U.S. patent application number 11/397851 was filed with the patent office on 2006-08-10 for treatment and diagnosis of infertitlity using tgfbeta or activin.
This patent application is currently assigned to Luminis Pty Ltd.. Invention is credited to Sarah Anne Robertson, Kelton Paul Tremellen.
Application Number | 20060177459 11/397851 |
Document ID | / |
Family ID | 3799811 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060177459 |
Kind Code |
A1 |
Robertson; Sarah Anne ; et
al. |
August 10, 2006 |
Treatment and diagnosis of infertitlity using TGFbeta or
activin
Abstract
A method of treating an infertility condition in humans or
mammals, by exposure of a prospective mother to TGF.beta. or
derivative or analog of TGF.beta.. The exposure is advantageously
in conjunction with one or more antigens of a prospective father so
that a hyporesponsive immune reaction is mounted to the one or more
antigens of the prospective father.
Inventors: |
Robertson; Sarah Anne; (St.
Peters, AU) ; Tremellen; Kelton Paul; (Vale Park,
AU) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1717 RHODE ISLAND AVE, NW
WASHINGTON
DC
20036-3001
US
|
Assignee: |
Luminis Pty Ltd.
Adelaide
AU
|
Family ID: |
3799811 |
Appl. No.: |
11/397851 |
Filed: |
April 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11211471 |
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11397851 |
Apr 5, 2006 |
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09380327 |
Sep 3, 1999 |
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PCT/AU98/00149 |
Mar 6, 1998 |
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11211471 |
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Current U.S.
Class: |
424/184.1 ;
514/8.9; 514/9.8 |
Current CPC
Class: |
A61K 2039/55522
20130101; G01N 33/689 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 38/484 20130101; A61K 35/19 20130101; A61K 38/484
20130101; G01N 33/74 20130101; A61K 35/19 20130101; A61K 39/001
20130101; G01N 2333/495 20130101; G01N 2800/367 20130101; A61K
38/1841 20130101; A61K 9/0034 20130101 |
Class at
Publication: |
424/184.1 ;
514/012 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 38/18 20060101 A61K038/18 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 11, 1998 |
WO |
WO 98/39021 |
Mar 6, 1997 |
AU |
PO 5508 |
Claims
1. A method of treating an infertility condition in a human or
mammal by exposure of the prospective mother to TGF.beta. or an
effective derivative or analog thereof before attempted conception
to elicit a transient hyporesponsive immune reaction to one or more
antigen of a prospective father to thereby alleviate symptoms of
the infertility condition.
2. A method of treating an infertility condition as in claim 1 by
exposure of a prospective mother to said one or more antigens of a
prospective father and to TGF.beta. or an effective derivative or
analog thereof before attempted conception to elicit a transient
hyporesponsive immune reaction to said one or more antigen to
thereby alleviate symptoms of the infertility condition.
3. A method of treating an infertility condition as in claim 2
wherein a mucosal surface of the prospective mother is exposed to
the one or more antigens.
4. A method of treating an infertility condition as in claim 3
wherein the mucosal surface is selected from the group comprising
an oral mucosal surface, a respiratory mucosal surface, a
gastrointestinal mucosal surface or a genital mucosal surface.
5. A method of treating an infertility condition as in claim 3
wherein the mucosal surface is a genital mucosal surface.
6. A method of treating an infertility condition as in claim 3
wherein the one or more antigens and TGF.beta. or derivative or
analog thereof is injected for systemic contact.
7. A method of treating an infertility condition as in claim 3
wherein the TGF.beta. or derivative or analog thereof and the one
or more antigens are adminstered at one site.
8. A method of treating an infertility condition as in claim 3
wherein the TGF.beta. or derivative or analog thereof and the one
or more antigens are each adminstered at a first site and a
different site respectively.
9. A method of treating an infertility condition as in claim 2
wherein the TGF.beta. or derivative or analog thereof and the one
or more antigen are adminstered temporarily spaced apart.
10. A method of treating an infertility condition as in claim 9
wherein the one ore more antigens are adminstered subsequent to
administration of the TGF.beta. or derivative or analog
thereof.
11. A method of treating an infertility condition as in claim 9
wherein the one or more antigens are administered first followed by
administration of TGF.beta. or derivative or analog thereof.
12. A method of treating an infertility condition as in claim 2
wherein the one or more antigens are chosen as a result of being
particularly antigenic and prominent either on the sperm, or on the
conceptus.
13. A method of treating an infertility condition as in claim 2
wherein the one or more antigens are present on cells taken from
the prospective lather that contain MHC antigens.
14. A method of treating an infertility condition as in claim 13
wherein the antigen is an MHC I antigen of the prospective
father.
15. A method of treating an infertility condition as in claim 2
wherein the one or more antigens are administered on leukocytes of
the prospective father.
16. A method of treating an infertility condition as in claim 2
wherein the one or more antigens are administrated on sperm cells
of the prospective father.
17. A method of treating an infertility condition is in claim 2
wherein the one or more antigens are administrated in the seminal
plasma of the prospective father.
18. A method of treating an infertility condition as in claim 2
wherein the one or more antigens are presented in purified or
semi-purified form.
19. A method of treating an infertility condition as in claim 18
wherein the purified or semi purified one or more antigens are
presented on inert or adjuvant carriers.
20. A method of treating an infertility condition as in claim 2
wherein humans are being treated, and the exposure of TGF.beta. is
to a mucosal surface and the level of TGF.beta. is greater than 50
ng/ml with a total dose of 150 ng/ml.
21. A method of treating an infertility condition as in claim 2
wherein the mucosal surface is exposed to a concentration of
TGF.beta. of between 100 and 400 ng/ml with a total dose of between
100 to 2000 ng.
22. A method of treating an infertility condition as in claim 2
wherein the TGF.beta. or derivative or analog thereof is supplied
in a slow release form.
23. A method of treating an infertility condition as in claim 2
wherein the exposure of the one or more antigens is to the
prospective mother's genital tract in the form of the prospective
father's ejaculate, and the level of exposure is determined by the
cell count and antigenic density on the surface of such cells.
24. A method of treating an infertility condition as in claim 3
wherein humans are being treated and the one or more antigens are
present on leukocytes, whereby between 10.sup.7 and 10.sup.9
leukocytes are adminstered to a mucosal surface.
25. A method of treating an infertility condition as in claim 2
wherein the TGF.beta. is selected from the group of
TGF.beta..sub.1, TGF.beta..sub.2 and TGF.beta..sub.3.
26. A method of treating an infertility condition as in claim 2
wherein the TGF.beta. is TGF.beta..sub.1.
27. A method of treating an infertility condition as in claim 2
wherein the TGF.beta. is modified.
28. A method of treating an infertility condition as in claim 27
wherein the modification is selected from the group comprising
substitution, deletion or addition mutants, peptide fragments of
TGF.beta. or derivative or analog thereof, and peptide fragments of
TGF.beta. or derivative or analog thereof which have been
incorporated into another protein.
29. A method of treating an infertility condition as in claim 2
wherein the TGF.beta. or derivative or analog thereof is a member
of the TGF.beta. superfamily.
30. A method of treating an infertility condition as in claim 29
wherein the member of the TGF.beta. superfamily is activin.
31. A method of treating an infertility condition as in claim 2
wherein TGF.beta. is administered in its active form.
32. A method of treating an infertility condition as in claim 2
wherein TGF.beta. is administered in precursor form.
33. A method of treating an infertility condition as in claim 2
wherein the prospective mother is incapable of converting
sufficient of the inactive form of TGF.beta. to active TGF.beta.,
and the method of treating includes administration of active
TGF.beta..
34. A method of treating an infertility condition as in claim 2
wherein the prospective mother is incapable of converting the
inactive form of TGF.beta. to active TGF.beta., and the method of
treating includes administration of a compound capable of
activating TGF.beta..
35. A method of treating an infertility condition as in claim 2
wherein the prospective mother is incapable of converting the
inactive form of TGF.beta. to active TGF.beta., and the method of
treating includes administration of plasmin, so as to increase the
level of active TGF.beta..
36. A method of treating an infertility condition as in claim 2
wherein TGF.beta. is administered in an unpurified form using a
biological source rich in TGF.beta..
37. A method of treating an infertility condition as in claim 36
wherein the TGF.beta. is administered in the form of platelets.
38. A method of treating an infertility condition as in claim 3
wherein humans are being treated and the exposure to TGF.beta. and
male antigen is a multiple exposure.
39. A method of treating an infertility condition as in claim 38
wherein the multiple exposure is preferably performed over a period
spanning at least three months prior to attempted conception.
40. A method of treating an infertility condition as in claim 2
wherein humans are being treated and exposure is at least one week
before conception is attempted.
41. A method of treating an infertility condition as in claim 2
wherein the exposure is before attempted conception.
42. A method of treating an infertility condition as in claim 2
wherein administration of TGF.beta. or derivative or analog thereof
and the one ore more antigen occurs at least once after the
prospective date of conception.
43. A method of treating an infertility condition as in claim 42
wherein the exposure continues over a period of the first 12 weeks
of pregnancy.
44. A method of treating an infertility condition as in claim 2
first including the step of diagnosing or testing whether the male
has adequate levels of TGF.beta. or the female has the capacity to
activate TGF.beta., or alternatively whether anti-sperm antibodies
exist.
45. A method of treating an infertility condition as in claim 2
used in conjunction with IVF treatment, whereby the transient
hyporeactive immune response is elicited before transfer of the
conceptus or genetics is attempted.
46. A method of diagnosing an infertility condition in males by
testing the level of TGF.beta. in seminal fluid.
47. A method of diagnosing an infertility condition in a female by
testing for the capacity of the female to convert the inactive form
of TGF.beta. to the active form.
