Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF INFERTILITY
FIELD OF THIS INVENTION
This invention relates to an improved method of in vitro fertilisation
(hereinafter des-
ignated IVF).
BACKGROUND OF THIS INVENTION
1o Since the first IVF pregnancy was delivered in 1978, this procedure has
resulted in
thousands of pregnancies and opened a vast new frontier of research and
treatment
for the infertile couples. Still, there is a significant need for improved
infertility treat-
ment modalities today. It is presumed that about one out of seven couples
experi-
ence problems with subfertility or infertility.
IVF of human oocytes has become commonly used for the treatment of female and
male subfertility. The standard IVF treatment includes a long phase of hormone
stimulation of the female patient, e.g. 30 days, which is initiated by
suppressing the
patient's own follicle stimulating hormone (hereinafter designated FSH) and
luteinis-
2o ing hormone (hereinafter designated LH) by gonadotropin releasing hormone
(here-
inafter designated GnRH), and this is followed by injections of exogenous
gonad-
otropins, e.g. FSH and/or LH, in order to ensure development of multiple
preovula-
tory follicles and aspiration of multiple in vivo matured oocytes immediately
before
ovulation. The aspirated oocyte is subsequently fertilised in vitro and
cultured, typi-
cally for three days before transferral back into the uterus at the 4-8 cell
stage. Con-
tinuous efforts have been made to optimise and simplify this procedure.
Neverthe-
less, the overall pregnancy rate cannot be increased significantly over about
20%
with the current treatment modalities. In a large European survey of IVF
patients, it
was found that 7.2 oocytes out of 11.5 aspirated oocytes per patient had
undergone
so resumption of meiosis immediately before fertilisation, only 4.3 oocytes
were fertil-
ised and only 2.2 oocytes reached the 8-cell embryo stage after fertilisation
and in
vitro culture (ESHRE, Edinburgh, 1997).
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Due to the very unpredictable quality of the state of the art embryos today,
more than
one embryo has to be transferred just to give a reasonable chance of success.
Therefore, it is common to transfer 2-3 embryos (up to 5 embryos in some
countries),
which carries the very large side effect of multiple pregnancies with great
discomfort
and risk to both patient and children. Moreover, it has been estimated that
the in-
creased health care expenses due to multiple birth (twins, triplets etc.) is
exceeding
the entire IVF expenses.
o Hence, there are several disadvantages with the current treatment, the four
most no-
table being:
1. the risk of ovarian hyperstimulation with injecting gonadotropins which is
a poten-
tial fatal condition that requires hospitalisation,
2. multiple pregnancies (50-1.000 times the normal frequency of twins and
triplets,
respectively),
3. the existence of considerable patient segments that do not tolerate the
current
method due to, e.g. polycystic ovarian syndrome and many diabetics,
4. a potential long-term cancer risk.
Furthermore, weight gain, bloating, nausea, vomiting, labile mood and other
2o patient discomforts together with patient reluctance to inject themselves
are reported
as disadvantages.
It is known from WO 96/00235 that certain sterol derivatives can be used for
regulat-
ing meiosis. An example of such a sterol is 4,4-dimethyl-5a-cholesta-8,14,24-
triene-
3~i-of (hereinafter designated FF-MAS).
Herein, the term MAS compounds designates compounds which mediate the meiosis
of oocytes. More specifically, MAS compounds are compounds which in the test
de-
scribed in Example 1 below has a percentage germinal vesicle breakdown
(hereinaf-
3o ter designated GVB) which is significantly higher than the control.
Preferred MAS
compounds are such having a percentage GVB of at least 50%, preferably at
least
80%.
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Examples of MAS compounds are mentioned in WO 96/00235, 96/27658, 97/00884,
98/28323, 98/54965 and 98/55498, more specifically in Claim 1 thereof.
