A PolScope evaluation of meiotic spindle dynamics in frozen–thawed oocytes

In mature human oocytes, the metaphase II (MII) spindle presence and birefringence signal detected through the PolScope may vary before and after freezing. In particular, spindle dynamics during the first few hours after thawing is still under study. In this study, oocytes from stimulated ovaries were cryopreserved in 1.5 mol/l 1,2-propanediol with 0.3 mol/l sucrose using a slow freezing–rapid thawing method. Oocytes were examined with the PolScope for the presence, intensity of signal birefringence and size of the meiotic spindle before freezing and at 0, 1 and 2 h post-thaw (where 0 h = the time of the end of the thawing procedure). Of the 173 surviving oocytes exhibiting a spindle before freezing, 82.7% (143/173) showed spindle birefringence within 1 h of thawing. However, at the end of the thawing procedure the intensity of spindle birefringence (retardance) and the spindle length were smaller in comparison to the pre-freezing condition. These parameters increased after 1 h, although were not restored to the values observed before freezing. No significant changes were observed by extending the culture to 2 h.     

Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope

OBJECTIVE: To examine whether spindle morphologic features imaged with the LC-PolScope (Cambridge Research and Instrumentation, Woburn, MA) in living human oocytes matured in vitro can be used to predict chromosome configuration and select oocytes with normal chromosomes. DESIGN: Morphological study. SETTING: Academic IVF clinic. PATIENT(S): Women undergoing oocyte retrieval for ICSI treatment. INTERVENTION(S): Oocytes were examined after in vitro maturation. MAIN OUTCOME MEASURE(S): The study examined meiotic spindle morphologic features and chromosome alignments. RESULT(S): After culture for 22 to 24 hours, 77.1% of oocytes reached metaphase II stage, with 51.9% of oocytes showing birefringent spindles. Confocal microscopy revealed that 71% of oocytes with the birefringent spindles had normal chromosome alignment, and 29% of oocytes with birefringent spindles and all oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment. CONCLUSION(S): The spindle images obtained with the PolScope in living human oocytes are coordinate with those in fixed oocytes as imaged by confocal microscopy. Spindle images with the PolScope can be applied to human in vitro fertilization to help predict chromosomally normal oocytes for insemination.     

Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

Purpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.     

Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program

OBJECTIVE: To assess the usefulness of the vitrification method in clinical practice. DESIGN: Clinical study of vitrification of human oocytes. SETTING: A university-affiliated hospital. PATIENT(S): Thirty-four patients who agreed to undergo additional IVF-ET after the failed fresh cycle using the previously vitrified/thawed oocytes. INTERVENTION(S): Surplus oocytes from the IVF-ET patients were vitrified for the next cycle. MAIN OUTCOME MEASURES: Morphologic normality of post-thawed oocytes, fertilization, cleavage, pregnancy, and implantation rate. RESULT(S): Overall morphological survival and fertilization rates of vitrified/liquefied oocytes were 68.6% (325/474) and 71.7% (142/198), respectively. Pregnancy rate and implantation rate per embryo transfer were 21.4% (6/28) and 6.4% (8/125), respectively. All pregnancies resulted in the delivery of healthy babies (1 twin and 5 singletons/6 pregnancies). CONCLUSION(S): The feasibility of the vitrification method for human oocytes was confirmed by our clinical results. Subsequent studies on vitrification and thawing procedures should be undertaken for further optimizing the vitrification method.     

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。     

PHYSIOLOGY: Evaluation of the Meiotic Spindle Apparatus in Metaphase II Human Oocytes Following Cytoplasmic Donation

Purpose: To determine if the removal of cytoplasm from metaphase II human donor oocytes damages the meiotic spindle apparatus.Materials and Methods: Cryopreservation of metaphase II human oocytes was performed using a fast-freeze, fast-thaw protocol. Upon thaw, oocytes were incubated for 3–4 h and then used for cytoplasmic donation (test oocytes). Oocytes thawed but not used for donation served as controls. Test and control oocytes were fixed using a microtubule-stabilizing buffer. Tubulin was localized using antitubulin monoclonal antibody. Chromosomes were identified by counterstaining with DAPI.Results: Forty-four oocytes had cytoplasm removed (test group) while 12 were not used for the procedure (controls). Twenty-three oocytes survived the donation procedure. Rates of normal spindle structure for the control and test groups were 21/23 (91.3%) and 12/12 (100%), respectively.Conclusion: The removal of cytoplasm from a metaphase II human donor oocyte does not appear to significantly increase the damage to chromosome alignment or to the spindle structure.     

Evaluation of the meiotic spindle apparatus in metaphase II human oocytes following cytoplasmic donation.