48. A composition for use in treating an infertility condition,
comprising TGF.beta. or derivative or analog thereof and one or
more paternal antigens, and a pharmaceutically acceptable carrier,
suitable for administration to a mucosal surface.
49. A composition for use in treating an infertility condition as
in claim 48 wherein the composition comprises a vaginal gel.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a diagnostic method for an
infertility condition giving rise to reduced ability to have
offspring and to a method of treating such a condition.
BACKGROUND OF THE INVENTION
[0002] An inability or reduced ability to have children can cause
great personal distress and has a high attendant social cost,
particularly in terms of the cost of medical intervention. A large
proportion of couples fall into this category. In the USA, for
example, it is said that some 10-15% of couples of reproductive age
are unable to have children, whereas in the United Kingdom this is
14%. In 1995 it was calculated that 5.1 million women had impaired
fertility in the USA alone, with this figure projected to increase
to 5.9 million by the year 2020 (56). In the US, the cost of a
pregnancy conceived by IVF varies between US$66.000 for the first
cycle to US$114.000 by the sixth cycle (60).
[0003] In the context of this patent an infertility condition is to
be understood to relate not only the capacity to conceive but also
to miscarriage, spontaneous abortion or other pregnancy related
conditions, such as pre-eclampsia, and includes sub fertility.
[0004] Recent studies have revealed that a major proportion of
infertile couples are childless because of a higher than normal
rate of early embryonic loss (70% miscarriage v. 21% miscarriage in
fertile controls, 57), rather than an inability to conceive. These
findings have initiated a search for reasons for the increased rate
of early embryonic loss in infertile couples, as well as potential
therapies to avert such losses.
[0005] In the last 20 years or so some hope has been held out to
infertile couples with the development of in vitro fertilisation
(IVF) techniques. These IVF techniques generally take the form of
stimulating the female to ovulate, contacting collected ova with
sperm in vitro and introducing fertilised ova into the uterus.
Multiple variations of this general process also exist. Despite
considerable research and technical advances in the IVF field the
rate of successful pregnancy following IVF treatment is still quite
low and is in the order of 15 to 25% per cycle.
[0006] Undertaking an IVF program often causes great anguish,
especially when there is no resultant successful pregnancy. It is
presently believed that the poor success rate in IVF treatment is
due to an extraordinarily high rate of early embryonic loss (58,
59), possibly related to the patient's impaired reproductive state
or the IVF process itself.
[0007] The low efficacy of IVF, together with its high cost and the
associated psychological trauma from repeated treatment failures
makes it desirable that alternative approaches to the problem of
infertility are sought. Current methods of increasing pregnancy
rates during IVF treatment include placing multiple embryos (2-5)
into the uterine cavity, but this is not always effective since
uterine receptivity is believed to be at fault at least as commonly
as embryonic viability. Furthermore, the ensuing high rates of
multiple pregnancy are associated with an increased maternal risk
of pre-eclampsia, haemorrhage and operative delivery, and fetal
risks including pre-term delivery with the attendant possibility of
physical and mental handicap.
[0008] Similarly, early pregnancy loss is a major constraint in
breeding programs for livestock and rare or threatened species.
Embryonic mortality during the pre- and per-implantation period is
viewed as the major reason for poor pregnancy outcome when assisted
reproductive technologies such as artificial insemination are used.
Even following natural mating, variability in litter size and in
the viability of offspring arc additional limitations with serious
economic implications.
[0009] The reasons for increased rates of early embryonic loss
following natural and assisted conception remain unknown.
Chromosomal studies on miscarried embryos have confirmed that at
least half of these embryos are genetically normal (61). Normal
embryos appear to be lost primarily because the environment
provided by the maternal tract during pre-implantation development
or at the time of implantation into the endometrium is insufficient
to nurture their growth and development. Embryos may lose viability
or developmental potential if the maternal tract milieu comprises
inappropriate or insufficient nutrients or peptide growth factors.
Moreover, a primary determinant of uterine receptivity is provided
by the maternal immune response to the conceptus, which is
perceived as foreign or semi-allogeneic due to expression of both
maternal and paternal antigens.
[0010] Medawar originally hypothesised that maternal immune
accommodation of the semi-allogeneic conceptus may be facilitated
by immunological tolerance to paternal transplantation antigens
(major histocompatibility [MHC] antigens) (70). This hypothesis
lost favour when it was found that pregnancy does not permanently
alter the capacity of mice to reject paternal skin grafts (5, 46).
However, the concept of transient hyporesponsiveness to paternal
MHC antigens (46) is now receiving renewed attention, as a recent
study by Tafuri et al (31) has provided clear evidence to show that
during murine pregnancy, T-lymphocytes reactive with paternal class
I MHC become `anergic`, or unable to recognise antigen due to
internalisation of T-cell receptors. This anergic state conferred
`tolerance` to paternal MHC antigen-expressing tumor cells, and was
functionally operative from as early as implantation (day 4 of
pregnancy) and lasted until shortly after parturition when
lymphocytes regained their reactivity. The data support the
hypothesis that a permissive maternal immune response to other
antigens expressed on the embryo, or the fetal-placental unit
(hereafter referred to as the conceptus) may similarly be due to
induction of a tolerant immune response specific to those
antigens.
[0011] Just precisely what is responsible for inducing this
tolerance of paternal MHC antigens and other conceptus antigens has
heretofore been unclear. Additionally the nature of the tolerance
was unclear.
[0012] The term tolerance in the context of this invention is taken
to mean inhibition of the potentially destructive cell-mediated
immune response against conceptus antigens, and/or inhibition of
synthesis of conceptus antigen-reactive immunoglobulin of
complement-fixing isotypes (for example the `Th1` compartment of
the immune response). This tolerance may or may not be associated
with induction of synthesis of non-destructive, conceptus
antigen-reactive immunoglobulin of the non-complement-fixing
isotypes and subclasses (for example the `Th2` compartment of the
immune response). The term tolerance should be taken to encompass T
cell anergy and other permanent or transient forms of
hypo-responsiveness or suppression of the maternal Th1
compartment
[0013] Tafuri et al (31) have shown that paternal antigen-specific
tolerance is active by the onset of blastocyst implantation on day
4 of pregnancy in mice. The pre-implantation embryo is a poor
antigenic stimulus since it usually comprises fewer than 100 cells
and is enveloped by a protective coat (zona pelluicida) until just
before implantation. Semen however is richly endowed with paternal
antigens present on and within sperm, somatic cells and the seminal
plasma itself, and comprises an effective priming inoculum for many
paternal antigens (5) known to be shared by the conceptus. Up until
now seminal plasma has been conventionally thought to function
primarily as a transport and survival medium for spermatozoa
traversing the female reproductive tract (21). The recent studies
described by the inventors in this specification have highlighted a
hitherto unappreciated role for this fluid in interacting with
maternal cells to induce a cascade of cellular and molecular events
which ultimately lead to maternal immune tolerance to paternal
antigens present in semen and shared by the conceptus, thereby
abrogating immune rejection during implantation.
[0014] Ejaculation during coitus provokes a leukocyte infiltrate at
the site of semen deposition termed the `leukocytic cell reaction`
in a variety of mammalian species, including man (1). In mice, the
cascade of cellular and molecular changes initiated by the
introduction of semen into the uterus, in many respects, resembles
a classic inflammatory response. Within hours after mating, a
striking influx and activation of macrophages, neutrophils, and
eosinophils occurs in the endometrial stroma (2-4), in association
with upregulated expression of major histocompatibility complex
(MHC) class II and CD86 antigens by endometrial dendritic cells,
followed by enlargement of draining lymph nodes (5.6). This
inflammatory response is transient and fully dissipates by the time
of embryo implantation on day 4 of pregnancy (2-4), when leukocytes
persisting in the endometrium are predominantly macrophages with an
immunosuppressive phenotype (7).
[0015] The temporal changes in trafficking and phenotypic behaviour
of endometrial leukocytes during the period between mating and
implantation are likely to be orchestrated principally by cytokines
emanating from steroid hormone regulated epithelial cells lining
the endometrial surface and comprising the endometrial glands (8).
Of particular importance are granulocyte-macrophage
colony-stimulating factor (GM-CSF) and interleukin-(IL)-6, the
synthesis of which are upregulated at least 20-fold and 200-fold
respectively in estrogen primed epithelial cells following
induction by specific proteinaceous factors in seminal plasma (8.9)
known to be derived from the seminal vesicle gland (10). Previous
studies have implicated the surge in epithelial GM-CSF release as a
key mediator in the post-mating inflammatory response since
injection of recombinant GM-CSF into the estrous uterus is
sufficient to produce cellular changes resembling those seen
following natural mating (11). The inventors have found, using
GM-CSF deficient mice, that the chemotactic activity of GM-CSF is
likely to be compensated or augmented by an array of chemokines,
the expression of which are transiently upregulated after mating
(12), and cytokines synthesised by activated endometrial
macrophages including IL-1 and tumour necrosis factor-.alpha.
(TNF-.alpha.)(4).
[0016] The present inventors have investigated the nature of the
seminal factor which acts to stimulate GM-CSF release from the
uterine epithelium. Previous experiments have shown that the
increase in uterine GM-CSF content is neither the result of
introduction of GM-CSF contained within the ejaculate, nor a
consequence of a neuroendocrine response to cervical stimulation,
and is independent both of the presence of sperm in the ejaculate
and MHC disparity between the male and female (8). A mechanism
involving induction of GM-CSF mRNA synthesis in epithelial cells by
proteinaceous factors derived from the seminal vesicle was
suggested by experiments showing that seminal vesicle-deficient
(SV-) males did not evoke GM-CSF release or a post-mating
inflammation-like response in females, and that trypsin-sensitive,
high molecular weight material extracted from the seminal vesicle
could upregulate GM-CSF release from uterine epithelial cells in
vitro (10).
