Subjects and samples
The study protocol was approved by the Bioethics Committee of the Hospital Universitario “Dr. José Eleuterio González”. These studies are in compliance with the Declaration of Helsinki principles. Patients were provided with written information about the study prior to giving informed consent. A total of 49 patients (Group A) consulting for infertility were included in this study (age 20-39). This group was further subdivided in two groups: patients who became pregnant after the treatment (group A1); patients that did not achieved pregnancy (group A2). Diagnosis of infertility was based on the patient’s medical history, clinical investigation, including transvaginal ultrasonography, a standard set of tests that included hormone analyses, hysterosalpingogram contrast sonography and semen analyses, as described previously [21]. A diagnosis of unexplained infertility was chosen when no explanation for infertility was found, women had normal ovarian function and normal tubal passage (demonstrated by hysterosalpingogram contrast sonography) and their partners had normal semen samples according to WHO criteria [22].
All of them were programmed for an assisted reproduction procedure (either in vitro fertilization or intra cytoplasmic sperm injection). In addition, 14 healthy women donors (Group B) that were recruited as part of our egg donor program were also included in this study (control group).
The following information was recorded for each patient: Age, concentration of estradiol during stimulation, duration of hormonal stimulation (days), dose of gonadotropins used in stimulation, number of follicles (>14 mm), number of eggs retrieved, number of embryos, number of transferred embryos and occurrence of biochemical pregnancy (positive of beta hCG). Each participant was subjected to 2 blood draws (10 ml each). The first one at day 3 of the cycle, prior to hormonal stimulation. The second one, the day of egg retrieval. The samples were used to quantify the concentration of estradiol, folic acid, homocysteine and 5-MTHFR polymorphism.
IVF procedure
All women started the ovarian stimulation treatment with daily injections of 300 IU recombinant follicle stimulating hormone (rFSH) s.c. on cycle day 2 (Puregon®, NV Organon, Oss, the Netherlands, or Gonal-F®, Serono Benelux BV, The Hague, the Netherlands). Administration of daily s.c. gonadotrophin releasing hormone antagonist (Orgalutran®, NV Organon, or Cetrotide®, Serono Benelux BV) was started when at least one follicle was ≥14 mm. To induce final oocyte maturation, a single dose of 5000 or 10 000 IU hCG s.c. (Pregnyl®, NV Organon) was administered as soon as the largest follicle reached at least 18 mm in diameter and at least one additional follicle of >15 mm was observed. Oocyte retrieval was carried out 35 h after hCG injection by transvaginal ultrasound-guided aspiration of follicles. Luteal phase supplementation of 600 mg/day micronized progesterone intravaginally was started on the evening following oocyte pick-up and continued for 12 days thereafter. On Day 3 after oocyte pick-up, a maximum of two embryos were transferred.
Determination of Folic acid, homocysteine and Estradiol
Blood samples were taken to determine plasma folate (PF) and intracellular folate (ICF). Immediately after collection, the blood samples were centrifuged at 3000 g for 10 min and the plasma stored at −20°C until analyzed. For ICF determination, 0.1 ml of blood was diluted with 2 ml of ascorbic acid in order to produce the lysis of red blood cells. Folate was measured radioimmunoassay Dualcont AF (Diagnostic Product Co, Los Angeles, CA, USA) according to the manufacturer’s instructions. Blood samples showing hemolysis were excluded from the statistical analyses because of the risk of misleading values. Measuring range: IFC 175-700 ng / mL, PF 3-17 ng / mL. The intra and inter assay coefficient of variation were 5.2% and 9.2%, respectively.
For homocysteine determination, immediately after collection, the blood samples were centrifuged at 3000 g for 10 min and the plasma stored at −20°C until analyzed. Blood samples showing hemolysis were excluded from the statistical analyses because of the risk of misleading values. Homocysteine was assessed by fluorescence polarization immunoassay (FPIA) adapted to the IMx analyzer (Abbott Laboratories), with the high-performance liquid chromatography (HPLC) method with fluorescent detection. Measuring range: 4.5-15.0 µmol/L. The intra and inter assay coefficient of variation were 2.7% and 3.4%, respectively.
Electrochemiluminescence immunoassay Elecsys Estradiol III Assay (ECLIA) was used for the in vitro quantitative determination of estradiol in human serum and plasma. Measuring range (25-3,000 pg/mL); Intermediate imprecision was 4.5%.
Determination of MTHFR polymorphisms
Peripheral blood was collected for genomic DNA analysis to determine the substitution of cysteine by thymine (C677T). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Berlin, Qiagen, Germany). DNA quality and quantity were assessed by a UV spectrophotometer at 260 and 280 nm. The following primers were used (5´-TGA AGG AGA AGG TGT CTG CGG GA-3´) and MTHFR-2 (5´-AGG ACG GTG CGG TGA GAG TG-3´). The assays were performed using TaqMan Genotyping Master Mix with 50 ng DNA. The PCR conditions were 35 cycles of denaturation at 94°C for 5 min and annealing/extension at 62°C for 1 min, as recommended by the manufacturer. The amplified products were digested with HinfI since the C667T mutation creates a restriction site for this enzyme (converting the 198 bp product in two fragments of 175 and 23 bp). Homozygous individuals were detected when a single product of 198 bp (C/C) or 175 (T/T) was detected. Heterozygous displayed the two fragments (C/T).
Calculations and statistical analysis
Data are expressed as mean ± standard deviation of the mean (SD). Data were normally distributed. Statistical comparisons were performed by paired Student t-test or Pearson correlation coefficient. A power study was conducted to calculate the number of samples for plasma folate, intracellular folate and homocysteine levels. (α=0.05 and the desired power = 0.80). Calculations were performed with Libre Office 4.3.2.2 spreadsheet and statistical analysis with GraphPad Prism version 4.00 for Windows, GraphPad Software (San Diego, CA, USA). A probability (p) value p<0.05 was considered statistically significant.