U.S. patent application number 14/026799 was filed with the patent office on 2017-04-13 for motility-contrast imaging for oocyte and embryo viability assessment.
This patent application is currently assigned to Purdue Research Foundation. The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Ran An, Zoltan Machaty, David D. Nolte, John J. Turek.
Application Number | 20170102376 14/026799 |
Document ID | / |
Family ID | 52668271 |
Filed Date | 2017-04-13 |
United States Patent
Application |
20170102376 |
Kind Code |
A9 |
An; Ran ; et al. |
April 13, 2017 |
Motility-Contrast Imaging for Oocyte and Embryo Viability
Assessment
Abstract
A method and system is provided for evaluating viability of
oocytes and embryos comprising imaging an oocyte or embryo using
motility contrast imaging; generating temporal contrast and spatial
contrast data for the cells; generating a cell viability value as a
function of the temporal and spatial contrast data; and comparing
the cell viability value to a predetermined value indicative of a
cell suitable for use in an in vitro fertilization program.
Inventors: |
An; Ran; (West Lafayette,
IN) ; Nolte; David D.; (Lafayette, IN) ;
Machaty; Zoltan; (West Lafayette, IN) ; Turek; John
J.; (West Lafayette, IN) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
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Assignee: |
Purdue Research Foundation
West Lafayette
IN
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20150079621 A1 |
March 19, 2015 |
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Family ID: |
52668271 |
Appl. No.: |
14/026799 |
Filed: |
September 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61700378 |
Sep 13, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5091 20130101;
G01N 2015/1006 20130101; G01N 15/1463 20130101; G01N 15/1475
20130101; G01N 33/689 20130101; G01N 33/5044 20130101; G01N
2015/1454 20130101; G01N 15/1434 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 15/14 20060101 G01N015/14 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The invention was made with government support under Grant
CBET-0756005 awarded by the National Science Foundation. The U.S.
Government has certain rights in the invention.
Claims
1. A method for evaluating viability of oocytes and embryos
comprising: reversibly immobilizing reproductive cells; detecting
intracellular motion within the immobilized cells; generating a
measure of dynamic activity of the cells; and comparing the measure
of dynamic activity to a threshold indicative of cell
viability.
2. The method for evaluating viability of oocytes and embryos of
claim 1, wherein the step of detecting intracellular motion is
performed using motility contrast imaging.
3. The method for evaluating viability of oocytes and embryos of
claim 1, wherein the step of generating a measure of dynamic
activity includes determining frequency content of speckle imaging
of the cell.
4. The method for evaluating viability of oocytes and embryos of
claim 3, wherein the step of generating a measure of dynamic
activity further includes determining the temporal variation of the
frequency content of speckle imaging of the cell.
5. The method for evaluating viability of oocytes and embryos of
claim 3, wherein the step of generating a measure of dynamic
activity further includes determining the spatial variation of the
frequency content of speckle imaging of the cell.
6. The method for evaluating viability of oocytes and embryos of
claim 5, wherein: the step of generating a measure of dynamic
activity includes; determining the temporal variation of the
frequency content of speckle imaging of the cell; and deriving a
viability figure in relation to the temporal and spatial variation
of the frequency content; and the step of comparing the measure of
dynamic activity to a threshold indicative of cell viability
includes comparing the viability figure to the threshold.
7. The method for evaluating viability of oocytes and embryos of
claim 2, wherein: the motility contrast imaging generates a
two-dimensional motility map at a fixed depth within the cell and a
volumetric motility map to construct a holographic motility image
of the cell; and the step of generating a measure of dynamic
activity includes determining the temporal variation of the
frequency content of speckle imaging of the cell in which the
temporal contrast is determined as a normalized standard deviation
of the temporal variation of the intensity of all pixels of the
holographic image at a predetermined depth within the image.
8. The method for evaluating viability of oocytes and embryos of
claim 1 further comprising changing a condition of the cell and
generating a measure of the change of the dynamic activity of the
cell during the changing condition.
9. The method for evaluating viability of oocytes and embryos of
claim 8, wherein the changing condition includes one or more of
introducing follicle stimulating hormone to the cell and changing
the temperature of the cell.
10. The method for evaluating viability of oocytes and embryos of
claim 1, wherein the step of detecting intracellular motion is
performed using tissue dynamics spectroscopy to measure low, mid
and high frequency spectral content changes over time and/or to
measure the dynamic fluctuation spectrum of selected groups of
spatially located pixels.
11. The method for evaluating viability of oocytes and embryos of
claim 1, wherein the step of detecting intracellular motion is
performed by the combined use of motility contrast imaging to
measure the temporal and spatial variation of the frequency content
of speckle imaging of the cell, tissue dynamics spectroscopy to
measure frequency spectral content changes over time, and tissue
dynamics spectroscopy to measure low, mid and high frequency
spectral content changes over time.
