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(11) EP 3 501 533 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 26.06.2019 Bulletin 2019/26 A61K 38/08 (2019.01) A61K 31/4045 (2006.01) A61K 31/57 (2006.01) A61K 31/565 (2006.01) (2006.01) (2006.01) (21) Application number: 19153560.8 A61K 38/12 A61K 9/00 A61K 45/06 (2006.01) A61K 38/095 (2019.01) (2006.01) (22) Date of filing: 22.12.2014 A61P 15/08

(84) Designated Contracting States: (72) Inventor: Arce, Joan-Carles AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 2791 Dragor (DK) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (74) Representative: V.O. P.O. Box 87930 (62) Document number(s) of the earlier application(s) in Carnegieplein 5 accordance with Art. 76 EPC: 2508 DH Den Haag (NL) 14199709.8 / 3 037 101 Remarks: (71) Applicant: Ferring B.V. This application was filed on 24.01.2019 as a 2132 JX Hoofddorp (NL) divisional application to the application mentioned under INID code 62.

(54) THERAPY IN THE LUTEAL PHASE FOR IMPLANTATION AND PREGNANCY IN WOMEN UNDERGOING ASSISTED REPRODUCTIVE TECHNOLOGIES

(57) The present invention relates to the use of an antagonist in females undergoing an assisted reproductive technology. Specifically, the antagonists are released in the luteal phase when the endometrium is receptive for embryo implantation. EP 3 501 533 A1

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Description patients with lower frequency of uterine contractions at the time of transfer (Zhu et al. 2014). FIELD OF THE INVENTION [0006] Uterine contractility in controlled ovarian stim- ulation cycles has been compared to normal menstrual [0001] The present invention relates to the use of an 5 cycles (Ayoubi et al. 2003). The frequency of uterine con- oxytocin receptor antagonist in females undergoing em- tractions was found to be similar between the timepoint bryo transfer as part of an assisted reproductive technol- of hCG administration in a controlled ovarian stimulation ogy. Specifically, the antagonists are released in the cycle and at the time of LH surge in a natural cycle. In luteal phase when the endometrium is receptive for em- the luteal phase, the frequency of uterine contractions bryo implantation. 10 was higher at hCG +4 days (corresponding to day 2 post- retrieval) in a controlled ovarian stimulation cycle com- BACKGROUND OF THE INVENTION pared to at LH +4 days in a natural cycle (Ayoubi et al. 2003). However, the frequency of uterine contractions at [0002] In vitro fertilization (IVF) is a method for estab- LH +6 days and hCG +6 days (corresponding to day 4 lishing pregnancy in a female subject. The procedure typ- 15 post-retrieval) was not different and in both situations was ically involves ovarianstimulation withone or various hor- low, indicating identical level of uterine quiescence at that mones, mainly folliculo-stimulating hormone (FSH), and time point in controlled ovarian stimulation and natural human chorionic gonadotropin (hCG) is usually admin- cycles (Ayoubi et al. 2003). In another study, uterine con- istered to trigger final follicular maturation. Oocyte re- tractility was assessed at the day of hCG administration, trieval takes place generally 2 days (around 36h) after 20 hCG +4 days (corresponding to day 2 post- retrieval) and hCG administration. The ooctyes are then fertilized in hCG +7 days (corresponding to day 5 post-retrieval) in vitro, cultured for several days, and are transferred into women undergoing a controlled ovarian stimulation cycle the uterus. IVF also encompasses the transfer of embry- (Fanchin et al. 2001). The frequency of uterine contrac- os originating from the eggs of first female (the donor) tions was highest at the day of hCG administration, de- into a second female (the gestational carrier).Embryos 25 creased slightly during the early luteal phase as as- may be placed in frozen storage and transferred after sessed at hCG +4 days, and reached nearly quiescent several months or even years (i.e., frozen embryo trans- status at hCG+7 days (corresponding to day 5 post-re- fer). trieval). Another study reported a decrease in the number [0003] Improving the implantation rate of transferred of junctional zone contractions in oocyte donors in the embryos is one of the major challenges in assisted re- 30 early luteal phase from day 2 to day 3 and also to day 4 productive technologies (ART) treatment. Approximately post-retrieval (Lesny et al. 1999). Similarly, evaluation of only one-third of the transferred embryos implant in wom- uterine contractility in oocyte donors who had undergone en undergoing controlled ovarian stimulation for in vitro controlled ovarian stimulation and received exogenous fertilisation (IVF) / intracytoplasmic sperm injection (IC- progesterone luteal phase supplementation indicated SI). Implantation and pregnancy rates are influenced by 35 that there is a significant decrease in the frequency of multiple factors related to the age and other characteris- uterine contractions from day 2 post-retrieval to day 5 tics of the patient, the magnitude of the response to ovar- post-retrieval (Blockeel et al. 2009). ian stimulation, the quality of the embryos obtained, the [0007] The highest level of uterine contractility is at the endometrial receptivity as well as the actual transfer pro- end of controlled ovarian stimulation (day of hCG admin- cedure. 