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United States Patent Application |
20110245592
|
Kind Code
|
A1
|
Schoolcraft; William B.
;   et al.
|
October 6, 2011
|
Secretome Profile-Facilitated In Vitro Fertilization
Abstract
Secretome profiling improves the pregnancy success rate of in vitro
fertilization processes, while reducing the risk of multiple births.
Inventors: |
Schoolcraft; William B.; (Englewood, CO)
; Katz-Jaffe; Mandy; (Denver, CO)
; McReynolds; Susanna; (Parker, CO)
|
Serial No.:
|
081463 |
Series Code:
|
13
|
Filed:
|
April 6, 2011 |
Current U.S. Class: |
600/34; 435/287.1; 435/287.2 |
Class at Publication: |
600/34; 435/287.1; 435/287.2 |
International Class: |
A61B 17/435 20060101 A61B017/435; C12M 1/34 20060101 C12M001/34 |
Claims
1. A system for enhancing the pregnancy success rate of in vitro
fertilization, comprising: means for determining the secretome profile of
an embryo to identify proteins implicated in implantation success; and
means for recommending whether to implant the embryo on the basis of the
secretome profile.
2. The system of claim 1, further comprising data representative of a
secretome profile of an embryo acquired by ELISA.
3. The system of claim 2, further comprising the secretome profile of an
embryo acquired using a secretome panel selected from the group
consisting of SEQ ID Nos. 1-403.
4. The system of claim 3, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 1-404.
5. The system of claim 3, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 261-404.
6. The system of claim 3, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334,
335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349,
350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
7. The system of claim 3, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350,
368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
8. The system of claim 3, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 317 and 383.
9. The system of claim 1, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 1-404.
10. The system of claim 1, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 261-404.
11. The system of claim 1, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334,
335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349,
350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
12. The system of claim 1, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350,
368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
13. The system of claim 1, further comprising the secretome profile of an
embryo acquired using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 317 and 383.
14. A method of in vitro fertilization, comprising: determining the
secretome profile of a candidate embryo to ascertain proteins implicated
in implantation success; modeling on basis of the secretome profile to
assess candidate embryo viability for recommending whether to implant the
candidate embryo on the basis of the viability assessment; and
conditionally implanting the candidate embryo on the basis of the
recommendation.
15. The method of claim 14, wherein the step of determining the secretome
profile includes using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 1-404.
16. The method of claim 14, wherein the step of determining the secretome
profile includes using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 261-404.
17. The method of claim 14, wherein the step of determining the secretome
profile includes using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334,
335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349,
350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
18. The method of claim 14, wherein the step of determining the secretome
profile includes using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350,
368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
19. The method of claim 14, wherein the step of determining the secretome
profile includes using a secretome panel selected as one or more members
of the group consisting of SEQ ID Nos. 317 and 383.
20. In an ELISA assay kit comprising a plurality of microwells for the
quantitation of protein content in a sample, the improvement comprising:
the microwells being constructed and arranged to quantitate for a
plurality of proteins selected from SEQ ID Nos. 1-404.
21. The kit claim 20, wherein the plurality of proteins are selected from
the group consisting of SEQ ID Nos. 261-404.
22. The kit claim 20, wherein the plurality of proteins are selected from
the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334,
335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349,
350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
23. The kit claim 20, wherein the plurality of proteins are selected from
the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350,
368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
24. The kit claim 20, wherein the plurality of proteins are selected from
the group consisting of SEQ ID Nos. 317 and 383.
Description
RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S. Provisional
Patent Application No. 61/321,448 filed Apr. 6, 2010, which is
incorporated by reference to the same extent as though fully replicated
herein.
SEQUENCE LISTING
[0002] This application includes a Sequence Listing, as set forth in an
ASCII-compliant text file named "CCRMProtein_ST25.txt", created on Apr.
6, 2011, and containing 2263 kilobytes, which is incorporated by
reference to the same extent as though fully replicated herein.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The invention relates to the field of in vitro fertilization (IVF),
which is a process by which mammalian egg cells are fertilized by sperm
outside the womb. More particularly, evaluation of a secretome profile is
used to enhance the pregnancy success rate when the fertilized egg is
implanted into a patient's uterus.
[0005] 2. Description of the Related Art
[0006] IVF infertility treatment offers infertile couples a chance to have
a biologically related child. IVF may overcome female infertility due to
problems of the fallopian tube or endometriosis. IVF also overcomes male
infertility due to problems with sperm quality or quantity. The IVF
process involves hormonally controlling the ovulatory process, removing
eggs (termed ova) from the woman's ovaries and permitting the sperm to
fertilize the eggs in a fluid medium. The fertilized egg, termed an
embryo, is subsequently transferred to the patient's uterus with the
intent of establishing a successful pregnancy. Due to expensive
procedural costs, IVF is only attempted after the failure of less
expensive fertility treatments.
[0007] FIG. 1 displays a schematic for mammalian blastocyst implantation.
Mammalian embryo implantation is a complex and intricate process
involving numerous biological changes at both the embryo and endometrial
level. The interaction between the blastocyst and the endometrium is a
function of both a receptive endometrial environment and a healthy
blastocyst. A blastocyst failing to implant or an endometrium failing to
sustain growth and differentiation will result in spontaneous abortion.
The prior art teaches very little about the embryo's role in the events
leading to the attachment of a viable blastocyst to a receptive uterine
luminal epithelium.
[0008] IVF treatment begins with administration of hormonal medications to
stimulate ovarian follicle production, such as gonadotropins hormones.
The prevention of spontaneous ovulation involves using other hormones,
such as GnRH antagonists or GnRH agonists that block the natural surge of
luteinizing hormone. With adequate follicular maturation, administration
of human chorionic gonadotropin hormone causes ovulation approximately 42
hours after the administration. However, the egg retrieval procedure
takes place just prior to ovulation, in order to recover the eggs from
the ovary. The egg retrieval proceeds using a transvaginal technique
involving an ultrasound-guided needle that pierces the vaginal wall to
reach the ovaries. After recovery of the follicles through the needle,
the follicular fluid is provided to the IVF laboratory to identify eggs.
Typically, the procedure retrieves between 10 and 30 eggs. The retrieval
procedure takes approximately 20 minutes and is usually done under
conscious sedation or general anesthesia.
[0009] For IVF, the fertilization of the egg (termed insemination)
proceeds in the laboratory where the identified eggs and semen are
usually incubated together in a culture media. The confirmation of
fertilization proceeds by monitoring the eggs for cell division. For
instance, a fertilized egg may show two pronuclei. In certain situations,
such as low sperm count or motility, a single sperm may be injected
directly into the egg using a method called intracytoplasmic sperm
injection (ICSI). In another option known as gamete intrafallopian
transfer, eggs are removed from the woman and placed in one of the
fallopian tubes, along with the man's sperm. In this example,
fertilization occurs within the women's body, a process termed in vivo
fertilization.
[0010] Selected embryos are transferred to the patient's uterus through a
thin, plastic catheter, which goes through the vagina and cervix.
Typically, transfer of 6-8 cell stage embryos to the uterus occurs three
days after embryo retrieval. Alternatively, embryos can be placed into an
extended culture system with a transfer done at the blastocyst stage at
approximately five days post-retrieval. Blastocyst stage transfers often
result in higher pregnancy rates. Additionally, embryonic
cryopreservation, or the storage of embryos in a frozen state, is
feasible until uterine transfer. For example, the first term pregnancy
derived from a frozen human embryo was reported in 1984.
[0011] Despite progressively improving IVF pregnancy rates, the majority
of transferred human embryos result in implantation failure. For example,
Canadian clinics reported an average pregnancy rate of 35% for one cycle,
but a live birth rate of only 27% in 2006. Moreover, implantation success
rates may decrease with the increasing maternal age, if donor eggs are
not used. Various factors are associated with implantation failure,
including embryo chromosome aneuploidies related to advanced maternal age
and maternal factors such as endometrium response failure to hormone
regulation.
[0012] To overcome low implantation success rate, multiple embryos are
commonly transferred during a single IVF procedure. The process for
selecting embryos for transfer often involves grading methods developed
in individual laboratories to judge oocyte and embryo quality. An
arbitrary embryo score, involving the number and quality of embryos, may
reveal the probability of pregnancy success post-transfer. For example,
an embryologist may grade embryos using morphological qualities including
the number of cells, clearness of cytoplasm, evenness of growth and
degree of fragmentation. However, embryo selection based on morphological
qualities is not precise. Often, several embryos selected for these
general qualities are implanted to improve the chance of pregnancy. The
number of embryos transferred depends upon the number available, the age
of the woman and other health and diagnostic factors.
[0013] The transfer of multiple embryos, however, often results in
multiple pregnancies, a major complication of IVF. In general, multiple
pregnancies, specifically, more than twins, hold maternal and fetal
risks. For example, multiple births are associated with increased risk of
pregnancy loss, neonatal morbidity, obstetrical complications, and
prematurity with potential for long term damage. Some countries
implemented strict limits on the number of transferred embryos to reduce
the risk of high-order multiples (e.g., triplets or more). However, these
limitations are not universally followed or accepted.
SUMMARY
[0014] In one embodiment, a system for enhancing the pregnancy success
rate of in vitro fertilization includes a means for determining the
secretome profile of an embryo to identify proteins, polypeptides,
oligopeptides or protein fragments implicated in implantation success and
a means for recommending whether to implant the embryo, such as a
blastocyst, on the basis of the secretome profile.
