Visible Spindle Research Articles

P-196 PIEZO-intracytoplasmic sperm injection (P-ICSI) can improve the clinical outcomes of ICSI

Abstract Study question Can P-ICSI improve the clinical outcomes of ICSI, such as degeneration, fertilization and development to blastocyst, compared to conventional ICSI (C-ICSI)? Summary answer P-ICSI can improve the clinical outcomes of ICSI by increasing the number of fertilized oocytes and decreasing the number of degenerated oocytes after ICSI. What is known already In human IVF-ET, some of oocytes are damaged after ICSI when retrieved oocytes are inseminated by ICSI. The application of P-ICSI has been limited although it was developed over 30 years ago. Recently, it has been reported that P-ICSI can decrease damage after ICSI and improve fertilization and embryonic development compared to C-ICSI. Study design, size, duration This study included 50 subjects undergoing IVF and was performed between April and October 2022. Sibling oocytes were randomly assigned to C-ICSI and P-ICSI. Participants/materials, setting, methods A total of 1,207 oocytes were retrieved from 50 patients. Among them, 852 oocytes with visible spindle were inseminated by C-ICSI or P-ICSI in sibling oocytes. Four hundred twenty-five oocytes were inseminated by C-ICSI and 427 oocytes were by P-ICSI. Fertilization, degeneration after ICSI and blastocyst development on day 5 or day 6 were compared between the P-ICSI and C-ICSI. The differences between C-ICSI and P-ICSI were statistically analyzed using Mann-Whitney U test. Main results and the role of chance Normal fertilization rate of P-ICSI (78.2 ± 16.5%) were higher than that of C-ICSI (71.2 ± 18.9%). The difference was statistically significant (P = 0.0447). The oocyte degeneration rates after ICSI were 9.3 ± 11.8% (C-ICSI) and 4.8 ± 9.4% (P-ICSI). The degeneration rate of C-ICSI was significantly higher than that of P-ICSI (P = 0.04338). Blastocyst formation rate of C-ICSI was 58.6 ± 25.0% and that of P-ICSI was 64.3 ± 26.3%. The blastocyst formation rate of P-ICSI was higher than that of C-ICSI but the difference was not statistically significant (P = 0.2113). The rates of blastocysts that were available for embryo replacement or cryopreservation were higher in P-ICSI (56.0 ± 25.4%) than in C-ICSI (50.4 ± 24.7%) although the difference was not significant (P = 0.26272). Limitations, reasons for caution Pregnancy outcomes data were not available. The data of this study were obtained from single fertility center. More large-scale multicenter studies will be needed to confirm the effectiveness of P-ICSI. Wider implications of the findings Consistent with other studies, these results show that P-ICSI reduces the damage rate of oocytes after ICSI. The implementation of P-ICSI has the potential to improve clinical outcomes of human IVF although it is more complicated and takes longer than that of C-ICSI. Trial registration number not applicable

Investigation of the deterministic value of presence of visible spindle on fertilization and blastocytes utilization potential in frozen/thawed oocyte cycle

Background and Aim: To date, characteristics of the meiotic spindles in human oocytes studied by several authors and shown that they are associated with fertilization, embryo quality, pregnancy and live birth rates. Also, it has been shown that, meiotic spindle structures are very susceptible to cryodamage and can be disassembled and re-assembled depending on the changes in temperature. Thus, studying spindle characteristics of the vitrified-warmed oocytes are important and can provide information regarding the full recovery of the oocytes as well as association with fertilization, embryo development and pregnancy, which the data is limited in literature.

