WO2002000013A2 - Imagerie de fuseau meiotique dans des ovocytes et utilisations associees dans la fertilisation in vitro - Google Patents

Imagerie de fuseau meiotique dans des ovocytes et utilisations associees dans la fertilisation in vitro Download PDF

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WO2002000013A2
WO2002000013A2 PCT/US2001/020259 US0120259W WO0200013A2 WO 2002000013 A2 WO2002000013 A2 WO 2002000013A2 US 0120259 W US0120259 W US 0120259W WO 0200013 A2 WO0200013 A2 WO 0200013A2
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oocyte
retardance
spindle
image
oocytes
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PCT/US2001/020259
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WO2002000013A3 (fr
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David L. Keefe
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Women And Infants Hospital Of Rhode Island
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/70—Determining position or orientation of objects or cameras
    • G06T7/73—Determining position or orientation of objects or cameras using feature-based methods
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/695—Preprocessing, e.g. image segmentation
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro

Definitions

  • This invention relates to in vitro fertilization (IVF) and to cloning of animals, and to methods for improving the efficiency of IVF and cloning as well as increasing fertilization rates in IVF
  • Reproductive wastage is a universal characteristic of biology, with all forms of life devoting enormous energies toward production of germ cells far in excess of the number that eventually develop into a new adult capable of repeating the life cycle.
  • Ovaries of mammals, including women and domestic animal species contain hundreds of thousands of germ cells at birth, the majority of which never are ovulated, being lost by atretic processes at various stages of follicle development, before puberty or in adult life.
  • blastocyst transfer has less clear value.
  • the majority of embryos do not survive five days in culture, and the embryos which do survive may not exhibit superior implantation rates compared to embryos transferred on the conventional third day.
  • even cases of blastocyst transfer present the dilemma of which blastocysts to transfer.
  • oocyte serves as the "stem cell" for the embryo, and because more than 80% of aneuploidies that appear in preimplantation embryos originate in the oocyte spindle structure, the evaluation of oocyte structure and determination of fertilization and developmental potential is important, and examination of an important structure in oocytes, the meiotic spindle, is key.
  • oocyte quality has been difficult in humans. Attempts to estimate oocyte development potential demonstrate a number of morphologic features associated with poor developmental potential, such as darkness, granularity, vacuoles, fragmentation and irregularity (Bolton, 1989, Weimer, 1993, Riley, 1991, Fleming, 1982), but in fact, such standard imaging techniques do not provide a sensitive method of diagnosing oocyte dysfunction. Moreover, the pathobiological basis of these morphological markers is unclear.
  • IVF offers the opportunity to study the role of the meiotic spindle in human oocyte developmental potential, because oocytes are ovulated at the Mil stage of development, when the chromosomes are poised on the metaphase plate, tethered by microtubules that are inherently unstable, and relative to other structures in the oocyte, highly birefringent .
  • the MI spindle also is available for analysis.
  • the imaging methods currently used in the IVE laboratory e.g., Hoffman, Nomarski or bright field microscopy, cannot image clearly the meiobic spindle.
  • Birefringence is an optical property that derives from the molecular order found in such macromolecules as membranes, microtubules, microfilaments, and other cytoskeletal components.
  • Polarized light microscopy has the unique potential to visualize and measure birefringent structures, such as spindles dynamically and non-destructively in living cells.
  • the low sensitivity of conventional polarized light microscopes makes them marginally suitable for application to mammalian experimental and clinical embryology. (Oldenbourg. 1995, Oldenbourg. 1996. Oldenbourg 1997) .
  • birefringence is measured as retardance.
  • the invention is based, at least in part, on the discovery that meitotic spindles in living mammalian oocytes, in particular living human oocytes, can be imaged non-invasively and without damage .
  • imaging of oocytes is achieved using an orientation-independent polarized light microscope referred to herein as a "polscope".
  • the polscope uses digital image processing to improve sensitivity, and nearly circularly polarized light combined with electro optical hardware to achieve orientation independence.
  • CCD technology, liquid-crystal compensator optics, and computer algorithms are used to quantify birefringence magnitude (called retardance) and orientation (called azimuth) at every image point in the field of view.
  • the polscope' s orientation-independence enables quantification of retardance magnitude and azimuth of spindle fibers within microtubules, because differences in these parameters result from the tissue itself rather than settings of the compensators and stages .
  • the polscope is described in, e . g. , U.S. Patent 5,521,705, which is incorporated herein by reference.
  • a method of measuring retardance using this polscope is disclosed in U.S. application serial no. (attorney's docket no. 34250-36) filed on June 18, 2001, also incorporated herein by reference.
