U.S. patent application number 12/418713 was filed with the patent office on 2009-07-30 for system and sequential culture media for in vitro fertilization.
Invention is credited to David K. GARDNER, Michelle T. LANE, Peter SVALANDER.
Application Number | 20090191534 12/418713 |
Document ID | / |
Family ID | 22746469 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191534 |
Kind Code |
A1 |
GARDNER; David K. ; et
al. |
July 30, 2009 |
SYSTEM AND SEQUENTIAL CULTURE MEDIA FOR IN VITRO FERTILIZATION
Abstract
Instead of immersing human reproductive cells in a single
culture medium throughout the various procedures used in IVF, a
process is provided by which the reproductive cells may be moved
through a sequence of distinct culture media as the various IVF
procedures are carried out. In one implementation, the culture
media specifically formulated to provide a physical environment
similar to that found within the female reproductive tract and
conducive to growth and development of human reproductive cells
during the various stages of the IVF process. In this regard,
specifically formulated culture media can be applied to support the
reproductive cells in one or more of the following procedures:
oocyte retrieval and handling; oocyte maturation; ordinary
fertilization; oocyte, zygote and embryo examination and biopsy;
embryonic development to the eight-cell stage; embryonic
development to the blastocyst stage; embryo transfer, and
cryopreservation.
Inventors: |
GARDNER; David K.;
(Highlands Ranch, CO) ; LANE; Michelle T.;
(Hawthorndene, AU) ; SVALANDER; Peter; (Molnlycke,
SE) |
Correspondence
Address: |
FOLEY & LARDNER LLP
150 EAST GILMAN STREET, P.O. BOX 1497
MADISON
WI
53701-1497
US
|
Family ID: |
22746469 |
Appl. No.: |
12/418713 |
Filed: |
April 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11937200 |
Nov 8, 2007 |
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12418713 |
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11400746 |
Apr 7, 2006 |
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11937200 |
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10895809 |
Jul 21, 2004 |
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11400746 |
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10322914 |
Dec 18, 2002 |
6838235 |
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10895809 |
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09201594 |
Nov 30, 1998 |
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10322914 |
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Current U.S.
Class: |
435/1.1 |
Current CPC
Class: |
C12N 5/061 20130101;
C12N 2501/105 20130101; C12N 2501/31 20130101; C12N 2500/25
20130101; C12N 5/0609 20130101; C12N 2501/11 20130101; C12N 5/0604
20130101; A01N 1/021 20130101; C12N 2500/60 20130101; C12N 2517/10
20130101 |
Class at
Publication: |
435/1.1 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Claims
1. A method for performing an in vitro fertilization process,
comprising a. placing a gamete, zygote or embryo in a first in
vitro fertilization medium during a first in vitro fertilization
procedure wherein the in vitro fertilization medium comprises: (i)
water, (ii) a buffer present in an amount to buffer the in vitro
fertilization medium with physiological pH, (iii) ionic
constituents present in amounts that support growth and development
growth and development of the gamete, zygote or embryo, wherein the
ionic constituents include sodium, potassium, calcium and
magnesium, and (iv) alanyl-glutamine and one or more non-essential
amino acids present in amounts to prevent osmotic shock of the
gamete, zygote or embryo.
2. The method of claim 1 wherein the in vitro fertilization media
further comprises: (v) one or more of glucose, lactate and pyruvate
present in an amount sufficient to provide a source of energy for
the gamete, zygote or embryo.
3. The method of claim 1 wherein the ionic constituents further
include phosphate.
4. The method of claim 1 wherein the in vitro fertilization medium
further comprises: (v) one or more essential amino acids.
5. The method of claim 1 wherein the in vitro fertilization medium
further comprises: (v) a hormone, one or more growth factors, an
antibiotic or one or more vitamins.
6. The method of claim 1 wherein the in vitro fertilization medium
further comprises: (v) one or more of human serum albumin,
hyaluronate, polyvinylpyrrolidine, cysteamine, hyaluronidase and
glutathione.
7. The method of claim 1 wherein the in vitro fertilization medium
comprises the components shown in Table 1 in concentrations shown
in column B of Table 1.
8. The method of claim 1 wherein the in vitro fertilization medium
comprises the components shown in Table 2 in concentrations shown
in column B of Table 2.
9. The method of claim 1 wherein the in vitro fertilization medium
comprises the components shown in Table 4 in concentrations shown
in column B of Table 4.
10. The method of claim 1 wherein the in vitro fertilization medium
comprises the components shown in Table 5 in concentrations shown
in column B of Table 5.
11. The method of claim 1 wherein the in vitro fertilization medium
comprises the components shown in Table 6 in concentrations shown
in column B of Table 6.
12. The method of claim 1 wherein the in vitro fertilization medium
comprises the components shown in Table 7 in concentrations shown
in column B of Table 7.
13. The method of claim 1 further comprising: (b) performing a
first in vitro fertilization procedure on the gamete, zygote or
embryo in the first in vitro fertilization medium.
14. The method of claim 1 further comprising: (b) transferring the
gamete, zygote or embryo from the first in vitro fertilization
medium into a second in vitro fertilization medium for a second in
vitro fertilization procedure, wherein the first and second in
vitro fertilization media have different compositions but
integrated formulations sharing a core group of ionic and
non-essential amino acid constituents thereby minimizing trauma to
the gamete, zygote or embryo when they are moved from the first in
vitro fertilization medium to the second in vitro fertilization
medium.
15. The method of claim 14 further comprising: (c) sequentially
transferring the gamete, zygote or embryo from the second in vitro
fertilization medium into one or more of a third, fourth, fifth,
sixth or seventh successive in vitro fertilization medium for one
or more of a third, fourth, fifth, sixth or seventh successive in
vitro fertilization procedure, wherein the second and third,
fourth, fifth, sixth or seventh successive in vitro fertilization
media have different compositions but integrated formulations
sharing a core group of ionic and non-essential amino acid
constituents thereby minimizing trauma to the gamete, zygote or
embryo when they are moved into the successive in vitro
fertilization medium.
16. The method of claim 1 wherein the first in vitro fertilization
procedure is oocyte retrieval and handling, oocyte maturation or
oocyte fertilization.
17. The method of claim 1 wherein the first in vitro fertilization
procedure is embryonic development to the eight cell stage or
embryonic development to the blastocyst stage.
18. The method of claim 1 wherein the first in vitro fertilization
procedure is embryo transfer.
19. The method of claim 14 wherein one of the first in vitro
fertilization procedure and second in vitro fertilization procedure
is cryopreservation of the gamete, zygote or embryo.
20. The method of claim 1 wherein the buffer is a MOPS buffer.
21. The method of claim 20, wherein the in vitro fertilization
medium is phosphate free.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/937,200, filed Nov. 8, 2007, which is a
continuation of U.S. patent application Ser. No. 11/400,746, filed
Apr. 7, 2006, now abandoned, which is a continuation of U.S. patent
application Ser. No. 10/895,809, filed on Jul. 21, 2004, now
abandoned, which is a divisional of U.S. patent application Ser.
No. 10/322,914, filed Dec. 18, 2002, now U.S. Pat. No. 6,838,235,
which is a continuation of U.S. patent application Ser. No.
09/201,594, filed Nov. 30, 1998, now abandoned.
FIELD OF THE INVENTION
[0002] The present invention relates generally to human in vitro
fertilization (IVF) and, in particular, to a sequential culture
media system and process to be used in oocyte retrieval, handling
and maturation, sperm preparation, fertilization, embryo
development and transfer, and cryopreservation. The invention
provides the gametes, zygote and developing embryo with a physical
environment adapted to their physiological needs, so supporting
their normal growth and development in vitro and increasing the
likelihood of successful pregnancy.
BACKGROUND OF THE INVENTION
[0003] In vitro fertilization seeks to duplicate, to a large
extent, the conditions and processes normally occurring within the
female reproductive system that are necessary to oocyte
development, fertilization and early embryonic development. In the
clinic and laboratory, IVF involves several discrete procedures,
such as collection of the oocytes from the ovary of the mother,
preparation of the sperm, fertilization, and, once fertilized eggs
are identified, a period of early embryonic development, and then
transfer of the embryo to the mother's uterus. Each of these steps
can take place over extended periods of time, during which the
individual cells involved have a continuing need for nutrients, and
are subjected to significant stress as a result of clinical
manipulation and changed environmental conditions.