[0015] The CDC data from 2006 shows that use of donor eggs in a 40 year
old woman will result in a live birth 54% of the time. This compares to
20.6% using the woman's own eggs. Thus, an opportunity exists for the
instrumentalities of this disclosure to facilitate success rates
approximating the use of donor eggs when using one's own eggs, and may
even permit higher success rates due to better selection of viable
embryos.
[0016] In an embodiment, a system is provided for enhancing the pregnancy
success rate of in vitro fertilization. The system includes an electronic
system to configured to gather secretome data as a secretome profile of
an embryo by quantitating proteins implicated in implantation success. A
model is provided for use in recommending whether to implant the embryo
on the basis of this secretome profile. The secretome date may be, for
example, provided by use of mass spectroscopy or ELISA measurements.
[0017] According to one aspect of the system, the secretome profile may be
provided by using proteins as embryo secretions in culture media that may
be linked to changed odds of implantation success, for example, as may be
found in one or more sequences found in SEQ ID Nos. 1-404. The sequences
of SEQ ID Nos. 261-404 are preferred for this use. Particularly preferred
are the sequences of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350,
368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404. The sequences
of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339,
340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391,
397, 398, 399, 402, 403, and 404 are associated with increased odds of
implantation success, whereas those of SEQ ID Nos. 317 and 383 are
associated with failure or aneuploidy.
[0018] In an embodiment, a method of in vitro fertilization entails
determining the secretome profile of a candidate embryo by use of these
sequences to ascertain proteins implicated in implantation success. This
provides data that may be submitted to a model that associates one or
more of these proteins with changes odds of implantation success or
failure. A recommendation for implantation of the embryo may then be
provided based upon the modeling outcome. The embryo may be conditionally
implanted on the basis of the recommendation.
[0019] In an embodiment, there is an improved ELISA assay kit with a
plurality of microwells for the quantitation of protein content in a
sample. The microwells are constructed and arranged to quantitate for a
plurality of proteins selected from these sequences.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a diagram illustrating the process blastocyst
implantation.
[0021] FIG. 2 is a table that illustrates the success rates from donor
oocyte IVF cycles.
[0022] FIG. 3 is a flow chart for a method of developing and using a
predictive model that may be used to assess embryo viability as a
candidate for use in an IVF implantation procedure.
[0023] FIG. 4 shows a system that analyzes the secretome of an embryo to
assess viability.
DETAILED DESCRIPTION
[0024] The following definitions are provided to facilitate understanding
of certain terms used herein and are not meant to limit the scope of the
present disclosure.
[0025] The term "secretome panel" refers to a collection of individual
proteins, polypeptides, oligopeptides or protein fragments that are
differentially expressed and secreted by an embryo. The proteins,
polypeptides, oligopeptides or protein fragments of the secretome panel
are selected based on predictions of developmental competence and
implantation potential of an embryo.
[0026] The term "spent media" refers to media surrounding an embryo that
accumulates proteins secreted from the embryo.
[0027] The term "secretome profiling" refers to qualitative and
quantitative analysis of a secretome panel collected from spent media.
[0028] There will now be shown and described a system for enhancing the
pregnancy success rate of in vitro fertilization involving non-invasive
secretome profiling of an embryo. Secretome profiling may be performed on
embryos at differing embryonic developmental stages, such as day one
through day six embryos.
[0029] Non-limiting examples of proteins comprising the secretome panel
include proteins involved in amino acid metabolism, lipid metabolism,
carbohydrate metabolism, signal transduction, apoptosis, transcription
and combinations thereof. In one example, an embryo secretes a protein
that is involved in amino acid metabolism, thereby indicating potential
for successful embryo implantation. In another example, an embryo
secretes a protein that is involved in apoptosis, thereby indicating a
reduced potential for successful blastocyst implantation. Nonlimiting
examples of the number of proteins comprising the secretome panel
includes .gtoreq.250 proteins, .gtoreq.100 proteins, .gtoreq.50 proteins,
.gtoreq.20 proteins or .gtoreq.10 proteins.
[0030] Secretome profiling involves assessment of a secretome panel.
Secretome profiling is a noninvasive method for predicting developmental
competence and viability of an embryo. Comparison of secretome profiles
between developing embryos and degenerating embryos, both at the same
developmental stage, reveals significant differences in protein
expression. Moreover, secretome profiling provides a molecular
perspective of the functioning biochemical pathways present during embryo
development. A noninvasive secretome profiling assay correlates embryonic
secretome to embryonic viability, thereby facilitating single embryo
transfer during in vitro fertilization.
[0031] FIG. 2 highlights the importance of embryo developmental competence
by showing high success rates from donor oocyte IVF cycles using young
reproductive age oocytes transferred to an advanced maternal age
endometrium. The data is provided for a Colorado clinic from 2004-2009
including outcomes for over 1000 donor oocyte cycles. These results
demonstrate a 66.6% implantation rate for IVF recipients with an average
endometrial age of 40.6 years and donor oocytes with an average age of
26.6 years.
[0032] In one embodiment, secretome profiling occurs by assessing a
secretome panel via Enzyme-Linked Immunosorbent Assay (ELISA). In another
embodiment, secretome profiling proceeds by assessing a secretome panel
via mass spectrometry.
[0033] Non-limiting examples of solid-state substrates used in ELISA
include microwell plates, such as 96-well plates, 384-well plates and
8-well strips, microarray slides and nitrocellulose membranes. In one
embodiment, glass microarray surfaces contain chemical functional groups
such as epoxy, amine or aldehyde. In one embodiment, microwell plate
material comprises polystyrene.
[0034] The term "capture antibody" refers to an antibody secured, either
covalently or non-covalently, to a solid-state substrate. The capture
antibody recognizes and binds to a specific antigen such as a protein, a
polypeptide, an oligopeptide, a protein fragment, a carbohydrate or a
small molecule. In one example, a solid-state substrate is functionalized
with capture antibodies by passive adsorption or by specific binding. For
example, specific binding of capture antibodies may occur using
biotinylated capture antibodies and streptavidin-coated solid-state
substrates. In another example, solid-state substrates are functionalized
with antigens via passive adsorption.
[0035] Non-limiting examples of ELISA types include direct, indirect,
competitive, and sandwich. In one example, proteins from the secretome
panel are detected on a solid-state substrate using either a primary
labeled antibody or a secondary labeled antibody. In another example,
proteins from the secretome panels are detecting using a sandwich ELISA
wherein a protein from the secretome panel is bound between two primary
antibodies, namely the capture antibody and the detection antibody.
Non-limiting examples of antibody labels include enzymes and
fluororphores. In one embodiment, an enzyme that is conjugated to a
detection antibody binds to a substrate producing either a chromogenic,
fluorescent or chemiluminescent signal that is proportional to the
quantity of protein from the secretome panel.
[0036] FIG. 3 shows a method 300 implementing the concepts described above
to identify those embryos having the greatest chanced for implantation
success rates. Step 302 entails ascertaining the secretome of proteins
secreted into culture medium by an egg, blastocyst or developing embryo
(hereinafter "embryo") that has been previously fertilized by methods
known to the art. Step 302 may be performed at any time, including before
an egg is fertilized or immediately thereafter. More typical times to
sample the media include form one to five days after fertilization
occurs. Step 302 is preferably conducted before step 304, but samples of
the culture medium may be frozen for later analysis. The embryo is then
used 304 in an IVF procedure attempting to implant the embryo in a woman
who desires to have a baby. The implantation successes and failures are
monitored 306 to collect data relating the successes and failures to one
or more proteins of the secretome. Once a sufficient number of instances
have transpired 308 to provide for statistical significance, the proteins
are mapped 310 to the instances of success and failure. The exact manner
of mapping is not of particular importance, as there a number of
biostatistical analysis software products are commercially available. For
example, this may be done using the JMP.RTM. 9 packages from SAS of Cary,
N.C. to implement methods as taught by Zar, Biostatistical Analysis, 5th
Ed., Prentice Hall (2007) to map the secretome profiles using also time
as a variable. The mapping may be multivariate, or by an artificially
intelligent algorithm, such as a neural network. This model may be then
used 312 as a predictive tool to select embryos for implantation based
upon their secretome profile. This is done by analyzing 314 the secretome
of candidate embryos for implantation with use of the model to predict
implantation success or failure. One or more of the selected embryos are
then implanted 316
[0037] FIG. 4 shows an analytical system 400 that may be used in step 314
according to one embodiment. A specimen 402 contains a sample of culture
medium that is collected for secretome analysis as described above. By
way of example, this specimen may contain culture medium in the form of a
single or multi-well ELISA assay, or in a form that has bee pre-processed
for mass spectroscopy analysis. A corresponding measurement device 404
obtains primary measurements from the specimen 402. The measurements are
representative of one or more proteins in the secretome that have been
mapped to incidences of implantation success of failure. The model for
this mapping is provided in step 312 of method 300 and resides on
analyzer 406, which may be a computer operating on program instructions.
The analyzer 406 receives signals from the measurement device 404 and
interprets these by computational analysis according to the model then
provides the results of computational analysis to an output device 408,
which may be printer or an optical display. The various components of
system 400 may be partially integrated, such as by combining the
measurement device 404, analyzer 406, and output device 408 into one
integrated system.
[0038] It will be appreciated that perceptive use of the instrumentalities
described herein may result in a better selection of healthy embryos,
such as blastocysts, for implantation. Thus, fewer blastocysts need to be
implemented, such that there is lower risk of multiple pregnancies while
achieving a higher overall pregnancy success rate.