[0017] It has, however, not been clear from previously published
work that this inflammatory response is related to the induction of
tolerance by the mother to the conceptus, or alternatively whether
the inflammatory response has a role in enhancing the immune system
to combat the influx of foreign matter such as potential pathogenic
bacteria is not clear. Nor is there any indication as to what the
trigger for the induction of tolerance is or indeed that tolerance
is mediated by semen.
[0018] One known relevant prior art document is U.S. Pat. No.
5,395,825 by Feinberg. This specification discloses a finding that
suggests that elevated TGF.beta. in the female reproductive tract
can facilitate production of fibronectin, a protein hypothesised to
assist implantation by promoting adhesion of the embryo to the
endometrial surface. The half life of TGF.beta. is only a few
minutes and its effect on fibronectin is very short term. Therefore
the administration of TGF.beta. in the above method can only be
contemplated to assist implantation if delivered at precisely the
time at which the pre-implantation embryo arrives in the uterine
cavity. The present invention does not require such temporal
precision in TGF.beta. delivery, nor does it purport that the
effect of TGF.beta. is mediated through fibronectin.
SUMMARY OF THE INVENTION
[0019] The inventors have identified TGF-.beta. as a principal
immune regulatory molecule within seminal plasma. TGM.beta.
produced in the latent form in the seminal vesicle gland is
activated within the female reproductive tract where it acts to
induce GM-CSF synthesis in uterine epithelial cells, thereby
initiating the post-coital inflammatory response.
[0020] Additionally the inventors have shown that TGF.beta., when
administered to the female reproductive tract together with sperm
or semen, can elicit tolerance towards male antigens, including
paternal MHC class I antigens. This state of tolerance is evidenced
by inhibition of Th1-type immune responses to paternal antigens,
including delayed-type hypersensitivity (DTH) responses primed by a
previous injection with sperm, production of complement-fixing
isotypes of immunoglobulin specific for sperm, and cell-mediated
immune rejection of tumor cells bearing the same MHC class I
antigens as contained in the priming sperm inoculum. It is proposed
that this tolerance might be achieved by exposure of the female to
TGF.beta. either with or without male antigen.
[0021] The significance of this is that it is highly likely that
certain infertility conditions will be related to the incapacity to
produce tolerance to antigens of the male and/or to provide a
suitable cytokine environment for growth and development of the
pre-implantation embryo, as a result of either a lack of TGF.beta.
in the seminal fluid of the male, an incapacity of the female to
process the TGF.beta. from an inactive to an active form, or an
absence or low levels of paternal antigens in the ejaculate. In
some instances infertility may be due to the inability of the
female to respond to TGF.beta., in which case direct application of
molecules induced by TGF.beta., such as GM-CSF, may be
warranted.
[0022] The TGF-.beta..sub.1 content of murine seminal vesicle
secretions, like that of human seminal plasma (22), was found to be
extraordinarily high and second only to that reported for platelet
distillate (23). In mammalian species the TGF-.beta. family
comprises at least three closely related polypeptides,
TGF-.beta..sub.1, -.beta..sub.2 and -.beta..sub.3 (24), which
exhibit 70-80% sequence homology and share many biological actions.
TGF.beta., is the dominant TGF.beta. isotype responsible for
increasing murine uterine GM-CSF output, since
TGF.beta..sub.1-specific neutralising antibody is now found to have
the ability to block 85% of seminal vesicle GM-CSF stimulating
activity (FIG. 2). Other members of the TGF.beta. superfamily, such
as TGF.beta..sub.2 and activin, have also now been identified as
capable of eliciting an increase in uterine GM-CSF output (FIG. 4).
These additional members of the TGF-0 family, complexed with other
carrier proteins such as the 250-300 kDa binding protein betaglycan
(25) may account for the higher molecular weight activity present
in murine seminal vesicle fluid and human seminal plasma (22).
[0023] The synthesis of TGF.beta. as a latent complex is believed
to have a stabilising effect (26) and focus its activity at the
target site by binding to extracellular matrix (27). Evidence for a
uterine mechanism for activation of latent TGF-.beta. was provided
by the present finding that in contrast to activity in the seminal
vesicle, the majority of the TGF.beta..sub.1 found in the uterine
luminal fluid after mating was in the active form (FIG. 5). Plasmin
or other proteolytic enzymes derived from uterine cells or the male
accessory glands (28, 29, 47) may contribute to the activation of
TGF.beta. after ejaculation.
[0024] The proposal that components of the ejaculate can indirectly
contribute to pregnancy success is supported by experiments in
accessory gland-deficient mice (36, 37) and the finding that poor
pregnancy outcome and dysregulated fetal and/or placental growth
after embryo transfer or during first pregnancy in various
livestock species (38-40) can be partially ameliorated by prior
exposure to semen (41, 42). Likewise, studies in humans now clearly
identify lack of exposure to semen due to limited sexual
experience, use of barrier methods of contraception, or in IVF
pregnancies with increased risk of implantation failure,
spontaneous abortion and pre-eclampsia (43-45).
[0025] In a broad form the invention could be said to reside in a
method of treating an infertility condition in a human or mammal by
exposure of the prospective mother to TGF.beta. or an effective
derivative or analog thereof before attempted conception to elicit
a transient hyporesponsive immune reaction to one or more antigen
of a prospective father to thereby alleviate symptoms of the
infertility condition.
[0026] In another broad form the invention could be said to reside
in a method of treating an infertility condition in a human or
mammal by exposure of a prospective mother to one or more antigens
of a prospective father and to TGF.beta. or an effective derivative
or analog thereof before attempted conception to elicit a transient
hyporesponsive immune reaction to said one or more antigen to
thereby alleviate symptoms of the infertility condition.
[0027] Preferably a mucosal surface of the prospective mother is
exposed to the antigen, and more preferably the mucosal surface is
the genital mucosal surface, however, it is feasible that exposure
at other mucosal surfaces can give rise to the transient paternal
antigen tolerance. There are two basic reasons that this might be
the case, firstly it is known that tolerance to external antigens
can be elicited at mucosal surfaces, thus it is known that women
that are exposed to seminal fluid orally show evidence of reduced
pre eclampsia effects to MHC antigens of the male partner (48).
Thus the exposure could be oral, respiratory, gastrointestinal or
genital. For example the surface antigen and TGF.beta. may be
presented as an oral or nasal spray, or as a rectal or vaginal gel.
Such a gel might for example be a gel such as used in the vaginal
gel sold under the brand name PROSTIN (Upjohn Pty Ltd).
Alternatively it might be desired to take the TGF.beta. and the
surface antigen in a form that gives exposure to the small and
perhaps large intestines, such as perhaps contained in a gelatin
capsule.
[0028] Whilst a mucosal exposure may be preferred because it is
likely to give rise to a transient tolerant immune reaction, it may
also be feasible to provide for another route of exposure. Thus the
surface antigen and TGF.beta. may be injected for systemic
contact.
[0029] It may be desirable to deliver the TGF.beta. and the antigen
together, for example where the two are combined in a gel, or
spray, alternatively, it might be desirable to provide a source of
TGF.beta. at the mucosal surface of interest, which might be the
genital tract, and the antigen could subsequently be deposited onto
the mucosal surface. It is also not yet clear whether the TGF.beta.
needs to be present at the same time as the antigen is present,
although it is believed to be preferable, however, it is proposed
that it may be possible to have a delay between the delivery of the
TGF.beta. and the surface antigen. Thus an alternative would be to
deposit the antigen first perhaps as an ejaculate and then deliver
the TGF.beta. as a pessary after intercourse.
[0030] The nature of the relevant surface antigens is not entirely
clear, but will presumably be those that are particularly antigenic
and prominent either on the sperm, or on the conceptus. The most
likely candidates are MHC antigens, and more preferably MHC class
I. The most efficient manner of presenting these antigens is in the
form that they are naturally present--on any appropriate cell of
the intended male parent that expresses them and those cells would
include sperm cells and may include leukocytes. The antigens may
also be presented in biological fluids such as seminal plasma which
is known to carry certain male antigens (49). This use or cells
other than sperm cells will be pertinent where the sperm count of
the prospective father is somewhat low. The use of cells other than
sperm cells may be preferred where a non-genital route is used.
Alternatively the antigens may be presented in purified or
semi-purified form, which may or may not be presented on inert or
adjuvant carriers, thus for example it may be presented in the
carriers known as ISCOMS. This latter approach however is likely to
be more technically complex and expensive. It is additionally
possible that the antigens may be encoded within sperm cells in the
form of mRNA (or other nucleic acid) and this RNA message is then
expressed by maternal genital tract cells. It may be that TGF.beta.
therefore plays a role in promoting the events leading to
presentation of paternal antigen to maternal lymphocytes through
activating genital tract antigen presenting cells to take up and
translate sperm mRNA.
[0031] The level of TGF .beta. may be varied, and will vary
depending upon which species is being treated. For humans the level
of TGF.beta. will preferably be greater than 50 ng/ml with a total
dose of 150 ng/ml and more preferably at a concentration of between
100 and 400 ng/ml with a total dose of between 100 to 2000 ng. The
level of TGF.beta. in normal male semen is in the order of 200
ng/ml. This level can be judged empirically when assessing other
animals, and thus for horses or cattle the preferred level is
expected to be in the order of 100 ng/ml. These levels may vary if
the TGF.beta. is supplied in a slow release depot, perhaps as a
patch or as a gel or latent TGF.beta. complex.
[0032] The level of exposure to surface antigens may vary, in a
preferred form the exposure will be to the prospective mother's
genital tract in the form of the prospective father's ejaculate,
and the level of exposure will be determined by the cell count and
antigenic density on the surface of such cells. Where cells are
administered other than in the above manner, a similar number of
cells might be used, however, the most effective manner may be
determined empirically. It is though that an exposure of leukocytes
in the order of 10.sup.7-10.sup.9 cells might be the appropriate
level of exposure to a mucosal surface.