[0004] During IVF, a culture medium is ordinarily used as a
substitute for the fluid secreted by the female reproductive tract
that would ordinarily surround the gametes, zygote, and developing
embryo. Most laboratories carrying out IVF use a single culture
medium throughout the various procedures involved. In a number of
laboratories, there has been a tendency to use tissue culture media
for IVF procedures, such as Ham's F-- 10, which is formulated to
support somatic cell growth, not gamete or embryonic cell growth.
Tissue culture media generally are complicated systems, containing
an array of amino acids, vitamins and other constituents. They can
contain components that significantly impair embryonic development
and viability after transfer. Further, to the extent tissue culture
media contain components that are generally needed by the gametes
and the embryo, the media are not formulated to provide the
components at levels appropriate to support healthy gamete and
embryonic development. Other laboratories have used simple culture
media, consisting of balanced salt solutions supplemented with
carbohydrate energy sources such as glucose, pyruvate and lactate.
Examples include Earle's, T-6, and human tubal fluid (HTF). These
media generally lack certain key components found in the female
reproductive tract, such as non-essential amino acids, and their
constituents are not formulated in concentrations that meet the
specific changing needs of the gametes and developing embryo at
various stages of their development.
[0005] The two types of culture media commonly used for IVF
generally are only capable of supporting embryonic development to
the eight-cell stage. Such media are ordinarily not capable of
supporting and producing a viable blastocyst stage embryo, complete
with an epithelium and competent inner cell mass. Accordingly,
embryo transfer, the return of the fertilized oocyte to the uterus
of the mother, usually occurs at around the four-cell stage (day
two) or around the eight-cell stage (day three). This is a time
when the four- or eight-cell embryo would not typically have
arrived in the uterus of the mother, if fertilization had occurred
in vivo. Embryo transfer at this time involves placing the cleavage
stage embryo in an environment oriented to a blastocyst stage
embryo. The cleavage stage embryo must then undergo further
development in a non-homologous environment to reach the blastocyst
stage, in which the embryo has trophectoderm cells capable of
implanting in the uterine lining.
[0006] Recent research and human trials have led to the development
of two new culture media, G1 and G2, which represent significant
advancements in adaptation of culture media to the physiological
needs of the cleavage stage embryo and the embryo in the eight-cell
through blastocyst stage of development. These media are described
in the following publications: Barnes, Crombie, Gardner, et al,
Blastocyst Development and Birth After In-vitro Maturation of Human
Primary Oocytes, Intracytoplasmic Sperm Injection and Assisted
Hatching, Human Reproduction, vol. 10, no. 12, pp. 3243-47
(December, 1995); Gardner and Lane, Culture and Selection of Viable
Blastocysts: A Feasible Proposition for Human IVF?, Human
Reproduction Update, Vol. 3, No. 4, pp. 367-82 (1997); Gardner,
Vella, Lane, et al, Culture and Transfer of Human Blastocysts
Increases Implantation Rates and Reduces the Need for Multiple
Embryo Transfers, Fertility and Sterility, Vol. 69, No. 1, pp.
84-88 (January 1998). Use of these media, and particularly the G2
medium, supports the growth and development of viable blastocyst
stage embryos in vitro. Accordingly, the development of these media
paves the way for new approaches to embryo transfer to the uterus
at the blastocyst stage, when the embryo is most adapted to
surviving in the uterine environment and has developed structures
and capabilities required for implantation to take place. Research
utilizing the G1 and G2 media, and embryo transfer at the
blastocyst stage, suggests that these media contribute to higher
pregnancy rates, and reduces the need for transfer of multiple
embryos and the risk of multiple births. Neither of these media,
however, is optimized for supporting the gametes, oocyte
maturation, or fertilization.
SUMMARY OF THE INVENTION
[0007] It has been recognized that IVF processes may be improved by
providing specific media and media sequences for supporting
gametes, zygotes and developing embryos relative to various phases
of the IVF process. In certain respects, such media and sequences
better reflect in vivo development. Within the female reproductive
system, the oocyte is developed within and released from the ovary
during ovulation, and proceeds through the oviduct towards the
uterus. During this journey, it experiences a dynamic physical
environment. The fluid of the oviduct contains a number of
components that provide nourishment to the oocyte and its
surrounding cumulus cells, and that also appear to interact with
the oocyte and its cumulus cells, so stimulating development.
Similarly, the fluid of the female reproductive tract provides
nourishment to sperm traveling through the oviduct, and also
stimulates certain changes in the sperm necessary to fertilization.
Once fertilization occurs, the resulting zygote travels down the
oviduct and enters the uterus approximately three days later,
undergoing internal transformation and experiencing a changing
environment.
[0008] As the zygote travels, cell division, or cleavage, occurs as
well as significant developmental changes. The cells of early
embryonic development have different capabilities and nutritional
needs from those of later embryonic development prior to
implantation. The zygote and cleavage stage embryo (up to the
eight-cell stage) are characterized by low levels of biosynthesis,
low respiratory rates, only limited ability to metabolize glucose,
and a capacity to utilize pyruvate. As the embryo develops, and
genome activation occurs, the embryo gains an increased capacity to
utilize glucose. At the blastocyst stage of development, when the
embryo is entering and within the uterus, the embryo's metabolic
system has developed and the embryo has a substantially greater
capacity to use and need for glucose, and less need for pyruvate.
The makeup of the fluid surrounding the developing embryo in the
female reproductive tract seems to be tailored to these changing
needs: in the oviduct at the time when the oocyte and developing
embryo are present, relatively low levels of glucose are found,
while pyruvate concentrations are high; at the time the embryo
enters the uterus, glucose reaches its highest level and the
pyruvate concentration is comparatively low. Cleavage stage
embryos, like the oocyte, are susceptible to loss of amino acids
through their cell membranes when surrounded by an environment
having a low concentration of such factors. Such loss of internal
amino acids can have devastating effects. Again, as if in response
to these needs of the osmolyte sensitive oocyte and cleavage stage
embryo, the female reproductive tract typically has high levels of
specific amino acids that are very similar to those found in the
oocyte and cleavage stage embryo.
[0009] In view of the foregoing, an important object of the present
invention is to further improve and enhance the culture of human
reproductive cells in vitro. The invention is intended to promote
the health and viability of the gametes, zygote and embryo at
various stages of the IVF process, thereby improving the overall
efficiency of the IVF process and increasing pregnancy rates.
[0010] In general, the present invention involves the application
of separate media specifically formulated to meet the physiological
needs of the gametes, zygote and/or developing embryo in various
stages of their development, and to support the processes necessary
to accomplish fertilization and embryonic development in vitro. The
present invention also generally contemplates a sequential culture
media system, in which the separate media utilized have integrated
formulations, intended to minimize trauma to the reproductive cells
as they are moved from one medium to another during the IVF
process.
[0011] In one aspect of the present invention, an oocyte retrieval
and handling medium is provided for use in the clinical procedure
of retrieving the oocyte from the mother. The medium may be used
for flushing, washing and holding the oocyte during the process of
removing the oocyte from the mother's ovary, and for storing the
oocyte for a period prior to fertilization. An optional use of the
medium envisioned by the invention is with procedures where
handling or manipulating the oocyte, zygote, or embryo is
necessary, such as examination of the oocyte to determine whether
fertilization has occurred, or examining the embryo to determine
the progression of its development. The present invention includes
use of an oocyte retrieval and handling medium comprised of water,
ionic constituents, and a buffer. Preferably the buffer used in the
medium is 4-Morpholinepropanesulfonic acid (MOPS) or
N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid (HEPES). In
addition, the medium may be supplemented with the carbohydrates
glucose, lactate and pyruvate. The medium may be supplemented with
non-essential amino acids. An optional formulation of the medium,
lacking calcium and magnesium, may be used in biospsy procedures.