40 istration) and has been attributed to the high serum es- [0004] Uterine contractility is considered a potentially tradiol and low serum progesterone concentrations at important factor affecting implantation and pregnancy that time point. The decrease in uterine contractility dur- rates in IVF/ICSI cycles (Fanchin et al. 1998; Schoolcraft ing the luteal phase is believed to be the result of the et al. 2001; Bulleti and de Ziegler 2005). A high frequency exposure to endogenous progesterone caused by the of uterine contractions at the time of transfer appears to 45 corpus luteum function in response to the hCG adminis- have a negative impact on outcome, possibly by expelling tration as well as exogenous progesterone luteal supple- the embryos in the uterine cavity or by displacing the mentation used in IVF/ICSI cycles. Although progester- embryos and thereby reducing implantation and preg- one supplementation is used for luteal phase support in nancy rates. IVF/ICSI patients and can reduce uterine contractility, [0005] Observational data indicated a decrease in clin- 50 there is elevated uterine activity during the early luteal ical pregnancy rates with increasing frequency of con- phase (day 2 or 3 post-retrieval) when transfer of cleav- tractions at the time of cleavage-stage embryo transfer age-stage embryos is performed. at hCG +4 days (i.e., four days after hCG administration, [0008] As uterine contractility is elevated during the corresponding to day 2 post-retrieval of oocytes) (Fan- earlyluteal phase (day2 or 3 post-retrieval) when transfer chin et al. 1998). A prospective controlled study also ob- 55 of cleavage-stage embryos is performed, investigations served that patients with a higher frequency of uterine assessing the impact of different interventions on uterine contractions on the day of cleavage-stage embryo trans- contractility for improving implantation have been con- fer (day 3 post-retrieval) had lower pregnancy rates than ducted in the early luteal phase (day 2 and 3 post-retrieval

2 3 EP 3 501 533 A1 4 transfer; hCG +4 days). Randomized controlled trials is still in cleavage stage such that the antagonist is re- (Moon et al. 2004; Bernabeu et al 2006; Kim et al. 2008; leased or continues to be released once the receptive Ng et al. 2014), quasi-randomized controlled trials (Mor- endometrium stage is reached and/or the embryo has aloglu et al. 2010), retrospective studies in fresh and fro- reached the blastocyst stage. zen embryo replacement cycles (Chou et al. 2011; Lan 5 [0012] In preferred embodiments, the receptive en- et al. 2012), or case studies in fresh and frozen embryo dometrium stage corresponds to: replacement cycles (Pierzynski et al. 2007; Liang et al 2009) reporting findings with compounds reducing uter- a) between LH+6 to LH+9, preferably between LH+6 ine contractility, like (Kim et al 2008; Moraloglu to LH+8, most preferably on day LH+7 of a natural et al. 2010; Ng et al. 2014), indomethacin (Bernabeu et 10 ovulation cycle; al. 2006) and piroxicam (Moon et al. 2004) have been all b) between hCG+6 to hCG+9, preferably between conducted on day 2 or 3 post- retrieval, i.e. at the time hCG+6 to hCG+8; most preferably on day hCG+7 of of cleavage-stage embryo transfer. an induced ovulation cycle; [0009] A recent randomised controlled trial (Ng et al. c) between day 4 to day 7, preferably between day 2014) compared the treatment outcome after adminis- 15 4 to day 6, more preferably on day 5 or 6, most pref- tration of atosiban or placebo in IVF/ICSI patients fol- erably on day 5, of luteal phase support, wherein lowed by cleavage-stage embryo transfer on day 2 or luteal phase support begins the day following oocyte day 3 post-retrieval. This large study was designed to retrieval in an IVF cycle, preferably wherein the fe- determine whether the anecdotal evidence found in the male has undergone ovarian stimulation; or previous smaller studies could be confirmed. This ade- 20 d) between day 4 to day 9, preferably between day quately-designed, large (N=800), double-blind, ran- 5 to day 7, more preferably on day 5 or day 6 of luteal domised, controlled trial found no significant increase in phase support preferably in preparation for frozen implantation or live birth rates with atosiban compared embryo transferor third partyIVF, preferably wherein to placebo, as illustrated by live birth rates of 39.8% ver- luteal support begins after the endometrium is sus 38.0%, respectively (Ng et al. 2014). Atosiban ad- 25 primed for at least 6 days with exogenous oestrogen. ministration on day 2 or day 3 post-retrieval therefore does not significantly increase implantation or live birth [0013] Preferably, luteal phase support comprises rates. supplementation with progesterone, human chorionic [0010] Consequently, improving implantation of trans- gonadotropin, estradiol and progesterone, progestins ferred embryos remains one of the major challenges in 30 and/or gonadatropin releasing