[0033] The specificity of TGF.beta. to be co-administered with the
male antigens is at present not entirely clear, and because
TGF.beta..sub.1 is thought to be responsible whereas
TGF.beta..sub.2,3 are less important, it is more likely that
TGF.beta..sub.1 is to be used. It will however also be understood
that various modification might be made to TGF.beta..sub.1 or
indeed TGF.beta.2, or TGF.beta..sub.3 which could be effective in
eliciting an effective transient tolerant immune reaction either
separately or in combination with another agent. Such modified
TGF.beta.'s might include substitution, deletion or addition
mutants, and might include peptide fragments, which may or may not
be incorporated into another protein to make a recombinant protein.
Alternatively other members of the TGF.beta. superfamily may also
be used or used as a starting point to developing an analog of the
TGF.beta. activity, one such member is known as activin.
[0034] Where unmodified TGF.beta. is used it will preferably be
administered as TGF.beta..sub.1. The TGF.beta..sub.1 may be
administered in its active form, however, where the prospective
mother is capable of activating TGF.beta..sub.1 it may also be
administered in its precursor form. An alternative "delivery"
option would be natural TGF.beta. such as in the form of platelets.
Thus instead of purified TGF.beta. a preparation of platelets or
other source rich in natural TGF.beta., such as milk or colostrum,
may be used.
[0035] The exposure is preferably a multiple exposure. The multiple
exposure is preferably performed over a period of at least three
months, with the mucosal surface being exposed to TGF.beta. during
each exposure to the prospective father's antigens. This period of
time could however be somewhat reduced, and it may be possible to
achieve improvement with one exposure but as a minimum it is
anticipated that exposure would be at least one week before
conception is attempted. It may also be preferred that non-barrier
contraceptive measures be taken prior to the planned conception,
where the antigens are associated with sperm cells and these are
administered to the genital tract, so that there is some certainty
of a period of exposure to the prospective father's antigens before
conception. This is particularly the case where the fertility
condition is of the type where conception takes place but either
miscarriage, spontaneous abortion or pre-eclampsia occurs after
conception.
[0036] It is also envisaged that the administration of TGF.beta. in
the presence or absence of the at least one surface antigen may
need to continue past the prospective date of conception perhaps
for the first 12 weeks of pregnancy.
[0037] In an alternative form the invention could be said to reside
in a method of diagnosing an infertility condition in males by
testing the level of TGF.beta. in seminal fluid.
[0038] Greater than 70% of the TGF-.beta..sub.1 in seminal vesicles
exists in the latent form. The infertility condition might
therefore not be due to a lack of TGF.beta. in the semen of the
male partner but it may be that the female cannot process the
inactive form of the TGF.beta.. The invention could therefore also
be said to include the method of exposing inactive form of
TGF.beta. to the genital tract of a female and testing for her
capacity to convert the inactive form of TGF.beta. to active
TGF.beta.. If this is found to be the case, the method of treating
the fertility condition will include administration of active
TGF.beta., or alternatively a compound capable of activating
TGF.beta. can be administered, such as plasmin, so as to increase
the level of active TGF.beta..
[0039] In a preferred form the method of treating infertility will
first include the step or diagnosing or testing whether the male
has adequate levels of TGF.beta. or the female has the capacity to
activate TGF.beta., or alternatively whether anti-sperm antibodies
exist.
[0040] The use of the present invention may be used in conjunction
with IVF treatment, whereby the transient tolerant immune response
is elicited before transfer of the conceptus or gametes is
attempted. It is expected however that where the infertility
condition is caused as a result of reduced TGF.beta. level in
semen, or capacity to activate TGF.beta., it is likely that the
trauma of IVF treatment may not be needed and that a `natural`
conception may be possible in its place.
[0041] It will be understood that this invention is not necessarily
limited to humans, but may also extend to treatment of other
mammals including livestock species.
[0042] Some specific disorders or procedures that may benefit from
the present invention are now discussed to some degree.
[0043] Recurrent miscarriage. It is known that approximately 2-5%
of couples are involuntarily childless due to recurrent
miscarriage. The aetiology of recurrent miscarriage is complex, but
in the vast majority of cases no chromosomal, hormonal nor
anatomical defect can be found and an immunological lesion is
implicated. A variety of therapies which attempt to modify the
mother's immune response to the semi-allogeneic conceptus have been
trialed with variable success. The predominant therapeutic approach
over the past 20 years has been to inject women with paternal
leucocytes in the hope of achieving `tolerance` to paternal
antigens. This therapy has had limited success with a meta-analysis
of 15 trials concluding that paternal leucocyte immunisation can
increase pregnancy rates by 8-10% (51).
[0044] Coulam & Stern (52) have administered seminal plasma
from a pooled donor source to the genital tract of women with
recurrent miscarriage and were able to produce a non-statistically
significant increase in live birth rates (60% v 48%, p=0.29 n=86).
This treatment differs significantly from a preferred therapeutic
regime in that seminal plasma was administered in the absence of
paternal antigen. It is not surprising that the success of this
therapy was limited, since no paternal antigen was
administered.
[0045] The data supporting the present invention provide
encouraging results which indicate that TGF.beta. may be a
beneficial treatment for recurrent miscarriage because of its
potent immune modulating capacity. It is expected that
administration of sperm in combination with TGF.beta. will help
produce a tolerant or `nurturing` immune response to a future
conceptus which would share some of the same MHC class I or other
antigens.
[0046] Adjunct to IVF treatment. It is currently believed that
premenstrual pregnancy wastage produces a significant negative
contribution to IVF success rates. One theory for this increased
early pregnancy loss is that IVF is an `unnatural` process that
separates the act of intercourse from conception. This would mean
that IVF recipients may not be exposed to seminal plasma and it's
associated antigens early in pregnancy. Several animal studies and
human investigations, including the randomised control trial
described herein, have suggested that exposure of the female
genital tract to semen at the initiation of a pregnancy, as well as
prior to a pregnancy, is beneficial to subsequent pregnancy
outcome. It is proposed that there will be some benefit derived
from giving women exogenous TGF.beta. in combination with partner's
sperm/leucocytes at or near the time of embryo transfer, especially
if the partner's seminal plasma TGF.beta. content is low or sperm
numbers are low.
[0047] Anti-sperm antibody therapy. A significant proportion of
infertility is due to the presence of anti-sperm antibodies in
either the male or female partner (53). Seminal plasma has been
shown to suppress the formation of anti-sperm antibodies in the
female serum and genital tract secretions of the mouse. One of the
active agents within seminal plasma responsible for suppressing
maternal production of potentially damaging, complement-fixing
isotypes or subclasses of immunoglobulin specific for sperm
antigens has been identified as TGF.beta.. It is expected that the
present invention may, in at least some instances, block anti-sperm
antibody formation. The relationship between maternal anti-sperm
antibody formation in women and their partner's seminal plasma
TGF.beta. concentration will be investigated to confirm this.
Current therapies for anti-sperm antibodies are not sufficiently
effective (for example oral steroids or the prolonged use of
barrier contraception) or require expensive assisted reproduction
therapy. It is proposed that administration of a
TGF.beta.-containing pessary following intercourse will abrogate
this anti-sperm antibody response and enable natural pregnancy to
ensue.
[0048] Pre-eclampsia and IUGR prophylaxis. Pre-eclampsia and some
forms of intra-uterine growth restriction (IUGR) are believed to be
an immunological disorder due to `shallow` placentation resulting
from a damaging, Th1-type immune attack on the invasive
trophoblast. There is epidemiological evidence showing that
repeated exposure of a woman to her partner's antigens through
intercourse in the absence of barrier contraception decreases her
chances of developing pre-eclampsia in a subsequent pregnancy to
that partner (54, 55). This may be brought about by the generation
of maternal `tolerance` towards paternal antigens as a consequence
of repeated exposure at intercourse, which facilitates placental
growth and invasion of the maternal decidua. Some women have a
propensity to develop pre-eclampsia or to suffer fetal growth
restriction every time they become pregnant. This may be due to
inadequate TGF.beta. content of their partner's semen, or an
inability to process latent TGF.beta. into a biologically active
form.
[0049] Priming with partner's antigens in combination with
TGF.beta. before conception and perhaps until 3 months of
pregnancy, by which time placental invasion is complete, may help
prevent the development of pre-eclampsia and IUGR in these high
risk women.
[0050] Prospective analysis of stud animal fertility in livestock
breeding industries. Variability in the productivity of stud males
is a major constraint in pig, cattle, sheep and other livestock
breeding programs. In many species there are substantial
differences between studs, particularly in the pre-implantation
mortality of embryos sired, even within a given herd. Currently,
reliable estimation of the fertility and fecundity of a stud male
is only possible after documentation of the outcome of multiple
pregnancies. Measurement of the TGF.beta. content of seminal plasma
of potential studs, for example by simple enzyme-linked
immunosorbent assay, is likely to be an effective tool in livestock
breeding management. Such measurements may need to be taken over
the course of some weeks and could be made in conjunction with
measurements of other factors known to inhibit the action of
TGF.beta., such as interferon-.gamma..