Another optional formulation of the medium includes antibiotics,
such as penicillin and/or streptomycin, to destroy bacteria that
might be introduced into the medium during the process of oocyte
collection.
[0012] Another aspect of the present invention involves the
provision and use of an oocyte maturation medium, for example, in
circumstances where the oocyte is removed from the mother before it
is mature. An example of a situation where application of this
medium may be desired arises when it is necessary to treat the
oocytes collected from the mother with hormones in vitro due to the
mother's intolerance of such hormones. The invention contemplates
holding the oocytes in the maturation medium for a period following
collection of the oocytes, to promote development prior to
fertilization. An optional use of the maturation medium in
accordance with the invention is for collection, although the most
cost effective approach will normally involve use of the retrieval
and handling medium for initial flushing, washing, collection and
storage, and then transfer to the maturation medium for an extended
period prior to fertilization. The present invention contemplates
use of a maturation medium comprised of water, ionic constituents,
and a buffer. Preferably, the maturation medium is supplemented
with the carbohydrates glucose, lactate and pyruvate. Specific
formulations in accordance with the present invention may involve
successive supplementation of the medium with one or more of the
following: non-essential amino acids; essential amino acids;
cysteamine; human serum albumin (HSA) and hyaluronate; one or more
growth factors such as insulin transferin selenium (ITS),
insulin-like growth factor (IGF), and epidermal growth factor
(EGF); and hormones follicule stimulating hormone (FSH) and human
chorionic gonadotrophin (hCG).
[0013] Another aspect of the invention involves the provision and
use of a sperm preparation and fertilization medium. This medium
may be applied to wash, prepare, and store sperm, to store the
oocyte in the period prior to fertilization, and to serve as the
medium in which the sperm and oocyte are placed together and
fertilization occurs. The present invention contemplates use of a
sperm preparation and fertilization medium that includes water,
ionic constituents, and a buffer. Preferably, the medium contains
an elevated concentration of sodium, as compared to the oocyte
retrieval and handling medium, to promote sperm function and
fertilization. In addition, the medium may be supplemented with an
elevated phosphate concentration, as compared to the oocyte
retrieval and handling medium. Even more preferably the medium is
supplemented with the carbohydrates glucose, lactate and pyruvate.
Specific formulations may involve supplementation of the medium
with one or more of bicarbonate; glutathione to promote sperm head
decondensation; non-essential amino acids; HSA and hyaluronate; and
antibiotics such as penicillin and streptomycin.
[0014] A further aspect of the invention relates to a medium for
intracytoplasmic sperm injection (ICSI) and related methodology.
The ICSI procedure may be necessary where there are obstacles to
normal fertilization, such as a thickened zona pellucida on the
oocyte hindering sperm head penetration. ICSI involves removal of
the cumulus cells and injection of the sperm into the oocyte,
ordinarily through a glass pipette. The invention contemplates
placing sperm in the ICSI medium, capturing the sperm by drawing
the medium containing sperm into the pipette, inserting the pipette
containing medium and sperm into the oocyte, and, following
insertion into the oocyte, transferring the medium containing sperm
from the pipette into the oocyte. The ICSI medium used in the
present invention includes the constituents water, ionic
constituents and a buffer. Preferably, in the present invention the
medium lacks phosphate. More preferably, the buffer used in the
medium is MOPS or HEPES. Additionally, the medium may be
supplemented with the carbohydrates lactate and pyruvate and the
medium may be further supplemented with one or more of the
nonessential acids most abundant in the oocyte: glutamine, glycine,
proline, serine, and taurine. In one formulation, the ICSI medium
used is supplemented with hyaluronate or polyvinylpyrolidone (PVP)
to slow or immobilize the sperm so that they may be captured by
pipette for the ICSI process. Further, an alternative formulation
of the ICSI medium referred to as denuding medium used in the
invention includes hyaluronidase, which is included in the portion
of the medium used to denude the oocyte prior to the ICSI
process.
[0015] Another aspect of the present invention involves the
provision and use of a medium for supporting initial cell cleavage
and embryonic development following fertilization, the medium
herein referred to as G1.2. The invention contemplates washing the
inseminated oocyte and zygote in the medium and placing the zygote
in; the medium for a period of about 48 hours to support cell
cleavage and development through about the eight-cell stage. The
present invention involves use of a medium that includes the
constituents water, ionic constituents, and a buffer. Preferably,
the medium is supplemented with the carbohydrates glucose, lactate,
and pyruvate. The medium may also be supplemented with
non-essential acids. Specific formulations in accordance with the
invention may include one or more of the following supplements:
EDTA; HSA; and hyaluronate. The form of glutamine used in the
medium is preferably alanyl-glutamine, which is particularly stable
and less prone to breakdown to the waste product ammonium, which is
toxic to the developing embryo.
[0016] A further aspect of the invention involves the provision and
use of a second medium for embryo development, herein referred to
as G2.2. The invention contemplates placing the embryo in the G2.2
medium for a period of about 48 hours, preferably at or after the
eight-cell stage, and continuing through the blastocyst stage of
development and up to the point of embryo transfer. This medium is
specifically adapted for and has as its preferred use support of
the embryo from the eight-cell stage through the time at which
implantation occurs, in tandem with the use of G1.2 for initial
embryonic development. The invention involves a G2.2 medium that
includes water, ionic constituents, and a buffer. Preferably the
medium is supplemented with the carbohydrates glucose, lactate and
pyruvate. More preferably, as compared to medium G1.2, medium G2.2
is supplemented with depressed levels of lactate and pyruvate, and
elevated levels of glucose. Additionally, the medium may be
supplemented with the non-essential amino acids, except taurine.
Specific formulations in accordance with the present invention
involve supplementing the medium with one or more of essential
amino acids, which stimulate development of the inner cell mass of
the blastocyst; vitamins, which further facilitate the function of
the blastocyst; HSA; and hyaluronate. An important aspect of the
G2.2 medium, in all formulations, is the absence of EDTA.
[0017] Another aspect of the invention is the provision and use of
an embryo transfer medium. The invention contemplates that this
medium will be used as a carrier for the embryo when it is
transferred back into the mother. The invention may involve the
same formulations of the medium for embryo transfer as are used
with medium G2.2. More preferably for embryo transfer, however, the
formulation of G2.2 is supplemented with a higher concentration of
hyaluronate, which supports implantation of the embryo in the
mother's uterus.
[0018] A further aspect of the invention is the provision and use
of a medium for cryopreservation of the embryo and/or oocyte. The
invention contemplates that the embryo may be placed in the medium
at either the one- to eight-cell stage or eight-cell to blastocyst
stage, and then frozen and stored in the medium. The invention also
contemplates that the medium may be used for cryopreservation of
the oocyte. The cryopreservation medium contains ionic
constituents, and a buffer. Preferably, it contains the MOPS or
HEPES buffer. More preferably, it contains the carbohydrates
lactate, pyruvate and glucose. Even more preferably, it contains
HSA. Most preferably, the medium contains certain additives such as
glycerol, ethylene glygol, DMSO, and/or sucrose.
[0019] According to a further aspect of the invention, different
media are used for two different phases of the IVF process, such as
oocyte collection and maturation, sperm preparation, fertilization,
embryo development and/or embryo transfer. One associated process
involves obtaining a gamete from a first medium and introducing the
gamete into a second medium different from the first medium,
wherein fertilization occurs in the second medium. The step of
obtaining a gamete from a first medium may include extracting an
oocyte from an oocyte collection medium or oocyte maturation medium
as described above. Additionally or alternatively, the step of
obtaining may involve extracting sperm from a sperm preparation and
fertilization medium as described above which, in turn, may be
different from the oocyte medium. The step of introducing the
gamete into the second medium may involve introducing the sperm
and/or oocyte into a fertilization medium, or injecting sperm into
an oocyte contained in the second medium. The various media may
have integrated formulations for minimizing trauma to the
reproductive cells.
[0020] Another associated process in accordance with the present
invention involves obtaining a zygote or embryo from a first medium
wherein fertilization has occurred and introducing it into a second
medium different from the first medium for a first growth phase.
