Visualization and Examination of the Meiotic Spindle in Human Oocytes with Polscope

PURPOSE: The effect of detectability/location of meiotic spindle to fertilization of human MII oocytes and outcome of clinical IVF/ICSI-ET cycles was studied. METHODS: The location of spindle relative to polar body positioned to 6 o'clock was detected with polscope prior to ICSI in oocytes. Data of 83 IVF/ICSI-ET cycle, including pregnancy results, and a total of 1033 oocytes examined were analyzed retrospectively. RESULTS: Polar body position does not always accurately predict spindle position. Similar fertilization was observed in the polscope detected and in the control group. The main number of oocytes and spindle positive oocytes collected was higher in the pregnant than in the non-pregnant group. Higher fertilization was found in that group where 2/3-d of the oocytes had detected spindle. The main number of oocytes and spindle positive oocytes obtained decreased with increasing age of patients. CONCLUSIONS: The presence of birefringent spindle predicts higher fertilization and development and greater probability of pregnancy.     

Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF

Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and apoptosis, a phenotype remarkably similar to that observed in oocytes and embryos from older women. A time-lapse video of a mouse oocyte imaged by polscope may be purchased for viewing on the internet at www.rbmonline.com/Article/824 (free to web subscribers).     

体内成熟与体外成熟的卵母细胞纺锤体位置及其与胚胎发育的关系

目的研究体内成熟与体外成熟卵母细胞的纺锤体位置及其与胚胎发育的关系。方法对134个体内成熟卵母细胞在卵母细胞胞质内单精子注射法（ICSI）操作时用纺锤体实时观察仪进行纺锤体位置的观察,体内成熟卵母细胞来自单纯因男性不育而进行ICSI治疗的患者15例（体内成熟组）。另外对45个体外成熟的卵母细胞观察纺锤体位置,体外成熟卵母细胞来自因多囊卵巢综合征致不孕而进行治疗的患者5例（体外成熟组）。纺锤体的位置按照其与第一极体之间的角度不同分为Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ级。并观察两组成熟卵母细胞的受精及其胚胎发育情况。结果体内成熟组和体外成熟组患者的卵母细胞中可观察到纺锤体的分别占83．6％（112／134）和82．2％（37／45）。体内成熟组患者卵母细胞纺锤体的位置Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ级分别为22．4％、55．2％、3．O％、3．O％、16．4％,体外成熟组则分别为17．8％、51．1％、8．9％、4．4％、17．8％,两组各级间分别比较,差异均无统计学意义（P〉0．05）。在体内成熟组卵母细胞中,纺锤体离第一极体较近（Ⅰ级）者受精率较高（93．3％）,显著高于其他各级（分别为73．0％、2／4、1／4、63．6％,P〈0．05）。结论体内成熟与体外成熟卵母细胞间纺锤体位置未见显著差异;纺锤体的位置与卵母细胞受精率有一定相关性。     

Evaluation of maturation competence of metaphase II oocytes in mice based on the distance between pericentriolar materials of meiotic spindle

Purpose  

To ascertain whether metaphase II (MII) spindle shape influences oocyte competence, we examined the meiotic spindle organization in in vivo ovulated (IVO) oocytes and in spontaneously matured or follicle stimulating hormone (FSH)-induced oocytes.     

The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes

OBJECTIVE: To image spindles in living human oocytes and to examine the relation between spindles and fertilization after ICSI. DESIGN: The LC polscope was used to examine spindles in an observational study of living oocytes. SETTING: Academic IVF clinic. PATIENT(S): Women being treated for infertility. INTERVENTION(S): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized oocytes after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods. MAIN OUTCOME MEASURE(S): Spindle structure in living oocytes and fertilization after ICSI. RESULT(S): Spindles could be imaged in 61.4% of oocytes. More oocytes with spindles than oocytes without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged oocytes were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. CONCLUSION(S): Spindles in living human oocytes can be imaged by using the polscope. A birefringent spindle in human oocytes may clinically predict the quality and age of oocytes. This method also can be used to monitor spindle position during ICSI.     

Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes

In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P < 0.001) and embryo quality (grade I embryos over total embryos: 36.7 and 22.2% with fresh oocytes in IVF and ICSI; 12.1% with frozen-thawed oocytes in ICSI; respectively P < 0.001 and P < 0.05) were statistically lower after oocyte cryopreservation. The significant decrease in meiotic spindle retrieval rate before freezing (62.4%) and after thawing procedures (43.4%; P < 0.001) suggests that cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed.     

