nmol/mg; P = 0.04), although not affected in the placenta, are also present in adult brain (160.2 ± 177 v. 76.2 ± 57 nmol/mg; P = 0.005). Altered oxidative status and lower level of 5HT in the placenta of mixed-sex fetuses are a result of an adaptative mechanism to balance fetal brain homeostasis; these adaptations can affect development in a sexually dimorphic manner. Alterations in the brain from IVC adult male offspring could be a consequence of insufficient adaptative mechanism, and highlight the importance of placental adaptations to compensate threats to the embryo. These results and further analysis of female adult offspring could help our understanding of how placenta responses to threats impact the placental-brain-axis.

This work was supported by National Science Centre, Poland, GA no. 2019/35/B/NZ4/03547, 2021/41/B/NZ3/03507, 2015/19/D/NZ4/03696, and UJ-PRA BIOS B.1.11.2020.34.