• 最初集中在前期核周圍的細胞器重新定位到外圍並均勻分佈在整個卵質中。
• 隨著成熟的進行，不同的分泌裝置似乎轉變為聚集在卵母細胞表面下方的皮質顆粒。
• 內質網可變元件和多個線粒體組成的具有原始形態的異源複合物逐漸形成。
• 在 GV 卵母細胞中，胞器在整個細胞質中表現出非均勻分佈。
• 亞細胞結構集中在核週區域，而油質下方的細胞質似乎缺乏細胞器。
• 在紡錘體陽性的 MI 和 MII 卵母細胞中，除減數分裂紡錘體區域外，細胞器均勻地分散在整個細胞體積中。
• GV 卵母細胞非常大的前期核（直徑 29μm）含有緻密的電子緻密核仁和相關的染色質質量，顯示出不同程度的濃縮。
• 在 MI 和 MII 卵母細胞中，在卵母細胞附近檢測到減數分裂紡錘體。
• 人類卵母細胞減數分裂紡錘體是在沒有典型中心體的情況下組裝的
• 所有成熟階段的卵母細胞中最豐富的細胞器是線粒體。
• 線粒體的形態及其數量在減數分裂成熟過程中似乎沒有顯著變化。
• 線粒體群與 ER 形成特徵複合物，作為細胞內鈣 (Ca2+) 的主要儲存庫

https://academic.oup.com/biolreprod/article/104/1/106/5909494?login=false

Cytoplasmic maturation in human oocytes: an ultrastructural study † 

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

• The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm.
• . As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface.
• The gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. 
• In all GV oocytes, detectable organelles displayed non-homogenous distribution throughout the cytoplasm. 
• The subcellular structures were concentrated in the perinuclear region, whereas the cytoplasm underneath the oolema appeared to be devoid of organelles.
• in spindle-positive MI and MII oocytes, the organelles were dispersed homogeneously throughout the entire volume of the cell except in the meiotic spindle territory. 
• The remarkably large prophase nuclei of GV oocytes (29.06 ± 1.63 μm in diameter) were found to contain compact electron-dense nucleoli and an associated mass of chromatin showing different degrees of condensation.
• In MI and MII oocytes, the meiotic spindle was detected close to the oolema. 
• In human oocytes, the meiotic spindle is assembled in the absence of typical centrosomes 
• 
  
• The cytoplasmic organelles detectable in our samples were mainly represented by mitochondria, endoplasmic reticulum (ER), and Golgi apparatus (GA). 
• The most abundant organelles in oocytes of all maturational stages were the mitochondria. 
• The morphology of the mitochondria, as well as their number, did not seem to change dramatically during meiotic maturation.
• The mitochondrial population formed characteristic complexes with ER, which serves as the primary storage for intracellular calcium (Ca2+) 
• Our observations raise questions regarding the functional significance of organelle rearrangements in preparation for fertilization and the morphological factors defining the quality of the human egg.

The morphometric analysis of organelle distribution in the cytoplasmic compartment of maturing human oocytes. The organelle density in the periphery versus central area of individual cells was compared (A), the average ratio was calculated in individual GV, MI, and MII oocytes and plotted for developmental categories (B). The number of analyzed oocytes is stated in brackets. *p-value ˂ 0.001.

• T

生殖功能的調節主要由糖蛋白激素，卵泡刺激素（FSH）、促甲狀腺激素（TSH）、黃體生成素（LH）和絨毛膜促性腺激素（hCG）進行。糖蛋白激素包含α 亞基 &  β 亞基

 hCG、FSH 和 LH 有相同的α 亞基，包含 92 個氨基酸，

 hCG、FSH 和 LH 之 β 亞基對於所有三種激素是特異性和獨特的，可引發不同的生物學和免疫學特性。

聚醣glycans在促性腺激素與其各自受體的結合中發揮著重要作用，識別相關聚醣及其藥理學特性非常重要。

蛋白質糖基化Protein glycosylation 是一種複雜的翻譯後修飾post-translational modification (PTM)，涉及glycans在特定位點的附著，最常見的是天冬酰胺（N 連接）或絲氨酸/蘇氨酸（O 連接）殘基。糖基化glycosylation模式在蛋白質的溶解度、穩定性和生物活性中起著至關重要的作用。