Does first polar body morphology predict oocyte performance during ICSI treatment?

Purpose

To gain insight into the morphology of the first polar body (1 PB) in ICSI patients and to explore whether it could predict mature oocyte viability and performance in this setting.

Methods

Seventy two consecutive women planned to perform ICSI treatment were prospectively recruited for this study. All oocytes retrieved underwent evaluation for nuclear maturity and accurate assessment of 1 PB morphology. MII oocytes were cultured in separate groups in each woman in accordance with two different categories of 1 PB morphology. Category A included normal intact round or ovoid 1 PB and category B included abnormal fragmented 1 PB. Each oocyte was followed throughout fertilization, embryo cleavage and embryo transfer. Cycles that reached embryo transfer, were divided into three groups in accordance with 1 PB morphology. Group I included only category A 1 PB embryos, group II included categories A and B 1 PB embryos, whereas group III included only category B 1 PB embryos.

Results

A total of 687 oocytes were aspirated and 553 MII oocytes underwent ICSI leading to 410 zygotes showing normal fertilization on day one. Three hundred ninety seven embryos cleaved on day two and a total of 176 embryos were replaced into the uterus. Clinical implantation and pregnancy rates were significantly correlated with the morphology of the 1 PB corresponding to 31%, 9% and 2% and 61%, 24% and 5%, in groups I, II and III respectively.

Conclusions

Our findings demonstrate that 1 PB morphology is related to mature oocyte viability and it has the potential to predict oocyte performance and pregnancy achievement in infertile women undergoing ICSI treatment.     

Meiotic spindle presence and oocyte morphology do not predict clinical ICSI outcomes: a study of 967 transferred embryos

With a view to correlating oocyte morphology and meiotic spindle presence to clinical intracytoplasmic sperm injection (ICSI) outcomes, 967 oocytes that led to 967 transferred embryos in 404 embryo transfers were studied. No relationship was found between oocyte morphology (ooplasm texture, perivitelline space largeness, perivitelline space granulation absence/presence and the first polar body shape) or meiotic spindle presence or absence and clinical pregnancy per transfer and implantation rates after ICSI. It was concluded that oocyte morphology and meiotic spindle presence or absence can only predict fertilization, cleavage rates and embryo quality, as previously described in the literature, but do not help in daily ICSI practice in the choice of the metaphase II oocyte that will lead to the embryo that starts clinical pregnancy.     

Effect of the position of the meiotic spindle on the outcome of intracytoplasmic sperm injection

The position of the meiotic spindle in relation to the injection site has been a matter of concern since intracytoplasmic sperm injection (ICSI) was developed. Disruption of the spindle may result in cell death or aneuploidy, and it is therefore essential to avoid injecting directly into the area of the spindle. Before the development of the PolScope, it was only possible to visualize the spindle after fixing and staining oocytes. The PolScope allows the spindle to be visualized in living oocytes, and thus their subsequent development can be followed. Studies have shown that the spindle is not, as had been assumed, always adjacent to the polar body. However, the proportion of oocytes in which the spindle is significantly displaced is low, and the displacement is not necessarily associated with poor oocyte and embryo development. Even oocytes in which no spindle is visualized may go on to fertilize and develop normally, and the evidence indicates that disruption of the spindle is not a significant factor in determining the likelihood of aneuploidy and developmental anomalies. Indeed, it has been suggested that injecting spermatozoa closer to the site of the spindle may in fact improve the outcome.     

不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。     

Metaphase II karyoplast transfer from human in-vitro matured oocytes to enuclueated mature oocytes