[0051] Optimisation of pregnancy outcome in livestock breeding
industries. A primary determinant of the productivity of livestock
breeding programs, particularly in species such as the pig where
litters are large, is variability in the litter size and weight of
offspring. As detailed above, these parameters are believed to be
influenced largely by the extent to which the mother's immune
response is `tolerised` to paternal antigens shared by the
conceptus. Pregnancy outcome is often further compromised where the
pregnancy is initiated by artificial insemination, particularly
when artificial semen extenders, as opposed to seminal plasma, are
employed as the carrier. Since the frequency of mating between
breeding females and studs is often limited, and variability in the
seminal plasma TGF.beta. content between males is probable,
pregnancy outcome is likely to benefit from exogenous
administration of TGF.beta. in many livestock species. TGF.beta.
could be given prior to, or at the initiation of a naturally-sired
pregnancy, or at the time of artificial insemination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1. Sephacryl S-400 size exclusion chromatography of (A)
GM-CSF stimulating activity and (B) TGF-.beta. immunoactivity in
murine seminal vesicle fluid. In A, uterine epithelial cells from
estrous mice were incubated for 16 h with untreated (o, =active
TGF-.beta.) or acid activated (.circle-solid.=active+latent
TGF-.beta.) fractions of seminal vesicle fluid. After a further 24
h culture, the GM-CSF content of supernatant % was determined by FD
5112 bioassay. Values are means of triplicate cultures and the
horizontal dashed line is GM-CSF production by epithelial cells
cultured with DMEM-FCS alone. In B, the content of immunoactive
TGF-.beta..sub.1 (.circle-solid.) in fractions of seminal vesicle
fluid was determined by ELISA. TOF-.beta. bioactivity was detected
by Mv-1-Lu cell bioassay. Fractions depicted by the hatched area
contained >300 pg/ml, and other fractions contained <50
pg/ml. Data is representative of similar results obtained from
three replicate experiments.
[0053] FIG. 2. The effect of neutralising antibodies specific for
TGF-.beta..sub.1,2,3 and TGF-.beta..sub.1 on GM-CSF stimulating
activity in murine seminal vesicle fluid. Uterine epithelial cells
from estrous mice were incubated for 16 h with 2% crude seminal
vesicle fluid or DMEM-FCS alone, in the presence or absence of
mouse anti-bovine TGF-.beta..sub.1,2,3 (20 .mu.g/ml) or chicken
anti-bovine TGF-01 (10 .mu.g/ml). After a further 24 h culture, the
GM-CSF content of supernatants was determined by FD 5/12 bioassay.
Values are mean.+-.SD of triplicate cultures. Data is
representative of similar results obtained from three replicate
experiments.
[0054] FIG. 3. The effect of TGF-.beta..sub.1 on GM-CSF production
by uterine epithelial cells in vitro. Uterine epithelial cells from
estrous mice were incubated for 16 h with 0.08-80 ng/ml recombinant
human TGF-.beta..sub.1. After a further 24 h culture, the GM-CSF
content of supernatants was determined by FD 5/12 bioassay. The
mean.+-.SD of triplicate wells is shown. Data is representative of
similar results obtained from four replicate experiments.
[0055] FIG. 4. The effect of TGF-.beta..sub.2, activin and inhibin
on GM-CSF production by uterine epithelial cells in vitro. Uterine
epithelial cells from estrous mice were incubated for 16 h with
0.05-50 ng/ml recombinant human TGF-.beta..sub.1, porcine
TGF.beta..sub.2, or human recombinant activin and inhibin. After a
further 24 h culture, the GM-CSF content of supernatants was
determined by FD 5/12 bioassay. The mean.+-.SD of triplicate wells
is shown. Data is representative of similar results obtained from
two replicate experiments.
[0056] FIG. 5. The effect of seminal composition on the
TGF-.beta..sub.1 content of uterine luminal fluid after mating.
TGF-.beta..sub.1 immunoactivity was determined by ELISA in
untreated (o=active TGF-0) or acid activated
(.circle-solid.=active+latent TGF-.beta.) uterine luminal fluids
collected from estrous mice, or from mice 1 h after mating with
intact, vasectomized (vas) or seminal vesicle deficient (SV-)
males. Symbols represent data from individual mice and median
values for treatment groups are scored. Data were compared by
Kruskal-Wallis one way ANOVA and Mann Whitney Rank Sum test. Data
sets labelled on the x-axis with different lower case letters
denote statistical significance between treatment groups
(p<0.01).
[0057] FIG. 6. The effect of intra-uterine TGF-.beta..sub.1 on the
GM-CSF content of uterine luminal fluid. Fluids were collected 16 h
after natural mating with intact males, or after administration of
0.4-40 ng recombinant human TGF-.beta..sub.1 in 50 .mu.l PBS/1%
BSA, or vehicle only, to the uterine luminal cavity of estrous
mice. Symbols represent data from individual mice and median values
for treatment groups are scored. Data were compared by
Kruskal-Wallis one way ANOVA and Mann Whitney Rank Sum test. Data
sets labelled on the x-axis with different lower case letters
denote statistical significance between treatment groups
(p<0.01).
[0058] FIG. 7. The effect of rTGF.beta..sub.1 and semen on GM-CSF
output from human reproductive tract epithelial cells. The GM-CSF
content of culture supernatants collected from (A) cervical
keratinocytes and (B) endometrial cell cultures was determined by
commercial ELISA. 12 hours after the addition of dilute whole semen
(10% vol/vol) or 10 ng/ml rTGF.beta..sub.1.
[0059] FIG. 8. The effect of intra-uterine priming with sperm and
TGF.beta. on induction of Th1-type immunity. Balb/c F1 female mice
were immunised by intra-uterine infusion with CBA sperm in the
presence of absence of 10 ng rTGF.beta.. Additional groups of
uterine-ligated mice were mated naturally with CBA males, or were
given sub-cutaneous immunisations with sperm in complete Freund's
adjuvant. Ten days later mice were assessed for DTH Lo sperm
antigens, or serum content of anti-sperm IgG2b immunoglobulin. Data
was compared by Kruskal-Wallis one way ANOVA, followed by Mann
Whitney rank sum test with different superscripts indicating
significant differences (p<0.05).
[0060] FIG. 9. Effect of prior immunisation with sperm and
TGF.beta. on fetal and placental weights during subsequent
pregnancy in mice. Balb/F1 female mice were immunised by
intra-uterine infusion with CBA sperm in the presence
(.+-.rTGF.beta..sub.1), and were mated naturally with CBA males 2
weeks later. Females were sacrificed on day 17 of pregnancy and
fetal (A) and placental weights (B) were determined. Comparisons
between groups were made according to the number of viable
fetal-placental units per uterine horn, by Kruskal Wallis one-way
ANOVA followed by Mann Whitney rank sum test (p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
Cell Lines, Media, Cytokines and Antibodies.
[0061] RPMI-1640 and low glucose Dulbecco's modified Eagle' medium
(DMEM, GIBCO) were supplemented with 10% fetal calf serum (CSL), 20
mM HEPES pH 7.2, 5.times.10.sup.-5 M .beta.-mercaptoethanol, 2 mM
L-glutamine and antibiotics (RPMI-FCS and DMEM-FCS). FD5/12 cells
(14), 3T3 fibroblasts, and JR-5 Balb/c fibrosarcoma cells were
cultured in RPMI-FCS and mink lung cells [Mv-1-Lu, CCL-64] and
uterine epithelial cells were cultured in DMEM-FCS. Human
ectocervical cells were cultured in 70% DMEM, 20% Hams F-12
(Gibco), 9% FCS, 1% Neutridoma-SP (Boehringer Mannheim), and 0.4
.mu.g/ml hydrocortisone (Upjohn. Rydalmere, NSW) (ECM-FCS), and
human endometrial cells were cultured in DMEM-FCS.
[0062] Recombinant human (rh)TGF-.beta..sub.1 was from R&D
Systems, recombinant murine GM-CSF was provided by N. Nicola, The
Walter and Eliza Hall Institute for Cancer Research, and
recombinant human activin and inhibin were provided by J. Findlay,
Prince Henry's Institute for Medical Research. Monoclonal
antibodies (mAb) used for immunohistochemistry were anti-CD45 (TIB
122), anti-Mac-1 (CD11b. TIB 128), anti-MHC class II (Ia antigen.
TIB 120; all from ATCC). F4/80 (15), and RB6-6C5 (16). Mouse
anti-bovine TGF-.beta..sub.1,2,3 mAb (which neutralizes all three
mammalian TGF-.beta. isoforms) was from Genzyme (Cambridge. MA) and
chicken anti-bovine TGF-.beta.1 mAb (neutralizes TGF-.beta.1,
<2% cross reactivity with TGF-.beta..sub.2 and -.beta..sub.3)
was from R & D Systems.
[0063] Mice and Surgical Procedures. Adult (8-12 week) female mice
of the [Balb/c X C57B1]F1, Balb/c or Balb/k strains, and adult male
mice of the [CBA.times.C57B1]F1, CBA, or Balb/c strains were
obtained from the University of Adelaide Central Animal House and
maintained in a minimal security barrier facility on a 12 hour
light/12 hour dark cycle with food and water available ad libitum.
Females were synchronised into estrus using the Whitten effect (17)
and cycle stage was confirmed by analysis of vaginal smears. For
natural mating, females were placed 2 per cage with individual
males and the day of sighting of a vaginal plug was nominated as
day 1 of pregnancy. Male studs used for collection of accessory
gland secretions were all of proven fertility and were rested for
one week prior to use.
[0064] For intra-uterine injections, uterine horns of estrus
females were exteriorised through a dorsal midline excision and
injected with 0.2-40 ng rhTGF-.beta..sub.1 in 50 ml of RPMI/0.1%
BSA, or vehicle only, prior to sacrifice of mice 16 hours later for
assessment of luminal cytokine content or collection of uterine
tissue for immunohistochemistry. Non-surgical administration of
sperm/TGF.beta..sub.1 to the uterine lumen was achieved by passing
a 3 French gauge Tom Cat.TM. catheter (Sherwood Medical, St. Louis,
Mo.) into the uterine lumen (proximal to the point of bifurcation)
of restrained females, after visualisation of the cervix with the
aid of an auriscope (Heine, Germany), and manual dilation of the
cervix with a fine wire. Each uterine catheter was loaded with 50
.mu.l of sperm/TGF.beta..sub.1, which was delivered to the uterine
cavity with the aid of a mouth pipette.