12. The method for evaluating viability of oocytes and embryos of
claim 11, further comprising generating a spectrogram fingerprint
of the cell complex averaged over predetermined portions of the
volume of the cell complex.
13. The method for evaluating viability of oocytes and embryos of
claim 12, wherein the step of comparing the measure of dynamic
activity to a threshold includes comparing the spectrogram
fingerprint of the cell to a reference library of fingerprints
indicative of grades of viability.
14. A method for evaluating viability of oocytes and embryos
comprising: reversibly immobilizing an oocyte or embryo in a sample
plate; placing the sample plate in a fixed mount; moving a
holographic detection system with negligible vibration across the
sample plate to generate a measure of dynamic activity of the
cells; and comparing the measure of dynamic activity to a threshold
indicative of cell viability.
15. The method for evaluating viability of oocytes and embryos of
claim 14, wherein the holographic detection system is a motility
contrast imaging system.
16. A method for evaluating the potential for success of oocyte
fertilization comprising: reversibly immobilizing a fertilized
oocyte; detecting intracellular motion within the immobilized
oocyte; generating a measure of dynamic activity of the oocyte; and
comparing the measure of dynamic activity to a threshold indicative
of cell viability indicative of a suitable potential for success of
fertilization.
17. The method of claim 16 in which fertilization success is based
on a number of metrics that relate to the dynamic activity of the
oocyte comprising, but not limited to, backscatter brightness,
spatial contrast, slope of spectrum, knee frequency, power
intensity at low frequency, ellipticity and temporal contrast.
18. The method of claim 17 in which the metrics are combined in a
principal component analysis.
19. The method of claim 18 in which the principal component
analysis is performed on the oocyte cell sample of interest and
compared with a set of reference samples to aid evaluation of the
viability of the sample.
20. The method of claim 16 in which the duration and magnitude of
oocyte activity prior to cleavage is compared with values from
reference samples of known viability to evaluate the reproductive
potential of the sample.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a utility conversion of and
claims priority to co-pending provisional application No.
61/700,378, filed on Sep. 13, 2012, the entire disclosure of which
is incorporated herein by reference.
FIELD
[0003] The present disclosure relates generally to the assessment
of oocytes and embryos for use in in vitro fertilization programs,
and particularly to systems and methods for using motility contrast
imaging techniques to assess cell viability.
BACKGROUND
[0004] Since the first successful pregnancy following in vitro
fertilization (IVF) was achieved in 1978, improvement in the
techniques of assisted reproductive technologies has led to an
increased success rate in the field of treating human infertility.
Success in IVF programs is typically dependent upon the selection
of the best embryo chosen for transfer to the uterus. Because of
uncertainty in the functional viability of oocytes or early
embryos, clinicians frequently transferred many embryos
simultaneously. While improving the rate of pregnancy after IVF,
this approach has also led to an increase in the rates of multiple
pregnancies. The dangers of multiple pregnancies for both the
mother and the neonates are well documented, and twin birth is now
considered an undesirable outcome of IVF.
[0005] In the early years of IVF, embryo viability was considered
to be a function of developmental progression during the
pre-implantation phase. Over the last decade, the focus has been on
the evaluation of certain morphological criteria of the oocyte or
embryonic cells. The current approach to determining oocyte/embryo
viability involves an indirect and subjective scoring system based
on morphologically observable traits under a microscope [1, 2].
Scoring oocytes/embryos requires a highly skilled technician that
takes the average clinical embryologist at least three months to
learn. Although this approach has had some predictive value, it has
been frequently criticized as imperfect and unreliable, generally
thought to yield only a 23% average success rate. In many cases a
selected oocyte or embryo may have normal morphology, as observed
by the embryologist, but possess undetected alterations in their
biochemistry that affect viability. For instance, oocytes invested
with cumulus granulosa cells, called cumulus-oocyte complexes
(COC), are not readily visible under a microscope because they are
obscured underneath the cumulus cells, making viability grading
difficult. The question of viability becomes more pronounced for
cryopreserved cells once thawed. Furthermore, after fertilization,
the combined sperm and egg form the zygote (a single cell embryo)
in which dramatic functional changes take place that are not
readily visible under conventional microscopes. One such current
microscope relies on Hoffman modulation contrast that is used to
visualize oocytes and zygotes.
[0006] However, currently there are no imaging modalities that
capture the functional viability of oocytes, or early embryos prior
to implantation inside the uterus.
SUMMARY
[0007] The present disclosure contemplates a system and a method is
provided for evaluating intracellular activity as a measure of
oocyte/embryo viability. In one aspect, the intracellular activity
is measured using motility contrast imaging. In certain examples
described herein, motility contrast imaging (MCI) is used prior to
and following stimulated maturation of a live oocyte in vitro to
provide a new type of functional biomarker for candidate oocyte
evaluation. In another aspect, the intracellular activity is
measured using biodynamic spectroscopy. In certain examples
described herein, biodynamic spectroscopy is used during embryo
development through first cleavage. The systems and methods
described herein can be used to evaluate viability of reproductive
cells, including oocytes and embryos.