In WO 95/000265, some potential meiosis regulating substances were tested on
im-
mature female mice. 48 hours before the test animal were killed by cervical
disloca-
tion, they were given a single injection of human menopausal gonadotropin
contain-
ing 20 IU FSH and 20 IU LH. The ovaries were removed, placed in a hypoxanthine
medium and freed of extraneous tissue. Then, the oocytes were punctured out of
the
1o follicles, freed from cumulus cells and cultured in a medium containing a
meiosis
regulating derivative.
At present, in vitro maturation in humans has proven highly unsuccessful
despite
substantial interest and clinical efforts.
One object of the present invention is to treat human infertility.
Another object of the present invention is to improve the maturation of hu-
man oocytes.
Another object of the present invention is to improve the synchrony of nu-
2o clear, cytoplasmic and/or membranous oocyte maturation.
Another object of the present invention is to improve the fertility of
oocytes.
Another object of the present invention is to improve the rate of implantation
of oocytes by human in vitro maturation and fertilisation.
Another object of the present invention is to diminish the incidence of human
preembryos with chromosome abnormalities (aneuploidy).
Another object of the present invention is to improve the cleavage rate of
human preembryos.
Another object of the present invention is to improve the quality of human
preembryos.
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SUMMARY OF THIS INVENTION
4
It has now, surprisingly, been found that the IVF can be improved
substantially when
a MAS compound is added at the stage in the usual method of performing in
vitro
fertilisation where one would expect that the maturation had taken place in
vivo.
Briefly, the present invention relates to a method for human in vitro
fertilisation
wherein a woman, within a consecutive period of 30 days, is treated with a
hypotha-
lamic hormone and/or a pituitary hormone or an agonist or antagonist thereof
or an
active derivative thereof where after oocytes are aspirated and actively final
matured
or the oocyte maturation is synchronised in vitro in contact with a MAS
compound.
Preferred embodiments of this invention are those stated in the sub claims
below.
~5 DETAILED DESCRIPTION OF THIS INVENTION
Referring to the female cycle, on way of performing the treatment of this
invention is
as follows:
2o Around day 21 in one cycle to around day 15 in the following cycle: The
eggs are
stimulated by treating the woman with GnRH, e.g. Synarel (400-600 ~g per day).
Around days 6-15 in the second cycle: The eggs are stimulated by treating the
woman with FSH, e.g. Gonal-F, Puregon or Humegon (150-400 IU per day).
Around days 15-16 in the second cycle: The eggs are stimulated by treating the
woman with hCG, e.g. Pregnyl or Profasi (2000-5000 IU per day).
Around day 18 in the second cycle: The eggs are retrieved from the woman.
Around day 18-19 in the second cycle: The eggs are maturated with a MAS com-
pound in order to stimulate the meiosis. In this additional maturation step,
the con-
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centration of MAS compound may be in the range about 0.1-100 ~.mol per litre,
e.g.
10-20 p,mol per litre. The time for this maturation step may be in the range
around 1-
60 hours. If the preovulatory follicles are induced to luthenise with a
lutenising hor-
mone or an agonist or antagonist thereof or an active derivative thereof
and/or hu-
5 man chorion gonadotropins or an agonist or antagonist thereof or an active
derivative
thereof, the maturation of the oocytes with the MAS compound is for a duration
of
about 1-15 hours, preferably about 6 hours. If, however, preovulatory
follicles are not
induced to lutenise with a lutenising hormone or an agonist or antagonist
thereof or
an active derivative thereof and/or human chorion gonadotropins or an agonist
or an-
o tagonist thereof or an active derivative thereof, the maturation of the
oocytes with the
MAS compound is for duration of about 15-60 hours.
Around days 19-21 in the second cycle: The eggs are fertilised in vitro.
From the day before aspiration, the woman will receive an oestrogen, e.g.,
oestrogen
valerate (2 x 10 mg daily). Two days later, she will also receive a
progestogen, e.g.,
Progestane vagetoria; daily, which will render the lining of the uterus more
prone to
receive the future embryos. The duration of this treatment will be
individually de-
signed per patient. The doctor can chose among a variety of oestrogens and pro-
2o gestogens.