  • the mammalian egg is arrested at metaphase II of the meiotic cell cycle, when the chromosomes are tethered by microtubule fibers of the mej otic spindle.
  • meiosis and fertilization the meiotic spindles are responsible for proper separation of the nuclear material, and abnormalities in this fragile structure can lead to infertility, miscarriage. and genetic diseases, such as Downs Syndrome .
  • Imaging the meiotic spindle in the unfertilized human oocyte offers a unique opportunity to assess the oocyte' s development potential, because up to 80% of aneuploidies found in embryos have their origin in the oocyte and early evidence suggests that the physical state of the spindle reflects its function. Moreover, increased maternal age, the single most important predictor of female fertility, is associated with disruption of the spindle architecture (Battagalia et al. , 1996) . Conventional methods of imaging the spindle
  • the first aim is to examine whether spindle birefringence can predict oocyte developmental ability. This will be accomplished by capturing polscope images of human Mil oocytes from infertile patients undergoing ICSI treatment, and polscope images of MI and II spindles from infertile patients undergoing in vitro maturation (IVM) and ICSI for clinical indications. After imaging, oocytes will be inseminated by ICSI and zygotes from oocytes with or without birefringent spindles will be cultured separately until embryo transfer. Development (fertilization, embryo development, implantation and pregnancy rates) between oocytes with or without spindles will be compared.
  • the second aim is to examine whether birefringence of MI and Mil spindles predicts aneuploidy in IVM human oocytes. Birefringence of MI and Mil spindles will be imaged with the polscope during IVM of human oocytes. The presence, shape and maximum retardance of spindles will be measured by the polscope and analyzed by image analysis. After imaging with the polscope, first polar body biopsy and multiprobe fluorescence in situ hybridization (FISH) will be performed on the first polar bodies, to examine the relationship between spindle structure and birefringence, and aneuploidy.
  • FISH multiprobe fluorescence in situ hybridization
  • the third aim is to examine whether spindles can be used to optimize the efficiency of IVM conditions, and therefore establish a more effective IVM technique for human IVF.
  • the invention is directed to determining the presence, position, and morphological characteristics of meiotic spindles in living mammalian oocytes, especially human oocytes. by detecting and measuring, non-invasively, and without detriment to the oocytes, the birefringence of the meiotic spindles. Accordingly, in one embodiment, the invention is a method for imaging the meiotic spindle in a living oocyte by detecting birefringence of the meitotic spindle.
  • the invention is a method for evaluating developmental potential (likelihood of fertilization, embryo development, implantation and pregnancy rates) of oocytes by detecting and quantifying the birefringence of the meiotic spindles in the oocytes to determine the presence, position and/or morphology of the spindles.
  • the invention is a method for increasing the fertility rates of oocytes in IVF procedures by detecting the presence and position of the meiotic spindle in oocytes.
  • the invention is a method of predicting oocyte aneuploidy.
  • the invention is a method for optimizing IVF conditions, for example temperature conditions for handling of oocytes during IVF, culture and implantation, to increase efficiency of IVF and the likelihood of fertilization, implantation and embryonic development.
  • the invention is a non-invasive method for enucleation of mammalian oocytes.
  • FIGS. 1A and IB are DIC images of human eggs
  • FIGS. 1A and IB' are polscope images of human eggs
  • FIGS. 2A-2C are polscope images demonstrating meiotic spindle separated from the polar body in living human eggs;
  • FIGS. 3A and 3B are polscope images of hamster eggs
  • FIGS. 4A-4C shown sequence of aged eggs on post retrieval days 1, 2 and 3;
  • FIGS. 5A and 5B show spindle images during metaphase I and metaphase II after in vitro fertilization;
  • FIGS. 6A and 6B show retardance vs. position in the spindle of a human metaphase II oocyte, along lines A and B.
  • the polscope uses digital image processing to improve its sensitivity, and nearly circularly polarized light combined with electro optical hardware to achieve orientation independence.
  • CCD technology, liquid-crystal compensator optics, and computer algorithms are used to quantify birefringence magnitude (called retardance) and orientation (called azimuth) at every image point in the field of view.
  • the polscop ' e's orientation-independence enables quantification of retardance magnitude and azimuth of spindle fibers within microtubules. because differences in these parameters results from the tissue itself rather than settings of the compensators and stages.
  • the polscope has been described previously, however, since its application to mammalian embryology is still in its infancy it will be reviewed briefly here .
  • the polscope To produce a retardance image, the polscope generates four intensity images (which are in perfect register because there are no moving parts) at ,, four liquid crystal compensator settings. This gives four numbers at each pixel of the 480 x 640 pixel image. These four values are used in a ratiometric calculation to determine the sample's retardance and azimuth at each pixel. An additional four images, without the sample in the optical field, are taken to serve as a background correction. Quantification of the specimen's retardance then can be carried out by grayscale thresholding. With this strategy, the polscope can measure retardances of as little as 0.05 nm. or 0.03 degrees of phase change (which corresponds to 10 ⁇ 4 wavelengths of light) .
