US 20160213749A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0213749 A1 Gopichandran et al. (43) Pub. Date: Jul. 28, 2016

(54) EMIBRYO MPLANTATION Publication Classification (71) Applicant: Ostara Biomedical Ltd, Liverpool (GB) (51) Int. Cl. A638/20 (2006.01) (72) Inventors: Nadia Gopichandran, Liverpool (GB); A6D 9/02 (2006.01) Nicolas Michel Orsi, Liverpool (GB); A6D 9/04 (2006.01) David Andrew Brooke, Liverpool (GB) A638/9 (2006.01) A619/00 (2006.01) (52) U.S. Cl. (21) Appl. No.: 15/007,795 CPC ...... A61K 38/208 (2013.01); A61K 38/193 (2013.01); A61 K38/19 (2013.01); A61 K 9/0036 (2013.01); A61D 19/04 (2013.01); (22) Filed: Jan. 27, 2016 A61D 19/022 (2013.01) (57) ABSTRACT The present invention relates to methods of and compositions Related U.S. Application Data comprising cytokines for, improving the Success rate of (60) Provisional application No. 62/108,222, filed on Jan. embryo implantation and the Success rate of pregnancy rates 27, 2015. in females, by providing an immunopermissive uterine envi ronment prior to insemination or implantation of embryos. (30) Foreign Application Priority Data The methods of the present invention are used to make the uterus more receptive or less hostile to, for example, trans Jan. 27, 2015 (GB) ...... GB 15O1302.2 ferred embryos, sperm or other allografted tissue. Patent Application Publication Jul. 28, 2016 Sheet 1 of 2 US 2016/0213749 A1

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Nf Gill. (i. I. (i. F.G. 1 Patent Application Publication Jul. 28, 2016 Sheet 2 of 2 US 2016/0213749 A1

US 2016/0213749 A1 Jul. 28, 2016

EMIBRYO MPLANTATION 100% effective and needs testing for each male, while the production of genetically sterile males generates unwanted RELATED APPLICATIONS Surplus females. 0006 Alternatively, pseudopregnancy can be induced by 0001. This application claims priority to British Applica simulating the normal vaginal stimuli attained by mating with tion No. GB 1501302.2, filed on Jan. 27, 2015 and U.S. artificial mechanical stimulation, for example by a vibrating Provisional Application No. 62/108,222, filed on Jan. 27. engraving tool (Kenney et al; J Reprod. Fert. 1977, 49, 305 2015. The contents of each of these applications are incorpo 309). It was found that the number and rate of intromissions rated herein by reference in their entireties. were crucial influences on reproductive Success (Diamond; Science, 1970, 169, 4, 995-997). Whilst this approach has FIELD seen Some Success in rats and mice mechanical stimulation had no effect on the induction of pseudopregnancy in the 0002 The present invention relates to methods of and Golden Hamster (Diamond et al J. Reprod. Fert. 1968, 17. compositions for, improving the Success rate of embryo 165-168). When the female is mated with an infertile male or implantation and the Success rate of pregnancy in females, by mechanically stimulated, the corpus luteum persists without providing an immunopermissive uterine environment prior to an embryo, leading to pseudopregnancy. The female will insemination or implantation of embryos. The methods of the develop mammary glands, lactate and build nests in the present invention are used to make the uterus more receptive pseudopregnant state. There is a need to improve the methods or less hostile to, for example, transferred embryos, sperm or of inducing a pseudopregnant state in laboratory test animals. other allografted tissue. The invention also includes interalia 0007 Although the protocols for embryo transfer in an compositions and formulations for use in the methods of the array of rodent species are relatively well-established, their invention. poor optimization means that there is a significant wastage of animals, raising a number of financial and ethical issues in BACKGROUND animal units worldwide. The prior art standard approach cur rently relies on mating recipient females with vasectomised 0003. The uterine environment, which, if hostile/non-re males to induce pseudopregnancy rather than mechanical ceptive, can be responsible for poor implantation rates of stimulation, where copulatory activity and seminal exposure good quality embryos in human and animals alike. It is of the maternal reproductive tract triggers a neuroendocrine believed that an inadequately primed uterine environment and localised (to the uterus, principally) inflammatory may also be responsible for many cases of reproductive fail response involving a complex cascade of cytokine and pros ure in terms of failed implantation and spontaneous abortion. taglandin-mediated events geared towards creating an immu Similarly, a failure in uterine priming is recognised in humans nopermissive environment in the uterus, thereby favouring as being causative to pregnancy complications such as pre pre-implantation embryo development and/or blastocyst eclampsia and foetal growth restriction by preventing appro implantation and the establishment of pregnancy. Even in the priate placental development. absence offertilisation, luteal development and progesterone 0004 Genetically altered or modified animals provide production are Supported, and the maternal physiology is valuable models for testing novel gene and drug therapies in orchestrated to render the uterus receptive to transferred Vivo and are the main reason the numbers of animal experi embryos for up to 10-13 days. This technique is routinely ments have been rising in the last decade. In the UK, over four used to Support the development of normal (cryopreserved times as many scientific procedures using genetically modi strain regeneration/re-derivation) or genetically modified fied animals were carried out in 2011 as compared to 1995. (transgenic/chimaeric/cloned) embryos. The use of genetically modified animals now represents over 0008. However, the efficacy of this approach is limited. 50% of all scientific procedures on animals. The largest cat Typically, four times as many females are prepared for the egory of use is breeding (to produce genetically modified procedure compared to those becoming pregnant. When animals), with rodents accounting for almost 1.8 million pro implanting fresh or frozen embryos this represents a consid cedures in the UK in 2012 alone, on a background trend for erable wastage of valuable biological material and effort. this number to increase annually. Embryo transfer in rodents Moreover, numbers of young vasectomised males also need underpins the development of transgenic approaches, re-deri to be maintained alongside the prospective recipients: these Vation of specific strains and facilitates the transport of ani can only mate 2-3 times a week and are typically replaced mal-lines across large distances. Typically, embryo transfer every 6-9 months in order to maintain performance. requires induction of pseudopregnancy in recipient females. 