[0039] The following descriptions will show and describe, by way of
non-limiting examples, a process for improving pregnancy success rates
with lower incidence of multiple births. The following examples describe
secretome profiling of spent media from human blastocysts to provide
implantation recommendation. It is to be understood that these examples
are provided by way of illustration and should not be unduly construed to
limit the scope of what is disclosed herein.
Example 1
Secretome Profiling of Human Embryos Using Mass Spectrometry
[0040] The following nonlimiting example teaches by way of illustration,
not by limitation, secretome profiling of a human embryo using mass
spectrometry (MS). Human cleavage-stage embryos were cultured in 10 .mu.L
drops of G1 supplemented with 2.5 mg/mL recombinant albumin under oil at
37.degree. C., 6% CO.sub.2, 5% O.sub.2 for 24 hours. The embryos were
washed twice in G2 culture media and further cultured in 10 .mu.L drops
of G2 supplemented with 2.5 mg/mL recombinant albumin under oil at
37.degree. C., 6% CO.sub.2, 5% O.sub.2 for 48 hours with a fresh drop of
G2 media added after 24 hours. Spent media samples of blastocysts were
transferred into 0.65 mL Eppendorf tubes. Control groups comprised media
cultured and collected under the same conditions but without embryos.
[0041] Micro-drops of spent media were depleted of human serum albumin
(HSA) using Cibracron Blue Activated SwellGel Discs (Themo Fisher
Scientific, Rockford, Ill.). The proteins in the spent media were
separated by 1D gel electrophoresis (Invitrogen, Carlsbad, Calif.),
followed by Coomassie staining. Twenty five individual bands were cut out
from each sample lane and a standard in-gel digestion protocol was used
based on previously used methods ((1.) Rosenfeld, J., Capdevielle, J.,
Guillemot, J. C. & Ferrara, P. In-gel digestion of proteins for internal
sequence analysis after one- or two-dimensional gel electrophoresis.
Anal. Biochem. 203, 173-179 (1992), (2.) Hellman, U., Wernstedt, C.,
Gonez, J. & Heldin, C. H. Improvement of an "In-Gel" digestion procedure
for the micropreparation of internal protein fragments for amino acid
sequencing. Anal. Biochem. 224, 451-455 (1995)). Iodoacteamide (IAM) was
used for cysteine alkylation. The samples were analyzed on a LTQ-FT Ultra
Hybrid Mass Spectrometer (Thermo/Finnigan; Waltham, Mass.) with a method
based on a previously described protocol from Hansen et al., (Hansen KC,
Kiemele L, Maller O, O'Brien J, Shankar A, Formetti J, Schedin P. An
in-solution ultrasonication-assisted digestion method for improved
extracellular matrix proteome coverage. Mol Cell Proteomics. 8(7):1648-57
(2009)).
[0042] Spent media samples were analyzed on a LTQ-FT Ultra Hybrid Mass
Spectrometer (Thermo/Finnigan; Waltham, Mass.). Peptide desalting and
separation was achieved using a dual capillary/nano pump HPLC system
(Agilent 1200, Palo Alto, Calif.). On this system 8 .mu.L of spent media
sample was loaded onto a trapping column (ZORBAX 300SB-C18, dimensions
5.times.0.3 mm 5 .mu.m) and washed with 5% acetonitrile (ACN), 0.1%
formic acid (FA) at a flow rate of 15 .mu.L/min for 5 minutes. At this
time, the trapping column was put online with the nano-pump at a flow
rate of 350 nL/min. An 85 minute gradient, from 8% ACN to 40% ACN, was
used to separate the peptides. The column was made from an in-house
pulled 360/100 nm (outer/inner diameter) fused silica capillary packed
with Jupiter C18 resin (Penomenex; Torrance, Calif.). The column was kept
at a constant 40.degree. C. using an in-house built column heater. Data
acquisition was performed using the instrument supplied Xcalibur (version
2.0.6) software. The LC runs were monitored in positive ion mode by
sequentially recording survey MS scans (m/z 400-2000), in the ICR cell,
while three MS.sup.2 were obtained in the ion trap via CID for the most
intense ions. After two acquisitions of a given ion within 45 seconds,
the ion was excluded for 150 seconds.
[0043] For data analysis, The Raw Distiller program (UCSF) was used to
create a de-isotoped centroided peak lists from the raw spectra into the
mascot format using the default settings. The peak lists were searched
against the SwissProt Human database (51.6, Homo sapiens 15720 sequences)
using Mascot.TM. server (Version 2.2, Matrix Science, Boston, Mass.). The
search parameters are the same as those followed in Hansen et al.,
(Hansen K C, Kiemele L, Maller O, O'Brien J, Shankar A, Formetti J,
Schedin P. An in-solution ultrasonication-assisted digestion method for
improved extracellular matrix proteome coverage. Mol Cell Proteomic.
8(7): 1648-57 (2009)). The Mascot results were loaded into Scaffold (v
2.06) and the runs were compared.
[0044] In one embodiment, MS analysis of spent media reveals a secretome
panel of 261 individual proteins (Table 1). In one example, secretome
profiling using the secretome panel of 261 proteins (Table 1), as
analyzed by MS, identifies the potential for developmental competence and
implantation success of a human embryo.
[0045] In one embodiment, analysis of spent media via MS reveals a
secretome panel of 37 individual proteins (Table 2). Secretome profiling
via MS of 37 individual proteins (Table 2) from spent media of human
embryos correlates with embryonic viability and euploidy. The term
"euploidy" refers to having a chromosome number that is an exact multiple
of the haploid number for a human embryo, namely 23 pairs of chromosomes.
[0046] In Tables 1 and 2 below, the entries for "Entry Name" and
"Accession Number" refer to identifiers for published sequence data that
is stored in bioinformatic databases including the Uniprot Knowledgebase,
Swiss-Prot and TrEMBL. The information is made freely available to the
world and is coordinated by the Swiss Institute of Bioinformatics, which
is centrally administered in Lausanne, Switzerland with offices in Bern,
Geneva and Zurich. The sequence information represented by these
identifiers together with the representative publications is hereby
incorporated by reference to the same extent as though fully replicated
herein. For those proteins having isoforms, the sequences for Table 1
include consensus sequences for the primary isoforms, while sequences for
Table 2 include also all other isoforms available at the time of filing.
TABLE-US-00001
TABLE 1
Proteins identified in spent media of human embryos.
Protein
SEQ ID Molecular Accession
NO. Protein Name Entry Name Weight Number Taxonomy
1 Serum albumin ALBU_HUMAN 69 kDa P02768 Homo sapiens
2 Serotransferrin TRFE_HUMAN 77 kDa P02787 Homo sapiens
3 Haptoglobin HPT_HUMAN 45 kDa P00738 Homo sapiens
4 Alpha-1-antitrypsin A1AT_HUMAN 47 kDa P01009 Homo sapiens
