Análise da variação da concentração de amônia, dos aminoácidos alanina e glutamina e do dipeptídeo alanil-glutamina no meio de cultura de embriões humanos e os efeitos na reprodução assistida / Analysis of the variation of the concentrations of ammonium, alanine, glutamine, and alanyl-glutamine in culture medium of human embryos and the effects on assisted reproduction / Analysis of the variation of the concentrations of ammonium, alanine, glutamine, and alanyl-glutamine in culture medium of human embryos and the effects on assisted reproduction / Análise da variação da concentração de amônia, dos aminoácidos alanina e glutamina e do dipeptídeo alanil-glutamina no meio de cultura de embriões humanos e os efeitos na reprodução assistida

Juliana Stevanato

Objective: to verify the relations between culture media concentrations of ammonia, alanine, glutamine, and alanyl-glutamine and embryo quality, pregnancy, and type of hormonal stimulation. Methods: a prospective case-control study was carried out including 62 embryos from 20 women submitted to assisted reproduction at the Sao Paulo Federal University. Inclusion criteria were age (up to 37 years old), one to four embryos transferred at day 3 of embryo culture, couples submitted to ICSI, and LH peak achieved through hCG administration. Experimental groups were subdivided according to embryo morphologic quality on days 2 and 3, homogenous (good embryos on days 2 and 3) or heterogeneous evolution, type of hormonal stimulation used (FSH or FSH and LH), and occurrence of pregnancy evaluated by serum Ò-hCG. Culture media aliquots were collected after the embryos were transferred. Analysis of alanine, glutamine, alanyl-glutamine, and ammonia concentrations was performed using HPLC. Numerical variables were compared between groups using unpaired Students T-test (heteroscedastic variables were transformed), and frequencies using Pearsons Chi-square or Fishers exact test. Logistic models were constructed using female age, type of hormone used for stimulation, embryo quality, and the measured metabolites, in order to maximize predictability of occurrence of pregnancy. Data are presented as mean; standard deviation. Results: In the embryos transferred to women who achieved pregnancy, when compared to those who did not achieve pregnancy, lower levels of normalized glutamine (1.4; 0.7 and 1.9; 0.7, respectively, p=0.004) and ammonia (0.2; 0.01 and 0.3; 0.1, respectively, p=0.008) were observed. Higher levels or normalized alanyl-glutamine were observed in higher quality embryos on days 2 and 3, and on those who presented homogeneous evolution. Embryos from patients who received FSH with LH, when compared to those from patients who received only FSH, presented higher absolute values of alanyl-glutamine (334.9; 95.2 and 282.9; 62.4, respectively, p=0.017). In logistic regression, the model which best predicted pregnancy included all the normalized values of alanine, glutamine, age, type of hormone, and evolution (77.4% correct, p=0.00005). Conclusion: Our results allow us to conclude that, in our conditions: (i) embryos with better quality on days 2 and 3 and with a homogenous evolution are associated with higher contents of alanyl-glutamine in their culture media, (ii) lower levels of ammonia and glutamine in the culture media are associated to higher pregnancy rates, and (iii) FSH with LH during hormonal stimulation is associated to higher levels of alanyl-glutamine in the culture media.

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