hormone (GnRH) ago- assisted reproductive technologies (ART) treatment. It is nists. an object of the present disclosure to improve implanta- [0014] Accordingly, the disclosure provides oxytocin tion rates, thereby increasing pregnancy rates and live receptor antagonists which can be used to prepare med- birth rates. icaments for increasing ongoing implantation rate, in- 35 creasing ongoing pregnancy rate, increasing clinical SUMMARY OF THE INVENTION pregnancy rate, and/or increasing live birth rate, in a fe- male subject undergoing embryo transfer as part of an [0011] One aspect of the disclosure provides an oxy- assisted reproductive technology. Also encompassed by tocin receptor antagonist for use in increasing ongoing the disclosure are uses of oxytocin receptor antagonist implantation rate, increasing ongoing pregnancy rate, in- 40 for the preparation of a medicament for use in a female creasing clinical pregnancy rate, and/or increasing live undergoing transfer of a blastocyst-stage embryo. Pref- birth rate, in a female subject undergoing embryo transfer erably the medicaments are administered such that their as part of an assisted reproductive technology, wherein effect overlaps with the receptive endometrium stage the antagonist is provided to the female such that the and/or when the embryo has reached the blastocyst- effect of the antagonist is present when the female is in 45 stage. Preferably, the antagonists in the medicaments (or otherwise overlaps with) the receptive endometrium are released when the female is in the receptive en- stage and/or when the embryo has reached the blasto- dometrium stage and/or when the embryo has reached cyst-stage. Preferably, the antagonist is provided such the blastocyst-stage. that it is released in the receptive endometrium stage [0015] The disclosure further encompasses methods and/or when the embryo has reached the blastocyst-50 for increasing ongoing implantation rate, increasing on- stage. Preferably, the antagonist is administered when going pregnancy rate, increasing clinical pregnancy rate, the female is in the receptive endometrium stage and/or and/or increasing live birth rate, in a female subject un- when the embryo has reached the blastocyst-stage. In dergoing embryo transfer as part of an assisted repro- certain embodiments, the antagonist is formulated for im- ductive technology, comprising administering to the fe- mediate release. In other embodiments, the antagonist 55 male an oxytocin receptor antagonist such that the effect is formulated as a sustained or delayed release formu- of the antagonist overlaps with the receptive endometri- lation, such as a depot, and is administered prior to the um stage and/or when the embryo has reached the blas- receptive endometrium stage and/or when the embryo tocyst-stage.In preferred embodiments, the methods fur-

3 5 EP 3 501 533 A1 6 ther comprise transferring an embryo into the uterus, the Figure 2: Odds ratio for ongoing implantation rate by uterinecavity or the fallopiantubes of afemale, preferably day of transfer for (BASIC) clinical trial wherein a blastocyst-stage embryo is transferred. Figure 3: Ongoing implantation rate by day of trans- The disclosure further provides methods of implanting fer for (BASIC) clinical trial an embryo in a female subject, comprising transferring 5 an embryo into the uterus, the uterine cavity or the fallo- DETAILED DESCRIPTION OF THE DISCLOSED EM- pian tubes of a female and administering to the female BODIMENTS an oxytocin receptor antagonist such that the effect of the antagonist overlaps with the blastocyst-stage and/or [0021] As used herein, "to comprise" and its conjuga- the female is in the receptive endometrium stage. 10 tions is used in its non-limiting sense to mean that items [0016] In preferred embodiments, the female is under- following the word are included, but items not specifically going transfer of a blastocyst-stage embryo and the an- mentioned are not excluded. In addition the verb "to con- tagonist is administered to the female such that the an- sist" may be replaced by "to consist essentially of’ mean- tagonist is released to the female on the same day that ing that a compound or adjunct compound as defined the embryo is transferred. Preferably, the antagonist is 15 herein may comprise additional component(s) than the administered between 2 hours prior to and 2 hours post ones specifically identified, said additional component(s) embryo transfer (for example, in an immediate release not altering the unique characteristic of the invention. formulation), preferably wherein the antagonist is admin- [0022] The articles "a" and "an" are used herein to refer istered twice, preferably wherein the first administration to one or to more than one (i.e., to at least one) of the occurs around 45 minutes prior to embryo transfer and 20 grammatical object of the article. By way of example, "an the second administration occurs around 60 minutes af- element" means one element or more than one element. ter the first administration. Preferably, a blastocyst-stage [0023] The word "approximately" or "about" when used embryo has an expansion and hatching status of 3, 4, 5, in association with a numerical value (approximately 10, or 6, more preferably wherein the blastocyst-stage em- about 10) preferably means that the value may be the bryo is a day 5 post-insemination embryo. 