新的聚醣 glycan是一種四觸角物種tetra antennary ， FSH 與受體的結合中發揮重要作用，具有更高的生物活性、更低的清除率和更長的半衰期。

由於糖基化的差異，糖蛋白的生物活性和半衰期可能不同。

質譜儀mass spectrometry MS技術的進步，可以鑑定微量的相關聚醣。MS 可進行定量和定性分析，例如分子量測定、結構闡明或序列測定。

 https://www.sciencedirect.com/science/article/abs/pii/S1642431X18300949

https://link.springer.com/article/10.1007/s00216-011-4923-5

Glycan mapping of recombinant human follicle stimulating hormone by mass spectrometry

Author links open overlay panel

• In humans, regulation of reproductive functions are carried out mainly by glycoprotein hormones namely follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). 
• Since glycans play an important role in binding of gonadotropins with their respective receptors, it is important to identify associated glycans and their pharmacological properties not only for the disease manipulation but also for making more efficacious and safer recombinant versions. 
• Protein glycosylation is a complex post-translational modification (PTM) involving enzyme mediated attachment of glycans at specific sites, most commonly at Asparagine (N-linked) or Serine/Threonine (O-linked) residues. The glycosylation pattern plays a critical role in the solubility, stability and bioactivity of proteins. 
• With the advancement of mass spectrometry, it is possible to identify minute quantity of associated glycans. 
• The new glycan was a tetra antennary species that may have important role in binding of FSH with receptor with higher biological activity as well as lower clearance rate and higher half-life.
• The α-subunit contains 92 amino acids and is identical in hCG, FSH and LH (Table 1) while β-subunit are specific and unique for all the three hormones for eliciting differential biological and immunological properties. 
• Due to differences in the glycosylation, glycoprotein may differ in bioactivities and half-life. 
• MS allows both quantitative and qualitative analysis like determination of molecular mass, structure elucidation or sequence determination. 

Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches

• Thorough analysis of the heterodimeric heavily glycosylated protein of rFSH is a prerequisite for the evaluation of production batches as well as for the determination of “essential similarity” of new biosimilars. 
• The concerted application of different liquid chromatography-mass spectrometry methods enabled the complete depiction of the primary structure of this pituitary hormone. 
• Sequence coverage of 100% for the α- as well as the β-chain was achieved with tryptic peptides. 
• Most of these peptides could be verified by tandem mass spectrometry. Site-specific analysis of all four glycosylation sites was, however, not possible with tryptic but with chymotryptic peptides. Quantification of the glycoforms of each glycopeptide was accomplished with the software MassMap®. Both protein subunits gave interpretable mass spectra upon S-alkylation and separation on a C5 reversed-phase column. Glycan isomer patterns were depicted by separation on porous graphitic carbon, using mass spectrometric detection for the evaluation of the glycopeptide liquid chromatography-electrospray ionization data. The currently marketed product Gonal-f™ and a potential biosimilar were compared with the help of these procedures.

