Patent Application: US-39785106-A

Abstract:
a method of treating an infertility condition in humans or mammals , by exposure of a prospective mother to tgfβ or derivative or analog of tgfβ . 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 .

Description:
rpmi - 1640 and low glucose dulbecco &# 39 ; s modified eagle &# 39 ; medium ( dmem , gibco ) were supplemented with 10 % fetal calf serum ( csl ), 20 mm hepes ph 7 . 2 , 5 × 10 − 5 m β - 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 μg / ml hydrocortisone ( upjohn . rydalmere , nsw ) ( ecm - fcs ), and human endometrial cells were cultured in dmem - fcs . recombinant human ( rh ) tgf - β 1 was from r & amp ; 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 &# 39 ; 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 - β 1 , 2 , 3 mab ( which neutralizes all three mammalian tgf - β isoforms ) was from genzyme ( cambridge . ma ) and chicken anti - bovine tgf - β1 mab ( neutralizes tgf - β1 , & lt ; 2 % cross reactivity with tgf - β 2 and - β 3 ) was from r & amp ; d systems . 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 × 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 . for intra - uterine injections , uterine horns of estrus females were exteriorised through a dorsal midline excision and injected with 0 . 2 - 40 ng rhtgf - β 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 / tgfβ 1 to the uterine lumen was achieved by passing a 3 french gauge tom cat ™ 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 μl of sperm / tgfβ 1 , which was delivered to the uterine cavity with the aid of a mouth pipette . 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 . 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 μl / g body weight injected i . p .]. 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 - β1 into the uterus by flushing each horn with 500 μl of rpmi - fcs . debris was sedimented at 2000 g and the supernatant stored at − 80 ° c . prior to cytokine assay . in experiments where uterine tgf - β1 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 (& gt ; 1 × 10 6 sperm per ml ). chromatography . approximately 1 ml of seminal vesicle fluid in 6 m guanidine hcl was applied to a sephacryl s - 400 column ( 40 cm × 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 - β assay half of each fraction was acid activated as previously described ( 18 ). 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 × 10 5 cells / ml in 500 μl of dmem - fcs . after 4 h incubation at 37 ° c . in 5 % co2 to allow cell adherence , a further 500 μl 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 . 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 μl of medium containing either rtgfβ 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 . 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 ° 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 ° c ., and repeated aspiration using a needle and syringe . keratinocytes were cultured in ecm - fcs , at a density of 1 - 2 × 10 − 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 μl of ecm - fcs containing 10 ng of rtgfβ 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 . 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 μci of [ 3 h ]- 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 - β 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 - β 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 - β1 immunoactivity was measured in a specific elisa ( r & amp ; d systems ) according to the manufacturers instructions . immunohistochemistry . uterine tissue was embedded in oct tissue tck ( miles scientific ) and frozen in isopropanol cooled by liquid n 2 , then stored at − 80 ° c . until use . six μm 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 - β1 and fixed in 96 % ethanol ( 4 ° 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 . 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 × 10 6 sperm / ml in pbs ) using a branson sonicator . 50 μl of sperm antigen suspension was added to polystyrene 96 well flat - bottomed elisa plates ( maxisorb ™, nunc ), and incubated overnight at 4 ° c . plates were blocked with pbs / 3 % bsa for 1 h , and stored at − 20 ° 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 α mouse antibody ( mouse typer ™, biorad ; 1 hr ), followed by biotinylated donkey α 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 2 so 4 . quantification of each immunoglobulin isotype ( igg 1 , igg 2a , igg 2b ) was performed in duplicate , and all incubations were at room temperature . the antibody titre of each serum was determined by plotting a 150 against titration . 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 tgfβ , 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 μl of sperm suspension ( 1 × 10 8 sperm 1 ml in hbss ). antigen - specific swelling was calculated by subtracting the thickness of contralateral footpads injected with hbss . human leukocyte chemotaxis assay : leukocyte populations were obtained from human peripheral blood using ficoll - paque ™ 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 × 10 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 μm polycarbonate mounted adjacent to an 8 μm polycarbonate sparse - pore filter ( nuclepore ). following 45 - 60 mins incubation at 37 ° c ., during which time pbmcs migrating through the 8 μm sparse - pore filter were trapped on the surface of the underlying 3 μm filter , cells were fixed by addition of 1 ml of 10 % formalin and quantified by manual counting after staining with mayer &# 39 ; s haematoxylin . mean cell numbers (± s . d .) of triplicate measurements were made for each test sample . seminal tgfβ initiates the post mating inflammatory response in mice and humans 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 . 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 tgfβ 1 , on the basis of findings that it &# 39 ; s gm - csf stimulating activity was enhanced by acid activation , that tgfβ 1 immunoactivity and bioactivity co - eluted in the same fraction , and that anti - tgfβ 1 neutralising antibody could block the gm - csf stimulating activity of this fraction ( fig1 ). 