Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.     

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.     

Optimal ICSI timing after the first polar body extrusion in in vitro matured human oocytes

BACKGROUND: This study was conducted to investigate the fertilization and embryo development of human oocytes injected at different time intervals after extrusion of the first polar body (PB) following in vitro maturation (IVM) in IVM cycles. Also, we evaluated whether spindle imaging could serve as a tool to determine the optimal ICSI time. METHODS: Oocytes were collected from 43 women with polycystic ovary syndrome. Metaphase I (MI) oocytes after in vitro culture for 24 h from germinal vesicle stage were subjected to ICSI according to time after first PB extrusion. The intervals were: within 1 h (n=38); 1-2 h (n=30); 2-4 h (n=26);4-6 (n=28) and 6-8 h (n=40). In some MI oocytes, viable spindle location was evaluated using Polscope microscopy at different time intervals after first PB extrusion. RESULTS: Fertilization rate of the MI oocytes injected within 1 h after first PB extrusion was low (15.8; 6/38) (P<0.01 versus all other times). In contrast, the fertilization rate was 80, 92.3, 82.1 and 85% for oocytes injected 1-2, 2-4, 4-6 and 6-8 h after first PB extrusion, respectively. Development of good-quality embryos was not significantly different among all the groups. Interestingly, all the oocytes injected within 1 h after first PB extrusion were in Telophase I. CONCLUSIONS: Human oocytes matured in vitro needed at least 1 h after first PB extrusion to complete nuclear maturation. Use of a live spindle imaging system can help to decide the timing of ICSI for oocytes matured in vitro.     

Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI

BACKGROUND: Meiotic spindles in living human oocytes can be visualized by the Polscope. This study investigated the relationship between the presence/location of the spindle in metaphase II (MII) oocytes and developmental competence of embryos in vitro. METHODS: The spindles in 626 MII oocytes were examined by the Polscope and divided into six groups (A-F) based on the presence or absence of the spindles and the angle between the spindle and the first polar body. After ICSI, the fertilization and embryo development were evaluated. RESULTS: Meiotic spindles were imaged in 523 oocytes (83.5%), while 103 (16.5%) did not have a visible spindle (group F). The majority of oocytes (68.8%) had the spindle directly beneath or adjacent to the first polar body (groups A and B: 48.2 and 20.6%). Oocytes in group C (11.2%) had the spindle located between 60 and 120 degrees angle away from the first polar body, those in group D (2.4%) had the spindle located between 120 and 180 degrees angle and those in group E (1.1%) had the spindle located at 180 degrees angle to the first polar body. The fertilization and embryonic development were similar in the oocytes with spindles regardless of spindle position. However, the rate of high quality embryos was significantly higher in the oocytes (64.2%) with visible spindles than in the oocytes (35.9%) without spindle and multipronuclear proportion showed a slight tendency to increase in oocytes without spindles. (10.7 versus 5.9%, P = 0.12; NS). CONCLUSIONS: the presence of a bi-refringent meiotic spindle in human oocytes by using the Polscope can predict a higher embryonic developmental competence. However, the relative position of the spindle within the oocyte doesn't appear to influence the developmental potential of embryos.     

The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation

BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.     

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

First polar body morphology and blastocyst formation rate in ICSI patients

BACKGROUND: It may be beneficial to identify, at a very early stage of development, concepti that will result in viable blastocysts by using a non-invasive technique. METHODS: Homogeneous groups in terms of first polar body (PB) morphology were analysed with regard to fertilization, embryo quality and blastocyst formation. The strategy was to transfer a maximum of two blastocysts with an adequate inner cell mass deriving from oocytes with identical first PBs in order to obtain information about the actual implantation potential. RESULTS: A significant relationship between first PB morphology and embryo quality was found. Fragmentation after 2 days was increased in embryos derived from oocytes with fragmented first PBs (P < 0.05) in comparison with those derived from oocytes with intact PBs. No similar correlation could be demonstrated for fertilization rate. Embryos in the intact first PB group showed an increased rate of blastocyst formation as compared with the fragmented first PB group (P < 0.05). In addition, a significant difference in implantation rate (48.6 versus 22.0%; P < 0.025) and ongoing pregnancy rate (68.4 versus 34.8%; P < 0.05) was observed for the intact versus fragmented groups respectively. CONCLUSION: In conclusion, the current study provides further evidence that preselection at a very early stage may be helpful in identifying a subgroup of preimplantation embryos with a good prognosis to form blastocysts and, consequently, to implant.     