Metaphase II karyoplast transfer is believed to be a useful method to rescue aged oocytes. This study attempted karyoplast transfer of in-vitro matured metaphase II (MII) oocytes, as a model of aged oocytes, into enucleated freshly ovulated metaphase II oocytes with visualization of their chromosomes under an inverted microscope. Recipient karyoplasts derived from immature oocytes were cultured in-vitro until first polar body extrusion. After 1–2 days culture, 52.1% extruded a polar body, 95.5% had PSC, aneuploidy was very low (4.5%) and none had structural aberrations. Donor oocytes were obtained from IVF or intracytoplasmic sperm injection (ICSI) patients. Chromosomes were easily confirmed in 92.3% and 95.0% of in-vivo and in-vitro matured oocytes respectively. Thirty-one karyoplasts were placed in the perivitelline space of enucleated donor oocytes, and 25 (80.6%) fused to form a reconstituted oocyte. Fertilization, cleavage and blastocyst formation rates following ICSI were 76.0%, 64.0% and 28.0% respectively for reconstructed oocytes and 59.2%, 48.0% and 3.1% respectively for control (in-vitro matured) oocytes. Chromosomal analysis of five embryos developed after karyoplast transfer and ICSI showed normal diploid sets of 46 chromosomes. In conclusion, this metaphase II karyoplast transfer technique can be applied to the solution of chromosomal abnormalities related to oocyte ageing.     

Correlation between human follicular diameter and oocyte outcomes in an ICSI program

Purpose: To determine the correlation between the follicular sizes and oocyte recovery, metaphase II oocyte recovery, fertilization rate and good embryo quality from mature and immature oocytes in an intracytoplasmic sperm injection (ICSI) program. Methods: 991 follicles obtained from 72 ICSI cycles were classified into three groups according to their diameters as measured by transvaginal ultrasound including group A (<10 mm), group B (10–14 mm), and group C (>14 mm). All obtained oocytes were classified according to their nuclear maturation: germinal vesicle (GV), metaphase I (MI) and metaphase II (MII). Mature oocytes underwent ICSI while immature oocytes were further cultured until maturity before ICSI was performed. The rates of fertilization and good quality embryos at day 3 were evaluated. Results: A progressive and significant increase in the rates of oocyte recovery and MII oocyte recovery were observed from group A follicles compared to the other groups (p < 0.001). The fertilization rate of mature and in vitro matured oocytes, as well as the rate of good quality embryos showed a tendency to increase from group A to group C follicles, but not significantly. The corresponding fertilization rates were 78 and 55.3% (p < 0.001) for mature and in vitro matured oocytes, respectively. Conclusion: Collection of oocytes from small follicles, especially with a mean diameter less than 10 mm, and in vitro maturation of immature oocytes before fertilization may allow the total number of good quality and transferable embryos to be increased.     

Timed IVM followed by ICSI in a patient with immature ovarian oocytes

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF-intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)-ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.     

Manipulation of the oocyte: possible damage to the spindle apparatus

Oocytes are structured, polarized cells. For high developmental potential, it is essential that the distribution of organelles and molecules, and the function of meiotic spindles remain intact during handling of oocytes in assisted reproduction. Spindles are dynamic cell organelles. Spindle formation depends on activity of motor proteins, energy supply and temperature. Disturbances in spindle function may predispose oocytes to aneuploidy or maturation arrest. Thus, perturbation of the cytoskeletal integrity of oocytes may critically influence the fate of the embryo. Recently, enhanced polarizing microscopy has been developed for non-invasive analysis of spindle morphology in living mammalian oocytes. Chemically induced dynamic alterations have been characterized in the spindle in individual mouse oocytes and it has been shown that spindle aberrations are predictive of risks for non-disjunction. Spindle imaging identified adverse, irreversible effects of handling in living human oocytes (for instance, the extreme susceptibility of human oocytes to cooling). Also, oocyte immaturity may be detected. Selection of metaphase II oocytes and an injection site for intracytoplasmic sperm injection (ICSI) that avoids spindle damage may increase the yield of euploid embryos. The detection of genetic, environmentally induced, or treatment-related defects in oocyte maturation by non-invasive spindle imaging can improve quality control and assist in the selection of morphologically normal oocytes for assisted reproduction.     

Expression and intracytoplasmic distribution of staufen and calreticulin in maturing human oocytes

Purpose

In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated.

Methods

We assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy.

Results

STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle.

Conclusions

The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.     

