The study was reviewed and approved by the Ethics Committee of the University of Pécs. Signed informed consent was obtained from all patients who participated in the study. The investigation conforms to the principles outlined in the Declaration of Helsinki.
Our case–control study was performed between March 24, 2011 and May 9, 2011 in the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, University of Pécs, Hungary. In this period we performed 44 unselected IVF cycles, in 42 cases we made transvaginal ultrasound guided aspiration of FF. In the remaining 2 cycles the stimulation was unsuccessful. The patients were aged 23–40 years (mean: 32.3±5.1 years) and had BMI of 17.3-34.7 (mean: 23.80±4.9).
The patients were recruited into this study according to the date of the procedure, so it was an unselected population. They presented with the following main infertility diagnosis: male factors (14, 33.3%), damaged or blocked Fallopian tubes (10, 23.8%), severe endometriosis (7, 16.7%) and unexplained infertility (11, 26.2%). These latter patients experienced six unsuccessful intrauterine inseminations previously.
Among the patients there were no diabetes mellitus (type I and II), or reduced glucose tolerance.
Superovulation treatment was started after the necessary examinations, such as cervical smear, serum hormone measurements (follicular stimulating and luteinizing hormones /FSH, LH/, prolactin, estradiol, progesterone, testosterone, thyroid-stimulating hormone) on the 3rd and 21st days of the unstimulated cycles, human immune-deficiency virus and hepatitis-B surface antigen screening, hysteroscopy and andrologic examination. Patient enrollment into IVF procedure was approved by two independent physicians.
During the study period samples were also obtained from 18 healthy women aged 25–40 years (mean: 33.4±5.2 years) and had BMI of 19.4-32.6 (mean: 25.01±4.7) admitted for minor elective surgery to serve as control for plasma carnitine profile.
Controlled ovarian hyperstimulation
Inducing IVF GnRh agonist triptorelin (Gonapeptyl; Ferring®, Germany) was used in a long or short protocol, and the stimulation was performed with individual dosages of rFSH (Gonal-F; Serono® Aubonne, Switzerland), varying from 100 to 225 IU per day depending on the follicular maturation. The starting dose was adapted according to the BMI and the age. For patients with a previously known low response it was increased to a maximum dose of 300–350 IU daily. The follicular maturation was determined by ultrasound examination from the 6th day of the cycle, every other day. We changed the amount of the administered gonadotropins individually according to the size of the follicles. Ovulation was induced by injection of 250 μg of hCG (Ovitrelle; Serono®Aubonne, Switzerland) when at least two follicles exceeded 17 mm in diameter, and aspiration of FF was performed 36 hours later by ultrasonography-guided transvaginal puncture under routine intravenous sedation.
Collection of follicular fluid
The oocyte collection was performed using Sonoace 6000C two dimensional real time ultrasound scanner equipped with 4–8 MHz endovaginal transducer. The oocyte collection was performed in G-MOPS™ medium (Vitrolife, Göteborg, Sweden).
FF from individual follicles was aspirated and after collecting the oocytes the fluid was centrifuged for 10 min at 1500 r.p.m. and the supernatants were frozen and stored at −70°C for future analysis.
We performed the fertilization with intracytoplasmatic sperm injection (ICSI) depending on the andrological status (sperm count less than 15M/ml), the maternal age (> 35) and the number of the previous IVF cycles the patient had before (>2). The oocytes selected for ICSI were denuded with hyaluronidase and were assessed for maturity. Only metaphase II oocytes, identified by the presence of the first polar body, were chosen for fertilization. ICSI was performed 3–6 h after oocyte recovery in the medium G-MOPS™. The remained oocytes were fertilized with the conventional IVF method in a bicarbonate buffered medium (G-IVF™, Vitrolife®, Göteborg, Sweden). Fertilization was assessed 24 hours later in the medium G-1™ v5 (Vitrolife®, Göteborg, Sweden), the presence of two pronucleus signed the fertilization.
Embryo transfers were done 3–5 days after the oocyte retrieval. From day 3 to blastocyst stage we use the medium G-2™v5 (Vitrolife®, Göteborg, Sweden). According to the patient request we transferred one, two or three embryos. Cryopreservation of the remaining embryos was performed at this stage according to the Hungarian law. Progestogen supplementation was provided using 300 mg of progesterone 3 times a day (Utrogestan; Lab.Besins International S.A.®, Paris, France).
To evaluate the success of the treatment transvaginal ultrasound examination was performed 21 days after the embryo transfer to detect gestational sac.
Measurements of FC and ACs
FC and all the ACs were determined by butyl-ester forms using isotope dilution mass spectrometry (MS) method in a Micromass Quattro Ultima (Manchester, UK) ESI triple-quadrupole mass spectrometer coupled with a Waters 2795 HPLC (Milford, MA, USA) system for sample introduction. For sample preparation 10 μl of serum or FF was used and a previously described procedure was followed . During the ESI/MS/MS analysis FC and ACs were measured by positive precursor ion scan of m/z 85, with a scan range of m/z: 200?550. The applied capillary voltage, cone voltage and collision energy were 2.54 kV, 55 V and 26 eV, respectively.
Our mass spectrometry facility is a registered participant in the International Newborn Screening Quality Assurance Program organized by the Center for Disease Control and Prevention, USA .
Routine hormone measurements were performed by using commercially available immunoassay kits. FSH and LH were measured with the electrochemiluminescent assay of Roche Ltd. (Elecsys 2010), while beta-HCG was measured with radioimmunoassay (Laborexpert, Hungary).
All statistical analyses were performed using SPSS, version 20 (SPSS Inc., Chicago, IL, USA). Normality of data was evaluated by Kolmogorov-Smirnov test. Variables are presented as median and interquartile range. Differences in carnitine ester concentrations between patient and control groups were analysed using the Kruskal-Wallis test. If changes were found to be significant, Mann–Whitney U test were performed. Data binning was applied in the case of oocyte and embryo number. This process is a data pre-processing technique used to reduce the effects of minor observation errors. Optimal binning thresholds, resulting into harmonic groups were offered by the statistical program. For correlation analysis between serum and follicular fluid values, the non-parametric Spearman’s bivariate test was used. A difference of p<0.05 was considered as significant.