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 - β 1 , a complex of 230 - 290 kda which comprises of the mature tgf - β dimer ( 25 kda ) non - covalently associated with a 75 - 80 kda latency associated protein and a 130 - 190 kda binding protein ( 23 ). the tgf - β 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 tgfβ 1 content , with the prostate and coagulating gland secretions containing only small amounts of tgfβ 1 . the addition of rtgfβ 1 to uterine epithelial cells in culture and in vivo was confirmed to increase uterine epithelial gm - csf output in a dose responsive manner ( fig3 ). the administration of rtgfβ1 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 fig6 ). this result further supports the proposal that tgfβ can fully replicate the post - mating inflammatory response induced in the natural situation by seminal plasma . in vitro experiments with human cervical keratinocytes and endometrial tissue indicated that both semen and rtgfβ 1 can elicit an increase in gm - csf production from reproductive tract tissues in women ( fig7 ). furthermore , the content of leukocyte chemotactic activity in supernatants from keratinocyte cultures was enhanced by treatment with either semen or rtgfβ 1 ( fig8 ), further supporting a principal role for seminal tgfβ in the post - mating inflammatory cascade in women ( 63 ). tissues were collected 16 h after natural mating with intact males , or after administration of 20 ng rhtgf - β 1 in 50 μl 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 & lt ; 0 . 01 ). seminal vesicle fluid modulates maternal reproductive performance and the maternal immune responsive to paternal antigens . 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 . 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 tgfβ at mating . 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 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 . 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 k ) female mice were mated with intact balb / k or congenic balb / c ( h - 2 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 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 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 × d4 pregnant 4 / 8 ( 50 ) + cba balb / k c57blk × d4 psuedo - 4 / 8 ( 50 ) + ( ut lig ) cba pregnant balb / c ( h - 2 d ) or balb / k ( h - 2 k ) female mice were mated with balb / c or c57blk × 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 5 ) were injected s . c . on day 4 , and tumor growth ( diameter , in two dimensions ) was measured on day 17 of pregnancy or psuedopregnancy (++++=& gt ; 8 mm ; +++=& gt ; 5 mm ; += 1 - 3 mm ). to assess the effect of tgfβ 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 rtgfβ , 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 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 tgfβ substantially diminished both of these parameters , and was comparable to the response elicited by natural mating ( fig8 ). 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 tgfβ in the immunising inoculum . in another experiment , the effect of tgfβ 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 rtgfβ 1 were not able to reject paternal mhc antigen - bearing tumour cells injected 4 days later , whereas tumours were rejected in naïve mice or mice given sperm alone ( table iv ). tumour rejection was also compromised in mice that administered tgfβ without sperm antigen , although tumours in this treatment group were not as large as those which grew in mice that received both antigen and tgfβ . both of these experiments show that delivery of paternal antigens in combination with tgfβ 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 tgfβ since antigen given alone elicits th1 immunity as opposed to tolerance . tgfβ given in the absence of antigen may confer a state of partial , non - antigen specific tolerance . table iv the effect of intra - uterine immunisation with balb / c sperm and tgfβ on rejection of balb / c jr - 5 fibrosarcoma cells in virgin balb / k mice . tumor growth median treatment at day 17 (%) tumor size # 5 × 10 6 balb / c sperm 3 / 8 ( 38 ) + 10 ng tgfβ 5 / 7 ( 71 ) +++ 5 × 10 6 balb / c sperm + 6 / 9 ( 67 ) ++++ 10 ng tgfβ control ( pbs ) 0 / 6 ( 0 ) − 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 × 10 6 balb / c sperm and / or 10 ng tgfβ in 100 ul of pbs ( 50 ul administered per horn ). balb / c tumor cells ( jr - 5 fibrosarcoma cells , 10 5 ) were injected s . c . 72 h after surgery , and tumor growth ( diameter , in two dimensions ) was measured 13 days later (++++=& gt ; 8 mm ; +++=& gt ; 5 mm ; += 1 - 3 mm ). 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 tgfβ - 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 rtgfβ 1 , two weeks before mating with intact cba male studs . immunisation with sperm plus tgfβ 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 tgfβ . this increase was still apparent after adjustment for different fetal numbers per uterine horn , thereby discounting an effect of litter size ( fig9 ). 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 / tgfβ immunisation to initiate th1 hypo - responsiveness against paternal antigens , or to deviate previously existing th1 immune responses in women , nor on the ability of tgfβ 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 tgfβ ) 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 tgfβ and seminal antigens at the time of intercourse . table v effect of prior immunisation with sperm and tgfβ on reproductive outcome in mice control sperm + tgfβ 1 sperm number 139 144 103 litter size 11 . 4 ± 1 . 0 a 10 . 4 ± 1 . 2 b 10 . 3 ± 0 . 9 b ( total ) litter size 11 . 25 ± 1 . 3 a 10 . 1 ± 1 . 5 b 10 . 1 ± 0 . 9 b ( viable ) # resorptions 0 . 167 ± 0 . 58 a 0 . 21 ± 0 . 58 a 0 . 20 ± 0 . 42 a fetal weight ( mg ) 645 . 2 ± 61 . 2 a 677 . 6 ± 56 . 6 b 646 . 1 ± 49 . 9 a placental weight 97 . 7 ± 12 . 1 a 105 . 2 ± 12 . 4 b 101 . 8 ± 9 . 8 b ( mg ) fetal : placental 6 . 69 ± 0 . 9 a 6 . 5 ± 0 . 8 ab 6 . 36 ± 0 . 8 b weight ratio balb / cf1 female mice were immunised by intra - uterine infusion with cba sperm in the presence or absence of 10 ng rtgfβ 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 & lt ; 0 . 05 ). 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 βhcg 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 . pregnancy outcome following thawed embryo transfer . patient characteristics were not significantly different between the two groups . an biochemical pregnancy was defined as one serum βhcg 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 . 2 . de et al ( 1991 ) j . leukocyte biol . 50 , 252 - 262 . 5 . beer & amp ; 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