Study design
It is a controlled retrospective study conducted in a private fertility center from the 2014 to 2020. In this study, 267 patients (AMA women) were included following the inclusion criteria. The PGT-A group (n = 53, study group), consisted of 53 patients who had undergone biopsy followed by euploid blastocyst transfer. Whereas the non-PGT-A group (n = 214, control group), comprised of 214 patients who had blastocyst transfer merely based on the morphological study of the embryo alone.
Inclusion Criteria
Women in the AMA group (≥ 35) were included in this study. Further, only frozen embryo transfer (FET) cycles were included. All the PGT-A cases where at least one or more euploid embryos were available for transfer were also included in this study. Informed consent was obtained from the patients to evaluate the data from the study.
Exclusion Criteria
All the PGT-A cases where no euploid embryos were available for transfer and with male factor were excluded.
Stimulation & Patient Preparation
All the patients were stimulated by following a down regulation stimulation protocol with GnRH antagonist (Gonal F, Merck Global, USA) for 10–12 days from D2/D3 of menstruation cycles to stimulate the ovaries to produce enough follicles. The growth of the Antral follicles was continuously monitored by Ultrasound guidance & blood estradiol (E2) levels. The dosage of the stimulation drug was individualized based on patient characteristics. Once the size of the follicles reaches 18–20 mm, a trigger injection was administrated (HCG 10,000, Serum Institute, India) and Oocyte retrieval was done 35–36 hours post trigger.
All interventions performed to the women during the infertility treatments were in accordance with relevant guidelines and regulations. All couples that opted for PGT underwent counseling session and an informed written consent was obtained about the benefits, challenges and pitfalls of the embryos biopsy technique and the results of PGT. Only couples that gave informed consent were recruited in this retrospective study. Since the data of this study was retrospective in nature and a prior ethical clearance was not applied. Nevertheless we applied for a waiver of ethical clearance to the institutional review board and obtained a waiver for this study in order to publish our findings in women with advanced maternal age.
Oocyte Collection, Insemination & Embryo Development
The ovum pick up (OPU) was done post 35–36 h of HCG trigger under trans vaginal ultrasonography (TVS) with the help of suction pressure. The follicular fluid was screened under a stereozoom microscope in the IVF laboratory by the embryologist into a 60mm petri dishes. The cumulus oocyte complexes (COCs) were separated from the follicular fluid and cultured in a fertilization media (Quinn’s advantage protein plus, Cooper surgical inc., USA) for 2–3 hours. The COCs was then denuded with enzymatic (Hyaluronidase 80U/ml, Cooper surgical, USA) & mechanical process. All the metaphase (MII) oocytes were used for intra cytoplasmic sperm injection (ICSI). The semen sample were obtained from their partners by masturbation and analysed for count & motility. Double density gradient was performed as per the count & motility of the sperm. Morphologically normal sperms were used for injection during ICSI. All MII oocytes were injected and cultured in a Single step culture medium till Day5 (Sage 1 step with HSA, Origio, Denmark) at 37 degrees in a humidified incubator with hypoxic culture conditions (5% oxygen). Normal fertilization was confirmed by observing two distinct pronuclei & two polar bodies 16–18 hours post-ICSI. The fertilized zygotes were cultured till Day5/6 using standard incubation protocol. The embryo development assessment was done on Day5/6 and grading of the blastocyst was done as per the standard grading system (The Istanbul consensus workshop on embryo assessment).
Biopsy, Tubing & Vitrification
Only grade 1& 2 fully expanded blastocysts were selected for biopsies. Number of embryos to be biopsied and consent forms were obtained from the patients. LASER assisted hatching was performed to facilitate the herniation of enough number of trophectoderm (TE) cells and once the appropriate number of cells were herniated, the biopsy was performed by aspirating six to eight TE cells by using a biopsy needle (Blastomere aspiration needle, Cook, USA). Aspirated TE cells were then transferred into a PCR tube that contained Phosphate buffer saline (PBS) provided by the genetic lab (Igenomix, India) carefully in the presence of an eyewitness The biopsied cells were then stored in a deep freezer at -21 degrees Celsius until they were shipped to the genetic lab.
The collapsed blastocysts were further cultured for 1–2 hours post biopsies for their expansion and then vitrified using a kitazato vitrification kit (Kitazato BioPharma, Tokyo, Japan) and stored on a cryotop in liquid nitrogen (LN2)
Frozen Embryo Transfer (Fet)
For subsequent FET, the endometrium was prepared by giving estrogen support. Once the endometrium attains adequate thickness (> 8mm), luteal phase support was started and, FET was planned. On the day of transfer, the thawing of one/two PGT-A euploid embryos was done by using the Kitazato Thawing kit (Kitazato BioPharma, Tokyo, Japan). The thawed blastocyst was cultured for 2 hours for survival confirmation in one step culture medium at 37 degrees Celsius. One or two thawed blastocyst was transferred into the uterus by using an ET catheter (Emtrac set, Gynetic, Belgium). Fourteen days after ET, the patient underwent a urine pregnancy test and pregnancy was confirmed by Beta Hcg levels in the blood (> 50mIU/ml).
Statistics
The variables were calculated and presented in absolute numbers and percentages. The chi-Square test was performed to analyze the difference between the variables. Differences were considered significant at P < 0.05.