Determination of APOB concentrations in blood-free FF samples, obtained from 61 female patients

A total of 61 female patients between 24 and 42 years of age (mean ± SD: 33.6 ± 4.8 years) and with a BMI between 16 and 35 kg/m² (mean ± SD: 22.5 ± 3.8 kg/m²) were included in our prospective study. Among these patients, 16 were overweight or obese (BMI >25 kg/m²), whereas 3 were underweight (BMI < 18.5 kg/m^2). These women underwent controlled ovarian stimulation (COS) followed by either IVF (n = 34) or ICSI (n = 27) procedure at the Assisted Reproduction Technology Department of the Dijon hospital (CHU de Dijon, France). Exclusion criteria included polycystic ovary syndrome or lipid metabolic disorders [21]. On average, the delay in conception was 3.6 ± 1.8 years. Forty three couples had primary infertility (70.5 %) and 18 couples secondary infertility (29.5 %). Fifty five couples were enrolled in IVF/ICSI procedure for the first or second time. Female infertility, sperm abnormalities, mixed and unexplained infertility were diagnosed in 49.2 %, 37.7 %, 8.2 %, and 4.9 % of the cases, respectively. Based on anti-müllerian hormone (AMH) and antral follicular count (AFC), all women had a normal ovarian reserve. The patients' clinical characteristics are summarized in Additional file 1: Table S1. Informed consents were obtained from all patients for the use of FF samples on the day of oocyte retrieval.

Sixty one patients underwent an agonist GnRH protocol including ovarian stimulation with recombinant FSH (r-FSH) (Puregon, Schering Plough, Courbevoie, France or Gonalf, Merck Serono, Lyon, France). Ovarian stimulation response was monitored by both, serum estradiol concentration and transvaginal ultrasound assessment of follicular and endometrial growth.

Ovulation was induced with 6500 UI of recombinant hCG (Ovitrelle, Merck Serono, Lyon, France) when at least three follicles had reached 17 mm or more in diameter. Cumulus oocyte complexes (COC) were retrieved by transvaginal ultrasound-guided aspiration, 36 h after ovulation triggering, rinsed twice and isolated for conventional IVF or ICSI protocol. In ICSI procedure, after denudation, oocyte maturity was assessed and mature oocytes were micro-injected. Oocytes were individually maintained in 30 μl microdrops of culture medium (Global Medium, LifeGlobal, USA) under oil at 37 °C in 6 % CO₂ and 5 % O₂. Normal fertilization was checked between 16 and 18 h after insemination or microinjection by the presence of two pronuclei (2PN) and the two polar bodies. Early cleavage (EC) was evaluated 25 or 27 h after microinjection (ICSI) or insemination (IVF), respectively. Two days after oocyte retrieval, embryo quality was scored according to morphological criteria: (i) cleavage stage, (ii) number and size of blastomeres, and (iii) degree of fragmentation. An embryo was considered as a top quality embryo if there were 4 to 5 blastomeres on day 2, less than 20 % of fragments, and no multinucleation [22]. On day 2, the embryo(s) (standard-of-care in the centre) was (were) transferred under transabdominal ultrasound guidance.

For the 61 patients, at the day of oocyte retrieval, all the follicles visualized by ultrasound were aspirated individually without flushing. Only blood-free FF samples (n = 201) were collected and centrifuged at 3000 g for 15 min. The supernatants were removed and stored at –80 °C for APOB quantification. APOB concentration in each FF sample was quantified using the Human APOB ELISA^prokit (no. 3715-1HP-2 Mabtech AB, Sophia Antipolis, France). Inter and intra-assay variation coefficients were 10.0 % (CV) and 2.0 % (CV), respectively.

Quartiles (Q) of APOB concentrations (ng/ml) measured in the FF (n = 201) samples of the 61 patients were defined as followed:

Q1: APOB <112 ng/ml

Q2: 112 <APOB<230 ng/ml

Q3: 230 <APOB<330 ng/ml

Q4: APOB>330 ng/ml

The patients’ characteristics in each defined quartile are presented in Additional file 2: Table S2.

See statistical analysis section for further information.