新型卵泡素製劑FSH：聚醣結構差異如何影響生化和生物學功能以及臨床效果

• 人工合成知FSH變體的1級~4級結構，包括糖基化差異。對於FSH知生化效果均產生不同效果
• 這些糖苷形式包括糖基化位點的數量、糖苷鏈的複雜性以及唾液酸化和硫酸化方面均有差異，
• 高唾液酸化，末端 N-乙酰半乳糖胺或半乳糖的 2,3 唾液酸封端通過阻斷肝臟中脫唾液酸糖蛋白受體 (ASGPR) 的結合，導致更長的循環半衰期。
• FSH 糖基化主要在 β 亞基上，隨著女性年齡的增長而變化，已成為FSH作用的關鍵修飾因子，在動物模型體內具有深遠的生物學效應。
•  FSH 在垂體中以兩個亞基的形式產生，一個是所有糖蛋白激素共有的亞基，即 α 亞基，另一個是每種激素特有的亞基，即 β 亞基。
• 尿液中的促卵泡素與天然垂體促卵泡素的不同之處在於其聚醣的程度和復雜性以及相關糖蛋白激素（黃體生成素）和其他蛋白質的污染
• FSH Alpha類包括   Gonal-F®、Bemfola®、Ovaleap® 、Cinnal-f®. 它是CHO細胞系生產的
• FSH Beta 類包括    Follistim® 和 Puregon®。它是CHO細胞系生產的
• Follitropin Delta 包括促卵泡素 Rekovelle®，它是使用人類細胞系生產的 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018285/

New Human Follitropin Preparations: How Glycan Structural Differences May Affect Biochemical and Biological Function and Clinical Effect

:

1. Pituitary follitropin is secreted into the circulation as a mixture of variants, which differ not in primary structure but rather at the level of glycosylation. 
2. These glycosidic forms vary in the number of glycosylation sites filled, complexity of glycosidic chains, and sialylation and sulfation. 
3. high sialylation, 2,3 sialic acid capping of terminal N-acetyl galactosamine or galactose leads to longer circulating half-life, by blocking binding of asialoglycoprotein receptor (ASGPR) in the liver. 
4. FSH glycosylation, primarily on the β-subunit, which varies as women age, has emerged as a key modifier of follitropin action, with profound biological effects in vivo in animal models.
5.  FSH is produced in the pituitary gland as two subunits, one subunit common to all glycoprotein hormones, the α-subunit, and another subunit specific for each hormone, the β-subunit. 
6. . Follitropin in urine differed from naturally occurring pituitary follitropin in degree and complexity of their glycans and contamination with a related glycoprotein hormone (luteinizing hormone) and other proteins
7.  Recombinant follitropin Gonal-F®, and biosimilars Bemfola®, Ovaleap® and Cinnal-f® are grouped into one class called follitropin Alpha. 
8. The follitropin Beta class includes Follistim® and Puregon®. 
9. Follitropin Delta included the follitropin Rekovelle® which is produced using a human cell line and results in a product that possesses sialic acid2,6 as well as sialic acid2,3 capped Gal.

Follitropin delta 促卵泡素 delta (樂可孕 Rekovelle)相關臨床報告

https://www.youtube.com/watch?v=uKM0Bwz8giA

• 每日 10 µg 促卵泡素 delta (Rekovelle)劑量可提供與 150 IU/天促卵泡素 α (Gona-F) 相似的卵巢反應。
• Rekovelle與Gona-F& Puregon 相比，胚胎髮育和妊娠率相似。
• Rekovelle妊娠率為 6 μg/d 的 19%、9 μg/d 的 20% 和 12 μg/d 的 25%。
• Rekovelle 6 μg/d、9 μg/d 和 12 μg/d 取卵數量為 7.0 ± 4.1、9.1 ± 5.6 和 11.6 ± 5.6，
• Rekovelle與Gona-F的臨床妊娠率（35.4% vs. 31.5%，P = 0.239）和活產率（31.0% vs. 25.5%，P = 0.101）相當。
• AMH ≥ 15 pmol/L 的女性注射Rekovelle與Gona-F 治療相比，Rekovelle回收的卵母細胞較少（10.3 ± 6.2 與 12.5 ± 7.5，P < 0.001）  
• Rekovelle與Gona-F 治療相比，早期卵巢過度刺激綜合徵 (OHSS) 和/或預防性干預的發生率較低（6.1% vs. 11.0%，P = 0.013）。
• 
  