PURPOSE: To determine if the removal of cytoplasm from metaphase II human donor oocytes damages the meiotic spindle apparatus. MATERIALS AND METHODS: Cryopreservation of metaphase II human oocytes was performed using a fast-freeze, fast-thaw protocol. Upon thaw, oocytes were incubated for 3-4 h and then used for cytoplasmic donation (test oocytes). Oocytes thawed but not used for donation served as controls. Test and control oocytes were fixed using a microtubule-stabilizing buffer. Tubulin was localized using antitubulin monoclonal antibody. Chromosomes were identified by counterstaining with DAPI. RESULTS: Forty-four oocytes had cytoplasm removed (test group) while 12 were not used for the procedure (controls). Twenty-three oocytes survived the donation procedure. Rates of normal spindle structure for the control and test groups were 21/23 (91.3%) and 12/12 (100%), respectively. CONCLUSION: The removal of cytoplasm from a metaphase II human donor oocyte does not appear to significantly increase the damage to chromosome alignment or to the spindle structure.     

Letters to the editor

Objective: To describe the birth achieved from frozen embryos after intracytoplasmic sperm injection (ICSI) of donor sperm into vitrified oocytes.

Patient: A 25-year-old woman whose husband was azoospermic undergoing IVF therapy.

Methods: Oocytes collected after ovarian stimulation were vitrified, thawed, and fertilized by frozen donor sperm.

Results: Nineteen oocytes were vitrified and all survived after thawing. Thirteen of the 19 oocytes that underwent ICSI with donors sperm were successfully fertilized. Twelve embryos were cryopreserved again by conventional slow-freezing protocol because of uterine bleeding on the day of transfer. Three thawed embryos were transferred, and a normal male with an infant karyotype of 46,XY was delivered.

Conclusion: This case report demonstrates effective oocyte cryopreservation by vitrification.     

Screening of conditions for rapid freezing of human oocytes: preliminary study toward their cryopreservation

One hundred and twenty-one freshly-collected human oocytes and 839 unfertilized human oocytes after insemination were cryopreserved by vitrification. The cryoprotectants used were dimethylsulfoxide (DMSO) and sucrose. Vital staining and morphological criteria were used to assess injuries to cells. Variation of the time exposure to DMSO and sucrose, and cryoprotectants concentrations, followed by extraction-dilution in sucrose without freezing made it possible to study chemical toxicities. Variation of cryoprotectant concentrations followed by immersion in liquid nitrogen, thawing, extraction, and dilution made it possible to choose optimal conditions for vitrification. The sucrose concentration upon extraction after freezing and thawing which was lower than that during soaking enhanced the oocyte survival rate as did the choice of duration and temperature of soaking. No parthenogenetical activation of these unfertilized ovum was observed. This study indicates that with a certain combination of DMSO and sucrose concentrations up to 80% of morphologically intact human oocytes can be recovered after rapid freezing and thawing.     

Congélation lente et vitrification des ovocytes humains matures et immatures

Performance and security questions in human oocyte cryopreservation have been taking researchers for about two decades. Oocytes are usually frozen at metaphase II. Immature oocytes cryopreservation is still a research alternative. Two techniques are currently available for oocyte cryopreservation: slow freezing and vitrification. Experimental data suggest that vitrification has less impact on oocyte physiology than classical slow freezing. After slow freezing of mature oocytes, survival and fertilization rates reach 70 to 80% whereas cleavage rates are around 90%, leading to five implantations and 1.2 births per 100 thawed oocytes. After vitrification of mature oocytes, survival and cleavage rates reach 90% leading to 11 implantations and 1.8 births per 100 thawed oocytes. The obstetrical and neonatal prognosis of these pregnancies is reassuring. No increased risk of congenital anomalies has been observed. However, further evaluation is needed to guarantee the safety of cryopreservation procedures. Immature oocyte cryopreservation is not currently perfected but some indications appear of great interest.     

Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using PolScope imaging

This study assesses meiotic spindle and zona pellucida characteristics using the PolScope, and analyses their relationship to embryonic development potential. A total of 205 matured oocytes retrieved from 25 patients undergoing ovarian stimulation were imaged for meiotic spindle and zona pellucida characteristics using the PolScope. After intracytoplasmic sperm injection, the oocytes were cultured and assessed for progression to blastocysts. Meiotic spindles were visualized in 78.0% of oocytes. Significantly more oocytes with visible spindles fertilized and progressed to blastocysts compared with oocytes without visible spindles. Oocytes with spindle retardance of >3 nm showed a greater progression to blastocysts compared with those with a retardance of 2-3 nm or <2 nm. More blastocysts were obtained from oocytes with spindle lengths of >12 nm than from oocytes with spindle lengths 10-12 nm or <10 nm. A difference in progression to blastocyst was observed in oocytes with a zona inner layer retardance of >3 nm compared with oocytes with retardance of 2-3 nm or <2 nm. Oocytes with an inner layer zona of 10-12 nm thickness showed better progression compared with those with a thickness of 8-10 nm or <8 nm. Quantitative measurement of length and retardance of the meiotic spindle and zona pellucida has a positive predictive value in relation to embryonic development.     