  • the polscope has been adapted for use in mammalian embryology.
  • the optical path of the polscope for imaging with an inverted microscope was optimized.
  • a number ot unique features of oocyte morphology were observed, including a laminar structure of the zona pellucida, as well as the meiotic spindle
  • the polscope' s noninvasive feature allows us to examine the same spindle over time to be able to study the kinetics of spindle polymerization and depolymerizatio .
  • Results As little as a 4°C drop in temperature completely depolymerizes the Mil spindle of hamster oocytes within six minutes. While transient exposure to cool temperatures allowed repolymerization upon return to physiological temperatures, prolonged (>6 minutes) exposure to 33 °C did not allow repolymerization. These findings have lead to changes in how eggs are handled in IVF laboratories to ensure that the egg temperature is maintained constantly at 37°C. Discussion: This finding suggests that the spindle can be used as an internal record of the thermal history of the egg.
  • Meiotic spindles are formed from microtubules comprised of polymerized tubulin and are extraordinarly temperature-sensitive.
  • an orientation-independent polarized light microscope (Polscope, CRI, Cambridge, Mass.) was used to image spindles non-invasively during human ICSI, a high proportion of oocytes exhibited abnormal or missing spindles and oocytes with abnormal spindles had decreased fertilization rates (Abstract #1, ASRM Annual Meeting, Oct.1999).
  • a thermistor to was used to measure temperature near the oocyte and thermal control with a conventional heating stage and a novel heating stage were compared.
  • the Delta T Bioptics, Butler, PA uses an indium tin oxide-coated coverslip and a thermostat to precisely regulate the temperature at the oocyte.
  • Egg temperature was also compared using the Delta T Bioptics unit with and without heating the 40 X objective with the Bioptics objective lens warmer.
  • the polar body does not predict the location of the meiotic spindle
  • the zona pellucida has a multilaminar structure Rationale: Since the zona pellucida is comprised of three related, filamentous glycoproteins (ZP 1. ZP2, ZP3) it was hypothesized that it would exhibit a high level of birefringence. The impairment of hatching identified in embryos after in vitro culture also begged the question of whether the artificial conditions associated with IVF might change the biophysical properties of zona proteins and thus alter the retardance and/or azimuth. Results: When imaged under Hoffman or Nomarski optics, the zona pellucida appears as a uniform, thick layer. However, when imaged with the polscope, the zona exhibits a multilaminar structure, with three layers differing in their degree of retardance and orientation (See Figures 3).
  • the Polscope does not disrupt pre-implantation mouse embryo development
  • oocytes without a birefringent spindle and 25% of oocytes with a birefringent spindle were abnormal and chromosomes were not configured normally. Discussion:
  • the polscope images of spindles are nearly identical to images generated confocal microscope.
  • the polseope provides nearly the same information about spindle structure as that by confocal microscope, but without damage of the oocytes.
  • Spindle microtubules can be quali tied by retardance measurements .
  • IVM may be of value to avoid ovarian hyperstimulation in gonadotropin-sensitive patients.
  • Specific Aim 1 Determine whether spindle birefringence, measured with the polscope, can predict subsequent preimplantation embryo development and pregnancy rates.
  • oocytes exhibiting spindle birefringence will be graded morpho et rically to determine whether the morphometric features of the spindles, characterized by Battaglia et al . in his study of immunofluorescently- labeled spindles, predict fertilization and embryo developmental potential.
  • shape a polygon, such as shown in Figure 6 aligned with the spindle poles. Measurement will consist of calculating the area between the standard shape perimeter and the perimeter of the spindle, as Figure 6.
  • ICSI then will be performed, and the resulting zygotes cultured in groups, based on the normalcy of their spindles, to day 3. 5, 6, depending on standard clinical protocols.
  • the rates of development in vitro and of implantation, and delivery of term babies after embryo transfer will be compared among groups .
  • oocytes with abnormal or absent spindles may be predisposed to develop aneuploidy. Therefore, we hypothesize that oocytes without spindles or with structurally abnormal spindles will be more likely to develop aneuploidy than oocytes with structurally normal spindles .
  • Ploidy will be determined by polar body biopsy and six probe fluorescent in situ hybridiza ion (FISH) (chromosomes 22, 21, 18, 16, and 13) .
  • FISH fluorescent in situ hybridiza ion
  • polar body biopsy and FISH have been used widely for preimplantation genetic diagnosis in human IVF clinics . Since polar bodies contain the complementary genotype to the oocyte. if the polar body contains abnormal chromosome distribution, it can be concluded that the oocvtes also contain abnormal chromosome distribution. Polar bodies are considered an unnecessary by-product of eiosis, so their biopsy does not interfere with the developing embryo itself.
