Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

Abstract

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.

A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator

“…The possibility to analyze the dynamic nature of embryo development gives rise to expectations of improved embryo selection. Time-lapse monitoring enables registration of dynamic events such as the precise timing and synchrony of cell-divisions, appearance and disappearance of nuclei and pro-nuclei; events that in descriptive studies have shown correlation with developmental competence of both animal and human embryos [18,30,41]. In this study, we demonstrate that time-lapse monitoring can be applied safely to human embryos, thereby enabling further investigation of a promising tool for improved embryo selection.…”

“…There is a well-documented correlation between embryo morphology evaluated at certain time-points and embryo competence as reviewed in a recent consensus paper by ALPHA and ESHRE [16]. However, the progressive and dynamic nature of cell cleavage and embryo development is also well known [13,18,30], and embryo scoring can change markedly within few hours [25]. In conventional morphological evaluation of embryo quality the number and duration of inspections outside the incubator must be restricted since changes in environment are known to induce stress [42].…”

Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number

“…Pronuclear fusion was not observed in an investigation using time-lapse cinematography from the appearance of 2PN until syngamy [24]. The following three patterns could be considered for the formation of 3PN zygotes: (1) polyspermy normally occurs only in IVF cases; however, it might be possible in rare cases that a diploid spermatozoon was chosen in ICSI; (2) failure of the 2nd PB extrusion in both IVF and ICSI cycles; and (3) fertilization of a diploid oocyte in IVF or ICSI [25][26][27][28].…”

Observation of human embryonic behavior in vitro by high‐resolution time‐lapse cinematography

“…In 1997, Payne et al [13] developed time-lapse video cinematography to overcome the limitations of intermittent observation using still images by providing continuous imaging, in which various cellular events could be distinguished in real time and monitored during the observation period. However, this initial system had a limited observation period of 17-20 h. Based on the original report of Payne et al [13], we developed a new in vitro culture system for high-resolution time-lapse cinematography (hR-TLC) by which clear images of embryonic development could be obtained for a long period of time (up to 5 days) by maintaining optimal culture conditions on the stage of an inverted microscope ( Fig.…”

“…However, this initial system had a limited observation period of 17-20 h. Based on the original report of Payne et al [13], we developed a new in vitro culture system for high-resolution time-lapse cinematography (hR-TLC) by which clear images of embryonic development could be obtained for a long period of time (up to 5 days) by maintaining optimal culture conditions on the stage of an inverted microscope ( Fig. 1) [14,15].…”