The first medium may be a fertilization medium as described above
and the second medium may be the G1.2 medium as described above.
The second medium may be used for supporting initial cell cleavage
and embryonic development. The method may further involve
transferring the resulting embryo from the second medium to a third
medium for a second growth phase. The third medium may be a G2.2
medium as described above.
[0021] A further associated process in accordance with the present
invention involves obtaining an embryo from a first medium and
introducing the embryo into a second medium different from the
first medium for transfer of the embryo into the mother for
implantation. The first medium may be a G2.2 medium as described
above and the second medium may be an embryo transfer medium as
described above.
BRIEF DESCRIPTION OF THE DRAWING
[0022] For a more complete understanding of the present invention
and further advantages thereof, reference is now made to the
following detailed description taken in conjunction with the
drawings, in which:
[0023] FIG. 1 is a flowchart illustrating an IVF process in
accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The following description discloses the composition of
various culture media in accordance with the present invention that
are particularly adapted for use with IVF. Each of these media is
specifically formulated to meet the physiological needs of the
gametes, zygote and developing embryo at key points in the
reproductive process. Also disclosed is a sequential culture media
system. While each of the separate media could be used
independently, the media also may be formulated together as a
system, sharing a core group of ionic and non-essential amino acid
constituents, with the objective of minimizing trauma to the
oocyte, and the resulting zygote and embryo, as they are moved from
one medium to another. The following description also discloses
methods of using the media and the sequential culture media system
in various clinical and laboratory procedures by which IVF is
carried out, as well as methods of making the culture media.
A. Composition of the Sequential Culture Media
[0025] 1. Oocvte Retrieval and Handling Medium
[0026] A preferred oocyte retrieval and handling medium is an
aqueous solution comprised of the ionic components sodium,
potassium, phosphate, magnesium, bicarbonate, and calcium, to
maintain an osmotic environment that does not stress the oocyte,
and a buffering system, preferably MOPS or HEPES, to maintain the
pH of the medium within the physiological range of 7.3 to 7.4. The
ionic components are included in the preferred amounts depicted in
column A of Table 1, and may be included in amounts described in
the ranges depicted in column B of Table 1.
TABLE-US-00001 TABLE 1 Composition of Oocvte Retrieval and Handling
Medium* A B Most Preferred Preferred Component Concentration Range
NaCl 90.08 75-105 KCl 5.5 3.5-7.5 NaH.sub.2PO.sub.4.cndot.2H.sub.2O
0.25 0.05-1.5 MgSO.sub.4.cndot.7H.sub.2O 1 0.2-4.0 NaHCO.sub.3 5
2.0-10.0 MOPS/HEPES 20 10.0-25.0 CaCl.sub.2.cndot.2H.sub.2O 1.8
0.8-2.8 Glucose 0.5 0.05-5.0 NaLactate (L-isomer) 10.5 5.0-20.0
NaPyruvate 0.32 0.1-1.0 Alanine (ala) 0.1 0.01-0.5 Asparate (asp)
0.1 0.01-0.5 Asparagine (asn) 0.1 0.01-0.5 Glutamate (glu) 0.1
0.01-0.5 Alanyl-Glutamine (ala-gln) 0.5 0.01-2.0 Glycine (gly) 0.1
0.01-0.5 Proline (pro) 0.1 0.01-0.5 Serine (ser) 0.1 0.01-0.5
Taurine (tau) 0.1 0.01-10.0 *Concentrations are in millimoles
unless otherwise indicated; the medium is aqueous.
[0027] It should be noted that Table 1 and the other tables
presented in this section also describe the preferred form of the
components used to make the respective culture media in practice.
The MOPS buffer has not been used before in IVF procedures, and is
preferred because it is not known to exhibit any toxic effects to
reproductive cells and does not require maintenance of a C02
atmosphere above the medium. HEPES may also be utilized, although
some research indicates a possible toxicity to reproductive cells.
Table 1 depicts the preferred amount and ranges for the MOPS or
HEPES buffer, although other buffering systems might be used. For
example, a bicarbonate buffering system may be used because it is
compatible with human reproductive cells. Such a system would not
ordinarily be practical with oocyte collection, because it requires
the maintenance of elevated levels of CO.sub.2 in the atmosphere
surrounding the medium, which is ordinarily accomplished by use of
a gassing incubator system that maintains a 3%-10% CO.sub.2
atmosphere. Oocyte collection is a clinical procedure, in which it
is typically not possible to maintain an elevated CO.sub.2
atmosphere. In some clinical environments, such as where a
humidicrib is available, it may be possible to perform oocyte
collection in an elevated CO.sub.2 atmosphere, and a bicarbonate
buffer accordingly may be used. In accordance with the present
invention, any buffering system used preferably maintains its
buffering qualities during exposure of the medium to the
atmosphere, and as well is preferably compatible with and not toxic
to human reproductive cells.
[0028] The oocyte retrieval and handling medium also includes the
carbohydrates glucose, lactate, and pyruvate, at levels similar to
those found in the female reproductive tract at the corresponding
point of ovulation. The preferred amounts and ranges in which these
are found in the medium are depicted in Table 1. In addition, the
preferred medium contains Eagle's non-essential amino acids (i.e.,
those not required for the development of somatic cells in culture)
alanine, aspartate, asparagine, glutamate, glycine, proline,
serine, and taurine, plus glutamine in the form of
alanyl-glutamine, at levels similar to those found in the female
reproductive system and in the oocyte. The preferred amounts and
ranges are depicted in Table 1. The inclusion of non-essential
amino acids and alanyl-glutamine in the medium is important to
preventing osmotic shock; a medium lacking these components may
drain the oocyte of its internal pool of amino acids, resulting in
considerable intracellular trauma. An optional formulation of the
medium which may be used in biopsy procedures, omits calcium and
magnesium. Another optional formulation of the medium may include
one or more antibiotics, such as penicillin and streptomycin, to
destroy any bacteria that might be present around the oocyte or
that might be introduced through the clinical procedure of oocyte
removal.
[0029] 2. Oocyte Maturation Medium
[0030] The oocyte maturation medium is adapted for use with
immature oocytes. Oocyte maturation is typically used with mothers
who are unable to withstand the hormonal treatment ordinarily
employed in IVF. Oocyte maturation generally involves treating the
immature oocytes in vitro with the hormones follicle stimulating
hormone (FSH) and human chorionic gonadotrophin (hCG) rather than
injecting these hormones into the mother. The preferred medium is
an aqueous solution that contains ionic constituents similar to
those used in the oocyte retrieval and handling medium, at similar
concentrations, although the magnesium level is increased and the
calcium level decreased to maintain a 2:1 magnesium to calcium
concentration. A buffer is included in the preferred medium to
maintain a physiological pH. Because oocyte maturation ordinarily
occurs in an incubator or isolette in which an elevated CO.sub.2
atmosphere can be maintained, a bicarbonate buffering system is
preferred. Other buffers may be used, provided they are compatible
with the oocyte and other components of the medium. Table 2
provides the most preferred amounts of each of these components, as
well as the preferred ranges of these components.