人类成熟卵母细胞慢速程序化冷冻复苏后培养时间对纺锤体和染色体的影响

目的 研究慢速程序化冷冻人类成熟卵母细胞复苏后培养时间对纺锤体和染色体的影响.方法 慢速程序化冷冻人成熟卵母细胞102枚,选择复苏后外观存活良好的卵母细胞共64枚,随机分为A组20枚、B组22枚、C组22枚,分别培养不同时间(1、3、5 h)后进行固定、荧光染色;另取新鲜成熟卵母细胞18枚固定、染色作为对照组,采用激光共聚焦显微技术,观察并分析纺锤体和染色体的形态学变化.结果 (1)A、B、C 3组正常纺锤体比例分别为10%(2/20)、46%(10/22)、41%(9/22),均明显低于对照组(83%,15/18),差异均有统计学意义(P＜0.05);A组分别和B组、C组比较,差异也有统计学意义(P＜0.05).纺锤体缺失率A组为45%(9/20),明显高于对照组(6%,1/18),差异有统计学意义(P＜0.01);A组分别和B组(14%,3/20)、C组(14%,3/20)比较,差异也有统计学意义(P＜0.05);B、C组之间比较,差异无统计学意义(P＞0.05).(2)A组正常染色体比例为30%(6/20),与B组(68%,15/22)、C组(64%,14/22)和对照组(78%,14/18)比较均明显降低,差异均有统计学意义(P＜0.05);B、C、对照组之间两两比较,差异均无统计学意义(P＞0.05).结论 慢速程序化冷冻复苏后培养3～5 h可以促进部分卵母细胞纺锤体和染色体形态的恢复.     

细胞松弛素B预处理人卵行玻璃化冻融后纺锤体与染色体变化的研究

目的:探讨细胞松弛素B(cytochalasin B,CCB)预处理对改善人卵玻璃化冷冻的效果。方法:收集常规IVF-ET或ICSI-ET周期中成熟卵母细胞71枚,随机分成4组行玻璃化冷冻。A组(17枚)、B组(20枚)、C组(17枚)分别用CCB预处理20min、30min、40min后行玻璃化冷冻,D组(17枚)不用CCB处理直接玻璃化冷冻。冻存3周后融冻卵母细胞,复苏的卵母细胞行荧光染色固定,在共聚焦显微镜下观察各组卵母细胞纺锤体及染色体形态结构。结果:A、B、C、D各组间的复苏率(70.59%、64.71%、50.00%和64.71%)无统计学差异(P>0.05),4组间的纺锤体及染色体正常形态率(27.27%,30.00%,45.46%及33.33%)亦无统计学差异(P>0.05)。结论:冷冻使成熟卵母细胞纺锤体及染色体的正常形态结构受到一定程度的破坏,CCB并不能保护卵母细胞纺锤体及染色体在冻融过程中的损伤。     

多囊卵巢综合征患者非酒精性脂肪性肝病发病情况分析

目的 分析多囊卵巢综合征(PCOS)患者中非酒精性脂肪性肝病(NAFLD)的发病情况.方法 选择PCOS患者60例为PCOS组、非PCOS患者60例为对照组,比较两组患者NAFLD的发病情况.结果 PCOS组丙氨酸转氨酶(AIJT)水平、空腹胰岛素水平和稳态模型胰岛素抵抗指数(HOMA-IR)分别为(29±15)U/L、(19±12)mU/L和0.47±0.29,均显著高于对照组[分别为(15±13)U/L、(11±8)mU/L和0.31±0.21],差异均有统计学意义(P＜0.05).PCOS组胰岛素抵抗、NAFLD的发生率分别为63%(38/60)和42%(25/60),与对照组的35%(21/60)和20%(12/60)比较,差异有统计学意义(P＜0.05).PCOS组ALT异常的发生率为40%(24/60),对照组为3%(2/60),两者比较,差异有统计学意义(P＜0.01).PCOS组伴有NAFLD患者与不伴NAFLD患者比较,体重指数显著增高,差异有统计学意义(P＜0.01);腰围臀围比值、ALT、C反应蛋白、空腹胰岛素、口服葡萄糖耐量试验2 h后胰岛素水平和HOMA-IR也显著升高,差异也有统计学意义(P＜0.05).结论 PCOS患者中NAFLD患病率升高,提示了两种疾病在临床上的相关性及对PCOS患者进行肝脏相关检查的必要性.     