P-126 Investigation of the deterministic value of presence of visible spindle on fertilization and blastocytes utilization potential in frozen/thawed oocyte cycle

Abstract Study question Does presence or absence of birefringent spindle after warming of the oocytes has deterministic value on fertilization and early embryonic development potentiaL? Summary answer The warmed oocytes with birefringent spindle seem to have a higher potential to fertilize and become good quality blastocytes. What is known already To date, characteristics of the meiotic spindles in human oocytes were studied by several authors by using a non-invasive method (PolScope) and shown that they are associated with fertilization, embryo quality, pregnancy, and live birth rates. Also, it has been known that meiotic spindle structures are very susceptible to cryodamage and can be disassembled and re-assembled depending on the change in temperature. Thus, studying spindle characteristics of the vitrified-warmed oocytes is important and can provide information about the full recovery of the oocytes, fertilization, embryo development, and pregnancy, which the data is limited in the literature. Study design, size, duration A retrospective study consisting 40 egg donation cycles by using vitrified-warmed oocytes between December 2017 and December 2019 at British Cyprus IVF Hospital. Participants/materials, setting, methods Spindle morphologies of vitrified-warmed oocytes were analyzed by PolScope (RI) combined with an image analysis system after approximately 1.5 hours post-warming. The data is divided into two according to the presence or absence of visible nucleus and correlation with embryonic development analyzed accordingly. To make a comparison in groups a new numerical scoring system for embryo quality on days 5 and 6 was created where embryos scored depending on the stage of expansion, Main results and the role of chance In total, 587 out of 648 vitrified/warmed oocytes survived (90.6%) and 447 of them were fertilized (75.0%). Among the survived oocytes, 503 (85.5%) oocytes were containing detectable spindle (Group I) while spindles were missing in 84 oocytes (Group II). The results showed that the fertilization rate (78.1% vs. 64.3%, p < 0.01), the proportion of good PN morphology (Z Score: Z1 and Z2 over other parameters: 50.4% vs 35.2%, p = 0.036), and viable embryo count (40.6% vs 15.1%, p < 0.01) which is defined as embryos that good enough to transfer or freezing, were higher in group I. Similarly, the degree of expansion on day 5/6 (p = 0.013, p = 0.015 respectively) and the quality of the cells in ICM and TE (p < 0.01) were found higher in group I. We further investigated the differences in embryo quality between groups by converting the embryo grades to numerical value starting from arrested (Score=0) to 6AA (Score=22) and found that embryo scores on day 5 (p = 0.01) and day 6 (p = 0.013) were higher in group I. Limitations, reasons for caution The main limitation of this study is the lack of data regarding the spindle status of the frozen oocytes before vitrification where some of the oocytes might not have visible spindle due to incomplete maturation rather than cryodamage. Wider implications of the findings The present data showed that the presence of birefringent meiotic spindles in warmed oocytes is a very important sign for a full recovery and early embryonic development potential, which can be useful for egg banks and IVF clinics. Trial registration number not applicable

Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes

ProposeThe presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI).MethodsThe study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern.ResultsThe visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded.ConclusionsThe study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage.

Effect of melatonin on developmental competence of denuded human oocytes during in vitro maturation

Melatonin is known to be a free radical scavenger and to exist at high concentrations in follicular fluid. It has been previously reported that supplementation of culture medium with melatonin improves oocyte maturation and embryo development in vitro in several animal models. It has been further reported that melatonin also has an effect on denuded bovine oocytes.We studied the effect of supplementing in vitro maturation (IVM) medium with melatonin on oocyte maturation and embryo development. This retrospective study was performed between July 2016 and December 2016. We analyzed 536 immature oocytes from 214 patients following clomiphene or letrozole stimulation. Oocytes were denuded with hyaluronidase then cultured in M199 supplemented with 25 μM dbcAMP and 50 ng/ml EGF (mM199; control). In the melatonin group (MT), mM199 was supplemented with 0.1 nM melatonin. After 27h IVM, mature oocytes were observed spindles by poloscope prior to ICSI. Fertilized embryos were cultured for 5 days. We studied the effect of melatonin on oocyte maturation, spindle visualization, fertilization, cleavage and blastocyst development. There were no significant differences between the control and MT groups in nuclear maturation rate or fertilization rate (51.4% vs. 45.6%, 86.0% vs. 75.9% respectively). While the number of mature oocytes with visible spindles in the MT group (74.5%) was significantly higher than in controls (61.3%), cleavage rate and blastocyst formation rates, per fertilized oocytes, were similar between the two groups (control vs. MT, 87.5% vs. 90.7%, 20.0% vs. 22.4% respectively). There was no significant difference in blastocyst developmental rate per IVM. Melatonin improved the spindle visualization rates of matured oocytes observed by polscope. However maturation rate, fertilization rate, and blastocyst development did not improve.