0009. Mating predominantly occurs when the recipient This phenomenon prepares the uterus for implanting female is in estrus. The estrus cycle lasts 4-5 days in the embryos, however, the Success rate of transferring genetically mouse and rat (equivalent to a woman's average 28 day men modified embryos, despite induction of pseudopregnancy, strual cycle), which leads to the need to rely on a large pool of remains relatively low. potential recipient females to take part in potential matings 0005 Mice are spontaneous ovulators and can become with vasectomised or otherwise sterile males. Typically, 75% pseudopregnant following an estrus in which the female is of recipients are not in estrus in randomly cycling popula mated with a genetically sterile male such as the T145H-Re tions, leading to large numbers of females and vasectomised strain (which is sterile due to a chromosomal translocation) or otherwise sterile males being kept and, in the case of the obtainable from Harlan Laboratories Inc or a vasectomised former, often not used as Surrogates in order to guarantee male. Both sets of males ejaculate seminal plasma devoid of adequate numbers of recipients for use in timed transfers. functional sperm. However, both genetically sterile and This is particularly evident in instances where the embryos to vasectomised mice are relatively costly. In the instance of be transferred are particularly valuable. Improvements to this vasectomised males, sterility cannot be guaranteed to be approach have relied on timedestrus induction via the Whit US 2016/0213749 A1 Jul. 28, 2016 ten effect in recipient females. This strategy relies on pher allograft tissue and/or for providing an immunopermissive emonal stimulation of recipient females, which typically uterine environment in females prior to implantation of an brings them into estrus 3 days after exposure to stud male embryo or prior to insemination. urine-Soiled bedding. However, the cycling stage of females 0016. According to a first aspect of the invention there is at the time of pheremonal exposure, proximity to stud cages provided a composition of matter for use in improving preg and the age of recipients can all have adverse effects on the nancy rates and/or for reducing maternal alloreactivity reliability of this approach, making it relatively inefficient. against Seminal fluid/sperm or embryos or other allograft 0010. The chances of females being in estrus (sexually tissue and/or for providing an immunopermissive uterine receptive) at the right time is 1:4 due to the length of their environment in females prior to implantation of an embryo or cycle (4 days). Thus, if 4 recipients are required, 16 females prior to insemination, the composition comprising IL-I2. will be mated to 16 males, which translates to a 25% success (0017 Preferably, the IL-12 is either IL-12 p40 or rate. This figure can be even lower as some females will refuse IL-12p70. to mate with their partner. The key point is that although most 0018 Preferably, the composition further includes GM breeders either select females in estrus, or induce estrus CSF. before mating with sterile males, still only a relatively low 0019 Preferably, the composition further includes any percentage (often about 50% but as low as 15% in some one two or three of TGFB, Eotaxin and RANTES. facilities) of oestrus females will become plugged and so 0020 Preferably, the composition further includes any assumed to be pseudopregnant. Furthermore, females also one, two, three, four, five six or seven additional cytokines have a very limited functional lifespan of a few months of age selected from the group comprising MIP, G-CSF, MCP-1, as embryo transfer recipients. Females rapidly accumulate IL-17, 1L-13, IL-9 and TNF-C. abdominal fat as they mature, making laparotomic embryo 0021. It is within the scope of the invention to provide a transfers (the most common and Successful method) techni number of specific combinations of the specified cytokines cally too challenging. using the basic IL-12 and a further, for example and without 0011. It is known that seminal fluid ejaculate is a complex limitation GM-CSF combination, or IL-12, eotaxin and mixture rich a variety of cytokines and prostaglandins, and RANTES combination, with or without additional further that some of the cytokines may have a positive effect on the cytokines for use in inducing a uterus to be more receptive or vaginal/uterine environment and others may have deleterious less hostile to transferred embryo, sperm or other allografted effects which both and ultimately affect receptivity to embryo tissue. implantation. For example, the pro-inflammatory cytokine 0022. Accordingly the compositions of the present inven GM-CSF (CSF-2) is present in seminal fluid ejaculate and is tion may include a variety of multiple cytokines as it is rec also known to be released by endometrial epithelial cells in ognised that high concentrations of a specific cytokine in response to seminal plasma within hours of coitus (Robertson seminal fluid do not necessarily reflect their biological sig et al. Reprod Fertil Dev, 1990. 2(4): p. 359-68) and that nificance. maternal GM-CSF is required for foetal viability and growth 0023 Preferably, the composition of the first aspect of the (Robertson et al. Biology of Reproduction Feb. 1, 1999 vol. invention further includes any one or more of the additional 60 no. 2 251-261). Furthermore WO1999067364 A1 dis cytokines selected from the group comprising IL-1C, IL-1B. closes media supplemented with GM-CSF to promote blas IL-1 ra, IL-2ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL10, tocyst growth and WO 2014087218 describes GM-CSF as IL-15, IL-16, IL-18, FGF, IFN-C2, IFN-y, IP-10, PDGF, sole acting Substance to prevent recurrent miscarriage. VEGF, CTACK, KC, GROC, HGF, ICAM-1, LIF, MCP-3, 0012. The cytokine IL-12 is also present in seminal fluid M-CSF, MIF, MIG, B-NGF, SCF, SCGF-B, SDF-1C, TNF-B, ejaculate and has been shown to be deleterious to pregnancy TRAIL and VCAM-1. (Reina et al. Am J Reprod Immunol. 2004 May: 51(5):345 0024 Table 1 below lists the acronyms for cytokines 51) and to be abortifacient in high concentrations, particularly referred to in the present invention: in Synergy with IL-18 and IL-2, via activation of maternal lymphocytes (Hayakawa et al. Am J Reprod Immunol. 