Around day 21 in the second cycle: One or more embryos are transferred to the
woman's uterus.
The description above is designated MAS add-on to the existing IVF protocol to
im-
prove efficacy by mediating a final or complete maturation or synchrony in the
oo-
cyte. Alternatively, MAS can be used to rescue oocytes in cycles that
otherwise
would be cancelled due to apparent FSH hyper response. In this instance, the
re-
sponsible clinician would consider cancellation based on the estradiol
profile, ultra-
so sonography (PCO like response) thus avoiding the hCG treatment. The oocytes
are
aspirated at the time around day 15-16 substituting hCG treatment.
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Apart from the additional maturation step with a MAS compound, the above IVF
is
performed the usual way. Since one expects that by the traditional IVF
procedure the
eggs had been matured sufficiently, one would not expect that it would have
any ad-
ditional effect to add this additional maturation step.
Most of the steps in the above treatment and procedure are performed in a
known
manner and the remaining steps are performed in a manner known per se. More de-
tails about the removal of the oocytes from follicles in the ovary, culturing
of the iso-
lated oocytes, the culture medium to be used, the fertilisation with sperm,
and the
o transfer of the embryo to the fallopian tube can be found in the literature,
for exam-
ple, in US patent specification No. 5,693,534 which is hereby incorporated by
refer-
ence.
According to this invention, the MAS compound is added to the culture medium
~5 used. In this medium, the amount of the MAS compound is in the range from
about
0.01 to about 100 ~,M, preferably in the range from about 0.1 to about 100
~.M.
A preferred reason for treating a woman, within a consecutive period of 30
days, with
a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist
2o thereof or an active derivative is to obtain multiple preovulatory
follicles.
Hypothalamic hormones are hormones present in the human hypothalamus.
Pituitary
hormones are hormones present in the human pituitary gland. Gonadotropic hor-
mones are hormones secreted by the anterior lobe of the pituitary in vertebras
and
25 by mammalian placenta, which control the activity of gonads. Chemically,
they are
glycoproteins. Examples of gonadotropic hormones are FSH, LH and chorion go-
nadotropin, e.g. human chorion gonadotropin (hereinafter designated hCG). FSH
stimulates growth of ovarian follicles and their oocytes in ovary and the
formation of
spermatozoa in testis. FSH can, e.g., be menopausal FSH or recombinant FSH. In
so females, LH activates the oestrogen-producing tissue of the ovaries to
produce pro-
gesterone, probably promotes the final stages of the development of ovarian
follicles,
initiates the final oocyte maturation, induces ovulation and in mammals
initiates cor-
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pus luteum development. These hormones are known. It is obvious for the
skilled art
worker that, alternatively, agonists or antagonists of these hormones can be
used. It
is also obvious for the skilled art worker that, alternatively, active
analogues of these
hormones can be used. Some of these agonists, antagonists and analogues are
s known and other can be prepared by process known per se. Examples of such
known processes are chemical synthesis and genetic engineering.
In a preferred embodiment, the present invention relates to a method or use
wherein
the consecutive period of 30 days within which the woman is treated with a
hypotha-
~o lamic hormone and/or a pituitary hormone or an agonist or antagonist
thereof or an
active derivative thereof is at least about 7 days, preferably at least about
10 days,
more preferred at least about 14 days.
In another preferred embodiment, the present invention relates to a method or
use
wherein the woman is treated for infertility, and/or for improving the
maturation of her
oocytes, and/or for improving the synchrony of nuclear, cytoplasmic and/or
membra-
nous oocyte maturation, and/or for improving the fertility of her oocytes,
and/or for
improving the rate of implantation by human in vitro maturation and
fertilisation
2o In another preferred embodiment, the present invention relates to a method
or use
wherein the consecutive period is one menstrual cycle.
In another preferred embodiment, the present invention relates to a method or
use
wherein the maturation of the oocytes with the MAS compound is for a duration
of
2s about 15 to about 60 hours.