5 Alpha-2-macroglobulin A2MG_HUMAN 163 kDa P01023 Homo sapiens
6 Hemopexin HEMO_HUMAN 52 kDa P02790 Homo sapiens
7 Apolipoprotein A-I APOA1_HUMAN 31 kDa P02647 Homo sapiens
8 Ceruloplasmin CERU_HUMAN 122 kDa P00450 Homo sapiens
9 Vitamin D-binding protein VTDB_HUMAN 53 kDa P02774 Homo sapiens
10 Alpha-1-antichymotrypsin AACT_HUMAN 48 kDa P01011 Homo sapiens
11 Plasma protease C1 inhibitor IC1_HUMAN 55 kDa P05155 Homo sapiens
12 Alpha-1B-glycoprotein A1BG_HUMAN 54 kDa P04217 Homo sapiens
13 Hemoglobin subunit beta HBB_HUMAN 16 kDa P68871 Homo sapiens
14 Antithrombin-III ANT3_HUMAN 53 kDa P01008 Homo sapiens
15 Afamin AFAM_HUMAN 69 kDa P43652 Homo sapiens
16 Angiotensinogen ANGT_HUMAN 53 kDa P01019 Homo sapiens
17 Transthyretin TTHY_HUMAN 16 kDa P02766 Homo sapiens
18 Inter-alpha-trypsin inhibitor heavy ITIH4_HUMAN 103 kDa Q14624 Homo
sapiens
chain H4
19 Complement factor B CFAB_HUMAN 86 kDa P00751 Homo sapiens
20 Hemoglobin subunit alpha HBA_HUMAN 15 kDa P69905 Homo sapiens
21 Ig alpha-1 chain C region IGHA1_HUMAN 38 kDa P01876 Homo sapiens
22 Ig gamma-1 chain C region IGHG1_HUMAN 36 kDa P01857 Homo sapiens
23 Heparin cofactor 2 HEP2_HUMAN 57 kDa P05546 Homo sapiens
24 Ig gamma-2 chain C region IGHG2_HUMAN 36 kDa P01859 Homo sapiens
25 Ig kappa chain C region KAC_HUMAN 12 kDa P01834 Homo sapiens
26 N-acetylmuramoyl-L-alanine PGRP2_HUMAN 62 kDa Q96PD5 Homo sapiens
amidase
27 Alpha-2-HS-glycoprotein FETUA_HUMAN 39 kDa P02765 Homo sapiens
28 Kininogen-1 KNG1_HUMAN 72 kDa P01042 Homo sapiens
29 Zinc-alpha-2-glycoprotein ZA2G_HUMAN 34 kDa P25311 Homo sapiens
30 Thyroxine-binding globulin THBG_HUMAN 46 kDa P05543 Homo sapiens
31 Corticosteroid-binding globulin CBG_HUMAN 45 kDa P08185 Homo sapiens
32 Attractin ATRN_HUMAN 159 kDa O75882 Homo sapiens
33 Pregnancy zone protein PZP_HUMAN 164 kDa P20742 Homo sapiens
34 Alpha-2-antiplasmin A2AP_HUMAN 55 kDa P08697 Homo sapiens
35 Vitronectin VTNC_HUMAN 54 kDa P04004 Homo sapiens
36 Ig lambda chain C regions LAC_HUMAN 11 kDa P01842 Homo sapiens
37 Apolipoprotein A-II APOA2_HUMAN 11 kDa P02652 Homo sapiens
38 Apolipoprotein C-III APOC3_HUMAN 11 kDa P02656 Homo sapiens
39 Insulin-like growth factor-binding ALS_HUMAN 66 kDa P35858 Homo
sapiens
protein complex acid labile chain
40 Complement C3 CO3_HUMAN 187 kDa P01024 Homo sapiens
41 Complement C4-B CO4B_HUMAN 193 kDa P0C0L5 Homo sapiens
42 Serum paraoxonase/arylesterase 1 PON1_HUMAN 40 kDa P27169 Homo sapiens
43 Plasma retinol-binding protein RETBP_HUMAN 23 kDa P02753 Homo sapiens
44 Carboxypeptidase N subunit 2 CPN2_HUMAN 61 kDa P22792 Homo sapiens
45 Beta-2-glycoprotein 1 APOH_HUMAN 38 kDa P02749 Homo sapiens
46 Inter-alpha-trypsin inhibitor heavy ITIH2_HUMAN 106 kDa P19823 Homo
sapiens
chain H2
47 Clusterin CLUS_HUMAN 52 kDa P10909 Homo sapiens
48 Protein AMBP AMBP_HUMAN 39 kDa P02760 Homo sapiens
49 Ig heavy chain V-III region BRO HV305_HUMAN 13 kDa P01766 Homo sapiens
50 Dynactin subunit 2 DCTN2_HUMAN 44 kDa Q13561 Homo sapiens
51 Inter-alpha-trypsin inhibitor heavy ITIH1_HUMAN 101 kDa P19827 Homo
sapiens
chain H1
52 Complement C2 CO2_HUMAN 83 kDa P06681 Homo sapiens
53 Serum amyloid P-component SAMP_HUMAN 25 kDa P02743 Homo sapiens
54 Lamin-A/C LMNA_HUMAN 74 kDa P02545 Homo sapiens
55 Phosphatidylinositol-glycan-specific PHLD1_HUMAN 92 kDa P80108 Homo
sapiens
phospholipase D
56 Alpha-1-acid glycoprotein 1 A1AG1_HUMAN 24 kDa P02763 Homo sapiens
57 Pigment epithelium-derived factor PEDF_HUMAN 46 kDa P36955 Homo
sapiens
58 Apolipoprotein C-I APOC1_HUMAN 9 kDa P02654 Homo sapiens
59 Transcription intermediary factor 1- TIF1B_HUMAN 89 kDa Q13263 Homo
sapiens
beta
60 Leucine-rich alpha-2-glycoprotein A2GL_HUMAN 38 kDa P02750 Homo
sapiens
61 Lumican LUM_HUMAN 38 kDa P51884 Homo sapiens
62 Ig kappa chain V-III region SIE KV302_HUMAN 12 kDa P01620 Homo sapiens
63 Actin, cytoplasmic 1 ACTB_HUMAN 42 kDa P60709 Homo sapiens
64 Apolipoprotein E APOE_HUMAN 36 kDa P02649 Homo sapiens
65 Kallistatin KAIN_HUMAN 49 kDa P29622 Homo sapiens
66 Myosin-15 MYH15_HUMAN 225 kDa Q9Y2K3 Homo sapiens
67 Carboxypeptidase B2 CBPB2_HUMAN 48 kDa Q96IY4 Homo sapiens
68 Serum amyloid A-4 protein SAA4_HUMAN 15 kDa P35542 Homo sapiens
69 Cholinesterase CHLE_HUMAN 68 kDa P06276 Homo sapiens
70 Apolipoprotein D APOD_HUMAN 21 kDa P05090 Homo sapiens
71 Tubulin beta-2C chain TBB2C_HUMAN 50 kDa P68371 Homo sapiens
72 Haptoglobin-related protein HPTR_HUMAN 39 kDa P00739 Homo sapiens
73 Hemoglobin subunit delta HBD_HUMAN 16 kDa P02042 Homo sapiens
74 Ig kappa chain V-IV region Len KV402_HUMAN 13 kDa P01625 Homo sapiens
75 Ig alpha-2 chain C region IGHA2_HUMAN 37 kDa P01877 Homo sapiens
76 Proteoglycan 4 PRG4_HUMAN 151 kDa Q92954 Homo sapiens
77 Histone H2A type 1-D H2A1D_HUMAN 14 kDa P20671 Homo sapiens
78 Biotinidase BTD_HUMAN 61 kDa P43251 Homo sapiens
79 Carboxypeptidase N catalytic chain CBPN_HUMAN 52 kDa P15169 Homo
sapiens
80 Plasma kallikrein KLKB1_HUMAN 71 kDa P03952 Homo sapiens
81 Ig kappa chain V-II region TEW KV204_HUMAN 12 kDa P01617 Homo sapiens
82 Apolipoprotein C-II APOC2_HUMAN 11 kDa P02655 Homo sapiens
83 DNA-dependent protein kinase PRKDC_HUMAN 469 kDa P78527 Homo sapiens
catalytic subunit
84 Heterogeneous nuclear HNRL1_HUMAN 96 kDa Q9BUJ2 Homo sapiens
ribonucleoprotein U-like protein 1
85 Apolipoprotein B-100 APOB_HUMAN 516 kDa P04114 Homo sapiens
86 Heterogeneous nuclear ROA2_HUMAN 37 kDa P22626 Homo sapiens
ribonucleoproteins A2/B1
87 Tubulin alpha-ubiquitous chain TBAK_HUMAN 50 kDa P68363 Homo sapiens
88 Gelsolin GELS_HUMAN 86 kDa P06396 Homo sapiens
89 Tetranectin TETN_HUMAN 23 kDa P05452 Homo sapiens
90 Alpha-1-acid glycoprotein 2 A1AG2_HUMAN 24 kDa P19652 Homo sapiens
91 Ig heavy chain V-III region GAL HV320_HUMAN 13 kDa P01781 Homo sapiens
92 IgGFc-binding protein FCGBP_HUMAN 572 kDa Q9Y6R7 Homo sapiens
93 Lactotransferrin TRFL_HUMAN 78 kDa P02788 Homo sapiens
94 Histone H4 H4_HUMAN 11 kDa P62805 Homo sapiens
95 Glutathione peroxidase 3 GPX3_HUMAN 26 kDa P22352 Homo sapiens
96 Histone H2B type 1-M H2B1M_HUMAN 14 kDa Q99879 Homo sapiens
97 Apolipoprotein M APOM_HUMAN 21 kDa O95445 Homo sapiens
98 E3 SUMO-protein ligase RanBP2 RBP2_HUMAN 358 kDa P49792 Homo sapiens
99 Ig kappa chain V-I region EU KV106_HUMAN 12 kDa P01598 Homo sapiens
100 Ig kappa chain V-I region Lay KV113_HUMAN 12 kDa P01605 Homo sapiens
101 Dermcidin OS = Homo sapiens DCD_HUMAN 11 kDa P81605 Homo sapiens
102 Leucine-rich repeats and LRIG2_HUMAN 119 kDa O94898 Homo sapiens
immunoglobulin-like domains
protein 2
103 Protein S100-A8 S10A8_HUMAN 11 kDa P05109 Homo sapiens
104 Glyceraldehyde-3-phosphate G3P_HUMAN 36 kDa P04406 Homo sapiens
dehydrogenase
105 Myosin-9 MYH9_HUMAN 227 kDa P35579 Homo sapiens