[0008] In one embodiment, the present invention provides a method
for evaluating viability of oocytes and embryos comprising imaging
using motility contrast imaging; generating temporal contrast and
spatial contrast data for the oocyte or embryo; generating a cell
viability value as a function of the temporal and spatial contrast
data; and comparing the cell viability value to a predetermined
value indicative of cells suitable for use in an in vitro
fertilization program.
DESCRIPTION OF THE FIGURES
[0009] FIG. 1 is a diagram of a floating-plate digital holography
system disclosed herein that protects weakly immobilized oocytes
and embryos from stresses induced by motion of the sample
wells.
[0010] FIG. 2 is a diagram of the motility contrast imaging system
for determining oocyte and embryo viability.
[0011] FIG. 3 is a series of motility maps of cumulus-oocyte
complexes examined using the system and methods disclosed
herein.
[0012] FIG. 4 is a histogram of the shell motility metric compared
to a histogram of the core motility metric.
[0013] FIG. 5 is a graph correlating the core motility metric with
the shell motility metric for many COCs including the COCs shown in
FIG. 2.
[0014] FIG. 6 is graph correlating the temporal and spatial
contrasts for the oocytes shown in
[0015] FIG. 7 shows histograms of a viability metric for matured
and immature COCs according to the systems and methods disclosed
herein.
[0016] FIG. 8 is graph correlating the motility metric (NSD) of
mature oocytes to the spatial contrast of the same oocytes.
[0017] FIG. 9 is a motility map of an immature granulosa-encased
oocyte as a function of temperature decreasing over time.
[0018] FIG. 10 is a spectral density graph comparing an active
oocyte to the cumulus shell.
[0019] FIG. 11 is a spectral density graph comparing a fertilized
oocyte to an unfertilized oocyte.
[0020] FIG. 12 is a principal-component analysis plot of oocytes in
fertilized and unfertilized groups.
[0021] FIG. 13 is a spectrogram of a zygote that undergoes
mitosis.
[0022] FIG. 14 is a spectral density graph of the zygote before,
during and after mitosis.
DETAILED DESCRIPTION
[0023] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments illustrated in the drawings and described in the
following written specification. It is understood that no
limitation to the scope of the invention is thereby intended. It is
further understood that the present invention includes any
alterations and modifications to the illustrated embodiments and
includes further applications of the principles of the invention as
would normally occur to one skilled in the art to which this
invention pertains.
Biodynamic Imaging
[0024] Biodynamic imaging (BDI) is the general term used to denote
a range of related approaches that extract motion information from
inside living tissue. BDI includes optical coherence imaging (OCI)
[3-5], motility contrast imaging (MCI) [6-8], and tissue dynamics
spectroscopy (TDS) [9-11]. All of these approaches have been
applied to macroscopic tissues containing up to a million cells and
down to about 50,000 cells, but never on a single cell like an
oocyte because the techniques require high light scattering to
provide detectable signal.
[0025] One of these approaches, named motility contrast imaging
(MCI), uses subcellular motion as a form of endogenous image
contrast [12]. MCI can be well suited for the evaluation of oocyte
and embryo quality because MCI non-invasively captures the
functional dynamics of the cells and tissues without exogenous
dyes. Motility Contrast Imaging is based on Optical Coherence
Imaging (OCI) which uses low-coherence Fourier-domain digital
holography [4, 5]. OCI collects and distinguishes back scattered
light deep from living tissue up to about 1 mm in thickness
[13].
[0026] A protocol for oocyte/embryo immobilization is an important
element of the disclosed system and method that allows oocytes and
embryos that are graded as "good" by MCI to be ultimately implanted
in utero. All previous applications of MCI to living tissue have
used irreversible immobilization of the sample. Because MCI is
motion-sensitive, the sample must be immobilized in the sample well
or Petri dish so that only intracellular motions are detected, and
not the gross motion of the sample.
[0027] In one approach for oocyte and embryo immobilization, a 2%
solution of gelatin suitable for cell culture is diluted 1:1 with
phosphate buffered oocyte culture medium and 300 .mu.l of the
solution added to each well of an 8-well Lab-Tek 1.times.3 glass
slide. The culture slide is placed covered on a warming tray and
held at 37-39.degree. C. for 3-4 hours. After incubation the
culture slide cover is removed and the medium is discarded. The
culture slide is placed back on the warming tray without the cover
inside a laminar flow hood with an ultraviolet light. The slide is
ready for use after 1-2 hours of drying. Oocytes in phosphate
buffered culture medium are placed in the 8 well plates and
incubated in a CO.sub.2 incubator at an appropriate physiological
temperature (37.degree. C. for humans, 39.degree. C. for pigs).
