2. Case presentation
The key patient, born in 1987, presented with a body mass index (BMI) of 20.34. She had a history of nulliparity (PARA: 1021) and had been actively trying to conceive for three years. After unsuccessful attempts at intrauterine insemination (IUI) in 2018, she was recommended for IVF. Table 1 provides an overview of the patient’s baseline characteristics. Preimplantation genetic testing for aneuploidy (PGT-A) was also carried out to investigate the ploidy status of embryos.
The IVF treatment followed a standardized protocol. Controlled ovarian stimulation was achieved through a GnRH-antagonist protocol, with daily administration of 150 IU of Gonal-F® and 150 IU of Pergoveris® (Merck Serono S.p.A, Italy). Cetrotide® (Merck KGaA, Germany) was introduced on the seventh day of stimulation. The patient received an intravenous administration of 10,000 units of hCG IVF-C (LG Chem, Korea).
Oocyte retrieval was performed on September 23 2021, followed by embryo vitrification on September 28 2021. The embryos were cultured in a Geri™ Time-lapse Incubator at 37°C, with a controlled atmosphere consisting of 6% CO2, 5% O2, and 89% N2. On the day of oocyte retrieval, her husband collected semen through masturbation. The sperm analysis yielded favorable result. The swim-up method was applied to prepare the sperm sample for fertilization. The sample was incubated in G-IVF PLUS media (Vitrolife, 10136) for 30 minutes to collect motile spermatozoa. Detailed information on semen quality is described in Table 2.
For oocyte retrieval, we used Modified HTF Medium with Gentamicin (FUJIFILM Irvine Scientific, 90126). Following retrieval, the oocyte-cumulus complexes (OCCs) were cultured using G-IVF PLUS media (Vitrolife, 10136). A total of 23 oocytes were retrieved, comprising 18 mature (MII) oocytes, 3 immature (MI) oocytes, and 2 germinal vesicle (GV) stage oocytes. Out of these, 18 oocytes underwent successful fertilization, resulting in the formation of 2 pronuclei (2PN) embryos.
Embryo development proceeded using Geri medium (Genea Biomedx, ONE-50), resulting in 13 blastocysts on the fifth day. Trophectoderm cells from 8 good-quality blastocysts were selected for aneuploidy screening using next-generation sequencing (NGS) technology. The screening identified five euploid embryos, indicating a favorable prognosis for successful implantation. Detailed information regarding the IVF with PGT-A cycle is provided in Tables 3 and 4. All embryos were preserved using the Vitrification Kit 101 (Cryotech, Japan).
Following endometrium preparation, the patient was recommended for frozen embryo transfer (FET). The embryologists thawed one embryo for transfer using the Warming Solution Set 205 (Cryotech, Japan). Unfortunately, the patient experienced three unexplained implantation failures. During the culture process, we observed cloudy particles in the petri dishes (Figure 1-A) and even at the blastocyst stage (Figure 1-B). Those particles proliferated aftterward, raising concerns about bacterial contamination. To investigate this, we collected IVF medium and sent it to the Department of Medical Microbiology for examination. The later result confirmed the contamination of Rhizobium radiobacter bacteremia - an infrequent cause of human infection. It’s important to highlight that the contamination occurred during the initial stages of embryo culture. Consequently, bacteria became cryopreserved along with the embryos. For the second and third FET attempts, we implemented a washing step for the embryos in culture medium before transfer. However, the patient continued to experience unsuccessful outcomes. In the final attempt, we employed the Octax NaviLase® Laser from Vitrolife to remove the zona pellucida. Following this intervention, the patient achieved pregnancy, and ultimately, a live birth was accomplished. The baby was born healthy, weighing 2608 g at 37.5 weeks’ gestation. Comprehensive details regarding the four FET cycles of the patient are presented in Table 5.