Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, >38 years; younger, ≤38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean ± SD: 24.1 ± 2.5 μm vs 23.1 ± 3.3 μm; p = 0.01) but ZP density was equal (2.8 ± 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 ± 2.2 μm vs 21.7 ± 1.6 μm; p = 0.28) and density (2.9 ± 0.7 nm vs 2.8 ± 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 ± 2.7 μm vs 19.1 ± 5.0 μm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 ± 0.5 nm vs 3.3 ± 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 ± 0.5 nm vs 2.9 ± 0.7 nm, p < 0.001; younger: 3.3 ± 1.0 nm vs 2.8 ± 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.

A liquid crystal polarized light microscope (LC PolScope) was used to examine spindle dynamics in living mouse oocytes. Immature oocytes were cultured for 0–48 h and spindles were imaged with the PolScope at various time points of culture. Oocytes at metaphase I (M-I) and metaphase II (M-II) were also exposed to shifts of temperature from 25 to 41 °C to examine the effects of fluctuations of temperature on spindle dynamics. After examination with the PolScope, some oocytes were fixed and examined by immunocytochemical staining and confocal microscopy. After culturing for 6 h, 76% and 2% of the oocytes reached M-I and M-II stages and all oocytes had birefringent spindles. When the oocytes were cultured for 14–16 h, 88% and 6% of oocytes were at M-II and M-I stages respectively and all oocytes had birefringent spindles. However, when the oocytes were cultured for 22–48 h, the proportions of oocytes with birefringent spindles decreased as culture time was increased. Exposure of oocytes to 25 °C induced spindle disassembly within 10–20 min in both M-I and M-II oocytes. Most (93–100%) oocytes reassembled spindles after warming at 37 °C. Furthermore, exposure of oocytes at M-I stage but not at M-II stage, to 30 °C also induced significant microtubule disassembly. However, exposure of oocytes to 38–41 °C did not obviously change the quantity of microtubules in the spindles, which was measured by retardance. This study indicates that the PolScope can be used to examine spindle dynamics in living oocytes, and it has the advantage over the routine fluorescence microscope in that images can be obtained in the same individual oocyte and the quantity of microtubules can be measured by retardance in living oocytes. These results also indicate that the M-II spindle in mouse oocytes is sensitive to oocyte ageing and cooling, but not heating, and M-I spindle is more sensitive to temperature decline than M-II spindle.

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24–36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 °C for up to 20 min. After heating, oocytes were returned to 37 °C and then imaged for another 20 min at 37 °C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 °C during 40 min of examination and their maximum retardance (1.72–1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 °C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 °C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 °C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 °C and 40 °C, respectively. Heating to 40 °C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 °C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 °C is crucial for normal spindle morphology.