This retrospective study includes patients with unexplained infertility who have failed to become pregnant, after 4 IUI, despite normal semen parameters of World Health Organisation (WHO) criteria  after sperm capacitation. These women were treated in our assisted fertilization program from 2008 until 2015. We included women between 18 and 43 years of age. We began by analysing only the first cycles of women in whom more than 4 oocyte cumulus complexes (OCC) were retrieved, after stimulation. Sibling oocytes were randomly divided between the two fertilization methods: classical IVF (IVF) and ICSI. We observed cycles in which a single embryo transfer was performed and excluded all others.
We then analysed the second stimulation cycle in a small cohort of similar patients who had been treated with complete classical fertilization technique according to the result of the first trial.
On the day of oocyte retrieval, sperm analysis was performed according to the WHO laboratory standards of human semen and sperm. Motile spermatozoa were selected using a Sil Select two layer gradient centrifugation (FertiPro, Beernem, Belgium) for 15 min followed by two 5 min cleansings in G-IVF (Vitrolife, Goteborg, Sweden).
Our sperm criteria required in order to perform the 4 first IUIs were the obtaining of at least 1 million motile spermatozoa, at least 4% of normal sperm and ≥32% progressive motility based on strict sperm morphology criteria (WHO), in the final sperm preparation aliquot (500 μl).
Our criteria for the final sperm preparation to perform classical IVF fertilization are 1–1, 5.105 motile spermatozoa per millilitre and >4% morphologically normal spermatozoa. If the final preparation does not meet these criteria during the first IVF cycle, ICSI is performed and this couple is excluded from the study.
During IVF trials, stimulation is achieved with 100 to 300 IU of gonadotrophins depending on age, body mass index (BMI) and the Anti-Mullerian hormone (AMH) level (FSHrec: Puregon,; MSD, Oss, the Netherlands; GonalF: Merck-Serono, Geneva, Switzerland) or human Menopausal Gonadotrophin (Menopur, Ferring, Alost, Belgique). Ovulation was controlled by either gonadotropin-releasing hormone (GnRH) antagonist (Cetrorelix, Merck Serono or Ganirelix, MSD) or GnRH agonist (Buserelin: Suprefact; Sanofi-Avantis, Diegem, Belgium or triptoréline: Gonapeptyl, Ferring) respectively in 68% (170 cycles) and 32% (81 cycles) of cases. The GnRH antagonist is administered according to a flexible protocol which is once daily, when at least one follicle has reached a diameter of 14 mm and / or the oestradiol (E2) level is at 400 pg / ml. When a GnHR agonist is used, the long protocol is applied in the majority of such cycles. Triggering was obtained with 5000 IU of human chorionic gonadotrophin (hCG) (Pregnyl, MSD, Bruxelles, Belgium).
For conventional fertilization, OCC were inseminated over-night with a sperm suspension at a concentration of 100–150.103 motile spermatozoa/ml. Oocytes selected for ICSI fertilization were stripped of their cumulus using Hyase (Vitrolife, Goteborg, Sweden) 40 IU/ml. Sperm injection was performed only on metaphase II oocytes, four hours after pick up.
Fertilization check, embryo culture and transfer
Between 16 and 18 h after injection or classical insemination, normal fertilization was confirmed by the presence of two pronuclei (PN). Abnormal fertilization was determined when 0, 1 and 3 PN were obtained. We excluded immature oocytes (0 polar body (PB) and germinal vesicle (GV)) from the count of conventional fertilization after removal of cumulus cells on day 1. The fertilization rate was expressed per mature oocytes injected or inseminated. Total fertilization failure (TFF) is defined as the absence of two pronuclei (2PN) embryos on day 1 after insemination. Embryos were cultured in an incubator at 37 °C, under 5% of CO2 and 6% of O2 and were assessed each morning until their transfer (day 3 or day 5) based on Istanbul consensus criteria . The embryo with the best morphological score was selected for transfer and only one embryo was transferred (SET). Supernumerary embryos of good quality were vitrified. The fertilization method is not a criterion for embryo selection in this study. When more than one embryo obtained the same morphological score, we analysed their kinetic development and selected the one which had the best one [10,11,12,13,14].
An hCG blood test was performed 15 days after the oocytes pick-up (OPU) and then 7 days after that. A developing pregnancy was confirmed by observation of an intrauterine gestational sac and foetal heart beat 10 days after the second blood test.
The analysis of the results was carried out using Fisher’s test and the Chi-square test when it was appropriate. Statistical significance was accepted when p-value < 0, 05. The analysis was performed on GraphPad© Prims v5.