[0065] Vasectomised mice were prepared by bilateral ligation of the
vas deferens through a transverse incision in the abdomen (Hogan et
al., 1986), and seminal vesiculectomised mice were prepared by
removal of the seminal vesicles through a transverse incision in
the abdomen following ligation and severing of the proximal tubule
at the base of the gland. The body wall and skin were sutured and
the mice were allowed to recover for at least two weeks prior to
mating.
[0066] All surgical procedures were performed under anaesthesia
using Avertin [1 mg/ml tribromoethyl alcohol in tertiary amyl
alcohol (Sigma) diluted to 2.5% v/v in saline; 15 .mu.l/g body
weight injected i.p.].
[0067] Collection of Reproductive Tract Fluids. Seminal vesicle
secretions were extruded from intact glands and solubilised in 6 M
guanidine HCl (1:4 v/v), then desalted into DMEM using 5 ml
Sephadex G-25 desalting columns (Pharmacia) before application to
epithelial cell cultures. Prostate and coagulating gland secretions
were extracted by homogenisation of intact glands in 0.5 ml of
PBS/1% BSA, followed by sedimenation of debris at 5000 g. Uterine
luminal fluid was collected 16 h after mating or instillation of
rhTGF-.beta.1 into the uterus by flushing each horn with 500 .mu.l
of RPMI-FCS. Debris was sedimented at 2000 g and the supernatant
stored at -80.degree. C. prior to cytokine assay. In experiments
where uterine TGF-.beta.1 was measured, flushings of the right horn
were made with 6 M guanidine HCl/0.1% BSA, and desalted into
PBS/0.1% BSA prior to cytokine assay. For matings with intact and
seminal vesicle deficient males the left horn was flushed with DMEM
to enable confirmation that adequate insemination had occurred
(>1.times.10.sup.6 sperm per ml).
[0068] Chromatography. Approximately 1 ml of seminal vesicle fluid
in 6 M guanidine HCl was applied to a Sephacryl S-400 column (40
cm.times.16 mm; Pharmacia) equilibrated in 6 M guanidine HCl/0.05 M
Hepes pH 7.4. Fractions of 1 ml were collected, desalted into DMEM
and assayed for GM-CSF-stimulating activity. Before addition to
uterine culture or TGF-.beta. assay half of each fraction was acid
activated as previously described (18).
[0069] Murine uterine epithelial cell cultures. Uterine epithelial
cells were prepared as previously described (19) and plated in 1 ml
culture wells (Nunc) at 1-2.times.10.sup.5 cells/ml in 500 .mu.l of
DMEM-FCS. After 4 h incubation at 37.degree. C. in 5% CO2 to allow
cell adherence, a further 500 .mu.l of desalted seminal vesicle
fluid in DMEM-FCS, cytokines in DMEM-FCS, or DMEM-FCS alone, were
added. Culture supernatants were collected and replaced with fresh
medium at 16 hours, then collected again 24 hours later, at which
time adherent cells were quantified as previously described (19).
All treatments were performed in duplicate or triplicate.
[0070] Human endometrial cultures. Human endometrial cell cultures
were prepared under sterile conditions using a modification of the
procedure described by Bentin-Ley (64). Briefly, stromal cells were
embedded in a collagen matrix, covered by a thin layer of Matrigel
(Collaborative Biomedical Products. Bedford, Mass.), which in turn
was over-laid with uterine epithelial cells. Uterine epithelial
cell supernatants were collected at 12 hrs (basal), replaced with
400 .mu.l of medium containing either rTGF.beta..sub.1, semen, or
fresh culture medium, and supernatants were collected 12 h later.
The GM-CSF content of 24 h supernatants were normalised to the
GM-CSF content of corresponding 12 h (basal) supernatants.
[0071] Human cervical keratinocytes. Human cervical keratinocytes
were cultured using a modification of the technique described by
Rheinwald and Green (65). Cervical biopsies were obtained from
consenting women undergoing hysterectomy for non-malignant
gynaecological indications. All women were pre-menopausal, but no
distinction was made regarding stage of menstrual cycle at the time
of surgery. The cervical biopsies were placed in ice-cold HBSS for
transport to the laboratory, washed twice in antibiotic containing
medium, and incubated overnight at 4.degree. C. in DMEM containing
5 U dispase (Boehringer Mannheim). Large sheets of keratinocytes
were mechanically stripped from the biopsy using sterile forceps
after a subsequent 1 h incubation at room temperature.
Disaggregation into single cells was facilitated by incubation in
DMEM/0.25% trypsin/0.05% collagenase for 30 minutes at 37.degree.
C., and repeated aspiration using a needle and syringe.
Keratinocytes were cultured in ECM-FCS, at a density of
1-2.times.10.sup.-5 cells/ml, over monolayers of murine 3T3
fibroblasts rendered mitogenically inactive by exposure to 4%
mitomycin C (Sigma). Keratinocytes were incubated for 5-7 days to
enable attachment and displacment of the 3T3 fibroblasts, when the
media was replaced with fresh ECM-FCS. Supernatant was collected 12
h later (basal) and replaced with 500 .mu.l of ECM-FCS containing
10 ng of rTGF.beta..sub.1, 10% semen or culture medium only
(control), which in turn was collected 12 hrs later. The GM-CSF
content of 24 h supernatants were normalised to the GM-CSF content
of corresponding 12 h (basal) supernatants.
[0072] Cytokines and Cytokine Assays. GM-CSF was assayed using the
GM-CSF dependant cell line FD5/12, essentially as previously
described (19). Cell proliferation was determined by the addition
of Alamar Blue (Alamar Biosciences) for the last 24 h of the assay
or by pulsing with 1 .mu.Ci of [.sup.3H]-thymidine per well for the
last 6 h of the assay. The minimal detectable amount of GM-CSF was
1 U/ml (50 U/ml defined as that producing half maximal FD5/12
proliferation). TGF-.beta. bioactivity was measured using Mv-1-Lu
cells as previously described (71), except that cell numbers were
quantified by the addition of Alamar Blue for the last 24 h of the
assay. The minimal detectable amount of TGF-.beta. in this assay
was 15 pg/ml. Cytokine bioassays were standardised against
recombinant cytokines and the specificity of the assays was
confirmed by the use of cytokine specific neutralising antibodies.
TGF-.beta.1 immunoactivity was measured in a specific ELISA
(R&D Systems) according to the manufacturers instructions.
[0073] Immunohistochemistry. Uterine tissue was embedded in OCT
Tissue Tck (Miles Scientific) and frozen in isopropanol cooled by
liquid N.sub.2, then stored at -80.degree. C. until use. Six .mu.m
semi-serial sections were cut from uteri collected at 1400 h on the
day of estrus or day 1 of pregnancy, or from mice injected with
rhTGF-.beta.1 and fixed in 96% ethanol (4.degree. C./10 min). For
mAb staining, sections were incubated with mAbs (neat hybridoma
supernatant containing 10% normal mouse serum [NMS]) and goat
anti-rat-horseradish peroxidase (HRP; Dako, 1:20 in PBS containing
10% NMS) as detailed previously (19). To visualise HRP or
endogenous peroxidase (to detect eosinophils), slides were
incubated in diaminobenzidine (Sigma)(5 mg/ml in 0.05 M Tris-HCl pH
7.2) plus 0.02% hydrogen peroxide for 10 min at room temperature.
After counterstaining in haematoxylin the sections were analysed
using a video image analysis package (Video Pro, Faulding Imaging,
Adelaide) in which the area of positive staining in the endometrial
stroma was expressed as a percentage of total cell staining.
[0074] Anti-sperm antibody ELISA: A solid phase ELISA technique
modified from the protocol of Okada (66) was used to quantify the
serum content of sperm-specific immunoglobulins in an
isotype-specific manner. Antigen was prepared by disruption of
freshly isolated CBA sperm (5.times.10.sup.6 sperm/ml in PBS) using
a Branson sonicator. 50 .mu.l of sperm antigen suspension was added
to polystyrene 96 well flat-bottomed ELISA plates (Maxisorb.TM.,
Nunc), and incubated overnight at 4.degree. C. Plates were blocked
with PBS/3% BSA for 1 h, and stored at -20.degree. C. until use.
Serum was diluted 1:4 in PBS, then serially diluted 1:2 to a final
dilution of 1:128, before 2 h incubation in the thawed sperm
antigen-coated plates. Bound immunoglobulin was detected with
rabbit .alpha. mouse antibody (Mouse Typer.TM., BioRad; 1 hr),
followed by biotinylated donkey .alpha. rabbit antibody (Amersham,
UK: 1:2000 in PBS/1% BSA; 1 hr) and streptavidin-HRP (Amersham;
1:4000 in PBS; 30 mins). HRP was visualised by the addition of
tetra methylbenzidine (TMB, Sigma; 20 mins) following acidification
of product with 1 M H.sub.2SO.sub.4. Quantification of each
immunoglobulin isotype (IgG.sub.1, IgG.sub.2a, IgG.sub.2b) was
performed in duplicate, and all incubations were at room
temperature. The antibody titre of each serum was determined by
plotting A.sub.150 against titration.
[0075] Sperm antigen delayed type hypersensitivity (DTH) response:
A footpad swelling assay (69) was employed to measure the DTH
response against sperm antigens. Balb/c F1 mice were primed on two
occasions separated by one month by intra-uterine inoculation with
sperm antigens in the presence or absence of TGF.beta., and 10 days
later, footpad thickness was measured using a micrometer gauge
(0.01 mm increments) (Mitutoyo, Tokyo, Japan) before and 24 h
following injection into the hind footpad of 25 .mu.l of sperm
suspension (1.times.10.sup.8 sperm 1 ml in HBSS). Antigen-specific
swelling was calculated by subtracting the thickness of
contralateral footpads injected with HBSS.