Intracytoplasmic sperm injection: position of the polar body affects pregnancy rate.

A prospective study on intracytoplasmic sperm injection (ICSI) was performed to evaluate the effect of the position of the polar body relative to the opening of the injection needle during sperm injection, and of the person who performs the injections on fertilization, cleavage, and pregnancy rates. This study included 173 couples undergoing 313 ICSI cycles from September 1995 to December 1997. All injections were performed by two persons. For each injected oocyte the person who performed the injection was recorded as well as the position of the polar body during injection (6 o'clock: animal pole towards the opening of the needle; 12 o'clock: animal pole away from the opening of the needle). Of 2630 oocytes retrieved, 2232 were injected. Significantly more oocytes developed two pronuclei after injection with the polar body at 6 o'clock versus 12 o'clock (P = 0.01; 51 versus 45% respectively) and after injection by person 1 versus person 2 (P = 0.02; 50 and 45% respectively). Higher pregnancy rate (P = 0.046) was found after transfer of embryos from oocytes injected with the polar body at 6 o'clock (36%) versus 12 o'clock (18%). This was the result of a significant interaction (P = 0.03) between the position of the polar body and the person performing the injections. Given the higher fertilization rate in the 6 o'clock group, it is recommended that oocytes be injected with the polar body at 6 o'clock. The higher pregnancy rate as a result of polar body position and the interaction between polar body position and the operator suggest variations in injection technique.     

卵母细胞第一极体形态与女性年龄及ICSI结局的相关性研究

目的探讨卵母细胞第一极体形态与女性年龄及ICSI结局的相关性。方法回顾性分析了2008年3月至2009年11月在我院行ICSI治疗的146个周期的临床资料,按MⅡ期卵母细胞第一极体形态分为完整极体组（1、2级极体）和异常极体组（3、4级极体）,比较不同形态第一极体与年龄及ICSI结局的相关性。结果 35岁以上女性1级和2级极体的卵母细胞比例显著低于小于35岁患者（P〈0.01）;极体完整组的受精率、卵裂率、早裂率、优胚率均显著高于极体异常组（P〈0.01）;移植完整极体胚胎组的着床率和临床妊娠率显著高于移植混合胚胎（含完整极体和异常极体胚胎）组（P〈0.01）。结论卵母细胞第一极体形态与年龄间存在一定的相关性,第一极体形态可作为选择移植胚胎的指标之一。     

Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation

The present study was conducted to determine if the cryopreservationof immature human oocytes has a deleterious effect on the meioticspindle following maturation in vitro. Oocytes were obtainedin excess from in-vitro fertilization patients and divided intofour groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immatureoocytes cryopreserved before or after maturation in vitro respectively.Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controlsand included oocytes matured in vitro and in vivo respectively.The meiotic spindle was identified after incubation in anti-tubulinmonoclonal antibody (1 h, 37°C) and fluorescein-conjugatedgoat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomeswere counterstained with 4‘, 6’-diamidino-2-phenylindole.Following cryopreservation, group 1 oocytes demonstrated a 63%survival rate and 68% maturation rate in vitro. In all, 58%of the oocytes in group 2 survived the thaw. The number of oocyteswith normal spindles in group 1 (81.0%) was not significantlydifferent from control groups 3 (83.8%) and 4 (88.9%), whilethe number of group 2 oocytes with normal structures (43.5%)was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002),and 4 (P = 0.025). These results suggest that cryopreservationof the prophase I human oocyte does not significantly increaseabnormalities in the resulting meiotic spindle.     