Oocytes adhere gently to the underlying surface after several hours
in the incubator and are then ready for fertilization. Oocytes
selected for fertilization based upon MCI viability scores are
maintained in the phosphate buffered medium until after
fertilization and the medium may be replaced with a HEPES buffered
medium for observation of the fertilized oocyte outside of the
incubator on a warming stage of the MCI instrument. The fertilized
oocytes with the best MCI viability scores are removed for
implantation by gentle pipetting the culture medium up and down
several times. The fertilized oocytes with the best MCI viability
metrics are now ready for implantation.
[0028] An important aspect of the protocol of the present
disclosure is the tunability of the immobilization strength. By
adjusting the percent gelatin and percent dilution by medium, the
"stickiness" of the underlying layer can be adjusted to release the
living samples more easily.
[0029] An embodiment of the present system and method strikes a
compromise between immobilization strength and perturbation of the
sample by system movements. Currently, the immobilized samples in
the stage are moved and the optics remain stationary. The moving
stage causes inertial forces on the samples that can make them
move. In this embodiment, the weakest immobilization that is
compatible with a stationary stage is used, and the detection
optics is moved rather than the sample stage. The use of the
weakest immobilization ensures that the reproductive samples are
removed after MCI assessment without any unnecessary stress.
[0030] The implementation of moving optics, instead of a moving
stage, requires special attention because of the use of digital
holography. Digital holography is highly sensitive to vibrations
and is performed on stationary plates or tables. To implement the
moving optics under digital holography, one embodiment contemplates
"floating plate" digital holography upon which the fixed-steering
optics 3 and digital holography optics 4 reside, as shown in the
exemplary system depicted in FIG. 1. A "floating plate" 2 is
mounted on low-friction sliders 5 that are moved with
computer-controlled actuators 1. The light source can be from a
fiber optic feed 7 that is free to move as the floating plate
moves. The floating plate digital holography disclosed herein
solves the problem of keeping the sample stationary, while scanning
from well-to-well, and while ultra-stable holography is
performed.
[0031] The optical elements of the holographic system of FIG. 1 is
shown in FIG. 2. In one experimental setup the system is based on a
Mach-Zehnder interferometer. Short coherence light is generated by
a superluminescent diode 10 having a center wavelength of
approximately 840 nm and a bandwidth of 50 nm in a specific
embodiment. The light is collimated by lenses L1, L2 and then
divided into two paths by a polarizing beam splitter to produce an
object beam with vertical polarization and a reference beam with
horizontal polarization. The object beam strikes the sample 12
which is in an environmentally controlled chamber with growth
medium. Wave plates and polarizing beam splitters control beam
intensity and ensure that most of the incoming light is directed
into the object path and that most of the back scattered signal is
directed to an EMC2 CCD camera 14. The CCD camera can be a 12-bit
CCD camera with one mega-pixel resolution and an exposure time of
10 msec. Lens L5 performs an optical Fourier transform to the image
plane, followed by a 4-f system L6, L7 that transfers the Fourier
domain to a CCD screen with a 1/3.times. magnitude. A delay stage,
including lens L4, is provided in the reference path to path-match
the interfering images and to perform volumetric scanning of the
living tissue. The interference fringes between the backscattered
object beam and the reference beam 13 are recorded by the CCD and
passed to a computer which is configured to construct a digital
hologram at successive times. Images at successive depths in the
target cell may be stacked to create volumetric images. Further
details of a suitable system and method for performing motility
contrast imaging are found in co-pending application Ser. No.
12/874,855, filed on Sep. 2, 2010, assigned to the assignee of the
present invention and published as US2010-0331672-A1, the
disclosure of which is incorporated herein by reference.
Oocytes and Embryos
[0032] To prepare for in vitro fertilization and early embryonic
development, oocytes must be matured through the reinitiation and
completion of the first meiotic division from the germinal vesicle
stage to metaphase II. Oocytes are encased (invested) in a
multilayer shell of cumulus granulosa cells that produce compounds
that are essential for normal oocyte development. Cumulus cells
transmit maturation signals to the oocyte. Part of the maturation
process is the expansion of the cumulus cells, meaning an increase
in the thickness of the investment, in response to exposure to
luteinizing hormone (LH) and follicle stimulating hormone (FSH).
Cumulus cells are capable of undergoing expansion in response to
FSH in vitro in which accumulation of hyaluronan (HA), an
extracellular matrix component of cumulus cells, brings about
expansion of cumulus-oocyte complexes (COCs).
[0033] In one experiment, cumulus-invested pig oocytes were
harvested immature and were matured in vitro. Ovaries from
slaughtered gilts were transported in a warm environment
(28-33.degree. C.) to the laboratory where they were washed in 0.9%
NaCl containing 100000 IU/L penicillin and 100 mg/L streptomycin.