106 Ig kappa chain V-III region VG KV309_HUMAN 13 kDa P04433 Homo sapiens
(Fragment)
107 Monocyte differentiation antigen CD14_HUMAN 40 kDa P08571 Homo
sapiens
CD14
108 Heterogeneous nuclear HNRH1_HUMAN 49 kDa P31943 Homo sapiens
ribonucleoprotein H
109 ATP-dependent DNA helicase 2 KU70_HUMAN 70 kDa P12956 Homo sapiens
subunit 1
110 Protein TFG TFG_HUMAN 43 kDa Q92734 Homo sapiens
111 Coagulation factor XII FA12_HUMAN 68 kDa P00748 Homo sapiens
112 Filaggrin-2 FILA2_HUMAN 248 kDa Q5D862 Homo sapiens
113 Prothrombin THRB_HUMAN 70 kDa P00734 Homo sapiens
114 Sex hormone-binding globulin SHBG_HUMAN 44 kDa P04278 Homo sapiens
115 Heat shock cognate 71 kDa protein HSP7C_HUMAN 71 kDa P11142 Homo
sapiens
116 Histidine-rich glycoprotein HRG_HUMAN 60 kDa P04196 Homo sapiens
117 Heterogeneous nuclear ROA3_HUMAN 40 kDa P51991 Homo sapiens
ribonucleoprotein A3
118 Ig kappa chain V-III region B6 KV301_HUMAN 12 kDa P01619 Homo sapiens
119 Testis-expressed sequence 15 TEX15_HUMAN 315 kDa Q9BXT5 Homo sapiens
protein
120 Polyadenylate-binding protein 1 PABP1_HUMAN 71 kDa P11940 Homo
sapiens
121 Lipopolysaccharide-binding protein LBP_HUMAN 53 kDa P18428 Homo
sapiens
122 Lamina-associated polypeptide 2, LAP2A_HUMAN 75 kDa P42166 Homo
sapiens
isoform alpha
123 Desmoplakin DESP_HUMAN 332 kDa P15924 Homo sapiens
124 Protein S100-A9 S10A9_HUMAN 13 kDa P06702 Homo sapiens
125 Far upstream element-binding FUBP2_HUMAN 73 kDa Q92945 Homo sapiens
protein 2
126 Platelet glycoprotein Ib alpha chain GP1BA_HUMAN 69 kDa P07359 Homo
sapiens
127 Zinc finger and SCAN domain- ZSC29_HUMAN 97 kDa Q8IWY8 Homo sapiens
containing protein 29
128 Heterogeneous nuclear HNRH3_HUMAN 37 kDa P31942 Homo sapiens
ribonucleoprotein H3
129 Histone H3.3 H33_HUMAN 15 kDa P84243 Homo sapiens
130 Stress-70 protein, mitochondrial GRP75_HUMAN 74 kDa P38646 Homo
sapiens
131 Ig kappa chain V-I region AG KV101_HUMAN 12 kDa P01593 Homo sapiens
132 Elongation factor 1-alpha 1 EF1A1_HUMAN 50 kDa P68104 Homo sapiens
133 RNA-binding protein FUS FUS_HUMAN 53 kDa P35637 Homo sapiens
134 DNA repair and recombination RA54B_HUMAN 103 kDa Q9Y620 Homo sapiens
protein RAD54B
135 Hornerin HORN_HUMAN 282 kDa Q86YZ3 Homo sapiens
136 Tubulin beta chain TBB5_HUMAN 50 kDa P07437 Homo sapiens
137 Heterogeneous nuclear HNRPC_HUMAN 34 kDa P07910 Homo sapiens
ribonucleoproteins C1/C2
138 RecQ-mediated genome instability RMI1_HUMAN 70 kDa Q9H9A7 Homo
sapiens
protein 1
139 Desmocollin-1 DSC1_HUMAN 100 kDa Q08554 Homo sapiens
140 Xaa-Pro dipeptidase PEPD_HUMAN 55 kDa P12955 Homo sapiens
141 Junction plakoglobin PLAK_HUMAN 82 kDa P14923 Homo sapiens
142 U3 small nucleolar RNA-associated UT14C_HUMAN 87 kDa Q5TAP6 Homo
sapiens
protein 14 homolog C
143 Obscurin OBSCN_HUMAN 868 kDa Q5VST9 Homo sapiens
144 Elongation factor Tu, mitochondrial EFTU_HUMAN 50 kDa P49411 Homo
sapiens
145 Heterogeneous nuclear HNRPF_HUMAN 46 kDa P52597 Homo sapiens
ribonucleoprotein F
146 Desmoglein-1 DSG1_HUMAN 114 kDa Q02413 Homo sapiens
147 Heterogeneous nuclear ROA1_HUMAN 39 kDa P09651 Homo sapiens
ribonucleoprotein Al
148 ELAV-like protein 1 ELAV1_HUMAN 36 kDa Q15717 Homo sapiens
149 Pyrroline-5-carboxylate reductase 1, P5CR1_HUMAN 33 kDa P32322 Homo
sapiens
mitochondrial
150 Ig lambda chain V-I region WAH LV106_HUMAN 12 kDa P04208 Homo sapiens
151 Zinc finger and BTB domain- ZBT10_HUMAN 92 kDa Q96DT7 Homo sapiens
containing protein 10
152 Cystatin-C CYTC_HUMAN 16 kDa P01034 Homo sapiens
153 Major vault protein MVP_HUMAN 99 kDa Q14764 Homo sapiens
154 Phosphatidylcholine-sterol LCAT_HUMAN 50 kDa P04180 Homo sapiens
acyltransferase
155 Sulfhydryl oxidase 1 QSCN6_HUMAN 83 kDa O00391 Homo sapiens
156 Far upstream element-binding FUBP3_HUMAN 62 kDa Q96I24 Homo sapiens
protein 3
157 Ig delta chain C region IGHD_HUMAN 42 kDa P01880 Homo sapiens
158 Plasma serine protease inhibitor IPSP_HUMAN 46 kDa P05154 Homo
sapiens
159 Heterogeneous nuclear HNRPR_HUMAN 71 kDa O43390 Homo sapiens
ribonucleoprotein R
160 Hemoglobin subunit gamma-1 HBG1_HUMAN 16 kDa P69891 Homo sapiens
(+1)
161 TAR DNA-binding protein 43 TADBP_HUMAN 45 kDa Q13148 Homo sapiens
162 Neural cell adhesion molecule L1- CHL1_HUMAN 135 kDa O00533 Homo
sapiens
like protein
163 Bifunctional polynucleotide PNKP_HUMAN 57 kDa Q96T60 Homo sapiens
phosphatase/kinase
164 Fibrocystin PKHD1_HUMAN 447 kDa Q8TCZ9 Homo sapiens
165 Heterogeneous nuclear ROAA_HUMAN 36 kDa Q99729 Homo sapiens
ribonucleoprotein A/B
166 Peroxiredoxin-1 PRDX1_HUMAN 22 kDa Q06830 Homo sapiens
167 40S ribosomal protein S16 RS16_HUMAN 16 kDa P62249 Homo sapiens
168 Heat shock 70 kDa protein 1 HSP71_HUMAN 70 kDa P08107 Homo sapiens
169 40S ribosomal protein S25 RS25_HUMAN 14 kDa P62851 Homo sapiens
170 Fetuin-B FETUB_HUMAN 42 kDa Q9UGM5 Homo sapiens
171 Transferrin receptor protein 1 TFR1_HUMAN 85 kDa P02786 Homo sapiens
172 Myelin basic protein MBP_HUMAN 33 kDa P02686 Homo sapiens
173 Aldo-keto reductase family 1 AK1C1_HUMAN 37 kDa Q04828 Homo sapiens
member C1
174 Uncharacterized protein C19orf21 CS021_HUMAN 75 kDa Q8IVT2 Homo
sapiens
175 TATA-binding protein-associated RBP56_HUMAN 62 kDa Q92804 Homo
sapiens
factor 2N
176 ATP-dependent DNA helicase 2 KU86_HUMAN 83 kDa P13010 Homo sapiens
subunit 2
177 Sulfide:quinone oxidoreductase, SQRD_HUMAN 50 kDa Q9Y6N5 Homo sapiens
mitochondrial
178 Interleukin enhancer-binding factor ILF2_HUMAN 43 kDa Q12905 Homo
sapiens
2
179 Prostaglandin F2-alpha receptor PF2R_HUMAN 40 kDa P43088 Homo sapiens
180 Peptidase inhibitor 16 PI16_HUMAN 49 kDa Q6UXB8 Homo sapiens
181 Actin, alpha cardiac muscle 1 ACTC_HUMAN 42 kDa P68032 Homo sapiens
182 E3 SUMO-protein ligase RanBP2 RBP2_HUMAN 358 kDa P49792 Homo sapiens
Mitochondrial import inner
membrane translocase subunit
183 TIM50 TIM50_HUMAN 40 kDa Q3ZCQ8 Homo sapiens
184 Heterogeneous nuclear HNRPD_HUMAN 38 kDa Q14103 Homo sapiens
ribonucleoprotein D0
185 Interleukin enhancer-binding factor ILF3_HUMAN 95 kDa Q12906 Homo
sapiens
3
186 Heterogeneous nuclear HNRPG_HUMAN 42 kDa P38159 Homo sapiens
ribonucleoprotein G
187 40S ribosomal protein S4, X isoform RS4X_HUMAN 30 kDa P62701 Homo
sapiens
188 Caspase-14 CASPE_HUMAN 28 kDa P31944 Homo sapiens
189 Annexin A2 ANXA2_HUMAN 39 kDa P07355 Homo sapiens
190 Isocitrate dehydrogenase [NAD] IDH3A_HUMAN 40 kDa P50213 Homo sapiens
subunit alpha, mitochondrial
191 Cysteine-rich secretory protein 3 CRIS3_HUMAN 28 kDa P54108 Homo
sapiens
192 Myeloperoxidase PERM_HUMAN 84 kDa P05164 Homo sapiens
193 Transformation/transcription TRRAP_HUMAN 438 kDa Q9Y4A5 Homo sapiens
domain-associated protein
194 Isocitrate dehydrogenase [NAD] IDH3B_HUMAN 42 kDa 043837 Homo sapiens
subunit beta, mitochondrial
195 Collagen alpha-2(V) chain CO5A2_HUMAN 145 kDa P05997 Homo sapiens
