preferentially localise to the uterus. This preferential localisation, termed the `uterine first-pass effect’, was identified because of the marked uterine response to vaginally administered progesterone despite low serum progesterone concentrations. Subsequent studies showed that the endometrial concentration of vaginally administered drugs was substantially higher than following other routes of delivery, including intramuscular and oral. The mechanism of preferential localisation to the uterus is not clear, although it seems likely that drugs administered via the vagina are absorbed into veins 17785458 within the upper third of the vagina and then transported by counter-current exchange to the uterine arteries. In women, Mirin distributor Vaginal application has been used as a delivery method for contraceptives that primarily target the ovary although these are anti-progestins which are very small. PEGLA is estimated to be,60 kDa. It is not known whether such a large molecule can be absorbed by the vagina to enter the blood and reach the uterus. To the best of our knowledge, the efficacy of vaginally applied contraceptives that target the uterus has not been reported. Numerous vaginally delivered microbicides, which inhibit sexually transmitted infections, including HIV, are currently undergoing clinical trials. Recently, a clinical trial of using a vaginal gel formulation of tenofovir, a nucleotide reverse transcriptase inhibitor, showed significant inhibition of HIV incidence, suggesting that tenofovir could be vaginally administered in tandem with a contraceptive such as PEGLA, making a `dual-role’ contraceptive, capable of preventing both pregnancy and STIs. We hypothesized that PEGLA would localise to the uterus more rapidly and at a higher concentration following vaginal delivery than following injection, making vaginal application a promising contraceptive delivery method for PEGLA in women. Our specific aims were to: 1. establish the contraceptive efficacy of PEGLA following vaginal delivery in mice; 2. determine whether bone and the central nervous system are non-uterine targets of PEGLA in mice and 3. to compare the tissue distribution and halflife of PEGLA following IP injection and vaginal application in mice. Materials and Methods Animals C57BL6/J mice housed under conventional conditions, had ad libitium food and water and were maintained in a 12 h: light-dark cycle. All procedures were approved by the Monash Medical Centre Animal Ethics Committee or the WEHI Animal Ethics Committee, and this study followed the NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Preparation 
of PEGylated proteins PEGLA was produced as previously described, except that Y-NHS-40K was used for PEGylation. The in vitro activity of PEGLA was measured using a LIFresponsive Ba/F3 line stably expressing human LIFR and IL6ST as previously described. PEGLA produced with the new PEG compound exhibited a 2-fold increase in the inhibition of LIFinduced proliferation of Ba/F3 cells compared to the original PEGLA. Control PEGylation reagent was generated by incubating YNHS-40K in Milli-Q water for at least 24 h. PEGylated bovine serum albumin or mouse serum albumin were prepared as for PEGLA except that an anion exchange step was performed. Vaginal application of PEGLA Tissue localization of PEGLA. Mated female mice were given a single vaginal application of PEGLA dissolved 1:2 in a placebo gel at 9am on day 3 and killed after 3 h or 24 h or