第一极体位置对多卵胞浆内单精子显微注射结局的影响

ICSI技术目前在辅助生殖领域已经成为一项不可或缺的常规技术，但总体上讲IVF／ICSI的成功率在30—40％左右，结果并不令人十分满意，笔者从事实验室ICSI技术5年的时间，在这几年中，随着患者数量及经验的增长，对此技术的掌握日趋稳定和成熟、通常我们会假设纺锤体的位置在第一极体附近，故我们在进行ICSI操作时会将极体置于6点或是12点的位置，然后在3点处进针，但在工作中发现似乎在6点处会获得更多的优质胚胎，于是前瞻性的将获卵数＞6个的病人分半，一半将极体放在6点一半放在12点，以比较两者的受精率．卵裂率．优胚率及不同ET患者妊娠率，期待会时临床工作有一定的指导作用．从而获得更高的成功率，这无论是对患者或是对一个生殖中心来讲都是非常重要的。     

Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF

BACKGROUND: Attempts to 'rescue' by ICSI oocytes that remained unfertilized 24 h after conventional IVF have generally resulted in poor outcomes. The aim of the present study was to compare the outcome of rescue ICSI performed on one group of patients 6 h after initial insemination with those of another group where rescue ICSI was performed 22 h after initial insemination. METHODS: Twenty-five patient IVF cycles provided the oocytes for rescue ICSI 6 h after initial insemination, and 20 cycles provided the oocytes for rescue ICSI 22 h after initial insemination in this retrospective study. Fertilization and cleavage rates, embryo quality, implantation, and pregnancy rates after rescue ICSI were the main outcome measures. RESULTS: A fertilization rate of 70.3% was achieved with 6 h rescue ICSI compared with 48.5% with 22 h rescue ICSI (P < 0.0001). From 6 h rescue ICSI, 12 clinical pregnancies (48.0%) resulted in three sets of twins, eight singletons and one abortion. From 22 h rescue ICSI there was one (5.0%) singleton pregnancy and delivery of a healthy baby. Likewise, the implantation rate was 20.2% from 6 h rescue ICSI compared with 1.72% from 22 h rescue ICSI (P < 0.02). CONCLUSIONS: Rescue ICSI after 6 h post-insemination (46 h post-HCG) gave better fertilization, pregnancy and implantation rates compared with rescue ICSI after 22 h when oocytes have become aged.     

Fertilization and early embryology: Consequences of non-extrusion of the first polar body and control of the sequential segregation of homologues and chromatids in mammalian oocytes

Absence of polar body formation, or premature chromatin condensation(PCC) in human oocytes can cause infertility. We studied in-vitromaturing mouse oocytes in order to identify risk factors forsuch conditions, and for the precocious segregation of homologuesor chromatids. Treatment with the actin-binding drug cytochalasinD (10 µg/ ml) arrested oocytes in metaphase I. Upon exposureto Ca²⁺-ionophores, anaphase I was triggered in the absenceof cytokinesis. Chiasmata resolved and homologues separatedinstantaneously. In some oocytes predivision of all chromatidsoccurred. Homologues or chromatids never separated even afterexposure to Ca²⁺-ionophores when microtubules were depolymerized,although bivalents could eventually decondense. Thus, in meiosisI checkpoints exist which ensure that homologue separation onlytakes place when a metaphase I spindle is present but cytokinesisand anaphase progression can be uncoupled. Cycloheximide induceda sequential separation of homologues in oocytes with intactmetaphase I spindle, resulting in metaphase II chromosomes andbivalents in individual cells as also found in some human oocytesof aged females. In oocytes which progressed to metaphase IIbut failed to extrude a first polar body, the two sets of chromosomeseventually aligned on a common spindle (‘diploid’metaphase II). PCC of one set was never observed. Ageing invitro of cytochalasin D-blocked metaphase I oocytes had no pronouncedeffect on chromosome segregation.     

Differential effects of repeated ovarian stimulation on cytoplasmic and spindle organization in metaphase II mouse oocytes matured in vivo and in vitro

The effects of four rounds of ovarian stimulation spaced 1-6 weeks apart on the normality of metaphase II (MII) spindle formation, chromosomal alignment and cytoplasmic organization were examined in intact ovulated mouse oocytes and at MII for oocytes obtained at the germinal vesicle stage from the same ovaries and matured in vitro. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay was used to identify DNA strand breaks in chromosomes, and histological studies of ovaries between and at each round of ovarian stimulation were performed. The results demonstrate a progressive and significant increase in the frequency of spindle defects with each round of ovarian stimulation, including those spaced weeks apart. Oocytes with spindle defects were also characterized by the occurrence of detached chromosomes and cytoplasmic asters. In contrast, in-vitro matured oocytes derived from the same ovaries were normal. No evidence of DNA strand breaks with repeated rounds of ovarian stimulation was detected in ovulated or in-vitro matured oocytes. The development and persistence of nodules of hypertrophied granulosa in regions where follicular growth occurs suggest that a progressively increasing proportion of oocytes in the ovulatory pathway may experience an intrafollicular milieu that has negative consequences for competence. The results are discussed with respect to ovarian and oocyte biological ageing and possible adverse implications for human oocyte competence with repeated hyperstimulation.     