TABLE-US-00002 TABLE 2 Composition of Oocyte Maturation Medium A B
Most Preferred Preferred Component Concentration Range NaCl 90.08
80.0-100 KCl 5.5 3.5-7.5 NaH.sub.2PO.sub.4.cndot.2H.sub.2O 0.25
0.05-1.5 MgSO.sub.4.cndot.7H.sub.2O 2 0.2-4.0 NaHCO.sub.3 25
15-30.0 CaCl.sub.2.cndot.2H.sub.2O 1 0.8-2.8 Glucose 3.15 0.5-5.5
NaLactate (L-isomer) 5.87 2.0-20.0 NaPyruvate 0.1 0.01-1.0 Alanine
0.1 0.01-0.5 Asparate 0.1 0.01-0.5 Asparagine 0.1 0.01-0.5
Glutamate 0.1 0.01-0.5 Alanyl-Glutamine 1 0.01-2.0 Glycine 0.1
0.01-0.5 Proline 0.1 0.01-0.5 Serine 0.1 0.01-0.5 Cysteamine 0.5
0.1-2.0 L-Arginine-HCl 0.6 0.1-1.2 L-Cystine 2HCl 0.1 0.05-0.25
L-Histidine-HCl-H2O 0.2 0.1-0.4 L-Isoleucine 0.4 0.1-0.8 L-Leucine
0.4 0.1-0.8 L-Lysine-HCl 0.4 0.1-0.8 L-Methionine 0.1 0.05-0.25
L-Phenylalanine 0.2 0.1-0.4 L-Threonine 0.4 0.1-0.8 L-Tryptophan
0.5 0.1-0.9 L-Tyrosine 2Na 0.2 0.1-0.4 L-Valine 0.4 0.1-0.8 D-Ca
Pantothenate 0.002 0.001-0.004 Choline Chloride 0.007 0.003-0.01
Folic Acid 0.0023 0.001-0.0045 i-Inositol 0.0111 0.005-0.02
Niacinamide 0.0082 0.004-0.016 Pyridoxal HCl 0.0049 0.002-0.01
Riboflavin 0.0003 0.0001-0.0006 Thiamine HCl 0.003 0.001-0.006 HSA
5 mg/ml 1-10.0 Hyaluronate 0.25 mg/ml 0.05-0.5 ITS 10 ng/ml 1-100
IGF-I 100 ng/ml 10-1000 EGF 100 ng/ml 10-1000 FSII 0.1 U/ml 0.01-10
hCG 0.1 U/ml 0.01-10 * Concentrations are in millimoles, unless
otherwise indicated; the medium is aqueous.
[0031] The carbohydrates glucose, lactate and pyruvate are also
included in the preferred maturation medium. Because of the
presence and importance of cumulus cells that surround the
developing oocyte, the glucose, lactate and pyruvate levels are
adapted to the needs of the cumulus cells. Non-essential amino
acids are preferably included in the medium to provide nutrients
and avoid subjecting the oocyte to osmotic stress. Essential amino
acids and vitamins may also be included to provide nutrients to the
cumulus cells. The medium also contains HSA and hyaluronate, which
act as a source of macromolecules. Insulin transferin selenium
(ITS), insulinlike growth factor (IGF), and epidermal growth factor
(EGF) are included to support the function of cumulus cells, which,
in turn, nourish and stimulate the oocyte. FSH and hCG are added to
stimulate the cumulus and oocyte to undergo changes associated in
vivo with ovulation. It should be noted that, when the maturation
medium is prepared, ITS, IGF, EGF and FSH and hCG are preferably
the last-added ingredients. The preferred amounts and ranges of
these components are found in Table 2.
[0032] 3. Sperm Preparation and Fertilization Medium
[0033] Current methods of in vitro fertilization employ the same
medium for sperm preparation and fertilization as is used for
embryo development. No attempt has been made to develop a separate
medium for preparation of sperm that is also suitable for storage
and support of the oocyte, for promoting the process of
fertilization, and for supporting the zygotes formed when
fertilization occurs. In many laboratories, the fertilization
process is allowed to take place over an extended period which
ranges from two to three hours to up to about sixteen (16) to
eighteen (18) hours. During this time, the oocyte, sperm, and
zygotes produced have significant nutritional needs. In addition,
sperm function and fertilization tend to be encouraged when the
surrounding fluid contains certain constituents. The sperm
preparation and fertilization medium of the present invention is
formulated to meet these concerns.
[0034] A preferred sperm preparation and fertilization medium in
accordance with this invention has virtually the same composition
of ions and non-essential amino acids as the oocyte retrieval and
handling medium. The fact that these media share a similar ionic
and amino acid composition minimizes the stress experienced by the
oocyte when it is removed from the retrieval and handling medium
and placed in sperm preparation medium. Table 3 sets out the
preferred amounts and ranges of the ionic and non-essential acid
components.
TABLE-US-00003 TABLE 3 Composition of Sperm Preparation and
Fertilization Medium* A B Most Preferred Preferred Component
Concentration Range NaCl 100 75-100 KCl 5.5 3.5-7.5
NaH.sub.2PO4.cndot.2H2O 0.5 0.05-1.5 MgSO4.cndot.7H2O 1 0.2-4.0
Glucose 3.15 0.5-5.6 NaLactate (L-isomer) 5 2.0-20 NaPyruvate 0.32
0.1-0.5 NaHCO3 25 15-30 CaC12.cndot.2H20 1.8 0.8-2.8 Glutathione
1.0 mg/ml 0.5-5.0 Alanine 0.1 0.01-0.5 Asparate 0.1 0.01-0.5
Asparagine 0.1 0.01-0.5 Glutamate 0.1 0.01-0.5 Glycine 0.1 0.01-0.5
Proline 0.1 0.01-0.5 Serine 0.1 0.01-0.5 Taurine 0.1 0.01-10.0 HSA
5 mg/ml 1.0-10.0 Hyaluronate 0.1 mg/ml 0.02-0.5 Penicillin 0.06
mg/ml 0.01-.10 Streptomycin 0.05 mg/ml 0.01-.10 *Concentrations are
in millimoles unless otherwise indicated; the medium is
aqueous.
[0035] As will be seen, the sperm preparation medium contains
sodium at a higher concentration than the level found in the oocyte
retrieval and handling medium. This elevated concentration of
sodium promotes sperm function and fertilization, without causing
undue osmotic stress to the oocyte. There is also a higher
concentration of phosphate, as compared to the oocyte retrieval and
handling medium. The glucose concentration of the sperm preparation
and fertilization medium is elevated over that of the oocyte
retrieval and handling medium, because glucose is the primary
nutrient for sperm and cumulus cells around the egg. The lactate
concentration of the present medium is lower than that found in the
oocyte retrieval and handling medium, to compensate for the
tendency of sperm cells and cumulus cells to give off lactic acid
as a waste product. A buffering system is used to maintain the
physiological pH, and because sperm preparation and fertilization
largely occur within an incubator that can maintain an elevated
CO.sub.2 atmosphere, a bicarbonate buffer is preferred. Glutathione
(not present in the oocyte retrieval and handling medium) is
included, to assist in the process of sperm head decondensation.
Alanyl-glutamine (present in the oocyte retrieval and handling
medium) is omitted from the present medium because it can impair
sperm function and reduce fertilization. The same is true of the
chelating agent EDTA, which (as will be discussed later) is present
in the embryo development media. HSA, the most abundant
macromolecule in the Fallopian tube and uterus, is included to
support sperm and embryo function. Hyaluronate, which promotes
sperm motility, and works in tandem with HSA, is also included.
Because sperm tends to contain high levels of bacteria, one or more
antibiotic substances are also included. Penicillin, streptomycin,
and/or gentamycin are preferred antibiotics. Table 3 sets out the
preferred amounts and ranges for these various components.
[0036] 4. The ICSI Medium
[0037] In circumstances where it is desired to accomplish
fertilization by other than natural interaction of sperm and
oocyte, such as where the sperm is unable to fertilize the oocyte
due to a thickened zona pellucida surrounding the oocyte, or where
the sperm is from a male-factor patient, the sperm may be
transported into the oocyte by a technique called intracytoplasmic
sperm injection (ICSI). When the ICSI technique is used, the
cumulus cells are removed from the oocyte, and sperm is injected
into the oocyte's interior using a glass pipette. The present
invention contemplates use of a single medium to bathe the oocyte
and also to serve as a carrier for the sperm as it is transported
by injection into the oocyte. The medium, accordingly, is
preferably highly compatible with the interior and exterior of the
oocyte. The ionic constituents in the preferred medium are similar
to those found in the oocyte retrieval and handling medium, except
that phosphate is omitted, to avoid metabolic and homeostatic
stress, and the magnesium-to-calcium ratio is 2:1. This ratio of
magnesium to calcium is felt to be highly beneficial to the oocyte.