• 
•  A daily follitropin delta dose of 10 µg provides a similar ovarian response to 150 IU/day follitropin alfa in IVF/ICSI patients.
•  Similar embryo development and pregnancy rates compared with those of stimulation with follitropin alfa or beta.
• Vital pregnancy rate per started cycle with follitropin delta was 19% for 6 μg/d, 20% for 9 μg/d, and 25% for 12 μg/d.
• Oocytes retrieved in the 6 μg/d, 9 μg/d, and 12 μg/d follitropin delta groups was 7.0 ± 4.1, 9.1 ± 5.6, and 11.6 ± 5.6, 
• The clinical pregnancy rate (35.4% vs. 31.5%, P = 0.239) and live birth rate (31.0% vs. 25.5%, P = 0.101) were comparable between the follitropin delta group and the follitropin alfa group. 
• Overall, the individualized follitropin delta treatment resulted in fewer oocytes retrieved compared to follitropin alfa treatment (10.3 ± 6.2 vs. 12.5 ± 7.5, P < 0.001), which was mainly due to fewer oocytes (10.5 ± 6.4 vs. 13.9 ± 7.8) in women with AMH ≥ 15 pmol/L.
•  Accordingly there was a lower incidence of early ovarian hyper-stimulation syndrome (OHSS) and/or preventive interventions (6.1% vs. 11.0%, P = 0.013). 
• A daily follitropin delta dose of 10.2 µg (95% CI: 9.3-11.2 µg) was estimated to provide the same number of oocytes retrieved as a starting dose of 150 IU/d of follitropin

. 2020 Oct;41(4):616-622.

 doi: 10.1016/j.rbmo.2020.07.006. Epub 2020 Jul 15.Establishing the follitropin delta dose that provides a comparable ovarian response to 150 IU/day follitropin alfa

Research question: The objective of this investigation was to determine the daily follitropin delta dose (µg) providing a similar ovarian response to 150 IU/day follitropin alfa. Design: The study was a post-hoc analysis of ovarian response in 1591 IVF/intracytoplasmic sperm injection (ICSI) patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist protocol in two recent randomized, assessor-blind, controlled trials in the development programme for follitropin delta: a phase II dose-response trial with a reference arm of a fixed daily dose of 150 IU follitropin alfa throughout stimulation, and a phase III efficacy trial with a comparator arm of 150 IU/day follitropin alfa as a starting dose. Results: Daily follitropin delta doses of 10.0 µg (95% confidence interval [CI] 7.9-12.8) and 10.3 µg (95% CI 9.7-10.8) yielded the same number of oocytes as 150 IU/day follitropin alfa for all patients participating in the phase II and III trials, respectively. When analysing patients with either normal or high ovarian reserve (based on serum anti-Mullerian hormone ≥15 pmol/l) and no dose changes, the same number of oocytes was obtained with 150 IU/day follitropin alfa and daily doses of follitropin delta of 9.7 µg (95% CI 7.5-12.4) and 9.3 µg (95% CI 8.6-10.1) in the two trials. Daily follitropin delta doses in the range 9.5-10.4 µg were consistently estimated to correspond to 150 IU/day follitropin alfa for serum oestradiol concentration and number of follicles ≥12 mm at the end of stimulation across analysis populations in the phase III trial.Conclusions: A daily follitropin delta dose of 10 µg provides a similar ovarian response to 150 IU/day follitropin alfa in IVF/ICSI patients.

F S Rep . 2020 Dec 14;2(1):30-35.  doi: 10.1016/j.xfre.2020.12.002. eCollection 2021 Mar.

In vitro fertilization cycles stimulated with follitropin delta result in similar embryo development and quality when compared with cycles stimulated with follitropin alfa or follitropin beta

Objective: To study the impact of follitropin delta for ovarian stimulation on embryo development and quality compared with that of follitropin alfa or beta in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.