Relationship between pre-ICSI meiotic spindle angle, ovarian reserve, gonadotropin stimulation, and pregnancy outcomes

The goals of this study are to analyze the clinical pregnancy rate as a function of the pre-intracytoplasmic sperm injection (ICSI) oocyte spindle angle and determine factors which can be associated with different spindle angles, if clinically relevant. Fifty-eight patients, who underwent their first ICSI cycle from January to December 2013, were included. Eight hundred thirty oocytes were collected, and 648 were metaphase II (MII) on retrieval day. Spindles were characterized in terms of visibility and position in relation to the first polar body (PB). Oocytes were separated into four groups based on angle: (group 1, n = 297) 0°-29°; (group 2 n = 212) 30°-89°; (group 3, n = 72) ≥90°; and those with no visible spindle (group 4, n = 67). The rate of blastocyst development was associated with the spindle angle (p = 0.002). The rate of good quality blastocysts were as follows: group 1 (42%), group 2 (30%), group 3 (35%), and group 4 (19%) (p = 0.02). Pregnancy and live birth rates were also affected (p = 0.007 and p = 0.046, respectively). Antral follicle count (AFC) (p = 0.001), total FSH stimulating dose (p = 0.0001), and peak serum estradiol level (p = 0.0001) were associated with spindle angle grouping. Miscarriage rates trended different (p = 0.07). On the other hand, day 3 follicle-stimulating hormone (FSH) levels and female and male age were not associated with spindle angle grouping. Embryos resulting from oocytes with pre-ICSI spindle angles between 0° and 29° were associated with better blastocyst, pregnancy, live birth, and miscarriage rates when compared to oocytes that had no visible spindle. Low ovarian reserve and excessive stimulation were also associated with lack of spindle and therefore lower pregnancy outcomes.

Is oocyte meiotic spindle morphology associated with embryo ploidy? A prospective cohort study

To explore whether an association exists between oocyte meiotic spindle morphology visualized by polarized light microscopy at the time of intracytoplasmic sperm injection and the ploidy of the resulting embryo. Prospective cohort study. Private IVF clinic. Patients undergoing preimplantation genetic screening/diagnosis (n = 113 patients). Oocyte meiotic spindles were assessed by polarized light microscopy and classified at the time of intracytoplasmic sperm injection as normal, dysmorphic, translucent, telophase, or no visible spindle. Single blastomere biopsy was performed on day 3 of culture for analysis by array comparative genomic hybridization. Spindle morphology and embryo ploidy association was evaluated by regression methods accounting for non-independence of data. The frequency of euploidy in embryos derived from oocytes with normal spindle morphology was significantly higher than all other spindle classifications combined (odds ratio [OR] 1.93, 95% confidence interval [CI] 1.33-2.79). Oocytes with translucent (OR 0.25, 95% CI 0.13-0.46) and no visible spindle morphology (OR 0.35, 95% CI 0.19-0.63) were significantly less likely to result in euploid embryos when compared with oocytes with normal spindle morphology. There was no significant difference between normal and dysmorphic spindle morphology (OR 0.73, 95% CI 0.49-1.08), whereas no telophase spindles resulted in euploid embryos (n = 11). Assessment of spindle morphology was found to be independently associated with embryo euploidy after controlling for embryo quality (OR 1.73, 95% CI 1.16-2.60). Oocyte spindle morphology is associated with the resulting embryo's ploidy. Oocytes with normal spindle morphology are significantly more likely to produce euploid embryos compared with oocytes with meiotic spindles that are translucent or not visible.