Because ICSI is a clinical procedure performed outside the
incubator, a buffering system that is effective in a normal
atmosphere is used. MOPS and HEPES are accordingly preferred
buffers for this medium. Because the cumulus cells have been
removed from the oocyte, and the sperm is at the conclusion of its
independent life, glucose, the main energy source for cumulus cells
and sperm (but not the oocyte) is omitted from the medium. Pyruvate
and lactate levels are increased, as these are a primary energy
source for the oocyte. Only the non-essential amino acids most
abundant in the oocyte--glycine, proline, serine and taurine--and
glutamine (in the stable form alanyl-glutamine) are retained in the
medium to avoid osmotic and pH stress and to nourish the oocyte.
Preferably, the ICSI medium also includes hyaluronate or
polyvinylpyrollidone (PVP), to immobilize or slow the sperm so that
they may be captured in the ICSI pipette. Table 4 sets out the
preferred amounts and the ranges of these components in the ICSI
medium. Moreover, an alternative formulation of the ICSI medium
includes hyaluronidase, which alternative formulation is used to
pretreat the oocyte, to break down the hyaluronate gel holding the
cumulus cells around the oocyte. This medium is referred to above
as denuding medium, and lacks hyaluronate and PVP but includes
hyaluronidase. The composition of the denuding medium includes the
constituents of the ICSI medium (except hyaluronate and PVP) in the
preferred amounts and ranges shown in Table 4 plus hyaluronidase in
a preferred about of 40 IU/ml and a preferred range of Oct-80.
Optionally, HSA may be included in the denuding medium in the
preferred amount of 5 mM and the preferred range of 1.0-110 mM.
TABLE-US-00004 TABLE 4 Composition of Medium ICSI* A B Most
Preferred Preferred Component Concentration Range NaCl 90.08
75.0-105 KCl 5.5 3.5-7.5 MgSO.sub.4.cndot.7H.sub.2O 2 0.4-4
NaHCO.sub.3 5 2.0-10 MOPS/HEPES 20 10-25.0
CaCl.sub.2.cndot.2H.sub.2O 1 0.5-2.0 NaLactate (L-isomer) 10.5
5.0-20 NaPyruvate 0.32 0.1-1.0 Alanyl-Glutamine 0.5 0.1-2.0 Glycine
0.5 0.1-2.0 Proline 0.1 0.05-2.0 Serine 0.1 0.05-2.0 Taurine 0.1
0.05-5.0 HSA 5 mg/ml 1-10.0 Hyaluronate 0.1 mg/ml 0.02-0.5 PVP 10%
1-20% *Concentrations are in millimoles unless otherwise indicated;
the medium is aqueous.
[0038] 5. Embryonic Development Medium G1.2.
[0039] The present invention includes an embryonic development
medium G1.2. The preferred application of this medium is to support
development of the early one-to eight cell embryo. As depicted in
Table 5, the preferred medium has a backbone of ionic constituents
and non-essential amino acids that is similar to that found in the
oocyte retrieval and handling medium. Unlike the oocyte retrieval
and handling medium, the G1.2 medium contains the component EDTA,
which supports embryonic development and is believed to bind and
disable toxins that might have a deleterious effect on the early
embryo, and which also suppresses glycolysis. In addition, this
medium includes HSA and hyaluronate, in concentrations that are
thought to support early embryonic development. The preferred
formulation of medium G1.2 differs from the previously published
medium G1 in several important respects. First, research has shown
that an elevated phosphate concentration may not provide optimal
conditions for growth of the developing embryo. Accordingly, the
phosphate concentration has been decreased. Second, hyaluronate has
been added to work in tandem with HSA. Third, alanyl-glutamine has
been substituted for glutamine. A significant problem for embryo
culture with amino acids is the natural decomposition of amino
acids to ammonium, which decomposition is accelerated at higher
temperatures such as the physiological temperature (37 degrees
Celsius) used in IVF procedures. Ammonium can be toxic to embryos.
Moreover, glutamine is especially prone to decomposition to
ammonium within solution. Since embryos are generally cultured in
medium G1 or G1.2 for an extended period of up to about 48 hours, a
significant quantity of ammonium can develop in the medium and be a
significant inhibitor to embryo development. Accordingly, the use
of alanyl-glutamine provides substantial advantages; it is a
particularly stable form of glutamine and is not prone to breaking
down in solution. Also, the concentration of alanyl-glutamine in
G1.2 has been reduced to 0.5 mM. These three modifications make
G1.2 a significantly improved medium for early embryonic
development over medium G1. The most preferred amounts and
preferred ranges of the components of medium G1.2 are depicted in
Table 5.
TABLE-US-00005 TABLE 5 Composition of Medium G 1.2* A B Most
Preferred Preferred Component Concentration Range NaCl 90.08
80.0-100 KC1 5.5 3.5-7.5 NaH.sub.2PO.sub.4.cndot.2H.sub.2O 0.25
0.05-1.5 MgSO.sub.4.cndot.7H.sub.2O 1 0.2-2.0 NaHCO.sub.3 25
15.0-30 CaCl.sub.2.cndot.2H.sub.2O 1.8 0.8-2.8 Glucose 0.5 0.05-5.0
NaLactate (L-isomer) 10.5 5.0-20. NaPyruvate 0.32 0.1-1.0 Alanine
0.1 0.01-0.5 Asparate 0.1 0.01-0.5 Asparagine 0.1 0.01-0.5
Glutamate 0.1 0.01-0.5 Alanyl-Glutamine 0.5 0.1-1.0 Glycine 0.1
0.01-0.5 Proline 0.1 0.01-0.5 Serine 0.1 0.01-0.5 Taurine 0.1
0.01-10.0 EDTA 0.01 0.005-0.20 HSA 5 mg/ml 1-10.0 Hyaluronate 0.1
mg/ml 0.02-0.5 *Concentrations are in millimoles unless otherwise
indicated; the medium is aqueous.
[0040] 6. Embryonic Development Medium G2.2
[0041] Medium G2.2 is also formulated to support embryonic
development. Its preferred use is with embryos from the eight-cell
to the blastocyst stage (around 100 cells) to around one-hundred
cell stage. The backbone of ionic constituents and non essential
amino acids preferably found in medium G2.2 is essentially the same
as used with medium G1.2, except that the concentration of
alanyl-glutamine has been increased. This reduces the risk of
subjecting the embryo to osmotic stress as it is moved from medium
G1.2 to medium G2.2. Taurine is omitted because its benefits to the
embryo appear to be confined to the period prior to compaction.
Glucose, lactate and pyruvate are included as carbohydrates, except
that the concentration of glucose is increased, while lactate and
pyruvate are decreased, as compared to medium G1.2. This
modification in carbohydrate levels is in response to the
increasing ability of the developing embryo to metabolize glucose
as an energy source, and reflects also the observed composition of
the female reproductive tract. Eagle's essential amino acids are
included in medium G2.2 because they are necessary to stimulate the
growth of the inner-cell mass of the blastocyst. Vitamins are added
as a group because in animal studies they tend to facilitate the
function of the blastocyst, including fluid accumulation in the
cavity of the blastocyst. Importantly, this medium lacks EDTA. The
preferred amounts and ranges of the components of medium G2.2 are
depicted in Table 6.
