ИСТИНА

Study question: To investigate the association of serum folate and total magnesium (Mg) and calcium (Ca) levels before in vitro fertilization with the outcomes of ART. Summary answer: Using multivariable models, we suggested that baseline higher serum folate (≥33.0 ng/ml) and lower Ca/Mg ratio (4.78) were associated with worse ART outcomes in normogonadotropic women. What is known already: Women of reproductive age are recommended to consume folic acid and other supplements before and during pregnancy, aiming to prevent harmful complications of its deficiency. Some studies have demonstrated that higher serum folate before ART was associated with higher live births. However, other studies have shown that women with unexplained infertility had significantly higher median plasma folate than fertile women, and higher concentrations of serum folate can modulate the ovarian response to gonadotropin during ART by influencing oestradiol levels. Notably, a relationship between oestradiol and progesterone levels and the Ca/Mg ratio was shown in healthy women of reproductive age. Study design, size, duration: A subanalysis of data obtained from a multicentre, randomized, therapeutic equivalence study of follitropin alpha biosimilar. A total of 110 normogonadotropic women were enrolled using the following inclusion criteria: 20-35 years old, tubal and/or male causes of infertility factors, first or second attempt at IVF/ICSI; 18 ≤ BMI ≤ 30 kg/m2; baseline FSH <10 IU/l and oestradiol level 50 pg/ml; and AMH≥1.0 ng/ml. No restrictions for folic acid and other food supplements/vitamins were applied before, during and after ART. Participants/materials, setting, methods: Single ovarian stimulation (OS) with recombinant human follicle-stimulating hormone (r-hFSH) and GnRH antagonist in one fresh cycle was used. Over the 5-day fixed-dose regimen, the women received 150 IU/day of r-hFSH, followed by dose adaptation. Baseline serum folate, total Ca and Mg were measured before OS. Multivariable generalized linear models were used with Poisson distribution and log link function for adjusted earlier ART outcomes and a binomial distribution and log link function for adjusted late ART outcomes. Main results and the role of chance: A total of 110 women were included aged 30.72.8 years, and the mean duration of infertility due to tubal (35.5%), male (43.6%), and tubal and male (20.9%) factors was 41.6±28.2 months. The baseline levels of hormones were FSH (6.611.87 IU/l), LH (5.243.71 IU/l), AMH (5.023.43 ng/ml), and antral follicle count 11.282.87. The medians (interquartile range (IQR) baseline serum folate were 20.55 ng/ml (10.8, 32.9); Mg – 19.4 mg/L (18.7, 20.7); Ca – 94 mg/L (91.2, 96.4); Ca/Mg ratio - 4.78 (4.55, 5.02) and B12 – 350 pg/mL (239, 471). For each outcome, we fit a full multivariable model, including all covariates with univariate p≤0.20 for that outcome, and reduced it to a final model, retaining covariates with p < 0.10. Women with higher serum folate concentrations (Q4≥33.0 ng/ml) had significantly lower total numbers of oocytes retrieved and lower odds ratios of 0.24 (0.04, 1.37) for biochemical pregnancy, 0.12 (0.02, 0.79) for clinical pregnancy and 0.10 (0.01, 0.70) for live births compared with women in the lowest quartile, all p<0.05. Moreover, women in the highest Ca/Mg ratio quartile (≥5.02) had ORs of 6.58 (95% CI: 1.31, 33.04) for biochemical pregnancy, 4.85 (95% CI: 1.02, 23.08) for clinical pregnancy and 4.07 (95% CI: 0.83, 19.9) for live birth rate compared with women in the lowest quartile, all p<0.05. Limitations, reasons for caution: No assessment of diet or supplements was performed before ART. The level of folates was not measured in the red blood cells. Further preconception large-scale studies with known nutritional and hormonal status of both parents are needed. Wider implications of the findings: Our findings might be useful to choose a safe dosage of supplementation needed for both fertile women and women undergoing infertility treatment. The interplay of serum levels and additional consumption of important nutrients and microelements with sex steroid hormones should be taken into account before and during IVF procedures. Study funding/Competing interest(s): No specific funding was obtained. There are no conflicts of interest. Key words: folate, folic acid, ART, in vitro fertilization, nutrition supplements, ovarian stimulation, calcium, magnesium, pre-conceptional nutrition, Ca/Mg ratio, safe dose