1999; TABLE 1 41: 320-329). Other studies have shown that IL-12 injections (100 ng i.p. daily on days 5, 6, 7 and 8 of pregnancy) into Cytokines analysed using bio-plex assays CBA/J mice caused increased abortion rate when compared L-1C. interleukin-1C. to controls (Zenclussen et al Scand J Immunol. 2002 June; L-1B interleukin-1B 55(6):560-9). L-1 ra interleukin-1 receptor antagonist L-2ra interleukin-2 receptor antagonist 0013 By the compositions and methods of the present L-2 interleukin-2 invention it is envisaged that the need for vasectomised or L-3 interleukin-3 otherwise sterile male mice can be dramatically reduced L-4 interleukin-4 along with a significant reduction of female mice usage. L-5 interleukin-5 L-6 interleukin-6 0014. The present invention aims to improve the preg L-7 interleukin-7 nancy rates in mammalian females in terms of established L-8 interleukin-8 pregnancies and/or increased litter number following artifi L-9 interleukin-9 cial or natural insemination or following transplantation of L-10 interleukin-10 L12 (p40) interleukin-12 (p40) fresh or frozen or otherwise preserved embryos. L-12 (p70) interleukin-12 (p70) L-13 interleukin-13 BRIEF SUMMARY OF THE DISCLOSURE L-15 interleukin-15 L-16 interleukin-16 0015 The present invention resides in the use of IL-12 for L-17 interleukin-17 improving pregnancy rates and/or for reducing maternal L-18 interleukin-18 alloreactivity against Seminal fluid/sperm or embryos or other US 2016/0213749 A1 Jul. 28, 2016

TABLE 1-continued hamster, primate (monkey, ape), canine, feline, porcine or any other laboratory animal or endangered species into which Cytokines analysed using bio-plex assays embryos are placed. Eotaxin Eotaxin 0031 Preferably, the female is a mammal and more pref FGF Basic fibroblast growth factor erably still is a rare breed/species or a breed/species that is G-CSF Granulocyte-colony stimulating factor endangered. GM-CSF Granulocyte macrophage-colony stimulating factor IFN-O2 Interferon-C2 0032. Preferably, the female may be selected from the IFN-y Interferon-Y group comprising animals the orders of Artiodactyla, Car IP-10 IFN-Y inducible protein-10 nivora, Cetacea, Chiroptera, Dermoptera, Edentata, Hyra LEPTIN Hormone associated with weight control coidae. Insectivora, Lagomorpha, Marsupialia, Perissodac MCP-1 Macrophage chemotactic protein-1 MIP-1C Macrophage inflammatory protein-1C. tyla, Pholidata, Pinnipedia, Primates, Proboscidea, Rodentia, MIP-1B Macrophage inflammatory protein-13 Sirenia and Tubulidentata. PDGF Platelet derived growth factor 0033 Preferably, the amount of any one of the cytokines RANTES Regulated upon activation normal T cell expressed and secreted present in the composition is released, from any one of its TNF-ct, Tumour necrosis factor deliverable forms as described herein after, in situ either VEGF Vascular endothelial growth factor above or within their approximate physiological range found CTACK Cutaneous T cell attracting chemokine in seminal fluid. KC Ketatinocyte derived cytokine GROC. Growth regulated ongogene-C. 0034. Cytokines are measured as pg/ml as the standard HGF Hepatocyte growth factor ised recognised values in the art. ICAM1 Intercellular cell adhesion molecule 0035. The present invention resides in harnessing the LIF Leukaemia inhibitory factor properties of seminal agents, especially cytokines (which are MCP3 Monocyte chemoattractant protein-3 M-CSF Macrophage-colony stimulating factor protein modulators of the immune response and which pro MIF Macrophage migration inhibitory factor mote uterine receptivity), by providing a chemicophysical MIG Monokine induced by IFN-y formulation preferably in a form suitable for vaginal delivery B-NGF Basic-nerve growth factor for insertion into the female prior to mating/insemination or SCF Stem cell factor SCGF-B Stem cell growth factor-B prior to implantation of embryos. The introduced formulation SDF-1C. Stromal cell derived factor-1C. releases agents which enhance the receptivity of the uterine TGF-B1 Transforming growth factor f31 environment. TNF-B Tumour necrosis factor-f 0036. The approach used in the present invention is to TRAIL Tumour necrosis factor related apoptosis inducing ligand mimic the biochemical signalling mediated by seminal VCAM-1 Vascular cell adhesion molecule-1 plasma by using a pessary-based, gel-based, Solution-based, emulsion-based, powder-based or aerosol-based delivery 0025. The present invention resides in harnessing the system. Pessaries are already routinely used in an array of properties of seminal agents which promote uterine receptiv large domestic species (e.g. cattle) for the synchronisation of ity and in providing a chemicophysical alternative to vasec estrous cyclicity for embryo transfer/artificial insemination. tomised or otherwise sterile males, preferably in the form of However, to date, no pessary has been used with the compo a vaginal insert Such as a pessary, gel, spray or allied to any sitions of the present composition or for the specified function other dissolvable carrier. of promoting uterine receptivity and/or inducing a pseudopregnant state. 0026. The present invention, advantageously, given that 0037 Preferably, the cytokines are recombinant. That is to the demand for transgenic non-human animal models is set to say that they are made by genetically engineering a bacterium rise further, provides an alternative to vasectomised or other or other cell type using recombinant technology. wise sterile males inducing pseudopregnancy and also advan 0038. The present invention provides compositions for tageously reduces the number of females required by improv females comprising recombinant cytokine preparations, typi ing uterine receptivity to transferred embryos. cally in the form of a pessary placed in the vagina or an 0027. The present invention advantageously obviates the aerosol foam released in the vagina prior to insemination/ need for vasectomised or otherwise sterile males given that embryo transfer in order to reduce maternal immune allore their contribution solely relates to triggering the neuroendo activity against sperm/embryos, thereby improving preg crine and uterine inflammatory responses required to induce nancy rate/outcome. The use of this mode of delivery as a pseudopregnancy. strategy for improving endometrial receptivity is novel. 