偏光顯微鏡 (PLM) 檢查顯示 PB 的卵母細胞是否在 ICSI 之前真正重新組裝了減數分裂紡錘體。

https://www.youtube.com/watch?v=2XKPe457UFg

https://www.jove.com/t/60058/human-egg-maturity-assessment-and-its-clinical-application

• PB 擠出先於雙極 MII 紡錘體的形成。
• 這種不同步性使得僅 PB 的存在就不能成為卵母細胞成熟度的可靠標誌。
• 使用偏光顯微鏡 (PLM) 的無創紡錘體成像可以快速、輕鬆地檢查顯示 PB 的卵母細胞是否在 ICSI 之前真正重新組裝了減數分裂紡錘體。
• 在雙極 MII 紡錘體組裝和染色體對齊之前幾個小時，PB 變得可見。
• 如果無法檢測到 MII 紡錘體信號，則可以將精子注射推遲數小時，為 MII 紡錘體形成提供額外的時間。
• PLM 可用於避免晚熟卵母細胞過早受精的風險。
• 僅根據 PB 的存在分類為 MII 卵母細胞，可能包含尚未完成發育的晚熟卵母細胞，因此尚未準備好受精。
• 使用固定針和 ICSI 針轉動卵母細胞，使 PB 位於 12 點鐘位置並聚焦到 PB。
• 按照步驟 3.3−3.9 執行 PLM 重新檢查 ~2−3 小時後。 如果某些卵母細胞仍然缺乏 MII 紡錘體，則進一步延遲 ICSI 1−2 小時。
• 推遲 ICSI 可以為晚熟卵母細胞提供更多時間來組裝 MII 紡錘體，而 MII 紡錘體的存在與更好的臨床結果相關
• ICSI 應在取出當天進行，且不應超過 9 小時（hCG 後 45 小時），該時間段與胚胎質量下降相關36,37。
• 其他各種因素對生殖成功有重大影響（例如精子因子、線粒體、胚胎基因組激活、不規則卵裂、表觀遺傳學、子宮內膜、母體免疫力）。
• MII 紡錘體本身的檢測並不能保證 IVF 程序的積極臨床結果。
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• PB extrusion precedes the formation of the bipolar MII spindle. 
• This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity.
•  Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. 
• PB becomes visible a couple of hours before bipolar MII spindle is assembled and chromosomes are aligned1.
• If the MII spindle signal is undetectable, sperm injection can be deferred to a later time point providing extra time for the MII spindle formation. 
• PLM can be employed to avoid the risk of premature fertilization of late-maturing oocytes. 
• Classified as MII oocytes based only on the PB presence, might contain latematuring oocytes that have not yet completed their development and thus are not ready for fertilization.
• If the oocytes show no detectable MII spindle signal, place the PLM dish into the CO2- independent incubator and shift the ICSI into a later time.
• Perform PLM re-examination ~2−3 h later following steps 3.3−3.9. If some oocyte(s) still lack a MII spindle, further delay ICSI for additional 1−2 h.
• Use a holding and ICSI needle to turn the oocyte so the PB is in the 12 o’clock position and focus to the PB.
• Perform PLM re-examination ~2−3 h later following steps 3.3−3.9. If some oocyte(s) still lack a MII spindle, further delay ICSI for additional 1−2 h.
• Postponing ICSI provides late-maturing oocytes with more time to assemble an MII spindle whose presence is associated with better clinical results
• , ICSI should be performed on the day of retrieval and should not exceed 9 hours (45 hours post hCG), the period associated with a decline in resulting embryo quality36,37.
• Various other factors have significant impact on reproduction success (e.g., sperm factor, mitochondria, embryonic genome activation, irregular cleavage, epigenetics, endometrium, maternal immunity). 
• Detection of the MII spindle per se, does not guarantee a positive clinical outcome of the IVF procedure.

(A) prominent signal of bipolar spindle, (B) translucent apolar MII spindle spindle, (C) no detectable spindle, and (D) microtubule bridge.

Figure 3: Stages of MI to MII transition in oocyte maturation. The appearance of oocytes in bright field (top row), combined with a fluorescence signal of chromosomes (cyan) and microtubules (magenta) (middle row), and in polarized light (bottom row) is shown. Each oocyte was first PLM-examined and immediately fixed. Fixed oocytes were (immuno)labeled with Hoechst (DNA) and anti-α-tubulin antibody (microtubules). The yellow arrow indicates the presence of PB and the white arrow highlights the position of birefringent microtubules.

Figure 4: Correlation of chromosome-microtubule organization with birefringence pattern detected by polarized light microscopy. The appearance of oocytes in the bright field combined with a fluorescent signal for chromosomes (cyan) and microtubules (magenta) (top row), and in polarized light (bottom row) is shown. Each oocyte was fixed immediately after PLM-examination. DNA (Hoechst) and microtubule (α-tubulin) were stained. (A-C) oocytes with PLM-detectable spindle yet misaligned chromosomes (A, B) or loosely focused spindle poles (C); (D-F) abnormal oocytes without PLM-detectable MII spindle showing underdeveloped (D), apolar (E) or no spindle (F).

Figure 5: Birefringence structures in human oocytes. Representative examples of (A-D) birefringent structures detected by PLM in human oocytes. ZP = zona pellucida; PM = plasma membrane (oolema); RB = refractile bodies, vacuoles. White arrow, meiotic spindle in different stages of maturation. (E) MII spindle misalignment with polar body (PB). (F) Spindle detachment from plasma membrane. Scale bar = 20 µm.