Cumulus-oocyte complexes (COCs) were aspirated from 3-8 mm antral
follicles using a 10 mL syringe and an 18-gauge needle. For in
vitro maturation, cumulus-oocyte complexes were cultured in 500
.mu.L, Medium 199 (Earle's salts, L-glutamine, 2.2 mg/L sodium
bicarbonate) supplemented with 10% (v/v) fetal bovine serum, 0.5
mg/L follicle-stimulating hormone, 0.5 mg/L luteinizing hormone,
0.57 mM cysteine and 50 mg/L gentamicin sulfate under mineral oil,
at 39.degree. C. for 48 h in a 5% CO.sub.2 atmosphere.
[0034] In accordance with the present invention, MCI was applied to
two groups of COCs--47 immature and 48 in vitro matured COCs. When
collecting data, pig oocytes were placed in an S-well chamber slide
at 39.degree. C. filled with growth medium. For each oocyte, 500
successive images were captured at 25 fps at an exposure time of 10
msec using the MCI system shown in FIG. 1.
[0035] I.sub.H(x',y') is the intensity captured by the CCD camera
given by the expression:
I.sub.H(x',y')=|.psi..sub.R+.psi..sub.OF|.sup.2=|.psi..sub.R|.sup.2+|.ps-
i..sub.OF|.sup.2+.psi.*.sub.R.psi..sub.OF+.psi..sub.R.psi.*.sub.OF,
where .PSI..sub.R and .PSI..sub.OF are the reference wave and
objective wave. A spatial Fourier Transform was performed on each
raw image to reconstruct the digital hologram:
FT(I.sub.H)=FT(|.psi..sub.R|.sup.2+|.psi..sub.OF|.sup.2)+FT(.psi.*.sub.R-
.psi..sub.OF)+FT(.psi..sub.R.psi.*.sub.OF)
[0036] The first two terms represent the DC part (zero-order image)
of the hologram. FT(.PSI..sub.R*.PSI..sub.OF) and
FT(.PSI..sub.R.PSI..sub.OF*) are the holographic image and the
conjugate image. To obtain a good signal-noise ratio, the
zero-order is removed by subtracting the non-zero-path matched
image from the raw data. The holographic image part is chosen for
further analysis.
Results and Analysis
[0037] Motility Contrast Imaging (MCI) was used to generate a
two-dimension motility map at a fixed depth in the living tissue
(pig oocyte) and to generate a volumetric motility map from a
punctuated fly-through. Temporal contrast is calculated as the
normalized standard deviation (NSD) at each pixel given by the
expression:
N S D = MC ( x , y , z ) = 1 N t 0 ( I ( x , y , z ; t 0 ) - I ( x
, y , z ; t ) ) 2 / I ( x , y , z ; t ) ##EQU00001##
[0038] where I(x,y,z;t.sub.0) is the intensity value of an
individual pixel (x,y) of the holographic reconstructed image at
time t.sub.0 and at depth z. The depth z is fixed in a 2-D motility
map. High NSD values mean high temporal fluctuations indicating a
high motility, while low NSD values mean low motility.
[0039] Examples of the motility maps of two COCs are shown in FIG.
3, one with a high NSD core and the other with a low NSD core. Both
are immature oocytes surrounded by cumulous granulosa cells. The
diameter of the cellular structure is approximately 300 .mu.m and
the diameter of the central oocyte is approximately 100 .mu.m.
Color can be used to indicate the NSD value, with blue denoting low
motility and red denoting high motility. Optical coherence imaging
(OCI) and motility contrast imaging (MCI) are speckle-based imaging
techniques that use speckle contrast to measure physiological
properties of live tissue. Temporal contrast from MCI provides a
measure of dynamic physiology, while spatial contrast from OCI
provides a measure of structure and morphology.
[0040] FIG. 3 shows three mid-sections of the holographic data for
the same two immature cumulus-oocyte complexes. The mid-section
images on the left are the optical-coherence images (OCI), which
are the reconstructed holograms of the samples. The grayscale is
logarithmic, where a darker gray denotes high reflectance, and a
lighter gray denotes low reflectance. The middle images are the
temporal contrast (NSD) on a linear grayscale with dark denoting
high temporal fluctuations and light low fluctuations. The
mid-section images on the right are the spatial contrast on a
linear scale with darker denoting low spatial contrast and lighter
high spatial contrast.
[0041] The COC in the upper panels of FIG. 3 shows stronger motion
in the oocyte than the surrounding cumulous cells, indicating a
"good" oocyte. On the other hand, the oocyte in the bottom panels
of the figure shows an even weaker motion than the surrounding
cumulous cells, denoting a "bad" oocyte. As the images demonstrate,
there is a correlation between the temporal NSD map and spatial NSD
map. In particular, the oocyte in the upper panels with a high
temporal NSD core area has a corresponding low spatial NSD core
area. The oocyte in the lower panels of FIG. 3 without a low
temporal NSD core area has no corresponding low spatial NSD core
area. The volumetric motility maps of the oocytes reflect the same
activity/inactivity patterns.
[0042] The OCI results show no discernible oocyte within the
cumulus investment. This is believed to be because the relatively
long coherence of the light source (30 microns) and the low spatial
resolution (also 30 microns) produces high-contrast speckle with
little structural content. However, the same highly-developed
speckle provides high temporal contrast that shows a strong
activity in the core in the one COC but not in the other. The
spatial contrast shows a trend similar to the temporal contrast.