0028. According to a further aspect of the invention there 0039 Preferably, the composition further includes adju is provided a pharmaceutical composition comprising the vants such as preservatives, anti-oxidants, wetting agents, cytokines as herein before described in the form of a vaginal emulsifying agents and dispersing agents. Prevention of the insert or an intra-uterine device for improving pregnancy action of microorganisms may be ensured by the inclusion of rates and/or for reducing maternal alloreactivity against semi various antibacterial and antifungal agents, for example, nal fluid/sperm or embryos or other allograft tissue and/or for paraben, chlorobutanol or phenol sorbic acid. It may also be providing an immunopermissive uterine environment in desirable to include isotonic agents such as Sugars or sodium females prior to implantation of an embryo or prior to insemi chloride, for example. It may also be beneficial to include nation. waxes, water and co-solvents. 0040 Preferably the composition is in a form suitable for 0029 Preferably, the female is mammalian and more pref vaginal delivery such as a vaginal capsule, vaginal gel, vagi erably is human. nal tablet, vaginal powder, vaginal Solution, vaginal pessary, 0030 Preferably, the mammalian female is selected from vaginal cup, Vaginal sponge or vaginal foam or spray. Most the group comprising mouse, rat, rabbit, gerbil, guinea pig, preferably the composition is in the form of a vaginal pessary. US 2016/0213749 A1 Jul. 28, 2016

0041) Preferably, the vaginal formulation is dissolving or 0050. Preferably, in the instance of providing the compo non-dissolving, degradable or non-degradable. sition of the present invention as a vaginal pessary, tablet, 0042 Preferably, the compositions of the present inven bioadhesive tablet, capsule, powder, microparticle, bioadhe tion are prepared as a vaginal suppository, tablet, powder, sive microparticle, that the coating will be abrasive. This is an bioadhesive tablet, capsule, microparticle, bioadhesive appropriate modification to the deliverable composition, microparticle, microcapsule, microsphere, liposome, cream, especially if the penis of the male of the species to be prepared lotion, foam, spray, film, ointment, solution, gel, or a sus for uterine receptivity has a rough epidermis, keratinous tained release gel, tablet or capsule, or a sustained release spines, etc. In this instance it is desirable for the inserted Suppository administered to the vagina or incorporated into a Vaginal delivery vehicle to have a rough outer coating similar vaginal device. to that as on the penis in order to elicit a maternal inflamma 0043. In an alternative embodiment of the invention the tory response to improve penetration of the preparation into compositions of the present invention are prepared for oral or the mucosa, elicit an initial inflammatory response and aid rectal administration or as an enteric coated tablet for use in generic neuroendocrine stimulation. gastrointestinal tract delivery so that they may be absorbed 0051. In a particularly preferred embodiment of the inven from the mucosa of the gastrointestinal tract. The rationale for tion the composition is in the form of a pessary. Preferably the these modes of administration is that the mucosal immune pessary is suitably sized and shaped so as to be inserted into system in the digestive system is linked to that of the repro the vagina of the female. The pessary may have a solid core ductive tract. In this way, mucosal priming will occur, thereby and an outer porous layer. Typical dimensions of a pessary for facilitating embryo implantation, allograft/gamete/embryo mice are overall diameter of 4 mm and a length of 7 mm. The tolerance, self-immunotolerance or tolerance of the endog diameter and length of the pessary is dependent upon the size enous/exogenous microflora of both the reproductive and of the Vagina of the species into which it is inserted, it is digestive tracts. desirous that the pessary be sized and shaped so that when 0044 Preferably, the compositions of the present inven inserted into the vagina it is retained without discomfort. tion are prepared as multiwalled, multicored, microencapsu 0052 According to a further aspect of the invention there lated preparations. More preferably, the active components of is provided the composition of the first aspect of the invention the composition are when used as dried material encapsulated for the manufacture of a medicament for improving preg in a shell/coat like a gelatin capsule. nancy rates and/or reducing maternal alloreactivity against 0045 Compositions for vaginal administration are prefer seminal fluid/sperm or embryos or other allograft tissue and/ ably prepared by mixing the compositions of the present or for providing an immunopermissive uterine environment invention with suitable pharmaceutical ingredients or non in females prior to implantation of an embryo or prior to irritating excipients or carriers such as cocoa butter, polyeth insemination. ylene glycol or a suppository wax which are solid at room 0053 According to a further aspect of the invention there temperature but liquid at body temperature and therefore melt is provided a method of reducing maternal alloreactivity in a in the rectum or vaginal cavity and release the active com non-human female mammal against seminal fluid/sperm, pound. A typical example of a vaginal pessary would include embryos or other allograft (including existing microflora) the active ingredients and the following excipients: medium comprising exposing the vaginal/gastrointestinal tract chain try glycerides and hard fat. mucosa to a composition as hereinbefore described, the 0046) Alternatively, the composition can be incorporated method comprising: into an intravaginal device or a coating of such device, for 0054 (i) introducing at least one vaginal delivery example, a tampon or tampon-like device coating, or incor Vehicle comprising the composition of the present porated into a sponge, foam, strip, powder, pessary, or other invention into the vagina of the female; material. Absorbent material or matrix of such devices may 0055 (ii) optionally inserting further vaginal delivery be impregnated with a composition of the present invention as vehicle(s): a liquid solution, suspension lotion, powder, cream, micro 0056 (iii) allowing a sufficient period of time to elapse emulsions or suspension of liposomes, bioadhesive nanopar to allow the active components of the vaginal delivery ticles, or bioadhesive microparticles. Preferably, the vaginal Vehicle to be released and to penetrate into the vagina device is dissolving or non-dissolving, degradable or non and be absorbed transmucosally and/or diffuse and/or be degradable. transported into the uterus and/or through the gas 0047 Preferably, the compositions further include a trointestinal tract mucosa; and mucoadhesive agent, sorption promoter or penetration 0057 (iv) inseminating the female by either mating enhancer. The compositions of the present invention are with a male or by artificial insemination, donor gametes delivered by transmucosal vaginal delivery and comprise or introducing an embryo or other allograft into the contacting the vaginal mucosa with the compositions of the uterus for implantation. present invention. 