The current was a non-interventional, retrospective single centre review.
Institutional Review Board approval was not required since all couples included underwent a routine IVF/ICSI program in our Reproductive Unit, and no additional/experimental intervention was performed.
All IVF/ICSI cycles performed between April 2008 and November 2010 were reviewed.
All cycles characterized by the transfer of one embryo were selected and included in the final analysis. Were excluded cycles in which with more than one oocyte was fertilized and cleaved.
For any cycle we collected all available clinical and biological data. Specifically, we recorded the patients' characteristics, the controlled ovarian stimulation regimens, the drugs and protocols used for luteal phase support, the characteristics of oocytes retrieved and embryos transfer, the pronuclear and embryo morphology. Implantation, pregnancy, abortion and live-birth rates were also noted.
Ovarian stimulation and oocyte retrieval
In all cases the controlled ovarian stimulation was achieved using individualized protocols of recombinant-follicle stimulating hormone (rFSH; Gonal F, Serono, Rome, Italy) in down-regulated cycles. A serum estradiol concentrations <50 pg/mL and an absence of follicles having a mean diameter higher than 10 mm were considered as criteria for gonadotropin administration. The ovarian response was monitored by use of serum estradiol assays and serial ultrasonographies.
In presence of at least one follicle with a mean diameter equal or higher than 17 mm was observed, 10,000 IU human chorionic gonadotropin (hCG; Gonasi, IBSA, Milan, Italy) or 250 μg recombinant hCG (Ovitrelle, Serono, Rome, Italy) were intramuscularly administered. Oocyte retrieval was performed 34 to 36 hours after hCG administration by ultrasound-guided transvaginal aspiration.
Semen samples were collected by masturbation after 3–5 days of abstinence. The preparation for conventional IVF or ICSI was performed following the World Health Organization standard protocol [16, 17].
For the conventional IVF procedures, oocytes were cultured individually and inseminated in microdrops of fresh medium (Cook IVF, Melbourne, Australia) under mineral oil with 100,000 activated spermatozoa. For ICSI procedure, after the removal of the cumulus and corona cells, oocytes nuclear maturation assessment was performed using an inverted microscope to ensure the injection of metaphase II oocytes exclusively .
Assessment of fertilization/cleavage
Oocyte fertilization was assessed at 18–20 hours (day 1) from insemination/injection and confirmed by the presence of 2 pronuclei (2PN) and the alignment of nucleolar precursor bodies (NPB). At same time, the pronuclear morphological score was assessed . The observation of 2PN was performed using an inverted microscope with Hoffman modulation contrast at ×400 magnification (TE 2000 U, Nikon Corp., Japan). Zygotes with simultaneously juxtaposed and centralized PN, nucleoli of large size and orientated, and orientation of polar bodies in the longitudinal axis of PN were classified as Z1 and considered as the best zygote morphology [4, 5, 19]. Zygotes having all other configurations were classified as Z2.
ET was performed after 48 hrs (day 2) of embryo culture. During embryo observation five parameters were classified: cleavage symmetry, blastomere shape and size, cytoplasmic aspect, presence of fragmentation and of blastomere multi/micronucleation. Embryos characterized by symmetric cleavage, regular blastomeres sheep and size, absence of any cytoplasmic anomalies such as dark areas, granulations and vacuole, blastomeres multinucleation (≥ 2 nuclei per blastomere) or micronucleation were considered as grade I embryos. Grade II embryos presented slightly irregular blastomeres sheep and size, fragmentation ≤ 10%, and small cytoplasmic vacuoles. Grade III embryos were characterized by asymmetric cleavage, highly irregular blastomeres sheep and size, fragmentation among 10-30%, dark cytoplasm, cytoplasmic vacuoles and multinucleation. Finally, grade IV embryos displayed asymmetric cleavage, severe irregular blastomeres sheep and size, fragmentation among 30-50%, dark cytoplasm with massive presence of vacuoles. ETs of grades I and II embryos were defined as High Quality Embryo ET (HQE ET), whereas ETs of grades III and IV embryos as Poor Quality Embryo ET (PQE ET).
Embryo transfer and establishment of pregnancy
All patients received intramuscular (100 mg/day; Prontogest; IBSA, Milan, Italy) or transvaginal supplemental progesterone (600 mg/day; Prometrium, Rottapharm, Milan, Italy) for 15 days.
In each case, biochemical pregnancy was determined 12 days after ET by a positive quantitative serum β-hCG assay >10 IU. In case of positive pregnancy test, the hormonal support was continued until 35 days after the ET. Clinical pregnancy was defined as one embryo with heart beat revealed by transvaginal ultrasonography at 5 weeks after ET. Because of only one embryo was transferred in each ET, the clinical pregnancy was equivalent to the embryo implantation.
The normal distribution of continuous variables was evaluated with the use of the Kolmogrov-Smirnov test, and continuous data were expressed as the mean ± standard deviation (SD).
Continuous data were analyzed by Student t test for unpaired data. For categorical variables, the Pearson Chi square test was performed; Fisher’s exact test was used for the frequency tables when more than 20% of the expected values were lower than five.
To investigate which of the variables studied could best predict the chances of a clinical pregnancy and live-birth, a binary logistic regression was performed, using embryo quality, insemination technique and pronuclear morphology as predictors.
For all tests, the statistical significance level was set at P < 0.05.
Analysis was performed using SPSS version 20 for Microsoft Office.