Patients: A total of 403 IVF/ICSI cycles were conducted from September 1, 2018 to December 31, 2019. Cycles were grouped on the basis of stimulation with follitropin delta vs. follitropin alfa or beta.

Main outcome measures: Embryo parameters and clinical pregnancy and implantation rates.

Results: Ovarian stimulation using follitropin delta resulted in no statistically significant difference in day 3 embryo quality between the control group and follitropin delta group (median 0.50 vs. 0.54 for good quality embryos and median 0.25 vs. 0.20 for intermediate quality embryos). Although on initial analysis there was a lower proportion of good quality blastocysts in the follitropin delta group than in the control group (0.11 vs. 0.22), this difference was no longer present when day 3 after fertilization vitrification and transfer cycles were excluded (0.26 vs. 0.33 follitropin delta vs. control). The clinical pregnancy rates and clinical implantation rates were similar in both groups in fresh transfer cycles.

Conclusions: Stimulation with follitropin delta in IVF/ICSI cycles resulted in similar embryo development and pregnancy rates compared with those of stimulation with follitropin alfa or beta.

Clinical Trial

 

. 2021 Jun;115(6):1478-1486.

 doi: 10.1016/j.fertnstert.2020.10.059. Epub 2020 Dec 4. Randomized, assessor-blind, antimüllerian hormone-stratified, dose-response trial in Japanese in vitro fertilization/intracytoplasmic sperm injection patients undergoing controlled ovarian stimulation with follitropin delta

Objective: To establish the relationship between follitropin delta doses (recombinant follicle-stimulating hormone produced from the human cell line PER.C6) and ovarian response in Japanese women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment and to evaluate the influence of initial antimüllerian hormone (AMH) levels. 

Design: Randomized, controlled, assessor-blind, AMH-stratified (low 5.0-14.9 pmol/L; high 15.0-44.9 pmol/L) dose-response trial.

Patient(s): A total of 158 Japanese women (20-39 years of age).

Intervention(s): Controlled ovarian stimulation with 6, 9, or 12 μg/d of follitropin delta or 150 IU/d follitropin beta as a reference arm in a gonadotropin-releasing hormone antagonist cycle.

Main outcome measure(s): Number of oocytes retrieved.

Result(s): Among all women who started stimulation, the mean number (± standard deviation) of oocytes retrieved in the 6 μg/d, 9 μg/d, and 12 μg/d follitropin delta groups was 7.0 ± 4.1, 9.1 ± 5.6, and 11.6 ± 5.6, respectively, and a significant dose-relation was established, which also remained significant within each AMH strata. Significant dose-responses also were observed for serum estradiol, inhibin A, and progesterone at end-of-stimulation with follitropin delta. The vital pregnancy rate per started cycle with follitropin delta was 19% for 6 μg/d, 20% for 9 μg/d, and 25% for 12 μg/d. The rate of early moderate/severe ovarian hyperstimulation syndrome with follitropin delta was 8% for 6 μg/d, 8% for 9 μg/d, and 13% for 12 μg/d, with 82% of the cases in the high AMH stratum. Conclusion(s): This trial establishes the dose-response relationship between follitropin delta and ovarian response in Japanese women. 

Randomized Controlled Trial

 

. 2022 Oct 4;20(1):147.

 doi: 10.1186/s12958-022-01016-y.Comparative clinical outcome following individualized follitropin delta dosing in Chinese women undergoing ovarian stimulation for in vitro fertilization /intracytoplasmic sperm injection