The "active" COC in the upper panels of FIG. 3 has low spatial
contrast in the core, but the "inactive" COC has higher spatial
contrast.
[0043] To one versed in the art, the application of MCI to COCs
would not have been expected to yield the positive results shown in
FIG. 3. All previous applications of MCI were on multi-cellular
samples that have many cells per imaging volume. Cellular membranes
are sources of scattered light, which makes multi-cellular tissue
samples bright in backscattered light. The oocyte, on the other
hand, is a single large cell with a diameter of .about.100 microns.
There are many volume elements contained within the oocyte volume,
and hence the internal volume of the oocyte would not be expected
to be bright enough to detect using MCI. The internal structure and
motions of the oocyte generate a detectable MCI signal in spite of
the lack of extracellular membranes. It is apparent that high
density of mitochondria and internal membranes of the oocyte make
up the source of the MCI signal. Therefore, MCI is shown here for
the first time to be extended into the single-cell regime,
specifically for the application to oocyte viability
assessment.
[0044] It is demonstrated that the healthy oocytes have higher
cellular motion than the surrounding cumulus cells on average.
Histograms of the motility of the shell and the core (oocyte) are
shown in FIG. 4. The NSD values (calculated according to the
equation above) of oocyte (core area) plotted against the average
NSD values of the cumulus cumulus cells (shell area) are shown in
the temporal contrast graph of FIG. 5. The circle data points are
oocytes which were observed in their immature state, while the
square data points are oocytes which were observed after they were
matured in vitro in the presence of follicle stimulating hormone
(FSH) and luteinizing hormone (LH). Data points above the diagonal
line correspond to stronger motion in the oocyte than in the
cumulus investment. The average NSD value for the matured oocytes
is shifted to the right from the average value for the immature
oocytes, indicating that maturation in the presence of FSH and LH
increases the average sub-cellular motion of both the cumulus and
the oocyte.
[0045] The correlation of the spatial contrast of OCI with the
temporal contrast of MCI is shown in FIG. 6 for the immature and in
vitro matured oocytes. The correlation coefficient from the linear
fit is 0.80 for the immature oocytes and 0.73 for the matured
oocytes. It is important to note that while the nucleus is usually
in the center of the oocyte for viable oocytes, some non-viable
oocytes have asymmetric nucleus location and are believed to have
higher cytoplasmic heterogeneity which may explain the strong
correlation between high temporal and low spatial contrast, both of
which can be indicative of a viable oocyte for fertilization. As
the graph of FIG. 6 illustrates, the increase in average temporal
contrast from immature to maturation is greater than the increase
in average spatial contrast from immature to maturation. In
addition, as reflected in the graph of FIG. 4, the average increase
in temporal contrast of the cumulus shells upon maturation is
larger than the increase in temporal contrast values for the
oocytes. The shaded triangle in the upper right of FIG. 4 denotes
the oocytes that have high activity and that respond to hormonal
stimulation. Viable oocytes that are ready for IVF would be
selected from this region.
[0046] These data suggest a "viability" figure of merit V= {square
root over (T(1-S))}, where T is the temporal contrast value and S
is the spatial contrast value. Histograms of this figure of merit
are shown in FIG. 7 for immature (circles) and in vitro matured
COCs (squares), with the x-axis corresponding to the V value and
the y-axis corresponding to the number of oocytes. The skewed peak
in the matured histogram may be a potential indicator of oocyte
viability for subsequent fertilization. For the present example,
the "viability" line in FIG. 7 corresponds to the intersection of
the two histograms with oocytes having a V value to the right of
this line deemed to be viable.
[0047] FIG. 8 presents a graph in which the x axis is the spatial
contrast value of the core area of the mature oocytes and the y
axis is the NSD (temporal) value of the same oocytes. Circle data
points are the oocytes marked "good" by the technician, while the
square points are the oocytes marked "bad". The graph illustrates
that the oocytes with low spatial contrast values tend to have high
NSD values indicative of an active and healthy oocyte. Conversely,
oocytes with high spatial contrast tend to have lower NSD values,
indicative of an inactive or unhealthy oocyte. K-means clustering
can be applied to the data to reveal two clusters divided between
the centroids of the clusters by the line in the graph. One cluster
to the left of the line is the low spatial contrast, high NSD
group. Points in this cluster are dense and close to each other.
The other cluster to the right of the line is the high spatial
contrast, low NSD group. Points in this group distribute wider than
the other cluster. Note that for the "good" oocytes group, 80% of
the oocytes are in the low spatial contrast, high NSD cluster,
while for the "bad" group, the oocytes nearly equally distribute
between the two clusters (58%:42%).