25 given value of 10 more or less 1% of the value. [0017] In preferred embodiments, the female is under- [0024] When referring herein to a range, such as, e.g., going transfer of a cleavage-stage embryo and the an- a range of days, the range includes both end points. For tagonist is administered to the female such that the an- example, day LH+6 to day LH+9 encompasses day tagonist is released two or three days after the embryo LH+6, day LH+7, day LH+8, and day LH+9. is transferred. Preferably, a cleavage-stage embryo has 30 [0025] As used herein, the term "embryo" refers to a at least 6 blastomeres and fragmentation of 20% or less, zygoteup to eightweeks afterfertilization. "Embryotrans- preferably wherein the cleavage-stage embryo is a day fer" is the procedure in which one or more embryos are 2 or day 3 post-fertilization embryo. placed into the uterus, uterine cavity, or fallopian tubes [0018] In preferred embodiments, the antagonist is a of a female. selective oxytocin receptor antagonist or an vaso-35 [0026] As used herein, a female subject is a mammal pressin/oxytocin receptor antagonist. More preferably, which includes companion animals, e.g., dogs and cats; the antagonist is a selective oxytocin receptor antagonist. domestic livestock animals, such as pigs, horses, don- [0019] Preferably, the antagonist is . Prefer- keys, goats, sheep, llamas; as well as rare and threat- ably, barusiban is provided subcutaneously. Preferably, ened species. Preferably, the subject is human. between 30-80 mg, more preferably 50 mg of barusiban 40 [0027] Assisted reproductive technology (ART) refers is administered. In some embodiments, the female is un- to methods for achieving pregnancy using artificial dergoing transfer of a blastocyst-stage embryo and ba- means. Preferably, ART refers to methods in which an rusiban is administered to the female as a depot prior to in vitro fertilized embryo is transferred into a female sub- the day of embryo transfer. Preferably, the female is un- ject, for example using IVF/ICSI. dergoing transfer of a blastocyst-stage embryo and ba- 45 [0028] "Fresh embryo transfer" refers to the transfer of rusiban is administered to the female on the same day embryos without first freezing the embryos. as embryo transfer. Preferably, 40 mg barusiban is ad- [0029] The natural ovulation cycle ranges from 21 to ministered subcutaneously 45 minutes prior to blastocyst 35 days, with the average length being 28 days. The first stage embryo transfer and 10 mg barusiban is adminis- part of the cycle is referred to as the follicular phase in tered subcutaneously 60 minutes after the first adminis- 50 which the ovarian follicles mature. Ovulation follows by tration. which a mature egg is released into the oviduct. The luteal phase refers to phase of the ovulation cycle beginning BRIEF DESCRIPTION OF THE DRAWINGS with the formation of the corpus luteum at LH+1 and fin- ishing the day before the first day of menstruation. [0020] 55 [0030] "Ongoing implantation rate" as used herein re- fers to the number of intrauterine viable fetuses 10-11 Figure 1: Examples of oxytocin receptor antagonists weeks after transfer divided by number of embryos/blas- (Figure 1A and Figure 1B) tocysts transferred. Preferably, the administration of an

4 7 EP 3 501 533 A1 8 oxytocin receptorantagonist asdisclosed herein increas- [0038] Implantation is a critical process in which an em- es the ongoing implantation rate by at least 5%, more bryo apposes, attaches and invades the endometrium. preferably by at least 10% and most preferred by at least The uterus will accept the implanting embryo only during 20%. a limited time period of time described as the "window of [0031] "Ongoing pregnancy rate" as used herein refers 5 implantation" or "receptive window" (Makrigiannakis and to a pregnancy with at least one intrauterine viable fetus Minas 2006; Strowitzki et al. 2006). The window of im- 10-11 weeks after transfer . Preferably, the administra- plantation is a period of a few days in which the en- tion of an oxytocin receptor antagonist as disclosed here- dometrium acquires the receptive stage allowing embryo in increases the ongoing pregnancy rate by at least 5%, adhesion and invasion (Koot and Macklon 2013). This more preferably by at least 10% and most preferred by 10 stage is referred to herein as the "receptive endometrium at least 20%. stage". [0032] "Implantation rate" as used herein refers to the [0039] Successful implantation depends not only a re- number of intrauterine gestational sacs with fetal heart ceptive endometrium, but also a functional embryo and beat 5-6 weeks after transfer divided by number of em- the synchronized communication between the embryo bryos/blastocysts transferred. Preferably, the adminis- 15 and maternal tissues. Therefore, during the receptive tration of an oxytocin receptor antagonist as disclosed window of implantation, the embryo also needs to be at herein increases