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.     

Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes

The aim of this study was to evaluate the impact of different cryopreservation protocols on the repolymerization of metaphase (M)II spindles in human oocytes. Fresh aspirated donor oocytes were cryopreserved 3-4 h after retrieval using four different protocols: slow freezing using 1.5 mol/l 1,2-propanediol (PROH) + 0.2 mol/l sucrose (n = 36); 1.5 mol/l PROH + 0.3 mol/l sucrose (n = 34); 1.5 mol/l PROH + 0.3 mol/l sucrose with Na(+) depleted-choline replaced media (n = 27), and vitrification by the Cryotip method (n = 23). The control group comprised 34 fresh oocytes. Three hours after thawing, surviving and control oocytes were fixed for meiotic spindle/chromatin assessment. Survival rates were 63.8, 73.5, 74.1 and 86.9% respectively for the four protocols described above. Survival for vitrified oocytes was higher than that observed for slow freezing with 0.2 and 0.3 mol/l sucrose (P < 0.05). The proportion of oocytes showing normal spindle configuration was similar for the four protocols (81, 73.9, 88.9 and 81.3% respectively) and 88.5% for controls, showing that the MII spindle returns to its normal configuration after 3 h of post-thawing incubation under standard conditions, irrespective of the cryopreservation technique used.     

Effect of culture time on the spindle and chromosome configurations of human thawed oocytes with slow-cooling and raid-thawing protocol

OBJECTIVE: To examine the metaphase II spindle and chromosome configurations of human oocytes cultured for different times after thawing. METHODS: Using slow-cooling and raid-thawing protocol combined with 0.3 mol/L sucrose and 1.5 mol/L 1, 2-propanedio 1 (1, 2-PROH) to cryoprotect human mature oocytes (n = 102), the 64 survival oocytes without abnormal zona pellucida and cytoskeletal were randomly assigned to three groups after thawing: group A: culture 1 hour (n = 20), group B: culture 3 hour (n = 22), group C: culture 5 hours (n = 22), the fresh oocytes served as control group (n = 18). Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II spindle and chromosome. RESULTS: (1) The normal spindle rates of groups A, B and C were 10% (2/20), 46% (10/22) and 41% (9/22) respectively, significantly decreased compared with control group (83%, 15/18; P < 0.05). The rates of absent spindle in group A (45%, 9/20) was significantly higher than control group (6%, 1/18; P < 0.01). Also, the rates of absent spindle in group A was higher than groups B (14%, 3/20) and C (14%, 3/20; P < 0.05). However, no significant differences were observed in groups B and C (P > 0.05). (2) A significant increase in abnormal chromosome rate was observed in group A (30%, 0; 6/20) compared to groups B (68%, 15/22), C (64%,14/22) and control group (78%, 14/18; P < 0.05). No differences in chromosome morphology were observed in groups B, C and control group (P > 0.05). CONCLUSIONS: The cryoprotectant protocol leads to a deleterious effect on the organization of the meiotic spindle and chromosome at MII stage. The 3 - 5 hours post-thawing incubation could permit restoration of the meiotic spindles and chromosome.     

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.     

Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer

Objective: To establish an effective cryopreservation method.

Design: In vitro model study.

Setting: Infertility Medical Center, Pochon CHA University.

Animal(s): Four-week-old ICR mice superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin.

Intervention(s): Vitrified-thawed oocytes were fertilized and subsequently cultured in vitro.

Main Outcome Measure(s): Post-thawed development, chromosome/spindle normalities, and blastocyst quality.

Result(s): More cumulus-enclosed oocytes were fertilized and developed to the 8-cell stage after vitrification and thawing than denuded oocytes. However, cryopreserved oocytes of both types had lower spindle and chromosome normalities than fresh oocytes, which resulted in reduced developmental competence after thawing. The addition of 1 μM of Taxol™, a cytoskeleton stabilizer, to vitrification solution greatly promoted the blastocyst formation of vitrified-thawed oocytes, compared with no addition (24.0% vs. 58.6%). No difference in blastocyst quality, which was evaluated by blastomere and inner cell mass cell numbers and inner cell mass cell per trophoblast ratio, was found between fresh oocytes and oocytes vitrified with Taxol™.

Conclusion(s): A vitrification solution consisting of 5.5 M ethylene glycol, 1.0 M sucrose, 10% fetal bovine serum, and 1 μM Taxol™ greatly improved post-thawed development of vitrified oocytes.     

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.