[0076] Human leukocyte chemotaxis assay: Leukocyte populations were
obtained from human peripheral blood using Ficoll-Paque.TM. density
gradient centrifugation, according to the method described by Boyum
(68). Peripheral blood mononuclear cells (PBMC: lymphocytes and
monocytes) were suspended in HBSS containing 10% ECM-FCS at
5.times.10.sup.5 cells/ml. The chemotaxis assay was a modification
of a Boyden chamber protocol described by Bignold (69). Cervical
keratinocyte culture supernatants (diluted 1:1 with HBSS/10%
ECM-FCS), HBSS/10% ECM-FCS, or
N-formyl-methionyl-leucyl-phenylalanine (FMLP, Sigma) were added to
the bottom half of chambers and were separated from PBMCs by 3
.mu.m polycarbonate mounted adjacent to an 8 .mu.m polycarbonate
sparse-pore filter (Nuclepore). Following 45-60 mins incubation at
37.degree. C., during which time PBMCs migrating through the 8
.mu.m sparse-pore filter were trapped on the surface of the
underlying 3 .mu.m filter, cells were fixed by addition of 1 ml of
10% formalin and quantified by manual counting after staining with
Mayer's haematoxylin. Mean cell numbers (.+-.s.d.) of triplicate
measurements were made for each test sample.
EXAMPLE 1
Seminal TGF.beta. Initiates the Post Mating Inflammatory Response
in Mice and Humans
[0077] The cytokine GM-CSF, produced by the uterine epithelium
following contact with seminal vesicle secretions, is thought to be
pivotal to the generation of maternal tolerance since it is largely
responsible for initiating the leukocytic influx into the female
reproductive tract after mating and for increasing the antigen
presenting capacity of these cells.
[0078] Seminal vesicle fluid was fractionated by size exclusion
chromatography in order to identify GM-CSF-stimulating activity.
Two fractions were identified; a high molecular weight (650 kDA)
proteinacous moiety and a intermediate molecular weight, more
heterogenous moiety eluting between 150-440 kDa (10.62). The latter
moiety was identified as TGF.beta..sub.1, on the basis of findings
that it's GM-CSF stimulating activity was enhanced by acid
activation, that TGF.beta..sub.1 immunoactivity and bioactivity
co-eluted in the same fraction, and that anti-TGF.beta..sub.1
neutralising antibody could block the GM-CSF stimulating activity
of this fraction (FIGS. 1,2). The molecular weight of the GM-CSF
stimulating activity in seminal vesicle fluid (150-440 kDa) is
consistent with that of the latent form of TGF-.beta..sub.1, a
complex of 230-290 kDa which comprises of the mature TGF-.beta.
dimer (25 kDa) non-covalently associated with a 75-80 kDa latency
associated protein and a 130-190 kDa binding protein (23).
[0079] The TGF-.beta..sub.1 content of murine seminal vesicle
secretions, like that of human seminal plasma (22), was found to be
extraordinarily high and second only to that reported for platelet
distillate (23). Furthermore the seminal vesicle gland secretions
were identified as contributing in excess of 90% of total ejaculate
TGF.beta..sub.1 content, with the prostate and coagulating gland
secretions containing only small amounts of TGF.beta..sub.1. The
addition of rTGF.beta..sub.1 to uterine epithelial cells in culture
and in vivo was confirmed to increase uterine epithelial GM-CSF
output in a dose responsive manner (FIG. 3).
[0080] The administration of rTGF.beta.1 to the uterine lumen of
oestrus mice was observed to not only increase uterine GM-CSF
production, but also initiate an influx and activation of
inflammatory cells similar to that seen following mating (Table 1
and FIG. 6). This result further supports the proposal that
TGF.beta. can fully replicate the post-mating inflammatory response
induced in the natural situation by seminal plasma.
[0081] In vitro experiments with human cervical keratinocytes and
endometrial tissue indicated that both semen and rTGF.beta..sub.1
can elicit an increase in GM-CSF production from reproductive tract
tissues in women (FIG. 7). Furthermore, the content of leukocyte
chemotactic activity in supernatants from keratinocyte cultures was
enhanced by treatment with either semen or rTGF.beta..sub.1 (FIG.
8), further supporting a principal role for seminal TGF.beta. in
the post-mating inflammatory cascade in women (63). TABLE-US-00001
TABLE 1 The effect of intra-uterine injection with TGF-.beta..sub.1
on endometrial leukocyte parameters. treatment n CD45 F4/80 Mac-1
Ia RB6-8C5 peroxidase vehicle 5 15 (8-19).sup.a 15 (12-25).sup.a 9
(7-21).sup.a 20 (8-23).sup.a 11 (5-15).sup.a 4 (4-7).sup.a
rhTGF-.beta..sub.1 4 28 (13-39).sup.ab 37 (30-48).sup.b 23
(18-42).sup.a 25 (15-35).sup.ab 15 (4-20).sup.a 15 (11-19).sup.b
mated 4 41 (30-60).sup.b 31 (21-49).sup.b 48 (46-56).sup.b 32
(26-57).sup.b 36 (15-41).sup.b 13 (10-20).sup.b
[0082] Tissues were collected 16 h after natural mating with intact
males, or after administration of 20 ng rhTGF-.beta..sub.1 in 50
.mu.l PBS/1% BSA, or vehicle only, to the uterine luminal cavity of
estrous mice. The reactivity of endometrial tissue with mAbs
specific for all leukocytes (anti-LCA), macrophages (F4/80 and
anti-Mac-1), neutrophils (anti-Mac-1 and RB6-8C5), and activated
macrophages/dendritic cells (Ia), was determined by
immunohistochemistry and video image analysis. Eosinophils were
detected by staining for endogenous peroxidase activity
(peroxidase). Reactivity with mAbs are expressed as the median
(range) percent positivity. The number of mice in each experimental
group=n. Data were compared by Kruskal-Wallis one way ANOVA and
Mann Whitney Rank Sum test. Data sets labelled with different lower
case letters within columns denote statistical significance between
treatment groups (p<0.01).
EXAMPLE 2
Seminal Vesicle Fluid Modulates Maternal Reproductive Performance
and the Maternal Immune Responsive to Paternal Antigens.
[0083] Previously, exposure to semen at mating was found to cause
an intense but transient inflammatory response, and factors in
seminal plasma derived from the seminal vesicle were implicated in
this response. In studies in mice, the inventors have identified
seminal vesicle fluid as a pivotal determinant in optimal embryo
development and implantation. Furthermore, exposure to semen at
mating has been shown to have an important role in inducing
maternal tolerance prior to implantation, and factors present in
seminal plasma have been identified as necessary for induction of
this state, suggesting that the beneficial effect of seminal plasma
on pregnancy outcome may at least in part be due to the immune
deviating effects of this fluid.
[0084] To test the importance of exposure to seminal reside fluid
for pregnancy success. Balb/c F1 females were mated with CBA males
from which the seminal vesicles had been surgically removed (SV-
studs). No implantation sites were present in the uterus on day 17
of pregnancy (n=12 females). This total infertility was not due to
a lack of fertilisation, but rather was associated with
implantation failure or early fetal resorption. This may reflect
insufficient maternal tolerance of the semi-allogencic embryos due
to the lack or exposure to seminal reside TGF.beta. at mating.
TABLE-US-00002 TABLE II Effect of seminal plasma on embryonic
development of mice. Intact SV- Number of females with embryos 8/8
(100%) 8/8 (100%) on day 3 (%) # embryos @ day 3 (mean .+-. SD) 8.0
.+-. 2.1 9.0 .+-. 2.0 Number of females with implantation 10/10
(100%) 0/12 (0%) sites on day 17 (%) # implants @ day 17 (mean .+-.
SD) 7.5 .+-. 1.8 0
[0085] Balb/c F1 mice mated naturally with intact or seminal
vesicle-deficient (SV-) CBA males were sacrificed at 1600 h on day
3 to assess embryonic development, or on day 17 to determine number
of implantation sites.
[0086] To investigate the importance of semen, particularly seminal
vesicle fluid, on the induction of Th1 immune response to paternal
MHC antigens, Balb/k (H-2.sup.k) female mice were mated with intact
Balb/k or congenic Balb/c (H-2.sup.d) stud males, or Balb/c SV-
studs. To achieve psuedopregnancy, the uteri of Balb/k females were
ligated at the oviductal junction 2 weeks prior to mating. Immune
responsiveness to MHC class I (H-2.sup.d) antigen was assessed by
measuring the growth of tumor cells injected on day 4 of pregnancy
or psuedopregnancy. Tumor cells were rejected in most Balb/k
females mated with Balb/k males, but grew in pregnant or
psuedopregnant Balb/k females mated with Balb/c males. In contrast,
tumors did not usually grow in Balb/k mice mated with SV- Balb/c
males. These data demonstrate that exposure to semen is sufficient
to induce specific tolerance to paternal MHC class I antigens, even
in the absence of an ensuing pregnancy, and show that this
tolerance is dependent on factors derived from the seminal vesicle
(Table III). TABLE-US-00003 TABLE III Effect of pregnancy and
psuedopregnancy on rejection of Balb/c JR-5 fibrosarcoma cells in
Balb/k mice. status at tumor growth median tumor Female Male JR-5
injection at day 17 (%) size# Balb/c virgin 11/11 (100) ++++ Balb/c
Balb/c d4 pregnant 5/5 (100) ++++ Balb/k virgin 0/10 (0) - Balb/k
Balb/c d4 pregnant 13/14 (93) +++ Balb/k Balb/c (vas) d4 psuedo-
5/7 (71) ++ pregnant Balb/k Balb/c (SV-) d4 pregnant 4/11 (36) ++
Balb/k Balb/c d4 psuedo- 9/9 (100) +++ (ut lig) pregnant Balb/k
Balb/k d4 pregnant 5/15 (33) + Balb/k C57Blk .times. d4 pregnant
4/8 (50) + CBA Balb/k C57Blk .times. d4 psuedo- 4/8 (50) + (ut lig)
CBA pregnant
[0087] Balb/c (H-2.sup.d) or Balb/k (H-2.sup.k) female mice were
mated with Balb/c or C57Blk.times.CBA F1(H-2b/k) studs. In some
groups the uteri of Balb/k females were ligated at the oviductal
junction 2 weeks prior to mating (ut lig). Other groups of intact
Balb/k mice were mated with vasectomised Balb/c males (vas) or
Balb/c males from which the seminal vesicles were removed at least
2 weeks prior to mating (SV-). The day of finding a vaginal plug
was designated day 1 of pregnancy or psuedopregnancy. Balb/c tumor
cells (JR-5 fibrosarcoma cells, 10.sup.5) were injected s.c. on day
4, and tumor growth (diameter, in two dimensions) was measured on
day 17 of pregnancy or psuedopregnancy (++++=>8 mm; +++=>5
mm; +=1-3 mm).