the ongoing implantation rate by at least the appropriate stage. Implantation occurs after a blast- 5%, more preferably by at least 10% and most preferred ocyst hatches from the zona pellucida. Therefore, as is by at least 20%. well known if the field of ART, if a blastocyst stage embryo [0033] "Clinical pregnancy rate" as used herein refers 20 is transferred, the woman should ideally be in the recep- to a pregnancy with at least one intrauterine gestational tive endometrium stage, so that both the endometrium sac with fetal heart beat 5-6 weeks after transfer. Pref- and the embryo are synchronized for implantation. If a erably, the administration of an oxytocin receptor antag- cleavage stage embryo is transferred, then the woman onistas disclosed hereinincreases theclinical pregnancy should be in the pre-receptive stage. The endometrium rate by at least 5%, more preferably by at least 10% and 25 and embryo will both further develop such that when the most preferred by at least 20%. embryo reaches the blastocyst stage, the endometrium [0034] "Livebirth rate" refers tothe number oflive births will have reached the receptive stage. per women treated. Preferably, the administration of an [0040] Accordingly, the antagonists are administered oxytocin receptorantagonist asdisclosed herein increas- such that the effect of the antagonist overlaps with the es the live birth rate by at least 5%, more preferably by 30 receptive endometrium stage and/or the embryo reach- at least 10% and most preferred by at least 20%. ing the blastocyst-stage. Preferably, the antagonists are [0035] One aspect of the disclosure provides oxytocin provided such that the antagonist is released or contin- receptor antagonists for use in increasing ongoing im- ues to be released in the receptive endometrium stage plantation rate, increasing ongoing pregnancy rate, in- and/or the embryo reaching the blastocyst-stage. As dis- creasing clinical pregnancy rate, and/or increasing live 35 cussed further herein, the antagonists are usually formu- birth rate, in a female subject undergoing embryo trans- lated as immediate release compositions such that they fer. Preferably, the ongoing implantation rate is in- are administered during the receptive endometrium creased. stage. However, the disclosure also encompasses an- [0036] Previous large studies in the art report admin- tagonists formulated as control or delayed release for- istration of oxytocin receptor antagonists in the early40 mulations, for example as a depot, such that they are luteal phase (corresponding to day 2 or 3 post-retrieval) administered during the pre-receptive stage, but are re- when uterine contraction frequency is high. However, ox- leased during the receptive stage. ytocin receptor antagonists demonstrated no improved [0041] A number of cellular and morphological chang- effects on implantation when provided in the early luteal es are associated with the transformation of a pre-recep- phase (Ng et al. 2014). Thus, in certain embodiments, 45 tive endometrium to a receptive endometrium. Biomark- the present invention excludes immediate release or sub- ers have also been identified which can be used to eval- stantially immediate release formulations of oxytocin re- uate whether the endometrium is in a receptive stage. ceptor antagonists administered in the early luteal phase For example, the Endometrial Receptivity Array from Ig- (i.e., preceding the receptive endometrium stage). nomix™ analyses the expression of 238 genes in order [0037] The present disclosure demonstrates the effec- 50 to determined whether the endometrium is in the recep- tiveness of oxytocin receptor antagonists on embryo im- tive stage (see, WO2010010201 and WO2010010213). plantation when provided after the early luteal phase, or Preferably, the receptive endometrium stage is defined rather, at the receptive endometrium stage and/or the as having a normal receptive profile based on the ex- when the embryo has reached the blastocyst-stage (Ex- pression profile of the 238 genes of the Endometrial Re- ample 1). Since the frequency of uterine contractions has 55 ceptivity Array (ERA). returned or nearly returned to baseline at this stage, it is [0042] The receptive endometrium stage can also be surprising that a oxytocin receptor antagonist has an ef- characterized based on the stage of a normal ovulation fect on the implantation rate. cycle. Ovulationoccurs afterthe luteinizing hormone (LH)

