P-235 Lipid profile changes during neurodevelopment (ND) of mouse embryos cultured in vitro
Authors: Heber M  F , Gulzar H , Bisogno S , Musson R , Jędrzejczyk R , Rozpądek P , Depciuch J , Kędracka-Krok S , Zacchini F , Ptak G  E 
Published: July 2024

Abstract

Study question
Does in vitro culture (IVC) induce changes in lipid profile of the embryo?

Summary answer
IVC induces changes in embryonic lipid homeostasis that subsequently affect placenta-brain axis (PBA) function and are then maintained in the brain of adult mice.

What is known already
During ART, embryos are exposed to oxidative stress (OS)-prone environment. Cellular lipids are particularly sensitive to OS. Lipids are a crucial component for proper brain development and any aberrancies in lipid profile of the embryo can affect neurodevelopment (ND) through the PBA, leading to functional and behavioural deficits during adulthood. In particular, deficiencies of ether-linked glicerophospolipids (e.g. plasmalogens), which are highly abundant in the brain, and polyunsaturated fatty acids (PUFAs), have been associated with psychiatric and ND disorders.

Study design, size, duration
C57BL6 mouse embryos were cultured in vitro for 72hs, and analysed or transferred to 25 recipient females for further development. From 6 pregnant mice, conceptuses (placentae and brain) were collected at near-term pregnancy (19dpc, n &gt; 8/group). Remaining 8 pregnant mice were allowed to deliver and raise litters. Subsequently, brain was collected from male offspring at 4 month of age for lipid homeostasis evaluation. Control group consisted of embryos and offspring obtained from naturally mated C57BL6 mice.

Participants/materials, setting, methods
C57BL6 blastocysts (n &gt; 15/group) were fixed and subjected to lipid analysis by Fourier transform infra-red (FTIR) and Coherent antistokes Raman spectroscopies (CARS). To reveal lipid changes indicative for ND disorders, selected PUFAs were analyzed by liquid chromatography-mass spectrometry (LC-MS) (n &gt; 8/group) in placenta and brains from prenatal and adult mice. Additionally, placental expression of genes regulating biosynthesis of plasmalogens and proteome of adult males brain, was performed. Values were considered different when p &lt; 0.05, Mean±SEM; Mann-Whitney test.

Main results and the role of chance
Our results show that IVC induced lipid composition changes in blastocyst, characterised by increased proportion of phospholipids, fatty amides and cholesterol (59±8.2 vs.44.7±8.8; 149±4.5vs. 104±12; 44±2.9vs. 27±4.6 sum of absorbance, p &lt; 0.05); and lipid droplets (LDs) distribution changes, such as, increase size (2.3±0.11 vs 1.8±0.07 µm2, p = 0.0021), and number of large LDs (5.5±0.7 vs. 3.3±0.2, p = 0.006). LC-MS revealed increased levels of linolenic (LA), arachidonic (AA) and docosahexaenoic (DHA) acids in placenta (LA:15.49±1.7 vs. 6.92±2; AA:263.6±29.1 vs. 62.7±14.7 DHA:0±2.5 vs. 2.7±0.8 pmol/mg; p &lt; 0.03) and foetal brain (LA:13.6±5.5 vs. 1.5±0.9; AA:240.5±100 vs. 39.3±9.6; 13.9±6.9 vs. 2.11±0.6 pmol/mg; p &lt; 0.03) of IVC conceptuses, which may condition brain development and function. Conversely, levels of LA and AA were low in adult brain from IVC offspring (LA:4.4±1.5 vs 14.5±3.8; AA: 581±95 vs 1842±454 pmol/mg, p &lt; 0.04). Additionally, the expression of genes regulating biosynthesis of plasmalogens was reduced in IVC placentae (Gnpat:0.2±0.04 vs 1.2±03; Tmem189:0.3±0.1 vs. 1.3±0.3 fold change; p ≤ 0.01). Proteome analysis demonstrated that pathways regulating lipid homeostasis, such as fatty acid oxidation and phospholipid binding and those involved in IL-17a signaling and neurogenesis were upregulated in brain of adult IVC offspring.

Limitations, reasons for caution
Our results may not entirely reflect what occurs in human embryo, as mouse oocytes have a higher lipid content than human oocyte. Differences in lipid content may cause an increased or decreased penetrance and exhibition of developmental changes of the embryos.

Wider implications of the findings
Being of non-genetic background, lipid changes induced by IVC may similarly threaten embryos regardless of species causing ND disorders. Our study sheds new light on non-genetic origins of ND disorders and will likely ignite further studies on embryonic lipid changes and their consequences, relevant for human pregnancy and neuropsychiatry.

Trial registration number
not applicable