From January 2009 to August 2009 a total of 101 consecutive women underwent 101 IVF-ICSI cycles. All women studied, after ovarian stimulation, reached the ovum pick up stage, but only 86 managed to reach embryo transfer. All 101 women were allocated to one of the three groups according to their ff 25-OH vitamin D levels. Group A (n = 31), group B (n = 49), group C (n = 21) with less than 20 ng/ml, 20.1-30 ng/ml and more than 30 ng/ml ff vitamin levels, respectively. The biological rationale for this categorization lays in the fact that physiological serum 25-OH vitamin D levels range between 20 and 30 ng/ml . Upper or lower concentrations have been characterized as hypervitaminosis D and hypovitaminosis D, respectively.
Our primary end point was to measure serum and ff 25-OH vitamin D levels in combination with glucose concentrations in women undergoing IVF-ET treatment. Secondly, providing availability of embryological data, we investigated if circulating and ff 25-OH vitamin D levels correlate with the IVF outcome.
All women received the same ovarian stimulation protocol. Briefly, ovarian stimulation was performed by administration of recombinant FSH (Puregon®, Organon, or Gonal-F® Serono) in a short GnRH agonist protocol. Treatment started on day 2 of every woman's menstrual cycle with a FSH dose of 2-6 ampoules (75 IU each ampoule) depending on the age of every woman. This dose was given for 4 days and then it was modified according to the ovarian response. When at least three follicles reached a diameter of 17 mm, 5000 IU of Human Chorionic Gonadotrophin (HCG) was administered and 34-36 hours later, ovum pick up (OPU) was performed under light sedation.
Fertilization, cleavage, pregnancy rates and assessment of embryo quality
16-18 hours post-IVF/ICSI the fertilization percent, expressed as ratio of oocytes with two pronuclei (2PN) to the total number of injected oocytes, was scored. Fertilization failure was indicated by the absence of PN. Following cleavage of all normal fertilized oocytes, morphological grade of all embryos was assessed 3 days post-OPU. Morphological grade was scored numerically in terms of the blastomeres (4 to 8 blastomeres), grade of fragmentation (in scale 1 to 4 with the fourth scale representing no fragmentation) and irregularity of blastomeres (in scale 1 to 2 with the second scale representing regular blastomeres). For example, one embryo with 8 regular blastomeres and no fragmentation would be assigned a total sum of 14 embryo score, while an embryo with 5 irregular blastomeres and 10-20% fragmentation (scale 2) would be assigned a total sum of 8 embryo score. For every patient, the cumulative embryo score (CES) was calculated by adding embryo score of each embryo and the mean score of embryo quality (MSEQ) was obtained by dividing the CES with the number of embryos produced.
Three days post-OPU and after assessment of embryo quality a maximum of three selected embryos (depending to the age or to the number of available embryos) were transferred to the uterus.
Clinical pregnancy was defined when an intrauterine sac was seen by ultrasound 3-4 weeks post-hCG.
Vitamin D and glucose measurement procedure
Blood samples were collected for 25-OH vitamin D and glucose assessment during oocyte retrieval. All blood samples were centrifuged at 1,000 g for 15 min, and serum was stored at -20°C until assayed. Additionally, ff was aspirated from each follicle, pooled from all retrieved follicles and centrifuged (800 g for 15 min) after which it should be clear and not contaminated with blood. The ff was also stored at -20°C until assayed. Serum and ff 25-OH vitamin D was measured by the electrochemiluminescence immunoassay (ECLIA) with analytical sensitivity of 0.2 μU/ml, using a Modular Analytics E170 cobas e 601 Analyzer (Roche Diagnostics GmbH, D-68298 Mannheim), while serum and ff glucose levels by using a colourimetric enzymatic procedure, known as the exocinase method (Olympus Life and Material Science Europa GmbH (Irish Branch), Lismeeham, O' Callaghan's Mills, Co. Clare, Ireland).
Pregnancy rates were compared between the three groups with x2-test. Numeric data were normally distributed (one sample Kolmogorov-Smirnov test), and statistical analysis was performed by one way analysis of variance followed by Bonferroni post hoc testing. For correlations between the parameters Pearson's correlation was used. P value less than 0.05 was considered significant. All values are expressed as mean ± SD. The statistical software package used was NCSS 2001 (Number Cruncher Statistical Systems, Kaysville, UT).