5 9 EP 3 501 533 A1 10 surge, which normally takes place around day 14 of a US 20120252119. Culture media known in the art that normal ovulation cycle. The precise stage of the ovulation are suitable for use for the in vitro support of cell devel- cycle can be characterized based on the timing of the LH opment and growth includes human tubal fluid (HTF) (Ir- surge. The LH surge can be measured by taking blood vine Scientific), N-2-hydroxyethylpiperazine-N’-2- samples at various days of a woman’s cycle. The day of 5 ethane (HEPES) media (Irvine Scientific), IVF-50 (Scan- the LH surge is considered as day LH 0. LH+1 then usu- danavian IVF Science), S2 (Scandanavian IVF Science), ally corresponds to day 15 of the cycle and LH+7 usually G1 and G2 (Scandanavian IVF Science), UnilVF, ISM- to day 21. The endometrium becomes receptive to im- 1, BlastAssist, UTM media (sold as MEDICULT® media plantation at around day LH +7 in natural cycles and re- by Origio A/S), Modified Whittens medium, Wittinghams mains receptive for usually about 4 days (Bergh and Na- 10 T6 media, Ham’s F-10 media, Earle’s solution. G1 and vot 1992), although this timing varies for each woman. G2 media were specifically formulated to meet the phys- In preferred embodiments, the receptive endometrium iological needs of the cleavage stage embryo and the stage corresponds to between day LH+6 to day LH+9 of embryo in the eight-cell through blastocyst stage of de- a natural ovulation cycle, more preferably between LH+6 velopment. U.S. Pat. No. 6,605,468 discloses a medium to LH+8. The receptive window lasts normally only 2-3 15 for the propagation of early stage embryos to blastocyst days per ovulation cycle. However, as is well-known in stage. the art, there exists variability between women in both [0048] Embryos may also be subjected to morpholog- the length of the window and when it occurs. ical, kinetic and/or genetic testing. Preferably, visual ob- [0043] In women undergoing oocyte retrieval for fresh servation of the embryo by microscopy is used to deter- embryo transfer, the receptive window can be character- 20 mine if aberrant physical or morphological features are ized based on the day post-oocyte retrieval, the number present (see, e.g., WO2013078312). Preimplantation of days in luteal phase support following oocyte retrieval, genetic diagnosis is commonly performed to screen for and/or the number of days following hCG administration. inherited diseases. For this method, one or two cells are [0044] In a typical IVF procedure, ovarian stimulation removed from an embryo to test for genetic diseases. is used in order to stimulate the ovaries to produce mul- 25 [0049] Methods of embryo transfer are well known in tiple eggs. Gonadatropin releasing hormone (GnRH) ag- the art. One or more embryos may be aspirated into a onists and GnRH antagonists can be given to prevent catheter and inserted into the uterus, the uterine cavity premature ovulation while human menopausal gonado- or the fallopian tubes. tropin (hMG), follicle stimulation hormone (FSH), lutein- [0050] In some embodiments a cleavage-stage em- izing hormone (LH), and clomiphene citrate can be given 30 bryo is transferred. "Cleavage-stage" embryos range to stimulate the production of multiple eggs. Typically, from 2-cells to 16 cells and can be characterized based eight to fourteen days of stimulation are required before on, e.g., fragmentation, symmetry of division, and ab- the ovarian follicles are sufficiently developed. Human sence of multinucleation (see Prados et al. Human Re- chorionic gonadotropin (hCG) is usually then adminis- production 2012 27:50-71 for review). Fragmentation is tered to ensure the final stage of maturation and the eggs 35 generally characterized by the percent of embryo volume are retrieved prior to ovulation, usually around 36 hours that is replaced by fragments. Preferably, a cleavage- after hCG administration. The day of hCG administration stage embryo is characterized as having 4 blastomeres is defined as hCG+0 and oocyte retrieval is performed on day 2 post-insemination and 6-8 blastomers on day on hCG+2. 3 post-insemination. Preferably, the cleavage-stage em- [0045] In preferred embodiments, the receptive en- 40 bryo has at least 6 blastomeres and fragmentation of dometrium stage corresponds to between day hCG+6 to 20% or less. day hCG+9 (or rather, 4 to 7 days post-oocyte retrieval), [0051] Preferably, a day 2 or day 3 cleavage stage em- preferably between day hCG+6 to day hCG+8. bryo is transferred. In fresh embryo transfers where the [0046] Egg retrieval is a minor surgical procedure that female has undergone oocyte retrieval, the day 2 or day can be performed, for example, using transvaginal ultra- 45 3 cleavage stage embryo is then usually transferred 2 or sound aspiration. Theeggs maybe inspected microscop- 3 days post-oocytee retrieval, respectively. The blasto- ically and diagnosed to observe their morphological fea- cyst stage of the embryo and the receptive endometrium tures. Insemination is then performed in vitro, for example stage are reached, ideally simultaneously, several days by incubating oocytes together with sperm or by Intrac- after transfer. ytoplasmic sperm injection (ICSI) which implies injecting 50 [0052] Preferably, a blastocyst-stage embryo is trans- the sperm with a microscopic needle into the egg ferred. A "blastocyst-stage" embryo has an inner cell Fertilization" refers to the penetration of the ovum by the mass, an outer cell layer called the trophectoderm, and spermatozoa and combination of their genetic material a fluid-filled blastocele cavity containing the inner cell resulting in the formation of a zygote. mass from which the whole of the embryo is derived. An [0047] After fertilization, embryos are cultured in vitro. 55 embryo normally reaches this stage at day 5 or 6 post- Methods for culturing and staging embryos are well- retrieval. A blastocyst-stage embryo can be character- known in the art and are described in, e.g., USized based on its expansion and hatching status. Expan- 20140134632, US 20140017717, US 20120252119, and sion relates to the increasing volume of the cavity