Relationship between the meiotic spindle size in human oocytes and embryo developmental potential after intracytoplasmic sperm injection

Relationship between the meiotic spindle size in human oocytes and embryo developmental potential after intracytoplasmic sperm injection

Meiotic spindle normality predicts live birth in patients with recurrent in vitro fertilization failure

To investigate the impact of meiotic spindle normality on live birth rates in women with recurrent IVF failure. A retrospective comparative study. A large private sector IVF service in Sydney, Australia. Five hundred five intracytoplasmic sperm injection (ICSI)/ET cycles of patients who fulfilled the criteria for recurrent IVF failure, three or more previous fresh or frozen ETs with no ongoing pregnancy. Polarized light microscopy was used at the time of ICSI. Clinical pregnancy rates (PR) and live birth rates (LBR) were compared depending on the morphology of the meiotic spindle of the oocyte(s) from which the embryo(s) were transferred. Women receiving embryos where at least one was derived from a normally spindled oocyte had significantly increased clinical PR and LBR when compared with those who had only embryos derived from abnormally spindled oocytes (clinical PR: 31% vs. 7%; odds ratio [OR], 6.45; 95% confidence interval [CI] 3.65-11.40) and (LBR: 24% vs. 4%; OR, 7.24; 95% CI 3.62-14.49). Comparison between the abnormal spindle groups showed significantly higher clinical PR and LBR from the group of patients receiving embryos where at least one was derived from an oocyte with no visible spindle compared with the group receiving embryos from dysmorphic spindles only (clinical PR: 9% vs. 6%; OR, 0.58; 95% CI 0.22-1.57 and LBR: 8% vs. 1%; OR, 0.16; 95% CI 0.03-0.77). Normally spindled oocytes, as determined by polarized light microscopy, are associated with significantly higher clinical PRs in patients with recurrent IVF failure.

Influence of granulocyte colony stimulating factor to the rabbit inferior vena cava endothelial repair after balloon injury

Objective To research the affect of rabbit inferior vena cava vascular endothelial repair after balloon injury by granulocyte colony-stimulating factor (G-CSF).Methods 50 experimental rabbit randomly divided into second groups (n =25):Group A:rabbits were injected daily with 50 μg/kg G-CSF for 7 days shortly after inferior vena cava (IVC) injury.Group B:rabbits were injected daily with 2 ml N-S for 7 days shortly after IVC injury.The animals were killed at 7 and 30 days after balloon denudation.The samples of IVC were harvested for observation damaged vascular repair situation by hematoxylin and eosin (HE) dyeing,count the intima (I) area and membrane (M) area ratio to evaluate intima hyperplasia degree.Results The 7 days of A (0.0564 ±0.0303) and B group (0.0808 ± 0.0428) I/M ratio comparison,difference was not statistically significant (P ＞ 0.05).The 30 days of A (0.1462 ±0.0993)and B group (0.3098 ± 0.2154) I/M ratio comparison,difference have statistical significance (P ＜0.05).Pathological results observation:two groups of rabbit sacculus immediately after injury were visible artery endothelial stripped.7days after injury neointimal formation and thickening,the membrane can visible spindle smooth muscle cell arrangement disorder.30 days,group A of endometrial hyperplasia significantly suppressed.Conclusion It was found that the G-CSF treated can promote the rabbit inferior vena cava injury vascular re-endothelialization process,inhibit intima hyperplasia,for balloon injury has the repair effect,can prevent vein of restenosis after injury happened. Key words: Budd-Chiari syndrome; Balloon; Intervention injury; Granulocyte colony stimulating factor

Noninvasive Imaging of the Meiotic Spindle of In Vivo Matured Oocytes From Infertile Women With Endometriosis