In another preferred embodiment, the present invention relates to a method or
use
wherein preovulatory follicles are induced to lutenise with a luteinising
hormone (LH)
or an agonist or antagonist thereof or an active derivative thereof and/or
human
3o chorion gonadotropin (HCG) or an agonist or antagonist thereof or an active
deriva-
tive thereof.
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In another preferred embodiment, the present invention relates to a method or
use
wherein the maturation of the oocytes with the MAS compound is for a duration
of
about 1 to about 15 hours, preferably about 6 hours.
In another preferred embodiment, the present invention relates to a method or
use
wherein the dosage of MAS compound used is about 0.01 to about 100 ~.mol per
li-
tre, preferably about 0.1 to about 100 ~.mol per litre.
In another preferred embodiment, the present invention relates to a method or
use
o wherein the MAS compound is one of the compounds mentioned in WO 96/00235,
96/27658, 97/00884, 98/28323, 98/54965 and 98/55498, more specifically com-
pounds mentioned in Claim 1 thereof.
In another preferred embodiment, the present invention relates to a method or
use
s wherein the MAS compound is FF-MAS.
Additionally, the present invention relates to the use of a hypothalamic
hormone
and/or a pituitary hormone or an agonist or antagonist thereof or an active
derivative
thereof in the manufacture of a hormone product which is to be administered to
a
2o woman who, within a consecutive period of 30 days, is treated with a
hypothalamic
hormone and/or a pituitary hormone or an agonist or antagonist thereof or an
active
derivative thereof, and from whom, immediately after said period, one or more
oo-
cytes are aspirated, where after said oocyte(s) is/are cultivated in a
convenient me-
dium containing a MAS compound as defined herein, where after said oocyte(s)
2s is/are fertilised with human sperm, and where after the resulting embryos)
is/are
transferred to a woman.
Additionally, the present invention relates to the use of a hypothalamic
hormone
and/or a pituitary hormone or an agonist or antagonist thereof or an active
derivative
3o thereof and of a MAS compound for the manufacture of a medicament for the
treat-
ment of human in vitro fertilisation wherein a hypothalamic hormone and/or a
pitui-
tary hormone or an agonist or antagonist thereof or an active derivative
thereof is,
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within a consecutive period of 30 days, used to treat a women and, thereafter,
the
MAS compound is used in an in vitro oocyte maturation of the egg or eggs
retrieved
from this woman.
s Additionally, the present invention relates to a pharmaceutically kit in
unit dosage
form for use by in vitro fertilisation comprising separate unit dosages, said
kit com-
prising separate dosage units for sequential daily administration of a
hypothalamic
hormone and/or a pituitary hormone or an agonist or antagonist thereof or an
active
derivative thereof for sequential daily administration and 1 dosage units of a
MAS
o compound. This kit may have the preferred features described above.
The present invention is further illustrated by the following examples, which,
how-
ever, are not to be construed as limiting. The features disclosed in the
foregoing de-
scription, in the following examples and in the claims may, both separately
and in
s any combination thereof be material for realising the invention in diverse
forms
thereof.
Example 1
Method used for electing MAS compounds
Oocytes were obtained from immature female mice (C57BL/6J x DBA/2J F1, Bom-
holtgaard, Denmark) weighing 13-16 grams, that were kept under controlled tem-
2s perature (20-22°C), light (lights on 06.00-18.00) and relative
humidity (50-70%). The
mice received an intra-peritoneal injection of 0.2 ml gonadotropins (tonal-F,
Serono)
containing 20 IU FSH and 48 hours later the animals were killed by cervical
disloca-
tion. The ovaries were dissected out and the oocytes were isolated in Hx-
medium
(see below) under a stereomicroscope by manual rupture of the follicles using
a pair
of 27 gauge needles. Spherical oocytes displaying an intact germinal vesicle
(here-
inafter designated GV) were divided in cumulus enclosed oocytes (hereinafter
desig-
nated CEO) and naked oocytes (hereinafter designated NO) and placed in a-
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minimum essential medium (a-MEM without ribonucleosides, Gibco BRL, Cat. No.