[0048] Temperature is an important factor for the oocyte health. As
demonstrated in the images of FIG. 9, when the temperature
decreased from the pig physiological temperature (39.degree. C.) at
time 0, to room temperature (25.degree. C.) at time 160 min., the
motility of the cumulus-encased oocyte decreased over time until it
was relatively inert.
[0049] The power spectra of a cumulus shell and an oocyte are shown
in FIG. 10. There are characteristic differences in the knee
frequencies and in the frequency content. In particular, in this
quantitative comparison the oocyte power spectrum has a higher knee
frequency at about 0.11 Hz than the cumulus cell knee frequency of
about 0.04 Hz. The slope exponent for both oocyte and cumulus cells
is about 1.6.
[0050] A key step in the IVF process is fertilization of the oocyte
to form a zygote that develops into a blastocyst. The fertilization
step has many failure modes, and approximately only 50% of porcine
oocytes become correctly fertilized. For subsequent selection for
implantation, assessment of the fertilized zygote is valuable. A
dramatic shift in the power spectrum of a freshly-formed zygote
relative to an unfertilized oocyte is shown in FIG. 11, which is,
unexpected to one trained in the art. The unfertilized oocyte has a
higher knee frequency than the fresh zygote. The process of zygote
formation suppresses overall motion, with an enhancement in low
frequencies. In addition the slope exponent decreases from 1.9 to
1.5 representing the onset of "anomalous" transport inside the
zygote. The fluctuation spectra provide an accurate set of metrics
to distinguish fertilized from unfertilized specimens.
[0051] In accordance with the present invention, analysis of
experimental physical properties of the zygotes as well as
quantitative metric values obtained from the fluctuation power
spectrum provide a new approach to selecting fertilized oocytes. To
demonstrate this approach, the oocyte data used for principal
component analysis (PCA) contain two cohorts: one with 35
unfertilized oocytes and the other with 51 nominally fertilized
oocytes. It must be understood that the unfertilized oocytes are
all unfertilized, but while the fertilized oocytes have been
exposed to sperm they may not have been correctly fertilized. For
instance, the normal successful fertilization rate is less than
50%. Furthermore, the health of each fertilized oocytes is
different from each other. Therefore, the goal is to select the
best fertilized oocyte to pass through to the following steps in
IVF.
[0052] After calculating the principal components for both
unfertilized and fertilized oocytes, PCA is performed for all the
oocytes. The principal components are shown in Table. 1. It shows
that the backscatter brightness, the spatial contrast and the slope
of spectrum are the main contributors to the components. The total
contribution for these three is 63.5%.
TABLE-US-00001 TABLE 1 Principal components and their weight from
the PCA analysis Principal Component Weight 1 Backscatter
brightness 24.2% 2 Spatial contrast 22.3% 3 Slope of spectrum 17.0%
4 Knee frequency of spectrum 13.3% 5 Power intensity of the very
lowest frequency point 10.8% 6 Ellipticity 7.3% 7 Temporal contrast
5.1%
[0053] A two-dimensional PCA map is shown in FIG. 12. The circles
represent the unfertilized oocyte group and the asterisks represent
the fertilized oocyte group. The circles are close to each other
and cluster together, and the PCA map demonstrates the common
character of the unfertilized oocytes. However, the asterisks are
distributed over the entire space. About 2/3 of the asterisks
overlap with the circles and another 1/3 are well-separated from
the others. Therefore, this distribution demonstrates that 2/3 of
the oocytes in the fertilized oocyte group have the same character
as the unfertilized group. This result suggests that these
two-thirds of the so-called fertilized oocytes in fact had not
successfully been fertilized. This important step shows that
biodynamic spectroscopy can recognize unfertilized oocytes and pick
the healthy candidates among fertilized oocytes.
[0054] To further validate this approach, two groups of oocytes
were processed as test groups. One group was a set of healthy
oocytes that followed the normal fertilization process, designated
by filled stars in FIG. 12. The other group, designated by open
stars, consisted of unhealthy oocytes (treated with low temperature
before experiment). In FIG. 12, six normal fertilized oocytes
(filled stars) and five unhealthy fertilized oocytes (open stars)
are plotted on the 2D PCA map. Although all the open stars are in
the unfertilized zone, the healthy fertilized oocytes are split
into both zones. This primary validation shows the robustness of
the biodynamic imaging technology.
[0055] Spectrogram analysis is a powerful tool in biodynamic
imaging technology because it traces different kinds of cellular
motion as spectral fingerprints. FIG. 13 shows a biodynamic image
that has captured the mitotic spectral fingerprint of a fertilized
oocyte. The spectrogram in FIG. 13 shows a strong mid frequency
enhancement that begins about 4 hours after fertilization and is
sustained over the first 24 hours. This time period is consistent
with pronucleus formation which occurs prior to mitosis. After 24
hours, the enhancement shifts briefly to the high frequency range,
denoting a short term of high activity (cytokinesis), after which
it shifts to the low-frequency range.