The objectives of this prospective study were to evaluate the nuclear maturation stage and the presence and location of meiotic spindles of in vivo matured oocytes from infertile women with and without endometriosis (male or tubal causes of infertility) undergoing stimulated cycles for intracytoplasmic sperm injection (ICSI). We also compared the ICSI outcomes among groups. We analyzed the meiotic spindles of oocytes from 36 patients with endometriosis I/II, 24 with endometriosis III/IV, and 60 without endometriosis (male or tubal causes of infertility). The oocytes were imaged using polarization microscopy. There were no differences in the number of oocytes in telophase I (mean [standard deviation]: 0.1 [0.5], 0.2 [0.4], and 0.2 [0.5], respectively, in the endometriosis I/II, endometriosis III/IV, and control groups), in metaphase II with visible spindles (4.2 [2.5], 3.1 [2.0], and 3.6 [2.2], respectively, in the endometriosis I/II, endometriosis III/IV, and control groups), and in spindle location among groups. We can conclude from this study that noninvasive analysis of spindles from in vivo matured oocytes of infertile patients with endometriosis did not demonstrate significant differences in terms of the nuclear maturation stage, the percentage of oocytes in metaphase II with visible spindles, and the spindle localization when compared to the control group. However, it is important to state that there are no studies evaluating the accuracy of polarization microscopy for the detection of meiotic anomalies in human oocytes, which would need to be better evaluated in future studies using an appropriate methodology.

Oócitos aparentemente maduros injetados em telófase I apresentam piores resultados de reprodução assistida

To evaluate the nuclear maturation stage and the presence of meiotic spindles of in vivo matured oocytes from infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (ICSI) and compare intracytoplasmic sperm injection outcomes between oocytes in telophase I (TI) and metaphase II (MII), and the ones with and without visible meiotic spindle. A prospective and controlled study with 106 infertile patients who underwent ovarian stimulation for intracytoplasmic sperm injection purposes. Patients aged 38 years or less, with basal follicle stimulating hormone (FSH) less than 10 mIU/mL and body mass index (BMI) less than 30 kg/m². Were included patients presenting any systemic diseases, any active infection, smokers or patients who had been using hormonal medications and hormonal and nonhormonal anti-inflammatory drugs for the past two months prior to the assisted reproduction procedure were excluded. The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged by polarization microscopy immediately before intracytoplasmic sperm injection and characterized according to nuclear maturation stage (telophase I and metaphase II) and to the presence of a meiotic spindle. We analyzed the fertilization rates, cleavage, number of good quality embryos on the second day (D2) from oocytes on telophase I versus those in metaphase II, and metaphase II visible spindle versus non-visible ones. Data were analyzed comparatively by Fisher's exact test. The level of significance was set at 5% in all analyses (p<0.05). The meiotic spindles of 516 oocytes were imaged using polarization microscopy. From the 516 oocytes analyzed, seventeen were in telophase I (3.3%) and 499 (96.7%) in metaphase II. The oocytes injected in telophase I had significantly lower fertilization rates than those injected in metaphase II (53 and 78%, respectively) and produced no good quality embryos on day 2. When the oocytes with and without a visible meiotic spindle were compared, there was no significant difference in the intracytoplasmic sperm injection results. Oocytes injected in telophase I showed lower fertilization rates when compared to those in metaphase II. It is possible that the analysis of oocyte nuclear maturation by polarization microscopy can be used as a predictor of fertilization after intracytoplasmic sperm injection.

Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates?

To investigate the relationship between spindle normality and: 1) blastocyst formation; 2) implantation rates; and 3) live birth rates. Prospective observational study. A private fertility clinic. One hundred patients ≤40 years old undergoing intracytoplasmic sperm injection (ICSI). Meiotic spindles were imaged before ICSI with the use of Oosight microscopy. With the use of specific criteria, spindle normality was independently assessed by two researchers. Blastocysts were chosen for transfer by standard light microscopic morphologic criteria by researchers who were blinded to the spindle assessment. 1) Blastocyst formation; 2) implantation; and 3) live birth rates. A total of 808/920 oocytes were metaphase II. Of those, 711 (88%) had a visible spindle: 205 (29%) were normal spindles (NS) and 506 (71%) abnormal spindles (AS). Fertilization rates were significantly higher in NS oocytes. Although NS and AS oocytes both formed morphologically good-quality blastocysts, implantation and live birth rates were higher from NS oocytes. All ongoing pregnancies resulted from NS oocytes. Spindle assessment with polarized light microscopy provides an early predictor of the pregnancy potential of that oocyte. AS oocytes can form good-quality blastocysts, but these appear to have little chance of live birth. Therefore, spindle assessment should improve the selection of the best embryo for single embryo transfer.

