Wei-Hua Wang

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.

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.

Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates

OBJECTIVE To examine the effects of different thermodynamic control systems on the temperature stability of human eggs during in vitro manipulation, with the integrity of meiotic spindles imaged using the LC-PolScope (Cambridge Research & Instrumentation, Inc., Woburn, MA). DESIGN We performed intracytoplasmic sperm injection (ICSI) and/or imaging of eggs with the temperature regulated by three different systems: thermostated coverslip (system 1), thermostated coverslip combined with objective heater (system 2), and conventional stage warmer (system 3). SETTING Academic in vitro fertilization clinic. PATIENT(S) Oocytes were aspirated from stimulated ovaries of patients undergoing oocyte retrieval for ICSI. INTERVENTION(S) Measurement of temperature regulation in media surrounding eggs during in vitro manipulation and imaging. MAIN OUTCOME MEASURE(S) Rate of oocytes with spindles, fertilization rates, and clinical pregnancy rates after ICSI. RESULT(S) We imaged spindles in more oocytes with system 2 (81.2%) than with system 1 (61.4%). Spindles could not be imaged for system 3 because of technical limitations. Fertilization rates were significantly higher when oocytes were imaged and used for ICSI with system 2 (78.8%) than with system 1 (56.7%) or system 3 (64.0%). Most importantly, a significantly higher clinical pregnancy rate was observed when oocytes were manipulated with system 2 (51.7%) than with system 1 (25.0%) or system 3 (23.1%). No differences were found in average ages, number of previous cycles, number of eggs, or day 3 FSH or E2 levels among groups. CONCLUSION(S) Imaging meiotic spindles with the PolScope provides an intracellular thermostat during ICSI. Rigorous thermal control during ICSI stabilized spindles and increased the fertilization and clinical pregnancy rates achieved after ICSI. The presence of birefringent spindles in living human eggs served as a monitor for in vitro conditions.

Spindle Observation in Living Mammalian Oocytes with the Polarization Microscope and Its Practical Use

The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. Conventional methods to image spindles rely on fixation and transmission electron microscope or immunofluorescence staining and fluorescence microscope, so they provide limited value to studies of spindle dynamics and human clinical in vitro fertilization. A new orientation-independent polarized light microscope, the LC Polscope, was used to examine the bi-refringent spindles in living mammalian oocytes. It was found that spindles could be imaged with the Polscope in living oocytes in all mammals so far examined, including hamster, mouse, cattle, human, and rat. The first polar body did not accurately predict the spindle location in most metaphase II oocytes. Intracytoplasmic sperm injection (ICSI) could be performed by monitoring spindle position. Studies in humans indicated that, aftr ICSI, higher fertilization and embryonic developmental rates could be achieved in oocytes with than without bi-refringent spindles. Because spindles in most mammalian oocytes are extremely sensitive to slight changes in temperature, maintenance of temperature at 37 degrees C is crucial for normal spindle function. As chromosomes#10; are usually associated with microtubule fibers in the spindles, the position of chromosomes could be indirectly located by imaging spindles. Removing spindles under the Polscope can achieve an enucleation#10; efficiency rate of 100% in mouse oocytes. The Polscope can also be used to examine the spindle dynamics, detect spindle morphology, predict chromosome misalignment, and perform spindle transfer.

Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology.

Developmental Ability of Human Eggs with or Without Birefringent Spindles Imaged by Polscope Before Insemination

BACKGROUND Birefringent spindles imaged with the Polscope can predict fertilization rates after intracytoplasmic sperm injection (ICSI). The present study examined the development of human oocytes with or without birefringent spindles, imaged with the Polscope before ICSI. METHODS Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocyte retrieval for ICSI. Spindles were imaged with the Polscope combined with a computerized image analysis system. After imaging and ICSI, oocytes with or without spindles were cultured separately for examination of fertilization and embryo development. A total of 1544 oocytes from 136 cycles were examined with the Polscope and inseminated by ICSI. RESULTS Spindles were imaged in 82% of oocytes. After ICSI, more oocytes (P < 0.05) with spindles (69.4%) fertilized normally, forming 2 pronuclei, than oocytes without spindles (62.9%). At day 3, more oocytes (P < 0.01) with spindles (66.3%) developed to 4-11 cell stages than oocytes without spindles (55.4%). Significantly more (P < 0.001) oocytes with spindles developed to morula and blastocyst by day 5 (51.1 versus 30.3%) and day 6 (53.2 versus 29.3%) compared with oocytes without spindles. CONCLUSIONS The results indicate that the presence of a birefringent spindle in human oocytes can predict not only higher fertilization rate, but also higher embryo developmental competence.