  • Polar Body Biopsy Only first polar bodies will be biopsied, so as to not breech NIH' s embryo guidelines prohibiting invasive research on fertilized oocytes or embryos.
  • oocytes are held on a pipette with the polar body at the 12 o'clock position. Using a sharp needle, a slit is made in the zona pellucida tangentially to the polar body.
  • FISH Analysis - FISH is a method in which fluorescently labeled chromosome-specific probes are hybridized to metaphase or interphase chromosomes for aneuploidy. The structure of the spindle and it birefringence will be used to classify oocytes into normal and abnormal groups, and the rates of aneuploidy will be compared between groups.
  • N-FISH Nuclear fluorescent In situ Suppression Hybridization
  • a multicolor, five probe mixture for detecting chromosomes 13,21,18.16 and 22 (multivision PB multicolor probe panel, Vysis, downers Groove, 11 is applied to the slide under a coverslip ( region where the polar body if attached to the slide) and the probe and the nuclear DNA are denatured simultaneously at 73 degrees in hybrite (Vysis) for two minutes. After denaturation, the cover slip is sealed with rubber cement and incubated at 37 degrees in a humid chamber for approximately 12 to 16 hours (hybridization) . After hybridization, the rubber cement and covers] ip a e carefully removed and the slides are washed m 2X SSC for 10 minutes at 76 degrees.
  • oocytes are collected from gonadotropin-stimula ted women.
  • protocols for controlled ovarian hypersti ulation for IVF have been used in human IVF clinics for many years, some patients do not tolerate the controlled ovarian hyperstimulation used for IVF.
  • PCOS polycystic ovary syndrome
  • OHSS severe ovarian hyperstimulation
  • Imma ture oocytes will be obtained from young and old PCOS and other gonadotropin-sensitive patients who have consented to IVM in order to avoid the controlled ovarian stimulation component used during conventional IVF and from oocytes retrieved at CV stage despite gonadotropin stimulation.
  • follicles will be synchronized with oral contraceptives, then hCG, according to the protocol published by the McGill group (Chian et al . , 1999) .
  • Oocytes will be aspirated under low (50- 75 mm Hg) pressure and cultured in Tissue Culture Medium (TCM) 199 (Sigma) containing 75
  • TCM Tissue Culture Medium
  • ICSI of in vi tro ma tured oocytes Oocytes that reach metaphase II (release the first polar body) will be inseminated by ICSI.
  • the ICSI protocol has been used in human IVF clinics for several years world-wide and it is a standard ART technique in our IVF clinic. During the two past years ICSI was performed on just over 40% of the IVF cycles at our center and a 70-80% fertilization has been achieved. Embryo culture, cryopreservation and transfer will follow procedures standard to our IVF laboratory.
  • Embryo culture and track ing After ICSI, oocytes with or without normal spindles will be cultured separately in groups of two to three, and fertilization will be examined 16-18 h later. Normally fertilized eggs, with two pronuclei (zygotes) will be cultured in growth medium (P 1 medium with 10% SSS) . At Day 3, embryos will be graded and selected for transfer or continued in culture (in blastocyst medium) , depending on the numbers of developing embryos, following standard clinical protocols. We find grea er than 90% of zygotes cleave and 50% go on to form blastocysts in ICSI patients. Embryo transfer has resulted in an average 30% clinical pregnant rates, with variation about this overall average, depending on age.
  • Immature oocytes from the same patient will be separately cultured in TCM 199 with maternal serum (10%) or without serum. After maturation, oocytes will be examined by polscope and their spindle structure analyzed and compared to normal controls .
  • EGF epidermal growth factor
  • Immature oocytes from the same patients will be cultured in TCM 199 with or without 10 ng/ l EGF (a concentration has been found to stimulate oocyte maturation in mammals tested). After maturation, oocytes will be examined with the polscope and their spindles examined and compared to normal controls .
  • cysteine has been supplemented but the concentration is too low to provide enough cysteine for synthesis of intracellular glutathione (Yoshida et al . , 1993).
  • the addition of cysteine in pig and cattle IVM medium significantly increases the efficiency of IVM.
  • Immature oocytes from the sante patient will be cultured in medium with or without 0.6 mM cysteine (a concentration used in culture medium in other animals) . After maturation, oocytes will be examined with the polscope and their spindle structure compared to normal cAritrols . Experiment 5. Ma tura tion of cumulus-free immature oocytes for rescue insemina tion
  • cysteamine which also can be used to synthesize intracellular glutathione in oocytes without cumulus cells, stimulates cytoplasmic maturation in pig and cattle oocytes Yamauchi and Nagai, 1999; De Matos et al . , 1995) .
  • supplementation of cysteamine in medium will stimulate intracellular glutathione synthesis and maturation.
  • Cumulus cells will be collected and centrifuged to remove associated hyaluronidase in culture medium. Only cumulus cells from the same patient will be used for co-culture. Next, immature oocytes will be cultured in medium with or without cysteamine.
  • the spindles in matured oocytes will be examined with the polscope and inseminated by ICSI. Fertilization and embryo development will be compared between two groups . The viable embryos will be transferred to the patients or frozen. Immature oocytes from all ICSI patients