The radiosensitivity of the human oocyte

BACKGROUND: We determined the best model available for natural follicle decline in healthy women and used this to calculate the radiosensitivity of the human oocyte. METHODS: Ovarian failure was diagnosed in six patients with a median age of 13.2 years (range 12.5-16.0) who were treated with total body irradiation (14.4 Gy) at 11.5 years of age (4.9-15.1). We previously estimated the dose of radiation required to destroy 50% of the oocytes (LD(50)) to be <4 Gy. This estimate is an oversimplification, because decay represents an instantaneous rate of temporal change based upon the remaining population pool, expressed as a differential equation: dy/dx = -y[0.0595 + 3716/(11780 + y)], with initial value y(0) = 701 200. RESULTS: Solving the differential equation, we have estimated the number of follicles left after irradiation given as sol(51 - s + r), where r equals age at treatment, s equals age at diagnosis of ovarian failure, and 51 years is the average age of menopause. The surviving fraction of oocytes as a percentage is 100 times this value divided by sol(r). The mean surviving fraction for the six cases is 0.66%. We obtain a function, g(z), which decreases in value from 100% at zero dosage to mean value at dosage z = 14.4 Gy. We have g(z) = 10(mx+c), where c = log(10)100 = 2, and m = [log(10)(0.66) - c]/14.4. Solving g(z) = 50 gives an LD(50) of 1.99. CONCLUSIONS: Based on new data and a revised mathematical model of natural oocyte decline, we have determined the surviving fraction of oocytes following irradiation and estimate the LD(50) of the human oocyte to be <2 Gy.     

Comparative genomic hybridization analysis of human oocytes and polar bodies

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.     

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.     

Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte

At ovulation, the mouse oocyte is arrested at metaphase of thesecond meiotic division. Since microtubules are thermo-and chemosensitivestructures, the effects of 1.5 M dlmethylsulphoxide and 1.5M 1,2-propanediol were studied at room temperature on the morphologyof the meiotic spindle. Oocytes incubated at 37°C or atroom temperature served to estimate the effect of temperaturein the experiment. The meiotic spindle was visualized by immunogold-silverstaining of microtubules. In the control group at 37°C,88% of oocytes had normal spindles. After incubation at roomtemperature for the same time, 89% of oocytes showed abnormalspindles. In the oocytes exposed to dimethyl sulphoxide or 1,2-propanediolat room temperature a protective effect on spindle morphologycould be recognized. Subsequent incubation at 37°C resultedin partial restoration of the observed abnormalities after coolingto room temperature and after exposure to dhnethytsulphoxide.incubation at 37°C after exposure to 1,2-propanediol atroom temperature induced spindle absence in the majority ofoocytes. Although this latter condition allowed fertilizationwithout increased incidence of ploidy abnormalities, a rolefor 1 as an activating agent is hypothesized.     

The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos

Peptide nucleic acids (PNA) are synthetic DNA mimics based on an uncharged polyamide backbone, which hybridize with complementary DNA with high affinity and specificity. PNA have recently become recognized as efficient tools for in situ chromosomal identification. In the present study, this new approach has been tried on isolated human oocytes, polar bodies and blastomeres. Using centromeric PNA probes specific for chromosomes 1, 4, 9, 16, X and Y, we tested multicolour labelling PNA reaction on 27 oocytes and 23 blastomeres. Sequential PNA hybridization was performed on five oocytes and combined PNA and fluorescence in situ hybridization (FISH) reactions on two oocytes. Both the rates and the types of abnormalities observed are in agreement with results from previous FISH studies. This preliminary study indicates that PNA probes allow a reliable chromosomal analysis in isolated human oocytes and blastomeres and consequently might provide an interesting adjunct to FISH for diagnostic analysis.