6 11 EP 3 501 533 A1 12

(i.e.blastocoel), while hatching refers to the herniation or [0057] While embryos may be transferred into a female escape of the blastocyst from its membrane (i.e. zona within a few days post fertilization (fresh embryo trans- pellucida). The expansion and hatching status is charac- fer), it is also common to place the embryos in frozen terized as follows: storage for later use. Frozen embryo transfer (FET) is a 5 procedure that utilizes cryopreserved embryos from a 1. An early blastocyst, blastocoel being less than half previous cycle of in vitro fertilization or ICSI. The cryop- volume of that of the embryo reserved embryos are thawed and transferred into the 2. A blastocyst with a blastocoel whose volume is uterine cavity through a catheter. The disclosure also en- half of, or greater than half of, that of the embryo compasses the use of cryopreserving oocytes prior to 3. A blastocyst with a blastocoel completely filling 10 fertilization. In these embodiments, oocytes can be later the embryo thawed, fertilized, and cultured and transferred as de- 4. An expanded blastocyst with a blastocoel volume scribed herein. larger than that of the early embryo, with a thinning [0058] Rapid freezing can be used for these purposes, zona for example together with a cryoprotectant. Conventional 5. A hatching blastocyst with the trophectoderm15 cryoprotectants include glycols such as ethylene glycol, starting to herniate through the zona propylene glycol, and glycerol; 2-methyl-2,4-pentanediol 6. A hatched blastocyst, in which the blastocyst has (MPD); dimethyl sulfoxide (DMSO) and sucrose. Alter- completely escaped from the zona. natively, vitrification can also be used to freeze oocytes or embryos. In preferred embodiments, the blastocyst-stage embryo 20 [0059] FET, as well as "third-party IVF" (gestational for transfer has an expansion and hatching status of 3, surrogacy, ovum donation, embryo donation), may be 4, 5, or 6. performed during a natural ovulation cycle. The receptive [0053] In fresh embryo transfers where the female has window for these women can be determined based on a undergone oocyte retrieval, the blastocyst stage embryo natural ovulation cycle as described herein. In some em- is usually transferred to the female 5 or 6 days post-25 bodiments, ovulation is induced with the administration oocyte retrieval, preferably 5 days post-retrieval. of, e.g., hCG. Preferably, in these women the receptive [0054] For women undergoing fresh embryo transfer window corresponds to between hCG+6 to hCG+9, pref- following oocyte retrieval, the endometrium on day 2 and erably between hCG+6 to hCG+8. day 3 post-oocyte retrieval is a pre-receptive stage and [0060] In some embodiments, women undergoing FET is not conducive to implantation. In preferred embodi- 30 or third party IVF also receive luteal support as described ments, the receptive endometrium stage corresponds to above. Preferably in these women the receptive window between day 4 to day 7 post-oocyte retrieval, preferably corresponds to between day 4 to day 9, preferably be- between day 5 to day 6. If hCG is used to induce ovulation tween day 5 to day 7, of luteal phase support. Luteal or trigger final maturation, days 4 to 7 post-oocyte re- phase support is often used when FET or third party IVF trieval will normally correspond to hCG+6 to hCG+9. 35 is performed during an "artificial cycle". In these cases, [0055] Ovarian stimulation with fertility drugs usually the endometrium is prepared by administering estrogen leads to luteal phase deficiency. Therefore, it is generally and/or progesterone. Preferably, luteal phase support standard practice for luteal phase support to be used in begins after the endometrium is primed for at least 6 days women following oocyte retrieval. Luteal phase support with exogenous estrogen in order to induce an artificial refers to therapeutic interventions during the luteal phase 40 cycle. aiming at supplementing corpus luteal function for im- [0061] In an exemplary embodiment of FET or third proving the embryo implantation and the early pregnancy partyIVF, estrogen isprovided orally or vaginally in doses development. Luteal phase support usually comprises of 4-8 mg daily for 10 days, at which time luteal phase supplementation with progesterone, estradiol and pro- support is initiated with the administration of vaginal pro- gesterone, progestins, hCG, and/or a GnRH agonist, or 45 gesterone and blastocyst transfer occurs 6 days after rather the administration of exogenous progesterone, es- starting progesterone. tradiol and progesterone, progestins, hCG, and/or a Gn- [0062] In one embodiment of the disclosure, the effect RH agonist. Progesterone is normally administered in- ofthe oxytocinreceptor antagonist overlaps with the blas- tramuscularly or vaginally, while hCG is administered in- tocyst-stage of the embryo. Preferably, the