196 Baculoviral IAP repeat-containing BIRC6_HUMAN 528 kDa Q9NR09 Homo
sapiens
protein 6
197 Bleomycin hydrolase BLMH_HUMAN 53 kDa Q13867 Homo sapiens
198 Poly(rC)-binding protein 1 PCBP1_HUMAN 37 kDa Q15365 Homo sapiens
199 Tubulin beta-3 chain TBB3_HUMAN 50 kDa Q13509 Homo sapiens
200 60 kDa heat shock protein, CH60_HUMAN 61 kDa P10809 Homo sapiens
mitochondrial
201 ATP synthase subunit beta, ATPB_HUMAN 57 kDa P06576 Homo sapiens
mitochondrial
202 40S ribosomal protein S18 RS18_HUMAN 18 kDa P62269 Homo sapiens
203 DnaJ homolog subfamily A member DNJA1_HUMAN 45 kDa P31689 Homo
sapiens
1
204 RNA-binding motif, single-stranded- RBMS1_HUMAN 45 kDa P29558 Homo
sapiens
interacting protein 1
205 40S ribosomal protein S19 RS19_HUMAN 16 kDa P39019 Homo sapiens
206 Tubulin alpha-1 chain TBA1_HUMAN 50 kDa P68366 Homo sapiens
207 Protein Hook homolog 1 HOOK1_HUMAN 85 kDa Q9UJC3 Homo sapiens
208 Brother of CDO BOC_HUMAN 121 kDa Q9BWV1 Homo sapiens
209 Signal recognition particle 54 kDa SRP54_HUMAN 56 kDa P61011 Homo
sapiens
protein
210 Trinucleotide repeat-containing 6B TNC6B_HUMAN 194 kDa Q9UPQ9 Homo
sapiens
protein
211 Aldehyde dehydrogenase, dimeric AL3A1_HUMAN 50 kDa P30838 Homo
sapiens
NADP-preferring
212 40S ribosomal protein S3 RS3_HUMAN 27 kDa P23396 Homo sapiens
213 Fatty aldehyde dehydrogenase AL3A2_HUMAN 55 kDa P51648 Homo sapiens
214 Electron transfer flavoprotein ETFA_HUMAN 35 kDa P13804 Homo sapiens
subunit alpha, mitochondrial
215 RNA-binding protein EWS EWS_HUMAN 68 kDa Q01844 Homo sapiens
216 Histone H2A.V H2AV_HUMAN 14 kDa Q71UI9 Homo sapiens
Heterogeneous nuclear (+1)
217 ribonucleoprotein Q HNRPQ_HUMAN 70 kDa O60506 Homo sapiens
218 Pyrroline-5-carboxylate reductase P5CR2_HUMAN 34 kDa Q96C36 Homo
sapiens
219 Prohibitin PHB_HUMAN 30 kDa P35232 Homo sapiens
220 RuvB-like 1 RUVB1_HUMAN 50 kDa Q9Y265 Homo sapiens
221 Protein-glutamine gamma- TGM2_HUMAN 77 kDa P21980 Homo sapiens
glutamyltransferase
222 Ubiquitin-associated protein 2 UBAP2_HUMAN 117 kDa Q5T6F2 Homo
sapiens
223 Latent-transforming growth factor LTB1L_HUMAN 187 kDa Q14766 Homo
sapiens
beta-binding protein 1
224 Thioredoxin domain-containing TXND2_HUMAN 60 kDa Q86VQ3 Homo sapiens
protein 2
225 Leucine-rich repeat-containing LRC17_HUMAN 52 kDa Q8N6Y2 Homo sapiens
protein 17
226 Protein FAM105B F105B_HUMAN 40 kDa Q96BN8 Homo sapiens
227 Myosin-Ia MYO1A_HUMAN 118 kDa Q9UBC5 Homo sapiens
Ras GTPase-activating-like protein
228 IQGAP2 IQGA2_HUMAN 181 kDa Q13576 Homo sapiens
229 26Sproteasome non-ATPase PSMD1_HUMAN 102 kDa Q99460 Homo sapiens
regulatory subunit 1
230 Actin, alpha skeletal muscle ACTS_HUMAN 42 kDa P68133 Homo sapiens
231 Cleavage stimulation factor subunit CSTF3_HUMAN 83 kDa Q12996 Homo
sapiens
3
232 Clusterin CLUS_HUMAN 52 kDa P10909 Homo sapiens
233 Fibrocystin-L PKHL1_HUMAN 466 kDa Q86WI1 Homo sapiens
234 InaD-like protein INADL_HUMAN 196 kDa Q8NI35 Homo sapiens
235 Keratin, type I cuticular Ha1 K1H1_HUMAN 47 kDa Q15323 Homo sapiens
236 Keratin, type I cuticular Ha2 K1H2_HUMAN 50 kDa Q14532 Homo sapiens
237 Keratin, type I cuticular Ha6 K1H6_HUMAN 52 kDa O76013 Homo sapiens
238 Keratin, type I cytoskeletal 10 K1C10_HUMAN 59 kDa P13645 Homo
sapiens
239 Keratin, type I cytoskeletal 14 K1C14_HUMAN 52 kDa P02533 Homo
sapiens
240 Keratin, type I cytoskeletal 16 K1C16_HUMAN 51 kDa P08779 Homo
sapiens
241 Keratin, type I cytoskeletal 18 K1C18_HUMAN 48 kDa P05783 Homo
sapiens
242 Keratin, type I cytoskeletal 19 K1C19_HUMAN 44 kDa P08727 Homo
sapiens
243 Keratin, type I cytoskeletal 9 K1C9_HUMAN 62 kDa P35527 Homo sapiens
244 Keratin, type II cuticular Hb1 KRT81_HUMAN 55 kDa Q14533 Homo sapiens
245 Keratin, type II cuticular Hb5 KRT85_HUMAN 56 kDa P78386 Homo sapiens
246 Keratin, type II cytoskeletal 1 K2C1_HUMAN 66 kDa P04264 Homo sapiens
247 Keratin, type II cytoskeletal 1b K2C1B_HUMAN 62 kDa Q7Z794 Homo
sapiens
248 Keratin, type II cytoskeletal 2 K22E_HUMAN 65 kDa P35908 Homo sapiens
epidermal
249 Keratin, type II cytoskeletal 4 K2C4_HUMAN 57 kDa P19013 Homo sapiens
250 Keratin, type II cytoskeletal 5 K2C5_HUMAN 62 kDa P13647 Homo sapiens
251 Keratin, type II cytoskeletal 6B K2C6B_HUMAN 60 kDa P04259 Homo
sapiens
252 Keratin, type II cytoskeletal 7 K2C7_HUMAN 51 kDa P08729 Homo sapiens
253 Keratin, type II cytoskeletal 8 K2C8_HUMAN 54 kDa P05787 Homo sapiens
254 Lymphoid-restricted membrane LRMP_HUMAN 62 kDa Q12912 Homo sapiens
protein
255 Myosin light chain 1, skeletal muscle MLE1_HUMAN 21 kDa P05976 Homo
sapiens
isoform
256 Myosin-2 MYH2_HUMAN 223 kDa Q9UKX2 Homo sapiens
257 Protein Shroom3 SHRM3_HUMAN 216 kDa Q8TF72 Homo sapiens
258 Ras-related GTP-binding protein A RRAGA_HUMAN 37 kDa Q7L523 Homo
sapiens
259 Sushi, nidogen and EGF-like SNED1_HUMAN 152 kDa Q8TER0 Homo sapiens
domain-containing protein 1
260 Retinol-binding protein 4 RET4_HUMAN 23 kDa P02753 Homo sapiens
TABLE-US-00002
TABLE 2
Proteins identified in spent media of human embryos. *
SEQID Protein Molecular Accession Day of Embryo Nature of
NO. Protein Name Entry Name Weight Number Development Viability Marker
261 14-3-3 protein zeta/delta 1433Z_HUMAN 28 kDa P63104 D 1
262 6-phosphofructokinase, muscle K6PF_HUMAN 85 kDa P08237 D 1
type, isoform 1
263 6-phosphofructokinase, muscle K6PF_HUMAN 82 kDA P08237-2 D 1
type, isoform 1
264 Adenylate kinase isoenzyme 1 KAD1_HUMAN 22 kDa P00568 D 1
265 Alpha-actinin-2 ACTN2_HUMAN 104 kDa P35609 D 1
266 Beta-enolase, isoform 1 ENOB_HUMAN 47 kDa P13929 D 1
267 Beta-enolase, isoform 2 ENOB_HUMAN 44 kDa P13929-2 D 1
268 Beta-enolase, isoform 3 ENOB_HUMAN 42 kDa P13929-3 D 1
269 Creatine kinase M-type KCRM_HUMAN 43 kDa P06732 D 1
270 Fructose-bisphosphate aldolase A ALDOA_HUMAN 39 kDa P04075 D 1
271 Glycogen phosphorylase, muscle form PYGM_HUMAN 97 kDa P11217 D 1
272 L-lactate dehydrogenase A chain LDHA_HUMAN 37 kDa P00338 D 1
273 Myosin-1 MYH1_HUMAN 223 kDa P12882 D 1
274 Myosin-2 MYH2_HUMAN 223 kDa Q9UKX2 D 1
275 Myosin-3 MYH3_HUMAN 224 kDa P11055 D 1
276 Myosin-4 MYH4_HUMAN 223 kDa Q9Y623 D 1
277 Myosin-6 MYH6_HUMAN 224 kDa P13533 D 1
278 Myosin-7 MYH7_HUMAN 223 kDa P12883 D 1
279 Myosin-8 MYH8_HUMAN 223 kDa P13535 D 1
280 Phosphoglucomutase-1, isoform 1 PGM1_HUMAN 61 kDa P36871 D 1
281 Phosphoglucomutase-1, isoform 2 PGM1_HUMAN 64 kDa P36871-2 D 1
282 Phosphoglycerate kinase 1 PGK1_HUMAN 45 kDa P00558 D 1
283 Phosphoglycerate mutase 1 PGAM1_HUMAN 29 kDa P18669 D 1
284 Pyruvate kinase isozyme M2 KPYM_HUMAN 58 kDa P14618 D 1
285 Pyruvate kinase isozyme M1 KPYM_HUMAN 58 kDa P14618-2 D 1
286 Sarcoplasmic/endoplasmic reticulum AT2A1_HUMAN 110 kDa O14983 D 1
calcium ATPase 1, Isoform SERCA1B
287 Sarcoplasmic/endoplasmic reticulum AT2A1_HUMAN 109 kDa O14983-2 D 1
calcium ATPase 1, Isoform SERCA1A
