https://www.youtube.com/watch?v=zUxPnXgVAu4

 file:///C:/Users/user/Downloads/Science-2015-Holubcova.pdf

大多數胚胎染色體異常是由卵母細胞減數分裂過程中的染色體紡錘體分離錯誤造成的。

人類卵母細胞獨立於中心體或其他微管組織中心組裝減數分裂紡錘體。紡錘體組裝是由染色體和小鳥苷三磷酸酶 Ran 介導的，整個過程需要約 16 小時。

長的紡錘體組裝期的特點是紡錘體固有的不穩定性和異常的著絲粒-微管附著，

容易染色體分離錯誤&人類卵子中高非整倍體率 

人類卵母細胞的減數分裂比有絲分裂更容易出現染色體分離錯誤

核膜破裂[(NEBD)，設置為0小時

微管在大約 5 小時內首次被觀察到， 

染色質介導的紡錘體組裝由 三磷酸鳥苷 (GTP) Ran 驅動。

用二磷酸鳥苷鎖定突變體 Ran T24N 阻斷了(GTP) Ran 驅動功能， 可以觀察到Ran T24N 嚴重延遲微管成核的發生並損害紡錘體組裝

人類卵母細胞中的紡錘體組裝獨立於微管組織中心 (MTOC)，但由染色體介導並依賴於 Ran-GTP

染色體介導的微管成核（約 5 小時）和染色體排列的雙極紡錘體建立（約 16 小時）之間的時期顯示出相當大的紡錘體不穩定性。

人類卵母細胞中微管成核的主要位點是染色體，而不是 MTOC。

由於胞質分裂時染色體的不適當分配，後期落後的染色體增加了非整倍性的可能性。

紡錘體不穩定的卵母細胞也更有可能出現染色體排列缺陷。

染色體分離缺陷可能是由於進展到後期，著絲粒-微管附著異常。

約 80% 的著絲粒正確地附著在微管上，與單個紡錘體桿相連（兩性附著）。

20% 的動粒仍然附著在兩個紡錘體極上（merotelic 附著）

紡錘體的不穩定性可能會阻礙準確的動粒-微管附著的建立，從而促進染色體分離錯誤。

• Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg’s progenitor cell.
• Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. 
• Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours.
• This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.
• Meiosis in human oocytes is more prone to chromosome segregation errors than mitosis
• nuclear envelope breakdown [(NEBD),set to 0 hours
• Microtubules were first observed at ~5 hours, when they started to form a small aster within the chromosome aggregate
• Chromatin-mediated spindle assembly is driven by the small guanosine triphosphatase Ran. Guanosine triphosphate (GTP)
• We blocked its function with the guanosine diphosphate–locked mutant Ran T24N, which acts as a dominant-negative variant of Ran
•  Ran T24N severely delayed the onset of microtubule nucleation and impaired spindle assembly 
• spindle assembly in human oocytes is independent of  microtubule organizing centers (MTOCs) but mediated by chromosomes and dependent on Ran-GTP
• The period between chromosome-mediated microtubule nucleation (~5 hours) and establishment of a bipolar spindle with aligned chromosomes (~16 hours) displayed considerable spindle instability.
• Chromosomes, not MTOCs, serve as major sites of microtubule nucleation in human oocytes.
• Chromosomes that lag behind during anaphase increase the possibility of aneuploidy due to inappropriate partitioning of chromosomes upon cytokinesis.
• Oocytes with unstable spindles were also significantly more likely to have chromosome alignment defects. 
• The chromosome segregation defects could be due to progression into anaphase with abnormal kinetochore-microtubule attachments.
• only ~80% of kinetochores were correctly attached to microtubules, being linked to a single spindle pole (amphitelic attachment). 
• In contrast, 20% of kinetochores remained attached to both spindle poles (merotelic attachment)
• Spindle instability could hinder the establishment of accurate kinetochore-microtubule attachments and thereby promote chromosome segregation errors.

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。
• 
  

• 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.