EXAMPLE 3
Seminal TGF.beta. is an Immune Deviating Agent
[0088] To assess the effect of TGF.beta. on induction of Th1 and
Th2 immune responses against CBA sperm antigens, Balb/c F1 female
mice were immunised by intra-uterine infusion with CBA sperm, in
the presence or absence of rTGF.beta., on two occasions separated
by 4 weeks. Development of Th1 anti-sperm immunity was assessed two
weeks later by measuring the DTH response to a subcutaneous sperm
antigen challenge, and by measuring serum content of anti-sperm
reactive immunoglobulin of the IgG.sub.2b subclass. Whereas sperm
administered alone or in the presence of Freunds Complete Adjuvant
elicited a strong DTH response and a moderate IgG2b antibody
response, immunisation in the presence of TGF.beta. substantially
diminished both of these parameters, and was comparable to the
response elicited by natural mating (FIG. 8). In contrast,
synthesis of sperm-reactive immunoglobulin of the IgG1 isotype
(indicating induction of a Th2 response) occurred to a similar
extent in all treatment groups, regardless of the presence of
TGF.beta. in the immunising inoculum.
[0089] In another experiment, the effect of TGF.beta. on the
induction of `tolerance` to paternal MHC antigens associated with
sperm was investigated. Balb/k (H-2k) female mice that were given
intra-uterine infusions of sperm from Balb/c (H-2d) males together
with rTGF.beta..sub.1 were not able to reject paternal MHC
antigen-bearing tumour cells injected 4 days later, whereas tumours
were rejected in naive mice or mice given sperm alone (Table IV).
Tumour rejection was also compromised in mice that administered
TGF.beta. without sperm antigen, although tumours in this treatment
group were not as large as those which grew in mice that received
both antigen and TGF.beta..
[0090] Both of these experiments show that delivery of paternal
antigens in combination with TGF.beta. to the female reproductive
tract can generate systemic paternal antigen-specific tolerance,
specifically by inhibiting the Th1 compartment of the immune
response. This immune deviating effect is dependent on the
administration of TGF.beta. since antigen given alone elicits Th1
immunity as opposed to tolerance. TGF.beta. given in the absence of
antigen may confer a state of partial, non-antigen specific
tolerance. TABLE-US-00004 TABLE IV The effect of intra-uterine
immunisation with Balb/c sperm and TGF.beta. on rejection of Balb/c
JR-5 fibrosarcoma cells in virgin Balb/k mice. tumor growth median
Treatment at day 17 (%) tumor size# 5 .times. 10.sup.6 Balb/c sperm
3/8 (38) + 10 ng TGF.beta. 5/7 (71) +++ 5 .times. 10.sup.6 Balb/c
sperm + 6/9 (67) ++++ 10 ng TGF.beta. Control (PBS) 0/6 (0) -
[0091] Balb/k female mice were uterine ligated, and after two weeks
rest were synchronised into estrous by administration of GnRH
agonist. At 0900 h-1200 h on the day of estrous, mice were
anaesthetised and given intra-uterine injections of
5.times.10.sup.6 Balb/c sperm and/or 10 ng TGF.beta. in 100 ul of
PBS (50 ul administered per horn). Balb/c tumor cells (JR-5
fibrosarcoma cells, 10.sup.5) were injected s.c. 72 h after
surgery, and tumor growth (diameter, in two dimensions) was
measured 13 days later (++++=>8 mm; +++=>5 mm; +=1-3 mm).
EXAMPLE 4
Paternal Antigen-Specific Immune Deviation Improves Reproductive
Performance
[0092] The experiments described above show that seminal vesicle
secretions can elicit Th1 hypo-responsiveness which manifests as
`tolerance` in the maternal immune response specific for seminal
antigens, including but not likely to be limited to paternal MHC
antigens, deposited in the female reproductive tract at mating. The
data suggest that diminished reproductive outcome ensues when a
pregnancy has been initiated in the absence of exposure to seminal
plasma, perhaps because of inadequate induction of maternal
`tolerance` to conceptus antigens. An experiment was therefore
performed to test the hypothesis that a prior state of
TGF.beta.-mediated `tolerance` to antigens in paternal semen can
benefit reproductive performance. This experiment consisted of
immunisation by intra-uterine infusion of Balb/c F1 females with
CBA sperm, with or without rTGF.beta..sub.1, two weeks before
mating with intact CBA male studs. Immunisation with sperm plus
TGF.beta..sub.1 resulted in an increase in mean fetal and placental
weight (Table V), despite a small decline in litter size which was
evident in all females immunised with sperm regardless of the
presence of TGF.beta.. This increase was still apparent after
adjustment for different fetal numbers per uterine horn, thereby
discounting an effect of litter size (FIG. 9).
[0093] Induction of Th1 hypo-responsiveness against paternal
antigens has been reported to result in an improved pregnancy
outcome in women previously experiencing recurrent miscarriage
(102). While no data exist on the ability of paternal
antigen/TGF.beta. immunisation to initiate Th1 hypo-responsiveness
against paternal antigens, or to deviate previously existing Th1
immune responses in women, nor on the ability of TGF.beta. to
improve reproductive outcome, this is likely to be the case. The
inventors have been the first to conduct a large randomised,
controlled trial investigating the effect of semen exposure on IVF
treatment outcome. This trial has confirmed that women exposed to
semen (containing paternal antigen and natural TGF.beta.) around
the time of thawed embryo transfer have a reduced risk of early
embryonic loss compared to those instructed to abstain (Table VI).
This improvement in reproductive outcome is likely to be mediated
by maternal immune tolerance towards paternal antigens initiated by
TGF.beta. and seminal antigens at the time of intercourse.
TABLE-US-00005 TABLE V Effect of prior immunisation with sperm and
TGF.beta. on reproductive outcome in mice Control sperm +
TGF.beta..sub.1 sperm number 139 144 103 litter size 11.4 .+-.
1.0.sup.a 10.4 .+-. 1.2.sup.b 10.3 .+-. 0.9.sup.b (total) litter
size 11.25 .+-. 1.3.sup.a 10.1 .+-. 1.5.sup.b 10.1 .+-. 0.9.sup.b
(viable) # resorptions 0.167 .+-. 0.58.sup.a 0.21 .+-. 0.58.sup.a
0.20 .+-. 0.42.sup.a fetal weight (mg) 645.2 .+-. 61.2.sup.a 677.6
.+-. 56.6.sup.b 646.1 .+-. 49.9.sup.a placental weight 97.7 .+-.
12.1.sup.a 105.2 .+-. 12.4.sup.b 101.8 .+-. 9.8.sup.b (mg)
fetal:placental 6.69 .+-. 0.9.sup.a .sup. 6.5 .+-. 0.8.sup.ab 6.36
.+-. 0.8.sup.b weight ratio
[0094] Balb/cF1 female mice were immunised by intra-uterine
infusion with CBA sperm in the presence or absence of 10 ng
rTGF.beta..sub.1, and were mated naturally with CBA males 2 weeks
later. Females were sacrificed on day 17 of pregnancy and the
number of total, viable and resorbing implantation sites, as well
as fetal and placental weights of viable conceptuses, were
determined. Values are mean.+-.SD. Comparisons between groups were
by Kruskal Wallis one-way ANOVA followed by Mann Whitney rank sum
test (p <0.05). TABLE-US-00006 TABLE VI Effect of semen exposure
around the time of thawed embryo transfer on early pregnancy
outcome. signif- Intercourse abstain icance transfer cycles 59 56
NS embryos transferred 106 107 NS implantations (%) 11/106 (10.3)
11/107 (10.2) NS viable conceptus 10/106 (9.4) 7/107 (6.5) NS at 6
weeks (%) transfer cycles 9/59* (15.3) 7/56 (12.5) NS with
biochemical pregnancy biochemical 0 (0) 2/11 (8.2) NS pregnancy
loss clinical 1/11 (9) 2/11 (18.2) NS miscarriage total pregnancy
1/11 (9) 4/11 (36.4) 0.043 wastage Pregnancy outcome following
thawed embryo transfer. Patient characteristics were not
significantly different between the two groups. An biochemical
pregnancy was defined as one serum .beta.HCG exceeding 25 IU and a
clinical pregnancy as a conceptus/fetal pole seen at ultrasound at
6 weeks gestation. Statistical analysis was performed using the Chi
square calculation. NS = not significant. *= one twin
pregnancy.
[0095] Pregnancy outcome following thawed embryo transfer. Patient
characteristics were not significantly different between the two
groups. An biochemical pregnancy was defined as one serum .beta.HCG
exceeding 25 IU and a clinical pregnancy as a conceptus/fetal pole
seen at ultrasound at 6 weeks gestation. Statistical analysis was
performed using the Chi square calculation. NS=not significant.
*=one twin pregnancy.
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