Morphokinetic assessment of human embryo development using time-lapse imaging

Abstract

Background: The success rate of IVF (In vitro fertilization) is associated with the methods to select best embryos for uterine transfer thus facilitating the acceptance and adoption of single embryo transfer regime to avoid the risks of multiple pregnancies. Time-Lapse imaging is an emerging tool to evaluate the developmental potential of embryos noninvasively by continuous monitoring. Recent advancements in the TimeLapse monitoring have provided us with the knowledge of many new morphokinetic markers for embryonic potential. There is limited information available regarding the relationship between morphokinetics of human embryo, its chromosomal composition and the implantation potential hence the specific aims of the present study were to analyze the cleavage data of embryos from our population of infertile couples, visiting the IVF center, using Time-Lapse imaging system established first time in an IVF clinic in Pakistan and to compare this analysis with previously acquired data sets with the help of this novel technique. Moreover this study also aimed to compare the cleavage data of embryos from patients with different factors contributing to their infertility and also to follow their treatment outcome. Methods: The current study is the retrospective observe of the prospectively acquired data at Islamabad Clinic Serving Infertile Couples (ICSI), Islamabad, Pakistan. A total number of 200 patients undergoing ICSI treatment cycles at the clinic was selected for the study and divided into five age groups (< 26, 26-30, 31-35, 36-40, and > 40 years) for first analysis which focused on effect of female age on embryo morphokinetics. The second analysis comprehensively compared the morphokinetic markers among different pathology groups of patients presenting with the history of infertility. There were eight groups included in this analysis namely the endometriosis, advanced female age, high FSH, idiopathic, low AMH, male factor, PCOS, and tubal factor. The third analysis of the study compared the embryo morphokinetics in four groups of the drugs used for ovarian stimulation i.e. patients treated with Gonal-f, IVF-M, puregon and a combination of IVFM+puregon. Embryo culture was done at 37°C, 6% CO2 and 5% oxygen for 5-6 days. Ten kinetic markers were selected for analysis i.e. time for appearance of pronuclear stage (tPNa), time for 2-cell (t2), 3-cell (t3), 4-cell (t4), 5-cell (t5) and 8-cell (t8) cleavages, time for compaction i.e. morula formation (tM), start of blastulation (tSB), expanding blastocyst (tEB) and fully expanded blastocyst stage (tFEB). The cycle outcome was also compared among different groups in all sets of analyses to see the effect of embryo morphokinetics on embryonic competence. Results:The first analysis i.e. effect of female age on embryo morphokinetics showed that the number of retrieved, matured, fertilized and cleaved oocytes were significantly different (P≤ 0.0001) when compared among different age groups of females. There was no significant difference in average morphokinetic parameters in young vs. old women. Whereas timely cleaved embryos showed significant difference in tPNa i.e. time for pronuclear appearance (P≤ 0.001), t4 and t5 i.e. time for 4 and 5-cell cleavage (P≤ 0.05) when compared among different age groups. The quality of blastocysts formed and the clinical pregnancy rates showed a decline with increasing age. The comparison among different pathology groups also showed the number of retrieved, matured, fertilized and cleaved oocytes to be significantly different (P≤ 0.0001) among groups. Among the average morphokinetic parameters only t3 and tM showed significant difference (P≤0.05) when compared among different pathologies of patients. There was no significant difference in all other morphokinetic time-points among all study groups. Whereas the timely cleaved embryos showed significant difference in t3 (P≤ 0.01), t5 (P≤ 0.01) and t8 (P≤ 0.05) when compared among different pathology groups. The blastocyst formation rates were comparable among different groups while the clinical pregnancy rates were quite low in fresh cycle transfers in all study groups except PCOS and endometriosis. The frozen-thawed cycle showed comparable pregnancy rates in all groups except the patients with advanced age, high FSH and low AMH whose rates improved but not to an appreciable extent. The cell cycle intervals for second and third cycle i.e. cc2 and cc3 respectively were found to be in normal range for different groups in both analyses. Regarding the analysis of ovarian stimulation groups, the mean cleavage timings for 2cell (t2) and 8-cell cleavage (t8) showed a significant difference when compared among different groups. The rate of blastocyst formation is lowest in patients using IVF-M for ovarian stimulation. The clinical pregnancy rates in fresh cycle transfers were low in patients using puregon+IVF-M and the ones who used IVF-M only.Conclusions: Hence it is evident that advanced female age and other different pathological conditions affect the oocyte and embryo quality as well as the clinical outcome during an IVF/ICSI cycle when compared by using Time-Lapse morphokinetic evaluation. Similarly the high doses of ovarian stimulation drugs also affect the development of embryos adversely. Limitations and wider implication of findings:The current study is based on retrospective analysis of embryos from patients with different factors contributing to clinical indications for infertility so there were certain limitations for exclusion of cleavage data in order to highlight the effect of pathologies on embryonic competence. The prospective study design is although time taking and need more labor work but can overcome this limitation by selective analyses and further subgrouping among embryos showing mild, moderate or severe deviation from normal cleavage patterns. Hence it is suggested that the prospective approach in future can help to add in the already existing information of kinetic markers for selection of best embryo for transfer in patients with various factors contributing to infertility.