22561 ) supplemented with 3 mg/ml bovine serum albumin (BSA, Sigma Cat. No. A-
7030), 5 mg/ml human serum albumin (HSA, Statens Seruminstitut, Denmark),
0.23mM pyruvate (Sigma, Cat. No S-8636), 2 mM glutamine (Flow Cat. No. 16-801
),
5 100 IU/ml penicillin and 100 ~,g/ml streptomycin (Flow, Cat No. 16-700).
This medium
was supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377) and desig-
nated Hx-medium.
The oocytes were rinsed three times in Hx-medium and oocytes of uniform
size were divided into groups of CEO and NO. CEO and NO were cultured in 4-
well
1o multidishes (Nunclon, Denmark) in which each well contained 0.4 ml of Hx-
medium
and the compound to be tested in a concentration of 10 ~,M. One control well
(i.e.,
35-45 oocytes cultured in identical medium with no addition of test compound)
was
always cultured simultaneously with 3 test wells (35-45 oocytes per well
supple-
mented with test compound).
The oocytes were cultured in a humidified atmosphere of 5% C02 in air for
24 hours at 37°C. By the end of the culture period, the number of
oocytes with GV,
GVB and polar bodies (hereinafter designated PB), respectively, were counted
using
a stereo microscope (Wildt, Leica MZ 12). The percentage of GVB, defined as
per-
centage of oocytes undergoing GVB per total number of oocytes in that well,
was
2o calculated as:
GVB = ((number of GVB + number of PB)/ total number of oocytes) X 100.
Example 2
Procedure
All IVF patients can potentially receive this treatment, age range 20 to 45
year. The
hormonal treatment can be a short or long gonadotropin based treatment with or
without pituitary down regulation or with and without the use of GNRH
antagonist and
so with or without the use of hCG. Future appropriate hormonal therapies
designed for
IVF can also be used. Medium to full size follicles (size 10 to 25 mm,
preferential 16
to 20 mm follicles) will be aspirated under ultrasound guidance.
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The aspirated fluid will be searched for cumulus oocytes complexes (COC)
and once identified under the stereomicroscope (with or without the use of
embryo
filters), the COC will be placed in culture.
The exposure to FF-MAS can vary from 1 to 60 hours, preferentially from 4
s to 30 hours, and can be before, under and up to 24 hours after
fertilisation. A wide
variety of oocyte culture media or media components known to the skilled
worker can
be used. Human serum albumin (HSA) may or may not be added to the medium. If
added, it can be in a concentration of 0.1 to 100 mg/ml, preferentially 5 to
15 mg/ml
or 0.5 to 1.5 % volume/volume. The formulation of FF-MAS may be in the form of
an
1o ethanol stock solution, DMSO or other organic solvent solution or it may be
in form of
FF-MAS/HSA dry coated wells ready to use just by adding the appropriate
culture
medium. The concentration of FF-MAS may vary from 0.1 pM to 100 p.M, preferen-
tially 10 to 30 ~M.
Following or during in vitro culture with FF-MAS, the oocytes may be fertil-
15 ised by conventional IVF or by intracytoplasmatic sperm injection (ICSI) or
by future
appropriate fertilisation methods leading to fertilised zygotes. The
developing embryo
may be transferred on day 1 to day 6 after fertilisation, preferentially on
day 2 to 3,
either as single egg transfer or multiple egg transfer.
The patient can receive progesterone and/or oestrogen therapy before and
2o after the egg transfer in individually designed protocols to prime and
sustain appro-
priate receptive endometrial lineage.
Compared with the know procedures, better results were obtained using the
above procedure.