0058 According to further aspect of the invention there is 0048 Preferably, the compositions for vaginal delivery provided a method of improving pregnancy rate or outcome are for rapid delivery, controlled delivery, continuous deliv in a non-human female mammal prior to insemination or ery or pulsed delivery. implantation of an embryo comprising exposing the vaginal/ 0049. It is envisaged that the composition of the present gastrointestinal tract mucosa to a composition as hereinbe invention will be formulated in one embodiment as a pessary fore described, the method comprising: with a slow wax melt or a fast wax melt to achieve continuous 0059 (i) introducing at least one vaginal delivery or rapid delivery respectively. In an alternative embodiment, Vehicle comprising the compositions of the present the composition of the invention will be formulated into a invention into the vagina of the female; foam or gel with appropriate additives to permit controlled 0060 (ii) optionally inserting further vaginal delivery release. vehicle(s): US 2016/0213749 A1 Jul. 28, 2016

0061 (iii) allowing a sufficient period of time to elapse based upon the confidence analysis of the Bayesian result to allow the active components of the vaginal delivery (based on occurrence in >90% bootstrapping iterations). vehicle to be released and to penetrate into the vagina and be absorbed transmucosally and/or diffuse and/or be DETAILED DESCRIPTION transported into the uterus and/or through the gas (0072 Reference hereinto “embryo” is intended to include trointestinal tract mucosa; and a blastula, blastocyst, fertilized ovum or an organism in its 0062 (iv) inseminating the female by either mating early stages of development, especially before it has reached with a male or by artificial insemination, donor gametes a distinctively recognizable form that is to be implanted into or introducing an embryo or other allograft into the a female recipient. uterus for implantation. 0073 Reference hereinto an “improved pregnancy rate” is 0063. In a further embodiment of the invention, steps (iii) intended to include a positive pregnancy outcome or and (iv) are performed simultaneously/close chronological improved perinatal survival or general viability following sequence in the case of artificial or natural insemination. The artificial insemination with processed semen or natural methods of the invention uses a composition comprising insemination or following transplantation of fresh or frozen recombinant cytokine-containing pessaries, gel, spray or or otherwise preserved embryos. The term pregnancy as used foam placed in the vagina at the time of insemination/embryo hereinafter is to be interpreted as encompassing a pregnancy transfer to reduce maternal immune alloreactivity against resulting from natural or artificial insemination or following sperm/embryoS/gametes and endogenous or exogenous transplantation of a fresh or frozen or otherwise preserved microflora, thereby improving pregnancy rate. embryo(s) and gametes. 0064. The number of doses and period between doses can 0074 Reference herein to a "vaginal insert” or an “intra be varied according to requirements and may vary depending uterine device' is intended to include any pessary-based, on species or breed, Superiovulation status, seasonal effect, gel-based, solution-based, emulsion-based, powder-based or lactational status and number of previous failed pregnancies aerosol-based delivery system that is capable of delivering the or previous inseminations or IVF treatments. It may also vary compositions of the present invention into the vagina So as to depending on maternal age and whether the female is primi permit the compositions of the present invention to have a gravida. pharmacological effect on the vaginal/uterine environment. 0065 Preferably, the methods of the invention when used 0075 Reference herein to a "pessary” is intended as a for laboratory non-human animals further includes the step of means of delivery of the pharmaceutical substances of the synchronizing estrus in recipient females. present invention so that they are easily absorbed through the 0066. According to a yet further aspect of the invention mucosal Surfaces of the vagina, or intended to have action in there is provided a kit of parts and optionally a set of written the locality, for example against inflammation, or on the instructions therefore, the kit comprising a number of vaginal uterus. delivery vehicles containing the compositions of the inven 0076 “Pharmaceutical ingredient’ or “excipient’ means a tion and an apparatus for inserting said vaginal delivery pharmacologically inactive pharmaceutically acceptable vehicles into the vagina of the recipient non-human female compound added to a mucoadhesive composition of the mammal. invention. The ingredient or excipient does not have any 0067. It will be appreciated that features described for the pharmacological properties. first aspect of the invention are equally applicable to each and 0077 “Rapid delivery' means initial immediate rapid all aspects of the invention and apply mutatis mutandis. release and delivery of the components from the composition. 0068. The invention will be described by way of example The rapid delivery is typically followed by a time-dependent only with reference to the following figures wherein: reduction in release of the components from the composition or device and delivery of the drug to the plasma/uterine wall BRIEF DESCRIPTION OF THE DRAWINGS tissues (or gastrointestinal tract, where appropriate). 