TABLE-US-00006 TABLE 6 Composition of Medium G 2.2* A B Most
Preferred Preferred Component Concentration Range NaCl 90.08
80.0-100 KCl 5.5 3.5-7.5 NaH2PO4.cndot.2H2O 0.25 0.05-1.5
MgSO4.cndot.7H2O 1 0.2-4.0 NaHCO3 25 15-30.0 CaCl2.cndot.2H2O 1.8
0.8-2.8 Glucose 3.15 0.5-5.5 NaLactate (L-isomer) 5.87 2.0-20.0
NaPyruvate 0.1 0.01-1.0 Alanine 0.1 0.01-0.5 Asparate 0.1 0.01-0.5
Asparagine 0.1 0.01-0.5 Glutamate 0.1 0.01-0.5 Alanyl-Glutamine 1
0.01-2.0 Glycine 0.1 0.01-0.5 Proline 0.1 0.01-0.5 Serine 0.1
0.01-0.5 L-Arginine-HCI 0.6 0.1-1.2 L-Cystine 2HCl 0.1 0.05-0.25
L-Histidine-HCl-H2O 0.2 0.1-0.4 L-Isoleucine 0.4 0.1-0.8 L-Leucine
0.4 0.1-0.8 L-Lysine-HCl 0.4 0.1-0.8 L-Methionine 0.1 0.05-0.25
L-Phenylalanine 0.2 0.1-0.4 L-Threonine 0.4 0.1-0.8 L-Tryptophan
0.5 0.1-0.9 L-Tyrosine 2Na 0.2 0.1-0.4 L-Valine 0.4 0.1-0.8 D-Ca
Pantothenate 0.002 0.001-0.004 Choline Chloride 0.007 0.003-0.01
Folic Acid 0.0023 0.001-0.0045 i-Inositol 0.0111 0.005-0.02
Niacinamide 0.0082 0.004-0.016 Pyridoxal HCl 0.0049 0.002-0.01
Riboflavin 0.0003 0.0001-0.0006 Thiamine HCl 0.003 0.001-0.006 HSA
5 mg/ml 1-100 Hyaluronate 0.1 mg/ml 0.02-0.5 *Concentrations are in
millimoles unless otherwise indicated; the medium is aqueous.
[0042] 7. Embryo Transfer Medium
[0043] The preferred embryo transfer medium contains the same
formulation of constituents as medium G2.2 except that a much
higher concentration of hyaluronate is included. In the human
reproductive system, research indicates that there is a receptor on
the embryo for hyaluronate and that there is also a receptor for
hyaluronate on the endometrium of the mother. Hyaluronate is
thought to act like a biological glue that assists the embryo in
binding to the endometrium and, accordingly, supports implantation.
The preferred amount and ranges of the constituents of the embryo
transfer medium are depicted in Table 7.
TABLE-US-00007 TABLE 7 Composition of Embryo Transfer Medium A B
Most Preferred Preferred Component Concentration Range NaCl 90.08
80.0-100 KCl 5.5 3.5-7.5 NaH2PO4.cndot.2H2O 0.25 0.05-1.5
MgSO4.cndot.7H2O 1 0.2-4.0 NaHCO3 25 15-30.0 CaCl2.cndot.2H2O 1.8
0.8-2.8 Glucose 3.15 0.5-5.5 NaLactate (L-isomer) 5.87 2.0-20.0
NaPyruvate 0.1 0.01-1.0 Alanine 0.1 0.01-0.5 Asparate 0.1 0.01-0.5
Asparagine 0.1 0.01-0.5 Glutamate 0.1 0.01-0.5 Alanyl-Glutamine 1
0.01-2.0 Glycine 0.1 0.01-0.5 Proline 0.1 0.01-0.5 Serine 0.1
0.01-0.5 L-Arginine-HCl 0.6 0.1-1.2 L-Cystine 2HCl 0.1 0.05-0.25
L-Histidine-HCl-H2O 0.2 0.1-0.4 L-Isoleucine 0.4 0.1-0.8 L-Leucine
0.4 0.1-0.8 L-Lysine-HCI 0.4 0.1-0.8 L-Methionine 0.1 0.05-0.25
L-Phenylalanine 0.2 0.1-0.4 L-Threonine 0.4 0.1-0.8 L-Tryptophan
0.5 0.1-0.9 L-Tyrosine 2Na 0.2 0.1-0.4 L-Valine 0.4 0.1-0.8 D-Ca
Pantothenate 0.002 0.001-0.004 Choline Chloride 0.007 0.003-0.01
Folic Acid 0.0023 0.001-0.0045 i-Inositol 0.0111 0.005-0.02
Niacinamide 0.0082 0.004-0.016 Pyridoxal HCl 0.0049 0.002-0.01
Riboflavin 0.0003 0.0001-0.0006 Thiamine HCl 0.003 0.001-0.006
Hyaluronate 0.25 mg/ml 0.05-1.0 * Concentrations are in millimoles,
unless otherwise indicated; the medium is aqueous.
[0044] 8. Cryopreservation Medium
[0045] The present invention involves a separate medium to be used
in cryopreservation of the oocyte and embryo. The preferred
formulation to be used includes ionic constituents and a buffer,
preferably MOPS or HEPES, as well as the carbohydrates lactate,
pyruvate and glucose. Optionally, HSA may be included. In addition,
the medium may include certain additives, glycerol, ethylene
glycol, DMSO, propanedial, and/or sucrose. The preferred amounts
and ranges of the constituents of the cryopreservation medium are
depicted in Table 8.
TABLE-US-00008 TABLE 8 Composition of Crypreservation Medium* A B
Most Preferred Preferred Component Concentration Range NaCl 90.08
75.0-105 KCl 5.5 3.5-7.5 MgSO.sub.4, 7H.sub.2O 2 0.4-4
Na2P04.cndot.2H2O 0.25 0.1-1.5 NaHCO.sub.3 5 2.0-10 MOPS/HEPES 20
10-25.0 CaCl.sub.2.cndot.2H.sub.2O 1 0.5-2.0 NaLactate (L-isomer)
5.87 2.0-20 NaPyruvate 0.32 0.1-1.0 Glucose 1 0.5-5.5 HSA 5 mg/ml
1.0-10 ADDITIVES Glycerol and/or ethylene glycol and/or DMSO and/or
pkropanediol and/or sucrose Range for all except sucrdose is 2 to
20%; range for sucrose is 0.1 to 1M Concentrations are in
millimeters unless otherwise indicated; the medium is aqueous
B. Sequential Culture Media Process
[0046] Instead of immersing human reproductive cells in a single
culture medium throughout the various procedures used in IVF, the
present invention involves a process by which the reproductive
cells may be moved through a sequence of distinct culture media as
the various IVF procedures are carried out. In one aspect of the
invention, the culture media are specifically formulated to provide
a physical environment similar to that found within the female
reproductive tract and conducive to growth and development of human
reproductive cells during various stages of the IVF process. In a
further aspect of the invention, the specifically formulated
culture media can be applied to support the reproductive cells in
one or more of the following procedures: oocyte retrieval and
handling; oocyte maturation; ordinary fertilization; oocyte, zygote
and embryo examination and biopsy; embryonic development to the
eight-cell stage; embryonic development to the blastocyst stage;
embryo transfer; and cryopreservation. Most preferably, the media
will be applied sequentially during each of the applicable stages
of the IVF process to which the media have been adapted. It should
be noted that there is significant variation among clinics and
laboratories as to equipment and specific procedures used to
accomplish each of the principal steps in the IVF process. The
present invention contemplates that the sequential media and
process described herein may be utilized and/or readily adapted for
use with the wide variety of equipment and procedures employed in
IVF practice. What follows is a more detailed discussion of
exemplary applications of the media during IVF and related
methodology:
[0047] 1. Oocyte Retrieval and Handling: Embryo Handling
[0048] Referring to FIG. 1, an initial procedure in the illustrated
IVF process 100 is oocyte removal or retrieval (102) from the
mother's ovary. This is typically performed vaginally using a fine
needle attached to and guided by a transvaginal ultrasound probe.
The needle is ordinarily connected to fine Teflon tubing and thence
to an aspiration regulator controlled by a vacuum regulator. The
aspirate is collected in test tubes or other appropriate vessels,
containing medium. The medium may be used to preliminarily wash the
needle and tubing, and other equipment used in the procedure. In
some clinical settings, the medium may also be used with a
specially adapted needle to flush the follicle and aid in removal
of the oocyte. The medium, equipment used, and aspirate are
maintained, so far as possible, at 37 degrees Celsius. If a
bicarbonate buffer system is used in the medium, the procedure
ordinarily is carried out in a gassed humidicrib or isolette which
maintains a 3%-10% CO.sub.2 atmosphere. In the absence of such
atmospheric controls, the medium must contain a MOPS or HEPES
buffering system.