25 Example 3
Use of FF-MAS as adjunct to a standard FSH based IVF treatment
The patient underwent down regulation with recombinant FSH with an aver-
so age daily dose of 225 IU starting on Day 1 or 2 of the current cycle and
used a GnRH
antagonist, i.e., Cetrorelix (1 mg daily, subcutaneously, one shot), in the
current cy-
cle. At least 3 follicles of 17 mm or more were present at the time of
administering
hCG, i.e., Profasi (10.000 IU, one shot). Follicles were aspirated and
metaphase II
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oocytes were cultured in oocyte culture system containing standard in vitro
fertiliza-
tion (IVF) media (IVF 20 (which is available from Scandinavian IVF Science AB,
Gothenburg, Sweden)) supplemented with human serum albumin (0.8%) and FF-
MAS (5 p,M). All oocytes were cultured under normal conditions at 37°C
in the incu-
s bator. Each oocyte was cultured in one well in a four-chamber culture dish
as culture
media system. The duration of exposure to the culture media with treatment was
4 hours (~30 minutes) before intracytoplasmic sperm injection (hereinafter
desig-
nated ICSI) and 20 hours (~1 hour) after ICSI in the above IVF 20 medium.
Preem-
bryos were evaluated for cleavage stage and fragmentation / morphology at 1, 2
and
3 days post ICSI. After 3 days of culture, a selection of the best preembryos,
typically
two preembryos, was replaced to the female patient. The female patient
received an
estrogen, i.e., Estrofem (6 mg/daily), and a progesterone, i.e. Utrogestan
(600
mg/daily, micronized, vaginal suppository), to render the endometrium lining
the pa-
tient uterus responsive and ready for allowing implantation of the transferred
eggs.
The continuation of supporting steroid hormones was patient depending and was
seponated after 3-10 weeks depending on ultrasound scans and blood testing.
Compared with the know procedures, better results were obtained using the
above procedure.
Example 4
Use of FF-MAS as adjunct to a standard FSH based IVF treatment
2s The patient underwent down regulation with an GnRH analog, i.e., Synarel
(nasal
spray, two puffs daily), for at least 14 days (starting in the luteal phase of
the previ-
ous cycle or Day 1 or 2 in the follicular phase of the current cycle) and
ovarian stimu-
lation with recombinant FSH with an average daily dose of 225 IU. At least 3
follicles
of 17 mm or more were present at the time of administering hCG, i.e. Profase
(10.000 IU, one shot). Follicles were aspirated and metaphase II oocytes were
cul-
tured in oocyte culture system containing standard in vitro fertilization
(IVF) media
(IVF 20 (which is available from Scandinavian IVF Science AB, Gothenburg, Swe-
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den)) supplemented with human serum albumin (0.8%) and FF-MAS (5 pM). All oo-
cytes were cultured under normal conditions at 37°C in the incubator.
Each oocyte
was cultured in one well in a four-chamber culture dish as culture media
system. The
duration of exposure to the culture media with treatment was 4 hours (~30
minutes)
before intracytoplasmic sperm injection (ICSI) and 20 hours (~1 hour) after
ICSI in
the above IVF 20 medium. Preembryos were evaluated for cleavage stage and
fragmentation / morphology at 1, 2 and 3 days post ICSI. After 3 days of
culture, a
selection of the best preembryos, typically two preembryos, was replaced to
the fe-
male patient. The female patient received an estrogen, i.e., Estrofem (6
mg/daily),
1o and a progesterone, i.e., Utrogestan (600 mg/daily, micronized, vaginal
suppository),
daily to render the endometrium lining the patient uterus responsive and ready
for
allowing implantation of the transferred eggs. The continuation of supporting
steroid
hormones was patient depending and was seponated after 3-10 weeks depending
on ultrasound scans and blood testing.
Compared with the know procedures, better results were obtained using the
above procedure.
Example 5
Using the procedure described in Example 3 with the proviso that in stead of
using
FF-MAS in a concentration of 5 ~,M, FF-MAS was used in a concentration of 20
~,M,
better results were obtained than with the know procedures.
Example 6
Using the procedure described in Example 4 with the proviso that in stead of
using
FF-MAS in a concentration of 5 p,M, FF-MAS was used in a concentration of 20
~M,
3o better results were obtained than with the know procedures.
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