激光共聚焦显微镜观察小鼠卵母细胞及早期胚胎中微丝的分布

目的:观察微丝在小鼠卵细胞及早期胚胎中的分布情况,为进一步明确微丝在受精卵发育中的作用机制打下基础。方法:采用免疫荧光化学法结合激光共聚焦显微技术对卵细胞的微丝分布进行观察。结果:在小鼠MII期卵母细胞中微丝分布于卵细胞的皮质区,集中在纺锤体处。在小鼠1-细胞期受精卵中微丝集中分布于卵细胞与极体的连接处。在2-细胞期受精卵中的微丝则集中分布在卵细胞的分裂沟处。用松弛素B(CB)解聚小鼠体内受精卵的微丝,细胞形态及分裂受到严重影响。结论:微丝在小鼠卵母细胞及早期胚胎中有着独特的分布,其可能参与多种功能。     

Novel use of laser to assist ICSI for patients with fragile oocytes: a case report

An inadvertent consequence of intracytoplasmic sperm injection (ICSI) is the degeneration of some of the microinjected oocytes. Most patients may not suffer any disadvantage through losing oocyte(s) during micromanipulation; however, in some circumstances, this can result in a reduction of the chances for pregnancy. This study reports a clinical pregnancy obtained by a novel approach using laser-assisted micro-opening of the zona pellucida prior to ICSI to secure a non-traumatic microinjection that avoids degeneration of oocytes. A total of 12 oocytes were obtained from the 36 year old patient in her third IVF treatment cycle, following two previously failed attempts where very high degeneration rates of oocytes after ICSI were recorded, together with suboptimal embryo quality. Five of the 11 matured (MII) oocytes were submitted to conventional ICSI and the other six MII oocytes first underwent laser-assisted opening of the zona pellucida (5-7 microm hole size was created with a 1.48 microm diode laser) before microinjection (LA-ICSI). Three of the five conventionally microinjected oocytes degenerated while one oocyte fertilized normally and developed to a good quality embryo. After the LA-ICSI procedure, one of the six oocytes degenerated and four oocytes fertilized normally; of these, two developed to excellent quality embryos, one to a good quality embryo and one to a poor quality embryo. The three best embryos (LA-ICSI group) were transferred to the patient on day 3. Rising serum human chorionic gonadotrophin concentrations were measured 12 days after transfer and on week 7 two implantation sites were detected, together with regular heart activity. The results of the present report suggest that laser-assisted ICSI may provide a safer approach to non-traumatic microinjection of oocytes than conventional ICSI, thereby minimizing the risk of degeneration and possibly also improving embryo quality. Therefore, it is suggested that laser-assisted ICSI might be applied in all cases associated with difficult zona pellucida penetration or/and fragile oolemma, or where patients have very few oocytes available, to improve the chances for pregnancy.     

Maintenance of the meiotic spindle during vitrification in human and mouse oocytes

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37 degrees C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21-22 degrees C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37 degrees C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37 degrees C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37 degrees C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37 degrees C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.     

Fertility preservation for trans men: frozen-thawed in vitro matured oocytes collected at the time of ovarian tissue processing exhibit normal meiotic spindles

Purposes

At the moment of sex reassignment surgery (SRS), the ovarian tissue is sometimes cryopreserved as fertility preservation option for female-to-male trans men, also called trans men. During this preparation, cumulus-oocyte-complexes (COCs) can be found and in vitro matured. It is not known if these oocytes are developmentally competent. In order to use these oocytes for fertility preservation and subsequent fertilization, a normal spindle structure before and after vitrification is necessary.

Methods

A total of 680 COCs were collected from trans men (n = 16) at the time of SRS and after testosterone treatment. The COCs were subjected to in vitro maturation and those that reached the metaphase II stage (MII) were collected and split into two groups; group 1 was immediately fixed for spindle staining and group 2 was first vitrified and warmed followed by spindle staining. Statistical analysis was performed by Fisher’s exact test.

Results

After 48 h in vitro maturation, 38.1% of COCs were at MII stage. Those oocytes were split in two groups: (1) 126 MII oocytes in the noncryopreservation group and (2) 133 MII oocytes underwent cryopreservation through vitrification. The oocyte survival rate, after 2 h warming, was 67.7%. Both the noncryopreserved and the vitrified group showed comparable results concerning normal spindle structure and chromosomes alignment, 85.7% vs. 92.2% (P = 0.27).

Conclusions

Spindle structure analysis and chromosomal alignment after vitrification seem normal in in vitro matured COCs collected during the tissue processing of ovaries in trans men at the time of SRS. The MII oocytes do not seem to be morphologically affected by prolonged testosterone treatment.