[0049] The illustrated process 100 present invention contemplates
that the oocyte retrieval and handling medium may be used in each
phase of the retrieval process. The process of using the oocyte
retrieval and handling medium may involve washing any equipment
that may come into contact with the oocyte during removal from the
ovary, and that may be used to aspirate, flush and/or wash the
oocyte during the removal and collection process. Following removal
from the ovary, the oocyte may be washed with medium. Optionally,
the oocyte may be stored in the medium for a period.
[0050] In addition, it is contemplated that the medium may be used
during other clinical or laboratory procedures where the oocyte is
manipulated or handled, and also in procedures where the embryo is
manipulated or handled, especially where these occur outside the
isolette. Examples would include examination of the oocyte
following retrieval from the mother, examination of the oocyte
following the fertilization step, and examination of the embryo to
determine whether it has developed the eight-cell stage. In each of
these examples, the oocyte/embryo would be bathed in the medium as
it is withdrawn by pipette from the culture dish or test tube, and
would remain immersed in the medium while examined under a
microscope or with other equipment. The illustrated implementation
of the invention also contemplates that an alternative formulation
of this medium, which is calcium and magnesium free, may be used
during biopsy procedures.
[0051] 2. Oocyte Maturation
[0052] In the event the collected oocytes are immature, the
illustrated process 100 envisions that a second medium may be used
to support and promote development of the oocyte during maturation
(106). The oocyte maturation medium would ordinarily be used to
treat and mature the oocyte following a collection procedure, in
which the oocyte is retrieved from the ovary using oocyte retrieval
and handling medium. The retrieval and handling medium and
maturation medium have a very similar backbone of ionic
constituents and amino acids and glutamine, such that as the oocyte
is moved from one medium to another it experiences minimal ionic
shock. The illustrated process 100 includes immersing the oocyte
and surrounding cumulus cells in the maturation medium for a period
of about 30-48 hours, or until the oocyte is mature. The
illustrated process 100 then contemplates removing the oocyte from
the maturation medium and immersing it in either sperm preparation
and fertilization medium or ICSI medium for purposes of
fertilization.
[0053] In accordance with the invention, the oocyte maturation
medium may be applied to the oocyte retrieval process (102), in
place of the oocyte retrieval and handling medium described herein.
Additionally, a conventional culture medium, such as Ham's F-10 or
medium TCM-199 with or without a HEPES buffer, may be employed for
immature oocyte retrieval and handling, before immersion of the
oocyte in the maturation medium of the present invention. Once
maturation is complete, the oocyte will be immersed in a medium for
ordinary IVF fertilization procedure (110), or will be immersed in
an ICSI medium in preparation for assisted insemination through an
ICSI procedure (112).
[0054] 3. Sperm Preparation and Fertilization
[0055] The illustrated process 100 contemplates that the collected
oocytes will ordinarily be washed and immersed in, and allowed a
period of pre-incubation culture within, a first portion of the
sperm preparation and fertilization medium. This period of
pre-incubation culture (104) may last up to about six (6) hours.
Oocytes permitted a period of pre-incubation culture typically have
higher fertilization rates.
[0056] The process 100 also contemplates that the sperm may be
separately washed and stored in a second portion of the sperm
preparation and fertilization medium to purge it of bacteria and
any other contaminants that may be present. Sperm preparation (108)
may involve dilution of semen with the medium, centrifugation, and
resuspension of the concentrated sperm in a new portion of medium.
In the "swim up" method of sperm preparation, the medium containing
sperm is centrifuged, the medium is drained off, and a new portion
of medium is poured over the spundown sperm pellet. The sperm is
given a period to "swim up" into the fresh medium. That layer of
fresh medium, containing the more motile sperm, is then poured off
and centrifuged, and the process is repeated. In another aspect of
the invention, the sperm preparation and fertilization medium may
be used in one or more gradient separation procedures, such as the
Percoll procedure. The present invention envisions that the sperm
preparation and fertilization medium may be used as the medium in
any of the sperm preparation procedures that may be used for
IVF.
[0057] Once the sperm is prepared (108), the sperm is then examined
and counted while in medium, and a desired quantity is added to the
portion of medium which contains the oocyte. The sperm and oocyte
are permitted to remain together in the medium for a period of up
to several hours, and, in some laboratories, for a much longer
period, as long as about sixteen (16) to eighteen (18) hours. The
invention further contemplates that, following a period of
immersion in the medium with sperm, the oocytes will be removed and
examined (114) to determine whether fertilization (110) has
occurred. When removed for examination, the oocytes will continue
to be bathed in the sperm preparation and fertilization medium if
the examination can be conducted in an isolette. If not, then, as
noted above, the oocyte retrieval and handling medium may be used
for handling and examination of the oocytes.
[0058] 4. Fertilization by Direct Injection of Sperm into the
Oocyte (ICSITechnique)
[0059] In the ICSI process (112), sperm may be directly injected
into the cytoplasm of, the oocyte through a very fine pipette or
needle. The process 100 contemplates washing the sperm with a
portion of the ICSI medium containing hyaluronate and/or PVP, and
then placing the sperm in the medium. The process 100 further
involves drawing a microvolume of the medium containing sperm into
the pipette and then injecting the medium and sperm into the
interior of the oocyte.
[0060] The illustrated process 100 further contemplates that the
oocyte may be bathed in another portion of the ICSI medium during
the ICSI process. An alternative formulation of the ICSI medium may
be used, supplemented with hyaluronidase, for denuding pretreatment
(105) of the oocyte prior to the ICSI process. Pretreatment
involves immersing the oocyte in the ICSI medium supplemented with
hyaluronidase for a period until the oocyte becomes denuded of all
or most of its surrounding cumulus cells. Following pretreatment,
the oocyte is injected with sperm carried in a separate portion of
medium, using an ICSI pipette, as provided above.
[0061] After the ICSI injection process (112) is complete, it may
be necessary to examine (114) the oocyte to evaluate whether
fertilization has been effective and the oocyte is intact and
healthy. Examination may occur in the ICSI medium bathing the
oocyte, or may occur in the oocyte retrieval and handling medium as
described above.
[0062] 5. Embryonic Development to Eight-Cell Stage
[0063] Medium G1.2 is applied to the early embryo, following
formation of the zygote. After the zygote is identified, it is
washed with medium G1.2, and then immersed in G1.2 medium for a
culturing period (116) of up to about forty-eight hours. During
this time the embryo undergoes development from the one-cell to
around the eight-cell stage, and is removed at about the eight-cell
stage. Examination (118) of the embryo may occur in the G1.2
medium, or in the oocyte retrieval and handling medium, as
described above.
[0064] 6. Embryonic Development to Blastocyst Stage
[0065] The illustrated process 100 contemplates that medium G2.2
will be used to culture (120) the developing embryo to the
blastocyst stage, preferably from about the eight-cell stage to
about the one-hundred-cell stage. The process 100 also contemplates
that, once the embryo reaches the blastocyst stage, and assuming
that the embryo is judged on examination (124) to be viable, it is
removed from the G2.2 medium and prepared for transfer into the
uterus. In some laboratories, the G2.2 medium may, optionally, be
used for embryo transfer as well. Examination (124) of the embryo
may occur in the G2.2 medium or in the oocyte retrieval and
handling medium, as described above.
[0066] 7. Embryo Transfer
[0067] The process 100 contemplates that the embryo transfer medium
will serve as a carrier for the embryo as it is transferred (126)
back into the mother. The embryo will be bathed in the transfer
medium, the medium containing the embryo will be drawn into the
transfer catheter, the catheter will be inserted into the mother's
uterus, guided by an ultrasound probe, and the medium containing
the embryo will be injected into the uterus.
[0068] 8. Cryopreservation
[0069] The cryopreservation medium may be used for storing,
freezing, thawing, vitrification, and warming the oocyte, prior to
fertilization. The same medium may be used for storing, freezing,
thawing, vitrification, and warming the cleavage stage embryo, as
well as the embryo in the eight to one hundred cell stage.
[0070] While the present invention has been described in relation
to one embodiment, it will be appreciated that the invention may be
utilized in numerous additional embodiments and procedures. Such
additional embodiments and procedures are within the scope of the
present invention, as defined by the claims which follow.
* * * * *