This was a retrospective study reviewing the medical records of infertile patients undergoing IVF procedures from January 2016 to August 2017 at the Fertility Center of CHA Gangnam Medical Center. Patients who responded normally to ovarian stimulation were recruited. The normal responders were selected according to age, anti-Müllerian hormone (AMH) levels, follicle-stimulating hormone (FSH) levels, and number of aspirated oocytes. PGS at the blastocyst stage was offered to infertile patients of advanced age (≥35 years), and/or with ≥3 unexplained recurrent pregnancy losses and/or ≥3 recurrent implantation failures. Patients who did not undergo PGS and those who underwent 3-day embryo transfer were excluded from the study population. Study participants were grouped according to age: women aged <35 years and men aged ≤38 years (group 1), women aged <35 years and men aged >38 years (group 2), women aged ≥35 years and men aged ≤38 years (group 3), and women aged ≥35 years and men aged >38 years (group 4). The paternal age cutoff of 38 years was defined according to the most equilibrated distribution of cases in both age groups and on the basis of another study [9].

Gonadotropin-releasing hormone (GnRH) antagonist and agonist protocols were mostly used in this study. The following medications were used in these protocols: recombinant FSH (Gonal-F, Merck, Darmstadt, Germany; Pergoveris, Merck), a GnRH antagonist (Cetrotide, Merck), and a GnRH agonist (Lorelin; Dongkook Pharmaceutical, Seoul, Korea). Recombinant FSH doses were formulated according to the patient’s antral follicular count, AMH level, and previous response to stimulation.

Transvaginal ultrasound was performed to monitor the follicular response to stimulation, and gonadotropin doses were adjusted accordingly. Final oocyte maturation was triggered with recombinant human chorionic gonadotropin (hCG) (Ovidrel, Merck) when there were ≥2 follicles with a mean diameter ≥17 mm. Ultrasound-guided oocyte retrieval under conscious sedation was performed 34–36 hours after the trigger. Luteal phase support was provided with progesterone vaginal suppositories (Utrogestan; Han Wha Pharma, Seoul, Korea) starting on the day of ovum pick-up. The β-hCG level was checked 10–12 days after embryo transfer.

Intracytoplasmic sperm injection (ICSI) was used to fertilize all the embryos from fresh cycles. Of the thawed embryo transfer (TET) cycles, ICSI was used in 57%, half-ICSI (ICSI and conventional IVF combined) in 15.9%, and conventional IVF in 27.1%, since for some patients, clinicians decided to perform PGS after embryo cryopreservation. For ICSI, the collected semen was liquefied for 30 minutes on a 37°C hot plate, after which sperm motility was evaluated [10]. Semen analysis was done, and semen samples with a normal sperm count and motility were washed two times with 10% SPS including Ham’s F-10 media by centrifuge. Motile spermatozoa were collected using the swim-up method. The general condition of each spermatozoon was thoroughly checked by first inspecting its general motility and morphology (head, midpiece, and tail), and then its head shape was evaluated. A spermatozoon was considered to have normal morphology when its head length was 5–7 μm, its width was 2–3 μm, and the acrosome distribution was more than 70% [11]. After selecting normal sperm, ICSI was performed under microscopy with ×200 magnification. In total, ICSI was used in 73.7% of cycles, half-ICSI in 9.7%, and conventional IVF in 16.6%. Fertilization was confirmed when the two-pronucleus stage was observed after 16–20 hours. During the cleavage stage, embryos were cultured in cleavage medium (Cook, Eight Mile Plains, Australia), and then transferred to blastocyst medium (Cook) on day 3.

Embryos were cultured in incubators (HERAcell 240i; Thermo Fisher Scientific, Waltham, MA, USA) under 5% O2 and 6% CO2 at 37°C. Oil drop culture was done using a four-well dish (Nunc, Thermo Fisher Scientific). In 20-μL drops, a single round of embryo culture was carried out to facilitate the observation of embryonic development. Light paraffin oil (Ovoil; Vitrolife, Göteborg, Sweden) was dropped onto the media to prevent it from drying or undergoing a rapid pH change.

Morphological grading of the embryos after culture was done on day 5 (Figure 1). Embryo quality on day 5 was assessed according to the criteria of Gardner and Schoolcraft [12]. Good-quality embryos on day 5 were defined as those with a blastocoel equal to or greater than half the volume of the embryo and a good inner cell mass and trophectoderm (TE). Based on the morphological grade, the embryos were categorized as good-quality blastocysts (excellent, good, or average) and poor-quality blastocysts.

All embryos were biopsied on day 5, during the blastocyst stage. TE biopsy was done from blastocysts that were cultured after conventional IVF or ICSI using a pair of micromanipulators (NT- 88; Narishige, Tokyo, Japan) with a microscope (TE-2000; Nikon, Tokyo, Japan). The culture medium consisted of G-PGD (Vitrolife) with 5% human albumin solution (Vitrolife). For the TE biopsy, the embryos were immobilized with a holding pipette (left side), and the zona pellucida was perforated using laser pulses with a ZILOS-tk noncontact laser (Hamilton Thorne Biosciences, Beverly, MA, USA). Using the right-side biopsy pipette, gentle suction of the TE cells was performed through the zona pellucida perforation. The cells were eventually separated out of the zona pellucida. The biopsied TE cells were washed with Dulbecco’s phosphate-buffered saline (with Ca²⁺ and Mg²⁺ removed) and placed in RNase/DNase-free 0.2-mL polymerase chain reaction (PCR) tubes for referral to MG-MED Inc. (Seoul, Korea) for PGS. The biopsied blastocysts were then cultured in sterile oil-covered media under 6% CO₂ and 5% O₂ at 37°C, and observations were made at 24- to 48-hour intervals to check the development of the embryos.

Embryo transfer was performed on day 6 under transabdominal sonographic guidance. Surplus embryos that were left after transfer were cultured, and the good-quality blastocysts on day 5 underwent PGS. Only normal euploid embryos were cryopreserved on day 6.

The biopsied cells were washed and collected in a PCR tube with 2.5 μL of PBS. The DNA from the biopsied cells was extracted and amplified using a MG Flex single-cell whole genome amplification kit (MG Flex; MGmed, Seoul, Korea) following the manufacturer’s protocol. Approximately 3 μg of amplified DNA was used in the array comparative genomic hybridization (aCGH) experiments (MGmed). Briefly, the amplified DNA was labeled with Cy-3 and Cy-5 deoxycytidine triphosphate for 3 hours using a random priming method. The labeled DNA was purified, dissolved in hybridization buffer, and hybridized overnight. The slides were washed several times and dried. Images of the slides were acquired with a GenePix 4000B dual-laser scanner (Axon Instruments, Union City, CA, USA) and analyzed with MGViewer analysis software (MGmed).

Clinical pregnancy was defined as the presence of at least one gestational sac and a fetal heartbeat at week 7 after transfer. Women over 16 weeks of gestation were defined as having an ongoing pregnancy. Continuous data are expressed as mean±standard deviation unless stated otherwise, and categorical variables are presented as absolute numbers and percentage. The data were analyzed using the Student t-test and the chi-square test. The analysis of clinical outcomes between different paternal age groups was done using the Mantel-Haenszel chi-square test to adjust for maternal age differences. All statistical analyses were performed using IBM SPSS ver. 25.0 (IBM Corp., Armonk, NY, USA). The p-values <0.05 were considered to indicate statistical significance.

The study was approved by the Institutional Review Board of CHA Gangnam Medical Center (IRB No. GCI-17-40). Consent to participate was waived due to its retrospective nature and only medical records were used in this analysis.