Differences in amniotic amino acids concentrations between pregnancies obtained with transfer of vitrified/thawed IVP embryos and with natural mating in sheep

In vitro embryo production (IVP) and cryopreservation are associated with a high incidence of pregnancy complications and fetal abnormalities that may be linked with alterations of placental development. The amniotic fluid is partly derived from the transport of water and solutes across the placenta and provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiologic growth and development. To better understand the anomalies associated with IVP pregnancies, the present study was conducted to test the hypothesis that amniotic concentrations of AAs differ in pregnancies derived from vitrified/thawed (V/T) IVP embryos compared to gestations obtained with natural mating (NM) in sheep. Amniotic fluid was sampled in ewes that were pregnant after transfer of V/T IVP embryos and that had conceived with natural mating between day 60 and 65 (V/T n=6; NM n=11) and between day 80 and 85 (V/T n=5; NM n=14) of gestation via ultrasound-guided amniocentesis. Concentrations of 16 AAs in the amniotic fluid were measured using high-performance liquid chromatography. At day 60-65 of gestation concentrations of cystine, phenylalanine and isoleucine were lower in V/T compared to NM ewes. At day 80-85 of pregnancy the mean concentrations of cystine and lysine were lower in the V/T vs NM group. The total AA concentration per ewe was similar between groups at day 60-65 and 80-85 and decreased by 55% from day 60-65 to 80-85 of gestation in all ewes. The most abundant AA at day 60-65 of gestation was alanine in both groups, whereas at day 80-85 the most abundant AAs were alanine in NM and glycine in V/T ewes respectively; cystine was the less abundant detectable AA in all ewes at both stages of gestation. Results demonstrate that V/T IVP embryos have decreased concentrations of individual AAs in the amniotic fluid during the second trimester of gestation, possibly because of an impaired placental vasculogenesis or because of a reduced placental transport. These novel findings are relevant to unravel the mechanisms responsible for the issues of pregnancies achieved with the transfer of IVP and cryopreserved embryos.