A retrospective study recently showed that oocytes presenting with a high birefringence of the inner zona layer were more often associated with conception cycles. To further investigate these findings, a prospective study was conducted between September 2005 and September 2006 including intracytoplasmic sperm injection (ICSI) cycles presenting with at least two embryos for transfer. Using a polarization imaging system, oocytes were classified prior to ICSI treatment as having either a high zona birefringence (HZB) or a low zona birefringence (LZB) of the zona pellucida. Using zona birefringence as the only selection criterion, two fertilized oocytes, preferably derived from HZB oocytes, were selected for further culture and transfer. The required criteria were met by 135 ICSI cycles (124 patients; 34.9 +/- 4.1 years of age). Embryos for transfer were used in 20 cycles derived from HZB/HZB oocytes, in 50 cycles from HZB/LZB oocytes and in 65 from LZB/LZB oocytes. The corresponding implantation (P < 0.025), pregnancy (P < 0.005) and live birth (P < 0.025) rates were significantly different between HZB/HZB and HZB/LZB versus LZB/LZB group. Embryo development was superior in embryos derived from HZB oocytes. This study concludes that oocyte zona birefringence is a good selection criterion and a good predictive criterion for embryo implantation potential.

Recent studies using polarized light microscopy revealed a correlation between the presence of a spindle in human metaphase II meiotic oocytes and the fertilization rate following intracytoplasmic sperm injection (ICSI). Using a new spindle imaging system, it was possible to visualize the spindle image and the conventional light microscopic view of the oocyte simultaneously. Using this system, time-lapse studies of the meiotic cycle of human oocytes were performed. The video sequences showed that during the transition from metaphase I to metaphase II, the spindle completely disappears for approximately 40-60 min. These data support the idea that at least in some oocytes, the absence of the spindle is more likely an indicator for physiological progression through an important developmental stage of meiosis rather than a cellular disturbance. In view of the low fertilization rates of oocytes with absence of spindles as reported in the literature, the underlying problem could simply be the incorrect timing of ICSI.

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P!0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P!0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.

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To increase the efficiency of human IVP, noninvasive parameters to predict the developmental competence of oocytes and zygotes would be useful for selecting the right embryo to transfer. However, human oocytes and zygotes for experimental studies are rare. Therefore, using a bovine model with a precisely large sample size was the aim of the present study to investigate whether zona birefringence intensity (ZBI), measured by polarization light microscopy of metaphase II (MII) oocytes and zygote-stage embryos, was associated with subsequent development. In this study, ZBI was measured through 2 different parameters, the birefringence peak (PV, average signal strength of polarized light) and the birefringence peak combined with the surface of the signal (CV, average signal strength of polarized light multiplied by the surface of the signal), by using polarized light microscopy and OCTAX polarAIDE-software. Statistical analysis was done by the Tukey-Kramer test using SAS version 9.1. Cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries by aspiration. After in vitro maturation, MII oocytes were denuded and activated parthenogenetically (4-min exposure to 5 μm Ionomycin, followed by 3.5 h of incubation in 2 mm 6-DMAP) or fertilized in vitro before denudation. Subsequently, ZBI of MII oocytes (directly after activation) and zygotes (24 h after the beginning of IVF) were measured. To allow tracking of subsequent development, embryos were cultured individually in a well-of-well (WoW) culture system until Day 7 in CR1aa medium (5% CO2, 38.7°C, humidified air). When parthenogenetically activated embryos were cultured (n = 365), 287 (78.6%) cleaved and 115 (31.5%) reached blastocyst stage. The ZBI of MII-activated oocytes did not indicate any differences between those that cleaved and those that did not. On the contrary, we observed significant differences (P < 0.05) for mean values of PV (41.4 v. 43.3) and CV (21.3 v. 22.6) for MII-activated oocytes that subsequently reached the blastocyst stage at Day 7 and those that did not. When IVF zygotes (n = 415, 80.5% cleavage rate and 26.0% blastocyst rate) were analyzed, significantly (P < 0.05) lower mean values of PV (47.1 v. 51.5) and CV (24.7 v. 27.1) were observed for zygotes that cleaved within 48 h compared with zygotes that did not cleave. In addition, mean values of PV (45.5 v. 47.9) and CV (24.3 v. 25.4) for the zygotes that developed to the blastocyst stage at Day 7 were significantly lower in comparison with zygotes that did not develop to that developmental stage. In conclusion, we observed a connection between birefringence intensity at the MII/zygote stage and further development. This may offer benefits for the identification of favorable MII oocytes that could have implications for bovine somatic cell nuclear transfer as well as for human-assisted reproduction technology.