0069. Embodiments of the invention are further described (0078 “Controlled delivery” means a release wherein the hereinafter with reference to the accompanying drawings, in active agent is released from the material in a predesigned which: manner. The release of the active agent may be constant over 0070 FIG. 1 shows the Bayesian mathematical modelling along period, it may be cyclic over a long period, or it may be of cytokine networks in mouse seminal plasma. The nodes triggered by the environment or other external events. (cytokines) are colour-coded according to the conditional 0079. “Continuous delivery' means continuous and unin probability of corresponding mediator relative concentra terrupted release of the components from the formulation or tions being high (green), low (red) or medium (white) con device and delivering Such components in a continuous man centration given the state(s) of their parent nodes (the bar ner. Continuous delivery may be preceded by the rapid deliv charts adjacent to each node reflect underlying conditional ery. probabilities of categorization into one of the three concen 0080) “Pulsed delivery” means a release and delivery of tration bins from low on the left to high on the right). The the components in intermittent intervals. Such pulsed deliv normalized concentration (low or high) determines the inten ery may be provided, for example, by formulating the com sity of the node colour. Edges (causal connecting lines position in individual layers interspaced with inactive layers between nodes) represent causal directed interactions of dissolvable coatings or by using different pharmaceutical between nodes. These cytokines will interact with the mater ingredients. nal reproductive tract to induce immunopermissiveness to I0081. Seminal Fluid Cytokine Analysis paternal antigens. I0082 Sexually mature CD1 male mice (n=20) and Wistar 0071 FIG. 2 shows the Bayesian mathematical modelling rats (n=20) were sacrificed and seminal fluid collected from of cytokine networks in rat Seminal plasma (details discussed the seminal glands postmortem, a post mortem approach was above). Very high confidence level edges are coloured in red, chosen to avoid collecting samples by electroejaculation US 2016/0213749 A1 Jul. 28, 2016

since semen quality is variable by this method, and because predominant cytokine present. Of the cytokines analyzed the samples coagulate rapidly, making analysis problematic. only RANTES and GRO/KC had levels above 200 pg/ml. Seminal vesicle sampling is ideal as the fluid (rather than that IL-10 and IL-6 had levels above 100 pg/ml whereas several of the accessory glands) contains the maternal tract immuno cytokines such as MCP-1 had levels between 50-100 pg/ml modulatory factors investigated and because coagulating and several others such as IL-17 had levels below 50 pg/ml. gland secretions can more easily be avoided. 0083. Seminal fluid samples were weighed individually, suspended in phosphate buffered saline (PBS) supplemented TABLE 3 with 0.5% bovine serum albumin (BSA), and weighed again. Rat Mean SEM By inference to standard weight: Volume ratio of murine semi L-1C. 3.28 0.97 nal fluid, it was possible to determine the original volume L-1B 20.41 O.84 isolated and the dilution factor introduced by the PBS. This L-2 29.11 340 step was necessary because the fluid is too viscous to be L-4 20.17 1.13 pipetted accurately. Samples were spun and the Supernatant L-5 9.58 O.9S frozen at -80° C. until analysed simultaneously for the fol L-6 149.17 1.13 L-9 54.56 O.84 lowing 23 cytokines: interleukin (IL)-1C., IL-2, IL-3, IL-4, L-10 114.89 1.45 IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, L-12 p70 SS.14 4.31 IL-17, eotaxin, granulocyte-colony stimulating factor L-13 8.29 1.35 (G-CSF), granulocyte macrophage-colony stimulating factor L-17 15.8O 1.11 (GM-CSF), interferon (IFN)-y, keratinocyte-derived L-18 6.66 O.89 TNF-ct, 2.27 O16 chemokine (KC), monocytes chemotactic protein (MCP)-1, FN-y 2.93 O.39 macrophage inhibitory protein (MIP)-1C. MIP-1B, regulated Eotaxin 34.84 1.45 upon activation normal T cell expressed and Secreted GCSF 1.51 O.O7 (RANTES) and tumour necrosis factor (TNF)-C. This was GMCSF 40.53 2.10 achieved by custom 23-plex fluid-phase immunoassay kits MCP-1 61.56 2.21 LEPTIN 43.69 2.61 run on a Luminex-100TM equipped with StarStationTM soft MIP-1C O.13 O.O2 ware. Serum diluent was used in all cases to avoid false P-10 4.24 O.34 positive/negatives and dilution adjusted to 1:1 in order to GRO, KC 228.00 2.10 maximise sensitivity to baseline levels. Similar analysis was RANTES 287.31 2.21 VEGF O.OO O.OO performed on rat seminal fluid. TGF-B O.OO O.OO EXAMPLE1 0084 Table 2 below shows a variety of cytokines analyzed and measured in mouse seminal fluid. Eotaxin and RANTES EXAMPLE 2 appear to be the predominant cytokines present, with levels I0086 Eotaxin and RANTES appear to be the predominant above 500 pg/ml. IL-9, TNF-C. and MIP-1a had levels above cytokines each being present in an amount of more than 100 pg/ml whereas several cytokines such as G-CSF and 500pg/ml (see Tables 1 and 2). The cytokines IL-1C., IL-6, IFN-Y had levels between 50-100 pg/ml and several others IL-10, IL-12 (p40), IL-12 (p70), GM-CSF and MIP-1 B were such as IL-13 and TGF-B had levels below 50 pg/ml. present at levels below 20 pg/ml and cytokines IL-1B, IL-9, 1L-13, G-CSF, TNF-ct, MCP-1, KC, MIP-1C. and IFN-y were TABLE 2 present at levels above 20 and below 150 pg/ml. However, as Mouse Mean SEM stated hereinbefore the level of cytokines present does not necessarily correlate with the effect or potency. L-1C. 8.19 1.96 L-1B 87.48 9.04 I0087 Based on these analyses, a solution of cell culture L-2 3.03 O49 tested recombinant mouse cytokines was made up in PBS L-3 O3S O.04 using recombinant cytokines at the concentrations found in L-4 O.11 O.O1 L-5 O.S6 O.O7 seminal fluid (Table 3). This was stored at -80° C. until L-6 3.63 0.44 required for imbibing the pessary. L-9 135.14 33.47 L-10 1995 3.36 TABLE 4 L-12p40 5.25 O.S3 L-12 p70 10.91 1.08 Cytokine Concentrations in utero in a Mouse L-13 20.64 1.86 Pessary Preparation once solubilised. L-17 S.10 O.90 Eotaxin 857.22 73.85 Pessary solution G-CSF 4S.O3 3.33 concentration GM-CSF 4.16 O.39 Cytokine (pg/ml) FN-y 46.38 3.95 KC 37.17 3.56 IL-1C. 8.19 MCP-1 30.23 2.65 IL-1B 87.48 MIP-1C. 114.32 8.31 IL-6 3.63 MIP-1B 6.68 1.36 IL-9 135.14 RANTES 618.62 84.17 IL-10 19.95 TNF-C. 102.27 9.11 IL-12 (p40) 5.25 TGF-B 27.63 6.54 IL-12 (p70) 10.91 IL-13 20.64 G-CSF 45.03 0085 Table 3 below shows a variety of cytokines analyzed GM-CSF 4.16 and measured in rat seminal fluid. RANTES appears to be the US 2016/0213749 A1 Jul. 28, 2016