Background: To compare the efficacy and safety of follitropin delta in its individualized fixed-dose regimen with follitropin alfa in a conventional adjustable dosing regimen in Chinese women. METHODS: This was a subgroup analysis of the randomized, multi-center, assessor-blind, non-inferiority trial (GRAPE) including 759 Chinese women (aged 20-40 years) recruited in 16 reproductive medicine clinics in China. Women were randomized in a 1:1 ratio to be treated with either follitropin delta dose based on anti-Müllerian hormone (AMH) and body weight or conventional dosing with follitropin alfa following a gonadotropin-releasing hormone (GnRH) antagonist protocol. The primary outcome was ongoing pregnancy rate assessed 10-11 weeks after embryo transfer in the fresh cycle (non-inferiority margin -10.0%). Results: 378 in the follitropin delta group and 381 in the follitropin alfa group were randomized and exposed. Non-inferiority was confirmed with respect to ongoing pregnancy with rates of 31.0% vs. 25.7% for follitropin delta compared to follitropin alfa, estimated mean difference of 5.1% (95% confidence interval (CI) -1.3% to 11.5%). The clinical pregnancy rate (35.4% vs. 31.5%, P = 0.239) and live birth rate (31.0% vs. 25.5%, P = 0.101) were comparable between the follitropin delta group and the follitropin alfa group. Overall, the individualized follitropin delta treatment resulted in fewer oocytes retrieved compared to follitropin alfa treatment (10.3 ± 6.2 vs. 12.5 ± 7.5, P < 0.001), which was mainly due to fewer oocytes (10.5 ± 6.4 vs. 13.9 ± 7.8) in women with AMH ≥ 15 pmol/L. Accordingly there was a lower incidence of early ovarian hyper-stimulation syndrome (OHSS) and/or preventive interventions (6.1% vs. 11.0%, P = 0.013). A daily follitropin delta dose of 10.2 µg (95% CI: 9.3-11.2 µg) was estimated to provide the same number of oocytes retrieved as a starting dose of 150 IU/d of follitropin alfa. Conclusion: Follitropin delta in its individualized fixed-dose regimen showed similar efficacy and improved safety compared with follitropin alfa in a conventional adjustable dosing regimen in Chinese women.

Randomized Controlled Trial

 

. 2023 Jan;43(1):37-44.

 doi: 10.1007/s40261-022-01232-9. Epub 2022 Dec 7.Pharmacokinetics and Safety of Follitropin Delta in Gonadotropin Down-Regulated Healthy Chinese Women

Background: Follitropin delta, a novel recombinant follicle-stimulating hormone (rFSH) preparation derived from a human cell line, has different pharmacokinetic and pharmacodynamic properties compared with existing rFSH preparations expressed by Chinese hamster ovary cells (CHO). Objectives: The objective of this study was to assess the pharmacokinetic characteristics, dose proportionality, and safety of follitropin delta in healthy Chinese women. Methods: This was a phase I, randomized, open-label study. Twenty-four healthy Chinese women were randomized (1:1:1) to receive a single subcutaneous administration of follitropin delta 12, 18, or 24 μg. The pharmacokinetic parameters (maximum observed serum concentration [Cmax], time to reach Cmax [tmax], area under the serum concentration-time curve from dosing to infinity [AUC∞], and elimination phase half-life [t½]) of follitropin delta were derived using noncompartmental analysis. Results: Following a single subcutaneous administration of follitropin delta 12, 18, or 24 μg, mean Cmax (0.388, 0.677, and 0.825 ng/mL, respectively) and AUC∞ (41.3, 62.9, and 83.1 h·ng/mL, respectively) increased in a dose-proportional manner. The median tmax was 24 h, and the mean t½ was in the range of 50.5-60.9 h. All treatment-related adverse events were categorized as mild, except for one case of urticaria from the follitropin delta 18-μg dose group which was considered moderate. Only one woman presented with elevation of alanine transaminase and aspartate aminotransferase at the follow-up visit, which was reported as a treatment-emergent adverse event. There were no injection-site reactions and none of the participants showed any confirmed presence of treatment-induced anti-FSH antibodies. Conclusions: The administration of single doses of follitropin delta to healthy Chinese women demonstrated dose-proportional pharmacokinetics over the dose range of 12-24 μg, and these doses were well tolerated.

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.