288 Sarcoplasmic/endoplasmic reticulum AT2A2_HUMAN 115 kDa P16615 D 1
calcium ATPase 2, isoform 1
289 Sarcoplasmic/endoplasmic reticulum AT2A2_HUMAN 110 kDa P16615-2 D 1
calcium ATPase 2, isoform 2
290 Sarcoplasmic/endoplasmic reticulum AT2A2_HUMAN 110 kDa P16615-3 D 1
calcium ATPase 2, isoform 3
291 Sarcoplasmic/endoplasmic reticulum AT2A2_HUMAN 112 kDa P16615-4 D 1
calcium ATPase 2, isoform 4
292 Sarcoplasmic/endoplasmic reticulum AT2A2_HUMAN 110 kDa P16615-5 D 1
calcium ATPase 2, isoform 5
293 Triosephosphate isomerase, isoform 1 TPIS_HUMAN 27 kDa P60174 D 1
294 Triosephosphate isomerase, isoform 2 TPIS_HUMAN 27 kDa P60174-2| D 1
295 Tropomyosin alpha-1 chain, isoform 1 TPM1_HUMAN 33 kDa P09493 D 1
296 Tropomyosin alpha-1 chain, isoform 2 TPM1_HUMAN 27 kDa P09493-2 D 1
297 Tropomyosin alpha-1 chain, isoform 3 TPM1_HUMAN 33 kDa P09493-3 D 1
298 Tropomyosin alpha-1 chain, isoform 4 TPM1_HUMAN 33 kDa P09493-4 D 1
299 Tropomyosin alpha-1 chain, isoform 5 TPM1_HUMAN 28 kDa P09493-6 D 1
300 Tropomyosin alpha-3 chain, isoform 1 TPM3_HUMAN 33 kDa P06753 D 1
301 Tropomyosin alpha-3 chain, isoform 2 TPM3_HUMAN 29 kDa P06753-2 D 1
302 Tropomyosin alpha-3 chain, isoform 3 TPM3_HUMAN 29 kDa P06753-3 D 1
303 Collagen alpha-1(I) chain CO1A1_HUMAN 139 kDa P02452 D 1/D 3
304 Cystatin-A CYTA_HUMAN 11 kDa P01040 D 1/D 3
305 Filaggrin FILA_HUMAN 435 kDa P20930 D 1/D 3
306 Keratinocyte proline-rich protein KPRP_HUMAN 64 kDa Q5T749 D 1/D 3
307 Selenium-binding protein 1, isoform 1 SBP1_HUMAN 52 kDa Q13228 D 3
308 Selenium-binding protein 1, isoform 2 SBP1_HUMAN 45 kDa Q13228-2 D 3
309 Fatty acid-binding protein, epidermal FABP5_HUMAN 15 kDa Q01469 D 1/D
3/D 5
310 Galectin-7 LEG7_HUMAN 15 kDa P47929 D 1/D 3/D 5 Increased with
implantation
Success
311 Ubiquitin UBIQ_HUMAN X kDa P02248 D 1/D 3/D 5 Increased with
implantation
Success
312 Calmodulin-like protein 5 CALL5_HUMAN 16 kDa Q9NZT1 D 1/D 3/D 5
313 14-3-3 protein sigma 1433S_HUMAN 28 kDa P31947 D 1/D 3/D 5 Increased
with
implantation
Success
314 Histone H2A type 1-A H2A1A_HUMAN 14 kDa Q96QV6 D 1/D 3/D 5
315 Histone H3.1t H31T_HUMAN 16 kDa Q16695 D 1/D 3/D 5
316 Histone H1.3 H13_HUMAN 22 kDa P16402 D 3/D 5
317 Lysozyme C LYSC_HUMAN 17 kDa P61626 D 3/D 5 Increased with
Aneuploidy
318 Plakophilin-1, isoform 2 PKP1_HUMAN 83 kDa Q13835 D 3/D 5 Increased
with
implantation
Success
319 Plakophilin-1, isoform 1 PKP1_HUMAN 80 kDa Q13835-2 D 3/D 5 Increased
with
implantation
Success
320 Abhydrolase domain-containing AB12B_HUMAN 41 kDa Q7Z5M8 D 3/D 5
protein 12B, isoform 1
321 Abhydrolase domain-containing AB12B_HUMAN 32 kDa Q7Z5M8-2 D 3/D 5
protein 12B, isoform 2
322 Abhydrolase domain-containing AB12B_HUMAN 29 kDa Q7Z5M8-3 D 3/D 5
protein 12B, isoform 3
323 Abhydrolase domain-containing AB12B_HUMAN 13 kDa Q7Z5M8-4 D 3/D 5
protein 12B, isoform 4
324 Abhydrolase domain-containing AB12B_HUMAN 25 kDa Q7Z5M8-5 D 3/D 5
protein 12B, isoform 5
325 40S ribosomal protein SA RSSA_HUMAN 33 kDa P08865 D 5
326 Arginase-1, isoform 1 ARGI1_HUMAN 35 kDa P05089 D 5
327 Arginase-1, isoform 2 ARGI1_HUMAN 36 kDa P05089-2 D 5
328 Arginase-1, isoform 3 ARGI1_HUMAN 25 kDa P05089-3 D 5
329 Beta-2-microglobulin B2MG_HUMAN 14 kDa P61769 D 5
330 Carbonic anhydrase 2 CAH2_HUMAN 29 kDa P00918 D 5
331 Carboxypeptidase A4 CBPA4_HUMAN 47 kDa Q9UI42 D 5
332 Catalase CATA_HUMAN 60 kDa P04040 D 5
333 Cathepsin D CATD_HUMAN 45 kDa P07339 D 5
334 CD44 antigen, isoform 1 CD44_HUMAN 81 kDa P16070 D 5 Increased with
implantation
Success
335 CD44 antigen, isoform 2 CD44_HUMAN 3 kDa P16070-2 D 5 Increased with
implantation
Success
336 CD44 antigen, isoform 3 CD44_HUMAN 78 kDa P16070-3 D 5 Increased with
implantation
Success
337 CD44 antigen, isoform 4 CD44_HUMAN 77 kDa P16070-4 D 5 Increased with
implantation
Success
338 CD44 antigen, isoform 5 CD44_HUMAN 81 kDa P16070-5 D 5 Increased with
implantation
Success
339 CD44 antigen, isoform 6 CD44_HUMAN 77 kDa P16070-6 D 5 Increased with
implantation
Success
340 CD44 antigen, isoform 7 CD44_HUMAN 78 kDa P16070-7 D 5 Increased with
implantation
Success
341 CD44 antigen, isoform 8 CD44_HUMAN 74 kDa P16070-8 D 5 Increased with
implantation
Success
342 CD44 antigen, isoform 9 CD44_HUMAN 74 kDa P16070-9 D 5 Increased with
implantation
Success
343 CD44 antigen, isoform 10 CD44_HUMAN 53 kDa P16070-10 D 5 Increased
with
implantation
Success
344 CD44 antigen, isoform 11 CD44_HUMAN 47 kDa P16070-11 D 5 Increased
with
implantation
Success
345 CD44 antigen, isoform 12 CD44_HUMAN 39 kDa P16070-12 D 5 Increased
with
implantation
Success
346 CD44 antigen, isoform 13 CD44_HUMAN 46 kDa P16070-13 D 5 Increased
with
implantation
Success
347 CD44 antigen, isoform 14 CD44_HUMAN 43 kDa P16070-14 D 5 Increased
with
implantation
Success
348 CD44 antigen, isoform 15 CD44_HUMAN 32 kDa P16070-15 D 5 Increased
with
implantation
Success
349 CD44 antigen, isoform 16 CD44_HUMAN 73 kDa P16070-16 D 5 Increased
with
implantation
Success
350 CD44 antigen, isoform 17 CD44_HUMAN 76 kDa P16070-17 D 5 Increased
with
implantation
Success
351 Creatine kinase B-type KCRB_HUMAN 43 kDa P12277 D 5
352 Dermokine, isoform 1 DMKN_HUMAN 47 kDa Q6E0U4 D 5
353 Dermokine, isoform 2 DMKN_HUMAN 47 kDa Q6E0U4-2 D 5
354 Dermokine, isoform 3 DMKN_HUMAN 37 kDa Q6E0U4-3 D 5
355 Dermokine, isoform 4 DMKN_HUMAN 45 kDa Q6E0U4-4 D 5
356 Dermokine, isoform 5 DMKN_HUMAN 38 kDa Q6E0U4-5 D 5
357 Dermokine, isoform 6 DMKN_HUMAN 42 kDa Q6E0U4-6 D 5
358 Dermokine, isoform 7 DMKN_HUMAN 35 kDa Q6E0U4-7 D 5
359 Dermokine, isoform 8 DMKN_HUMAN 20 kDa Q6E0U4-8 D 5
360 Dermokine, isoform 9 DMKN_HUMAN 19 kDa Q6E0U4-9 D 5
361 Dermokine, isoform 10 DMKN_HUMAN 17 kDa Q6E0U4-10 D 5
362 Dermokine, isoform 11 DMKN_HUMAN 15 kDa Q6E0U4-11 D 5
363 Dermokine, isoform 12 DMKN_HUMAN 16 kDa Q6E0U4-12 D 5
364 Dermokine, isoform 13 DMKN_HUMAN 14 kDa Q6E0U4-13 D 5
365 Dermokine, isoform 14 DMKN_HUMAN 15 kDa Q6E0U4-14 D 5
366 Dermokine, isoform 15 DMKN_HUMAN 10 kDa Q6E0U4-15 D 5
367 Extracellular glycoprotein lacritin LACRT_HUMAN 14 kDa Q9GZZ8 D 5
368 Ferritin light chain (Ferritin L FRIL_HUMAN 20 kDa P02792 D 5
Increased with
subunit) implantation
Success
369 Fibrinogen gamma chain, isoform FIBG_HUMAN 52 kDa P02679 D 5
gamma-B
370 Fibrinogen gamma chain, isoform FIBG_HUMAN 49 kDa P02679-2 D 5
gamma-A
371 Gamma-enolase ENOG_HUMAN 47 kDa P09104 D 5 Increased with
implantation
Success
372 Gamma-glutamylcyclotransferase, GGCT_HUMAN 21 kDa O75223 D 5
isoform 1
373 Gamma-glutamylcyclotransferase, GGCT_HUMAN 13 kDa O75223-2 D 5
isoform 2
374 Heat shock protein beta-1 HSPB1_HUMAN 23 kDa P04792 D 5 Increased with
implantation
Success
375 Histidine ammonia-lyase HUTH_HUMAN 73 kDa P42357 D 5
376 Ig mu chain C region, isoform 1 IGHM_HUMAN 49 kDa P01871 D 5