TABLE 4-continued used with HEPES for embryo transfer. However, if embryos were kept for any length of time (>4 embryo transfers time), Cytokine Concentrations in utero in a Mouse they were transferred into a dish containing bicarbonate Pessary Preparation once Solubilised. buffered KSOM+Eagle's mix (amino acids)+1 mg/ml BSA Pessary solution and kept under 5% CO2 in an 37°C. incubator. Mice donors concentration are approximately 4-6 weeks of age and recipients are Cytokine (pg/ml) approximately 6-10 weeks of age. Specific weight ranges TNF-C. 102.27 were selected as weight is an important factor to consider and MCP-1 30.23 RANTES 618.62 mice should be 20-30gm, as less than 20gm and they may not Eotaxin 857.22 Support pregnancy and greater than 30gm and the abdominal KC 37.17 fat present will make embryo transfer more difficult. MIP-1C 114.32 MIP-1B 6.68 0092 Pessaries were inserted by holding the females in IFN-y 46.38 scruff and allowing them to calm/relax, then 35ul of the flush fluid is collected into a narrow-tipped Pasteur pipette and the fluid is flushed into the vagina. Fluid is flushed and collected EXAMPLE 3 4-5 times (ending on a flush). The pessary is then inserted by 0088 Additional formulation components of pessaries for hand after flush, with mouse still in scruff. The tail on the laboratory animals was dictated principally by toxicity (in pessary is used to push the pessary to the other side of the case of accidental ingestion), palatability (to dissuade inges vaginal muscles and it can be felt when the pessary will not go tion) and impact on luminal pH (the bioactivity of certain any further. Pessary retention is often short in some animals cytokines is promoted by vaginal pH). The size and shape of and is only monitored during the time taken to insert all the pessaries is largely determined by the species for which pessaries on the day. their use is intended. For example, pessaries of approximately 0093. Females are then returned to home cages with cage 4 mm in diameter are particularly Suitable for mice since the mates following embryo transfer. Surgery clips are removed size has been determined as appropriate for insertion without 5-7 days post-Surgery. When checking for implantations (not undue discomfort and is also of a suitable size to be retained live births), females are sacrificed 10 days after the embryo in the vaginal vestibule. Larger laboratory animals or indeed transfer and foetus number present in the uterine horn is larger breeds of mice may necessitate larger pessaries. Pes documented. At this stage, resorptions can be seen (Smaller saries were made from laser-etched nylon at a setting of and different in colour to other foetuses) and are also between 5-10 Watts, use of this technique makes it possible to recorded. For live births, females are group housed until manipulate porosity (which facilitates loading) and overall approximately 18 days into pregnancy. They may be visually shape and dimensions. It is envisaged that pessaries will be monitored for signs of pregnancy and they are weighed before provided in a range of sizes and that the Stalks may be Snapped embryo transfer and then weekly until birth. This helps to off from a central holding unit for use as desired and that a identify whether the female may have lost her litter or range of different sizes of pessaries may be provided to the destroyed it before the pups were seen. Pup body mass is user. Pessaries are prepared for use by soaking them over recorded at 7 days of age and at weaning (20-21 days). Pups night in 500 ul of 100 times the concentration of cytokine are also sexed at weaning. Solution so as to load the pessaries with the necessary active agents to guarantee a seminal fluid like final concentration in (0094) Table 5 below shows the protocol of days 1 to 6. the maternal reproductive tract. A pessary head is then removed from the stalk and inserted by means a suitable device directly into the mouse vagina. The pessary is then left Day Donor females Recipient females Comments in the mouse vagina for a period of time and then it is either 1 Male bedding removed at the time of embryo transfer when the animals are 2 PMSG Male bedding 5 IU 3 PMSG 2.5 IU anaesthetised, or it self-dissolves or the pessary self ejects 4 hCG and mate 5 IU once the active ingredients have been absorbed. with stud males 0089. It will be appreciated that the above embodiment is 5 Plug check hCG and mate with 2.5 IU only one example of a means of delivering the compositions vasectomised males. give pessary treatment of the present invention and that the pessary may be in the 6 Harvest embryos Plug check those 15 embryos transferred form of a slow or fast melt wax type formulation and that the mated with unilaterally into method of delivering the compositions of the present inven vasectomised males Oviduct by laparotomy tion may vary from species to species. The delivery means Embryo transfers may also be in the form of a biodegradable product and for example, in humans a vaginal Sponge may be a more conve nient method of delivering the compositions. 0.095 The treatment groups were as follows, vasectomised males (VSX) and pessaries consisting of a mixture MIP, IL-12, EXAMPLE 4 IL-13, G-CSF and GM-CSF (5 mix) plus other pessaries of the five cytokines as Solo components (not all data shown) and 0090 Embryo transfers into CD1 mice were investigated other pessaries of combinations, GM-CSF with IL-12, GM using the following protocol and combinations of cytokines. CSF with IL-12 and IL-13 and GM-CSF with IL-12 and MIP. 0091 Embryos were treated using M2 which is a common The total successful pregnancies with embryo transfer were media for in vitro culture of pre-implantation stage embryos. recorded. Table 6 shows the treatment (cytokine pessaries) M2 is a modified Krebs-Ringer bicarbonate solution and was Versus successful pregnancy outcome. US 2016/0213749 A1 Jul. 28, 2016

TABLE 6 cytokines selected from the group comprising MIP, G-CSF, MCP-1, IL-17, 11-13, IL-9 and TNF-C. Treatment Successful total 6. The composition according to claim 1 further compris WSX 1,3 ing any one or more of the additional cytokines selected from 5 mix 1.5 the group comprising IL-1C., IL-1B, IL-1 ra, IL-2ra, IL-2, IL-12 3.5 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL 10, IL-15, IL-16, IL-18, GM-CSF - IL-12 4f S GM-CSF - IL-12 - IL-13 2.5 FGF, IFN-C2, IFN-y, IP-10, PDGF, VEGF, CTACK, KC, GM-CSF - IL-12 - MIP OS GROC, HGF, ICAM-1, LIF, MCP-3, M-CSF, MIF, MIG, B-NGF, SCF, SCGF-B, SDF-1C, TNF-B, TRAIL and VCAM 1. 