Correlation of oocyte morphometry parameters with woman’s age

Aim of this study was to evaluate morphometric parameters of metaphase II oocytes, including cytoplasm diameter (CD), zona pellucida thickness (ZPT) and width of the perivitelline space (PS), in relation with zona pellucida birefringence, spindle presence and age of the woman. Oocytes were classified into groups according to zona birefringence (low or high zona birefringence, LZB and HZB, respectively) and presence or absence of a visible spindle (SP and aSP, respectively). HZB oocytes showed a thicker zona (17.7 ± 0.3 μm) than LZB oocytes (16.7 ± 0.3 μm, p < 0.01). Moreover, PS was narrower in HZB and SP oocytes than in LZB (p < 0,001) and aSP (p < 0,05) oocytes. Finally, we found that CD and ZPT linearly decrease with age of the woman (CD r = 0.028: p < 0.01; ZPT r = 0.050: p < 0.0001). Our results evidence an association in human oocytes between zona pellucida and spindle birefringence and defined morphometric parameters and a decrease of oocyte size and ZPT as a function of women's age.

Efficiency of polarized microscopy as a predictive tool for human oocyte quality

Conflicting results have been reported regarding the use of polarized microscopy as a predictive tool for human oocyte quality. Oocytes from 121 ICSI cycles were analysed with polarized microscopy. Both qualitative (spindle presence) and quantitative (retardance) data were correlated to the key assisted reproduction technology outcome parameters. Second, polarized microscopy was applied on in vitro matured (IVM) oocytes from germinal vesicle oocytes that matured after 24 or 48 h and from metaphase I oocytes matured after 3 or 24 h. These data were correlated with confocal analysis of spindle-chromosome complex. Spindles were detected in 82% of in vivo matured oocytes and in 64% adjacent to the first polar body (PB). Fertilization rate was higher in oocytes with a visible spindle (P = 0.0002). In patients aged over 35 years, the percentage of a visible spindle and mean spindle retardance was lower than in younger patients (P < 0.03). A higher number of spindles were located adjacent to the first PB in IVM matured oocytes (94%) versus in vivo matured oocytes (P < 0.0001). Confocal imaging revealed that spindle absent IVM metaphase II (MII) oocytes had a higher degree of aberrant spindle and chromosomal configurations versus IVM MII oocytes with a visible spindle (P = 0.002). Oocytes with absent spindles were associated with lower fertilization rates and advanced female age. Other important outcome parameters (embryo quality, pregnancy rates) were not correlated to spindle nor zona inner layer analysis. Interestingly, confocal imaging showed that polarized microscopy might be used as a qualitative predictive tool of human oocyte quality but no correlation could be demonstrated with quantitative polarized microscopy.

Relationship between meiotic spindle characteristics in human oocytes and timing of the first zygotic cleavage after intracytoplasmic sperm injection (ICSI)

OBJECTIVE: Early-cleaving zygotes are more likely to develop to the blastocyst stage than their late-cleaving counterparts. The mechanism causing variation in the timing of the first zygotic cleavage has not been identified. This study aimed to investigate the relationship between meiotic spindle characteristics in human oocytes and the timing of the first zygotic cleavage after ICSI. DESIGN: Retrospective clinical study. MATERIALS AND METHODS: We evaluated 150 patients undergoing ICSI cycles, resulting in 509 metaphase II (MII) oocytes. Meiotic spindles and zona pellucida characteristics were imaged using the PolScope before ICSI in all MII oocytes. After ICSI, the oocytes were cultured and assessed for early cleavage (EC). The first mitotic division was checked 25–27 hours after ICSI. Embryos that had reached the two-cell stage at this point were classified as EC embryos. Embryos with intact nuclei or nuclear membrane breakdown were classified as late cleavage (LC) embryos. We compared the meiotic spindle and zona pellucida characteristics of EC with LC embryos. RESULTS: The meiotic spindle was visualized in 468 oocytes (92.0%), but could not be visualized in the remaining 41 (8.0%). After ICSI, the fertilization rate in oocytes with a visible spindle was 384/468 (82.1%). From 384 embryos, 163 (42.4%) were classed as EC and 221 (57.6%) were LC. The zona pellucida characteristics and spindle angle to the first polar body were similar in EC and LC embryos. EC embryos had significantly greater spindle areas (108.0 ± 17.8 versus 89.8 ± 19.2 μm2) and lengths (14.7 ± 2.0 versus 13.4 ± 2.2 μm) compared with LC embryos (P < 0.01). Spindle retardance in EC embryos was significantly higher than that in the LC embryos (P < 0.01). CONCLUSION: Meiotic spindle characteristics were significantly associated with EC score. Quantitative measurement of the size and retardance of the meiotic spindle could be valuable for identifying human embryo potential in the IVF laboratory.