377 Ig mu chain C region, isoform 2 IGHM_HUMAN 52 kDa P01871-2 D 5
378 Interleukin-1 family member 7 IL1F7_HUMAN 224 kDa Q9NZH6 D 5
precursor, isoform B
379 Interleukin-1 family member 7 IL1F7_HUMAN 22 kDa Q9NZH6-2 D 5
precursor, isoform A
380 Interleukin-1 family member 7 IL1F7_HUMAN 20 kDa Q9NZH6-2 D 5
precursor, isoform C
381 Interleukin-1 family member 7 IL1F7_HUMAN 22 kDa Q9NZH6-2 D 5
precursor, isoform D
382 Interleukin-1 family member 7 IL1F7_HUMAN 17 kDa Q9NZH6-2 D 5
precursor, isoform E
383 Lipocalin-1 LCN1_HUMAN 19 kDa P31025 D 5 Increased with
Aneuploidy
384 Methionine synthase METH_HUMAN 141 kDa Q99707 D 5
385 Peroxiredoxin-2 PRDX2_HUMAN 22 kDa P32119 D 5
386 Proprotein convertase PCSK9_HUMAN 74 kDa Q8NBP7 D 5
subtilisin/kexin type 9, isoform 1
387 Proprotein convertase PCSK9_HUMAN 21 kDa Q8NBP7-2 D 5
subtilisin/kexin type 9, isoform 2
388 Protein POF1B, isoform 1 POF1B_HUMAN 69 kDa Q8WVV4 D 5
389 Protein POF1B, isoform 2 POF1B_HUMAN 68 kDa Q8WVV4-2 D 5
390 Protein POF1B, isoform 3 POF1B_HUMAN 35 kDa Q8WVV4-3 D 5
391 Protein S100-A7 S10A7_HUMAN 11 kDa P31151 D 5 Increased with
implantation
Success
392 Protein-glutamine gamma- TGM3_HUMAN 77 kDa Q08188 D 5
glutamyltransferase E
393 Pyruvate kinase isozymes M1/M2, KPYM_HUMAN 58 kDa P14618 D 5
isoform M2
394 Pyruvate kinase isozymes M1/M2, KPYM_HUMAN 58 kDa P14618-2 D 5
isoform M1
395 Receptor-type tyrosine-protein PTPRG_HUMAN 162 kDa P23470 D 5
phosphatase gamma, isoform 1
396 Receptor-type tyrosine-protein PTPRG_HUMAN 159 kDa P23470-2 D 5
phosphatase gamma, isoform 2
397 Serine protease inhibitor Kazal-type ISK5_HUMAN 121 kDa Q9NQ38 D 5
Increased with
5, isoform f1 implantation
Success
398 Serine protease inhibitor Kazal-type ISK5_HUMAN 104 kDa Q9NQ38 D 5
Increased with
5, isoform short implantation
Success
399 Serine protease inhibitor Kazal-type ISK5_HUMAN 124 kDa Q9NQ38 D 5
Increased with
5, isoform long implantation
Success
400 Serpin B3, isoform 1 SPB3_HUMAN 45 kDa P29508 D 5
401 Serpin B3, isoform 2 SPB3_HUMAN 39 kDa P29508-2 D 5
402 Uteroglobin UTER_HUMAN 10 kDa P11684 D 5 Increased with
implantation
Success
403 V-set and immunoglobulin domain- VSIG8_HUMAN 44 kDa Q5VU13 D 5
Increased with
containing protein 8, isoform 1 implantation
Success
404 V-set and immunoglobulin domain- VSIG8_HUMAN 25 kDa D 5 Increased
with
containing protein 8, isoform 1 implantation
Success
* All Proteins are from Homo Sapiens
Example 2
Secretome Profiling to Predict Implantation Potential of a Human Embryo
[0047] The following nonlimiting example teaches by way of illustration,
not by limitation, the fabrication and employment of a customized
immunoassay test kit for secretome profiling of a human embryo.
Immunoassay test kit fabrication occurs by modifying the well surfaces of
a 96-well microtiter plate. Each separate well of the microtiter plate is
incubated with an unlabelled capture antibody that recognizes one
specific protein from the secretome panel. Custom capture antibodies are
purchased from Rockland Immunochemicals, Inc. After incubation for 12
hours at 4.degree. C., capture antibodies passively adsorb to the well
surface. Subsequently, all wells of the 96-well plate are washed three
times with a buffer comprising 1.times. phosphate buffer saline and 0.1%
Tween-20 (PBST), blocked with 1% bovine serum albumin (BSA) for 1 hour at
ambient temperature and washed three times with PBST.
[0048] Human cleavage-stage embryos are cultured in 10 .mu.L drops of G1
supplemented with 2.5 mg/mL recombinant albumin under oil at 37.degree.
C., 6% CO.sub.2, 5% O.sub.2 for 24 hours. The embryos are washed twice in
G2 culture media and further cultured in 10 .mu.L, drops of G2
supplemented with 2.5 mg/mL recombinant albumin under oil at 37.degree.
C., 6% CO.sub.2, 5% O.sub.2 for 48 hours with a fresh drop of G2 media
added after 24 hours. Spent media samples of blastocysts are transferred
into 0.65 mL Eppendorf tubes. Control groups comprise media cultured and
collected under the same conditions but without embryos.
[0049] Secretome profiling proceeds using spent media culture in a
sandwich ELISA format. The spent media culture is diluted into phosphate
buffer saline (PBS) buffer and further distributed to separate wells of
said customized microtiter plate. After incubation for one hour at room
temperature, the samples are aspirated and all wells are washed four
times with PBST buffer. Primary antibodies, each recognizing a distinct
protein from the secretome panel, are distributed into wells that
correspond to appropriate capture antibodies and permitted to incubate
for one hour at room temperature. The wells are washed four times with
PBST and incubated with a secondary antibody conjugated with an enzyme.
The term "secondary antibody" refers to an antibody that binds to primary
antibodies and may be conjugated with detection probes such as enzymes or
fluorophores. In one embodiment, the secondary antibody contains the
horseradish peroxidase (HRP) enzyme that converts the chromogenic
substrate, 3,3',5,5'-tetramethylbenzidene (TMB), into a blue product
quantified at 655 nm on a spectrophotometer. In another embodiment, the
HRP enzymatic reaction is stopped with a solution containing sulfuric
acid and quantified at 450 nm.
[0050] In one embodiment, secretome profiling of a secretome panel of 261
individual proteins (Table 1), each selected for significant
developmental competence and implantation potential, are monitored using
said ELISA immunoassay test kit. In another embodiment, secretome
profiling of a secretome panel comprising 37 proteins (Table 2) are
monitored using said ELISA immunoassay test kit. Secretome profiling of
the 37 individual proteins (Table 2) from spent media of human embryos
correlates with embryonic viability and euploidy.
[0051] Changes may be made in the above methods and systems without
departing from the scope hereof. It should be noted that the matter
contained in the above description or shown in the accompanying drawings
should be interpreted as illustrative and not in a limiting sense. The
following claims are intended to cover all generic and specific features
described herein, as well as all statements of the scope of the present
method and system and reasonable variations thereof, which, as a matter
of language, might be said to fall therebetween.
Sequence CWU
0
SQTB
SEQUENCE LISTING
The patent application contains a lengthy "Sequence Listing" section. A
copy of the "Sequence Listing" is available in electronic form from the
USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110245592A1).
An electronic copy of the "Sequence Listing" will also be available from
the USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
* * * * *