0096. This data supports the present invention in showing 7. A pharmaceutical composition in the form of a vaginal that IL-12 alone or in combination can be used to Successfully insert or an intra-uterine device for use in improving preg increase the rate of implantation rate as compared to prior art nancy rates and/or for reducing maternal alloreactivity methods. against Seminal fluid/sperm or embryos or other allograft 0097. Throughout the description and claims of this speci tissue and/or for providing an immunopermissive uterine fication, the words “comprise' and “contain' and variations environment in females prior to implantation of an embryo or of them mean “including but not limited to’, and they are not prior to insemination, the composition comprising IL-12. intended to (and do not) exclude other moieties, additives, components, integers or steps. According to further embodi 8. The pharmaceutical composition according to claim 7. ments of the present invention, the compositions and methods wherein active ingredients are released in situ either above or described herein may also consist essentially of or consist of within the approximate physiological range as those found in components and/or steps recited therein. Throughout the seminal fluid. description and claims of this specification, the singular 9. The pharmaceutical composition according to claim 7 encompasses the plural unless the context otherwise requires. further comprising an additional cytokine. In particular, where the indefinite article is used, the specifi 10. The pharmaceutical composition claim 7, wherein the cation is to be understood as contemplating plurality as well intra-uterine device is a vaginal capsule, vaginal gel, vaginal as singularity, unless the context requires otherwise. tablet, vaginal powder, vaginal Solution, vaginal pessary, 0098. Features, integers, characteristics, compounds, Vaginal cup, Vaginal Sponge, Vaginal aerosol or vaginal foam chemical moieties or groups described in conjunction with a or spray. particular aspect, embodiment or example of the invention are 11. The pharmaceutical composition according to claim 7 to be understood to be applicable to any other aspect, embodi further including an adjuvant, excipient or carrier. ment or example described herein unless incompatible there 12. The pharmaceutical composition according to claim 7 with. All of the features disclosed in this specification (includ for rapid delivery, controlled delivery, continuous delivery or ing any accompanying claims, abstract and drawings), and/or pulsed delivery. all of the steps of any method or process So disclosed, may be 13. A pessary for transmucosal vaginal delivery compris combined in any combination, except combinations where at ing the composition according to claim 1 for use in promoting least Some of Such features and/or steps are mutually exclu uterine receptivity. sive. The invention is not restricted to the details of any 14. The pessary according to claim 13 for promoting uter foregoing embodiments. The invention extends to any novel ine receptivity in a rodent. one, or any novel combination, of the features disclosed in 15. The pessary according to claim 14, wherein the rodent this specification (including any accompanying claims, is a mouse or rat. abstract and drawings), or to any novel one, or any novel 16. A method of reducing maternal alloreactivity in a non combination, of the steps of any method or process So dis human mammalian female against Seminal fluid/sperm, closed. embryos or other allograft (including existing microflora) 0099. The reader's attention is directed to all papers and comprising exposing the vaginal/gastrointestinal tract documents which are filed concurrently with or previous to mucosa to a composition according to claim 1, the pharma this specification in connection with, this application and ceutical composition of claim 7, or the pessary of claim 13, which are open to public inspection with this specification, the method comprising: and the contents of all such papers and documents are incor (i) introducing at least one vaginal delivery vehicle com porated herein by reference. prising the composition according to claim 1, the phar 1. A composition of matter for use in improving pregnancy maceutical composition of claim 7, or the pessary of rates and/or for reducing maternal alloreactivity against semi claim 13, into the vagina of the female: nal fluid/sperm or embryos or other allograft tissue and/or for (ii) optionally inserting further vaginal delivery vehicle(s) providing an immunopermissive uterine environment in as necessary; females prior to implantation of an embryo or prior to insemi (iii) allowing a sufficient period of time to elapse to allow nation, the composition comprising IL-12. the active components of the vaginal delivery vehicle to 2. The composition according to claim 1, wherein the IL-12 be released and to penetrate into the vagina and be is either IL-12 p40 or IL-12p70. absorbed transmucosally and/or diffuse and/or be trans 3. The composition according to claim 1 further compris ported into the uterus and/or through the gastrointestinal ing GM-CSF. tract mucosa; and 4. The composition according to claim 1 further compris (iv) inseminating the female by either mating with a male ing any one two or three of TGFB, Eotaxin and RANTES. or by artificial insemination, donor gametes or introduc 5. The composition according to claim 1 further compris ing an embryo or other allograft into the uterus for ing any one, two, three, four, five six or seven additional implantation. US 2016/0213749 A1 Jul. 28, 2016

17. A method of improving pregnancy rate or outcome in a (iv) inseminating the female by either mating with a male non-human mammalian female prior to insemination or or by artificial insemination, donor gametes or introduc implantation of an embryo comprising exposing the vaginal/ ing an embryo or other allograft into the uterus for gastrointestinal tract mucosa to a composition according to implantation. claim 1, the pharmaceutical composition of claim 7, or the 18. The method according to claim 16, wherein steps (iii) pessary of claim 13, the method comprising: and (iv) are performed simultaneously or in close chronologi (i) introducing at least one vaginal delivery vehicle com cal sequence. prising the composition according to claim 1, the phar 19. The method according to claim 16, wherein the method maceutical composition of claim 7, or the pessary of further includes the step of synchronizing estrus in recipient claim 13, into the vagina of the female: (ii) optionally inserting further vaginal delivery vehicle(s) females. as necessary; 20. A kit comprising a vaginal delivery vehicle containing (iii) allowing a sufficient period of time to elapse to allow the composition according to claim 1, the pharmaceutical the active components of the vaginal delivery vehicle to composition of claim 7, or the pessary of claim 13 and an be released and to penetrate into the vagina and be apparatus for inserting said vaginal delivery vehicle into the absorbed transmucosally and/or diffuse and/or be trans vagina of the recipient non-human female and optionally a set ported into the uterus and/or through the gastrointestinal of written instructions therefore. tract mucosa; and k k k k k