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Abstract

Selon l'invention, on évalue le potentiel de développement d'un ovocyte mammifère, aux fins d'une implantation réussie, en produisant plusieurs images d'intensité de cet ovocyte, au moyen de surfaces optiques à lumière polarisée, et en calculant une image de retard comprenant une sensibilité de l'ordre de 3 nm ou moins. La présence, l'emplacement et les caractéristiques morphologiques du fuseau méiotique de l'ovocyte peuvent être alors déterminées en fonction de la structure du fuseau dans l'image de retard, et le potentiel de développement de l'ovocyte peut être évalué.
PCT/US2001/020259 2000-06-23 2001-06-25 Imagerie de fuseau meiotique dans des ovocytes et utilisations associees dans la fertilisation in vitro WO2002000013A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1783204A2 (fr) * 2006-04-04 2007-05-09 Cambridge Research & Instrumentation, Inc. Manipulation et imagerie d'un échantillon biologique
WO2007115297A2 (fr) * 2006-04-04 2007-10-11 Cambridge Research And Instrumentation, Inc. manipulation et imagerie d'échantillons biologiques
CN101974627A (zh) * 2010-10-13 2011-02-16 大连海洋大学 鱼类卵巢生殖细胞染色体的制备方法
CN110476839A (zh) * 2019-07-24 2019-11-22 中国农业大学 一种基于鱼类生长的优化调控方法及系统

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6331659B1 (en) * 1998-01-21 2001-12-18 University Of Hawaii Cumulus cells as nuclear donors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6331659B1 (en) * 1998-01-21 2001-12-18 University Of Hawaii Cumulus cells as nuclear donors

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1783204A2 (fr) * 2006-04-04 2007-05-09 Cambridge Research & Instrumentation, Inc. Manipulation et imagerie d'un échantillon biologique
WO2007115297A2 (fr) * 2006-04-04 2007-10-11 Cambridge Research And Instrumentation, Inc. manipulation et imagerie d'échantillons biologiques
EP1783204A3 (fr) * 2006-04-04 2007-12-26 Cambridge Research & Instrumentation, Inc. Manipulation et imagerie d'un échantillon biologique
WO2007115297A3 (fr) * 2006-04-04 2007-12-27 Cambridge Res And Instrumentat manipulation et imagerie d'échantillons biologiques
CN101974627A (zh) * 2010-10-13 2011-02-16 大连海洋大学 鱼类卵巢生殖细胞染色体的制备方法
CN110476839A (zh) * 2019-07-24 2019-11-22 中国农业大学 一种基于鱼类生长的优化调控方法及系统

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