Cytological studies on induced meiogynogenesis in Japanese flounderParalichthys olivaceus(Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major. After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.

Oocyte meiotic-stage specific differences in spindle disassembly and reassembly in response to temperature changes

OBJECTIVE: The objectives of this study were to compare the effect of temperature changes on meiotic disassembly in mouse MII and TI stage oocytes and to compare the reassembly of the spindle in MII and TI oocytes following vitrification and warming. DESIGN: Experimental. MATERIALS AND METHODS: Oocytes were collected from B6SJL-F2 female mice injected with 5 IU eCG followed 44 h later with 5 IU hCG. Oocytes were collected 12 h post-hCG and denuded. The first experiment TI and MII oocytes were placed into drops of HTFH at 37° C (control), room temperature (RT) and 4° C for 0, 5, 30 and 60 min before visualization of the spindle with the Oosight™ image analysis software. In the second experiment, TI and MII oocytes were vitrified and warmed. Oocyte spindles were measured at 0, 30 and 60 min post warming at 37° C. Results were analyzed using Chi-square analysis and considered significant at P<0.002. RESULTS: At 0 h all TI and MII oocytes had visible spindles. There were no differences in spindles in TI and MII oocytes after 5 min at RT. After 30 min at RT, there was a significant difference in presence of spindles between TI and MII oocytes (100, 43%), respectively. After 1h at RT a significantly higher percentage of TI oocytes had a spindle compared to MII oocytes (100, 25%), respectively. After 5 min at 4° C, significantly more TI oocytes had spindles compared to MII oocytes (100, 22%), respectively. After 30 and 60 min at 4° C, there were no spindles in MII oocytes while 100% of TI oocytes had a visible spindle (P<0.0005). No spindles were present in MII oocytes at 0h post warming compared to 100% in TI oocytes. After 30 min of warming there was a significantly higher percentage of TI oocytes with a spindle compared to MII oocytes (100, 57%), respectively. After 60 min there were no differences between spindle assembly in TI and MII oocytes. CONCLUSIONS: TI oocytes have a polar body present, making it impossible to distinguish from a MII oocyte under a light microscope. Selection of MII oocytes is essential in the success of ICSI and oocyte cryopreservation. This study demonstrates a striking difference in microtubule depolymerization in MII and TI oocytes. Further experiments will look into the differences in spindle properties between different stages of meiosis as well as the effect of vitrifying TI stage oocytes.

Preliminary study of embryo development following assessment of male and female gametes

Gamete characteristics are likely to contribute to embryo development. In this preliminary study, early parameters were assessed in embryos generated following microinjection of hyaluronan-bound and unbound spermatozoa into 176 sibling oocytes, which had themselves been assessed using a polarizing light microscope imaging system. The fertilization and early cleavage rates, and day 3 embryo quality did not differ when oocytes displaying similar characteristics were inseminated with either bound or unbound sperm. Regardless of the binding characteristics of the sperm, early embryos displaying good and poor quality derived from oocytes displaying visible spindles and similar characteristics. These results indicate that early embryo developmental characteristics were independent from the hyaluronic acid binding capacity of the sperm when oocytes displayed a visible spindle.

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.