[0056] It is important to note that the OCI image of the dividing
zygote had limited spatial resolution and hence did not show strong
evidence for division. However, from the spectrogram, the mitotic
event occurred after 24 hours. The power spectra in FIG. 14 show
the spectral difference before and after mitosis. The zygote shows
strong and rapid motions leading up to mitosis, but immediately
after cytokinesis the motions become much more quiescent. The
spectrogram contains high information content which pertains to the
specific physical processes involved in the first cleavage and can
be used as metrics or indicators of the potential of the sample for
implantation.
Embodiments
[0057] In vitro fertilization success relies on accurate assessment
of oocyte viability after harvesting, during maturation, and after
fertilization. Oocytes invested with cumulus granulosa cells,
called cumulus-oocyte complexes (COCs), are not readily visible
under a microscope, making viability grading difficult. As
described herein, one embodiment of the present invention
contemplates using motility contrast imaging (MCI) to measure
oocyte activity before and after maturation induced by follicle
stimulating hormone (FSH). MCI uses digital holography [14-17] as
the coherence gate for low-coherence interferometry [18-20] to
capture dynamic light scattering from intracellular motions. High
temporal contrast of the fluctuating speckle is correlated with low
spatial speckle contrast in the optical coherence imaging data.
Using these tools, the changes in intracellular activity induced by
FSH, and the differences in the fluctuation spectra between the
cumulus shell and the oocyte, can be evaluated to provide potential
new biomarkers for assisted reproductive technology.
[0058] Using the system and method described herein, assessing
reproductive cells, such as oocytes and embryos can be made routine
and can simplify the candidate selection process. In a "Domestic
Animal IVF Laboratory" hundreds of oocytes are available after
collection so it is easier and more economical to simply discard
oocytes that have no chance to form viable embryos. However, in a
"Human IVF Laboratory" the number of oocytes collected from a woman
is usually limited, maybe only up to 20 oocytes. The evaluation of
viable cells thus becomes much more critical. In one aspect,
assessing oocyte viability and quality can improve the human IVF
process. Going further, assessing embryo viability and quality can
be particularly valuable to determine whether a particular embryo
has the potential to develop to term. Traditional methods, such as
morphological assessment, are not reliable because an embryo can
look good under a microscope yet have low developmental potential.
For this purpose it is first important to use good quality oocytes
and then fertilize them to generate embryos. The embryos may then
be evaluated using the same biodynamic imaging techniques described
herein to select the "good" embryos with the highest likelihood of
developing to term.
[0059] In one aspect of the present invention, Motility Contrast
Imaging (MCI) is applied to measure the dynamic activity of
cumulus-invested oocytes (including immature oocytes and oocytes
matured in vitro) which are not readily visible under a microscope.
The physiological characteristics of the oocytes are measured
non-invasively by using motion as an endogenous imaging contrast
agent. MCI has interferometric sensitivity to cellular
displacements of a fraction of a wavelength by treating each
speckle as an independent interferometer. The ability to measure
sub-micron displacements over a field of view of a millimeter
represents a large dynamic range that could prove useful for key
indicators for reproductive cell viability applications, including
assessing viability of oocytes and embryos.
[0060] In one embodiment, the present invention provides a method
for evaluating viability of oocytes and embryos comprising imaging
oocytes and embryos using MCI; generating temporal contrast and
spatial contrast data for the cells; generating a cell viability
value as a function of the temporal and spatial contrast data; and
comparing the cell viability value to a predetermined value
indicative of a cell suitable for use in an in vitro fertilization
program.
[0061] In a further embodiment, the fluctuating light intensities
from the speckle images are analyzed by their frequency content and
how they change as a function of time. This method is called tissue
dynamics spectroscopy (TDS). From the analysis, the system measures
the low, mid and high frequency fluctuation spectral content
changes over the time development of the reproductive specimen
(e.g., oocytes, and embryos up to the blastocysts stage). The
resulting dynamic spectroscopy provides a matrix of measurement
values that are related to the health and viability of the
specimen.
[0062] A system is envisioned that combines MCI and TDS in a single
measurement system. This system provides quantitative measures of
specimen health and potential reproductive viability, as well as
time-evolving dynamics and dynamic maps that would show
heterogeneous properties of the reproductive specimen.
[0063] According to one method, the system performs statistical
analysis on the spectral content to generate spectrogram
fingerprints averaged over specified areas (volumes) of the
reproductive specimen. A library of these fingerprints can be
assembled that are cross-correlated with known pathologies of
reproductive specimens. In the present method, a specimen will be
measured and analyzed into a spectrogram fingerprint that is
compared to the reference library. In this manner, a specimen will
be graded as "viable" or "pathological" depending on the comparison
to the spectrogram library. Decisions to select or reject this
reproductive specimen may be made based on this comparison.
[0064] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same should
be considered as illustrative and not restrictive in character. It
is understood that only the preferred embodiments have been
presented and that all changes, modifications and further
applications that come within the spirit of the invention are
desired to be protected.
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