Fifty-three consecutive fresh IVF/intracytoplasmic sperm injection (ICSI) cycles were selected. All cycles were performed between 2014 and 2017 at Seoul National University Bundang Hospital. IVF/ICSI cycles were included if they were normal responders (i.e., 4 to 14 mature oocytes were retrieved) and at least one 2PN zygote was obtained. The median ages of women and their male partners were 35.0 (interquartile range: 32.0–39.5) and 38.0 (interquartile range: 35.0–44.0) years, respectively. Indications for IVF were unexplained (n=22), tubal (n=12), male (n=8), endometriosis (n=5), uterine (n=4), polycystic ovary syndrome (n=1), and old age (n=1). This retrospective study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB No. B-1806-474-106).

Ovarian stimulation was performed with recombinant folliclestimulating hormone (FSH) (Gonal-F, Merck-Serono, Darmstadt, Germany; or Follitrope, LG Chem, Seoul, Korea), highly purified human menopausal gonadotrophin (Menopur, Ferring, Saint-Prex, Switzerland), or recombinant FSH/luteinizing hormone (Pergoveris, Merck-Serono). The dose of gonadotropin was adjusted individually based on follicular response monitored by transvaginal ultrasonography. Pituitary downregulation was achieved with a flexible GnRH antagonist protocol (Cetrotide 0.25 mg/d, Merck-Serono) (n=49) or a midluteal long protocol using GnRH agonist (Decapeptyl 0.1 mg/d, Ferring) (n=3). Pituitary down-regulation was not employed in a single cycle. When the leading follicle reached a mean diameter of ≥18 mm or two follicles reached a mean diameter of ≥17 mm, 250 µg of recombinant hCG (Ovidrel, Merck-Serono) was injected subcutaneously. Oocyte retrieval was performed at 35–36 hours after hCG injection. If immature oocytes were retrieved, they were cultured in in vitro maturation media. Once matured, they were counted as mature oocytes.

Semen samples were collected on the day of oocyte retrieval. Standard sperm quality was assessed according to the World Health Organization guidelines (5th edition, 2010).18 Normal sperm was defined as semen volume ≥1.5 mL, concentration ≥15×10⁶/mL, motility ≥40%, and a percentage of normal form using strict criteria ≥4%.

After one washing, SDF was measured by SCD test using Halosperm assay (Halotech DNA, Madrid, Spain), as previously described in our institutional protocol.19 Briefly, an aliquot of the semen sample was diluted with Ham's F-10 medium (Sage, Trumbull, CT, USA) to 5×10⁶/mL to 10×10⁶/mL. Agarose gel from the kit was placed in a water bath at 90–100℃ for 5 minutes and then at 37℃ for 5 minutes. This was followed by adding and mixing 25 µL of semen in a marked Eppendorf test tube. The mixture of 15 µL was placed on a super-coated slide and covered with a coverslip. Slides were then placed in a refrigerator at 4℃ for 5 minutes to create a microgel with embedded sperm cells. After obtaining slides from the refrigerator, coverslips were gently removed, followed by immersing slides in acid denaturation solution (80 µL in 10 mL of distilled water) for 7 minutes. These slides were then immersed in a lysis solution from the kit and incubated for 25 minutes. After washing with distilled water, slides were dehydrated in increasing concentrations of ethanol (70, 90, and 100%) for 2 minutes and then air-dried. The slides were stained with Diff-quick solution I and II (7 minutes for each), rinsed with distilled water, and air-dried at room temperature.

Each slide was examined under a light microscopy at ×100 magnification. At least 300 sperms were scored. Each sperm was classified as large halo, medium halo, small halo, no halo, or degraded. A large halo meant that the halo width was similar to or higher than the minor diameter of the core. A medium halo meant that the halo size was between large halo and small halo. A small halo meant that the halo width was similar to or smaller than one-third of the minor diameter of the core. Degraded sperm was similar to sperm with no halo but weakly or irregularly stained. SDF level (%) was calculated as (the number of small halo+no halo+degraded form)/total sperm cell counted.

The remaining semen samples were processed by a discontinuous gradient (Sydney IVF Sperm Gradient, COOK Medial, Brisbane, Australia) as described in the kit instructions and our institutional protocol.819 Mature oocytes were inseminated by conventional methods (18 cycles), ICSI (29 cycles), or split insemination (6 cycles). Fertilization was confirmed by observing 2PN zygotes on day 1 after insemination.

Embryo quality was evaluated by morphological criteria based on the degree of fragmentation and the regularity of blastomeres on day 3 after insemination. Embryos were graded as follows: grade A, 0% anucleate fragments, regularity of blastomeres, no apparent morphological abnormality; grade B, <20% anucleate fragments, regularity of blastomeres, no apparent morphological abnormality; grade C, 20–50% anucleate fragments, irregularity of blastomeres, no apparent morphological abnormality; and grade D, >50% anucleate fragments, irregularity of blastomeres, and apparent morphological abnormalities.20 The top-quality embryo was defined if they showed seven cells or more with grade A. Day 3 embryo formation rate was always calculated per 2PN zygote. In the present study, three embryo formation rates were employed: top-quality embryo formation rate, grade A embryo formation rate, and grade A or B embryo formation rate.

The embryos were transferred at 3 days (n=37) or 5 days (n=10) after oocyte retrieval. Serum hCG levels were determined at 14 days after oocyte retrieval. Those with a positive hCG result underwent transvaginal ultrasound to confirm an intrauterine pregnancy. Clinical pregnancy was defined as the presence of one or more gestational sacs with a fetal heart beat.

All statistical analyses were performed using SPSS 22 (IBM Corp., Armonk, NY, USA). Spearman correlation rank test was used to analyze the associations of SDF levels with standard sperm quality and three embryo formation rates. A receiver operating characteristics (ROC) curve analysis was used to determine the specific cut-off value of SDF to predict specific embryo formation rate. Mann-Whitney U test was used to compare parameters between groups. Proportions between groups were compared using Pearson's chi-square test or Fisher's exact test if the observed cell number was less than five. All p values <0.05 were considered significant.