antagonist is tra-muscularly or subcutaneously. Preferably, luteal50 released when the embryo has reached the blastocyst- phase support begins the first day after oocyte retrieval, stage. Preferably, a blastocyst-stage embryo is trans- i.e., day 1 post-oocyte retrieval. ferred to said female and the antagonist is administered [0056] Preferably, the receptive endometrium stage on the same day that the embryo is transferred. corresponds to between day 4 to day 7, preferably be- [0063] The disclosure also contemplates the transfer tween day 4 to day 6, of luteal phase support in women 55 of a cleavage-stage embryo. In these embodiments, the who have undergone oocyte retrieval. In preferred em- antagonist may be provided as a sustained or controlled bodiments, the female has undergone ovarian stimula- release formulation for release several days after trans- tion in preparation for oocyte retrieval. fer. Alternatively, the antagonist may be administered as

7 13 EP 3 501 533 A1 14 an immediate release formulation several days after Mpa 3-mercaptopropionic acid residue transfer. As shown in the examples, administration of ba- IIe isoleucine residue rusiban (an oxytocin receptor antagonist) when an em- Asn asparagine residue bryo is in the blastocyst-stage results in an increase in Abu alpha-aminobutyric acid residue; ongoing implantation rate from 27% to 45%, a significant 5 increase. and wherein X is a D-aromatic alpha-amino acid; and Y [0064] Preferably, the oxytocin receptor antagonists is an aliphatic alpha-amino acid. Preferred oxytocin an- are selected from selective oxytocin antagonists and tagonists are listed in Figure 1. mixed /oxytocin receptor antagonists. A se- [0069] Further antagonists include OBE001/ AS- lective oxytocin antagonist has a Ki for the oxytocin re- 10 602305 (in particular oral formulations thereof), TT-235 ceptor which is at least one order of magnitude higher (Northwestern University), the selective oxytocin recep- than the Ki for the . tor antagonist ((3R,6R)-3-(2,3-dihydro-1H-in- [0065] Receptor antagonists include compounds den-2-yl)-1-[(1R)-1-(2,6-dimethylpyridin-3-yl)-2-(mor- which reduce the expression and/or activity of the recep- pholin-4-yl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]pipera- tor.A preferredvasopressin/oxytocin receptor antagonist 15 zine-2,5-dione); ((3R,6R)-6-[(2S)-butan-2-yl]- is atosiban (1-(3-mercaptopropanoic acid)- 2-(O-ethyl-D- 3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2-methyl-1,3- tyrosine)-4-L-threonine-8-L-ornithine-oxytocin). oxazol-4-yl)-2-(morpholin-4-yl)-2-oxoethyl]piperazine- [0066] The reduction of oxytocin receptor expression 2,5-dione); PF-3274167 (5-(3-(3-(2-chloro-4-fluorophe- can be achieved, for example, using RNA interference noxy)azetidin-1-yl)-5-(methoxymethyl)-4H-1,2,4-triazol- or RNA antisense molecules directed against said recep- 20 4-yl)-2-methoxypyridine); and L-368,899 hydrochloride tor. (CAS 148927-60-0); L-371,257 (1-[1-[4-(1-acetylpiperi- [0067] Preferably, the antagonist binds to the receptor din-4-yl)oxy-2-methoxybenzoyl]piperidin-4-yl]-4H-3,1- and prevents receptor activity. Such antagonists include, benzoxazin-2-one). Additional oxytocin antagonists are e.g., antibodies directed against the receptor (e.g., "neu- also described, e.g., in WO2004020414 and tralizing antibodies") as well as small molecules. Prefer- 25 WO2005/028452. Preferably, the antagonist is selected ably, the antagonist acts as a competitive antagonist and from barusiban, atosiban, OBE001/ AS-602305, PF- competes with oxytocin for binding to the oxytocin recep- 3274167 Epelsiban, and retosiban. tor. Preferably, the antagonists are small molecules that [0070] Preferably, the oxytoxin receptor antagonist is bind to the receptor and antagonize receptor activity. A formulated in a pharmaceutical composition. The com- preferred selective oxytocin antagonist is barusiban. 30 position may also include pharmaceutically acceptable [0068] Suitable oxytocin receptor antagonists are well- additives such as preservatives, diluents, dispersing known to the skilled person and can be easily identified agents, agents to promote mucosal absorption (exam- based on known screening methods which use, e.g., re- ples of which are disclosed by Merkus, F. W. H. M. et al., ceptor activation and/or receptor binding as a read-out. J. Controlled Release 24, 201-208, 1993, and which in- Suitable antagonists include those disclosed35 include surfactants, bile acids, fusidates, phospholipids US6143722, namely, heptapeptide analogues, or a phar- and cyclodextrins), buffering agents and flavourings. maceutically acceptable salts thereof, having oxytocin Such compositions may be formulated as solids (for ex- antagonist activity and consisting of a hexapeptide moi- ample as tablets, capsules or powders) or liquids (for ety S and a C-terminal beta-aminoalcohol residue Z example as solutions or suspensions), which is here tak- bound to the moiety S by an amide bond, wherein the 40 en to include creams and ointments, for oral or parenteral beta-aminoalcohol Z is: administration. Oral (including sublingual and buccal), in- tranasal, pulmonary, transdermal, rectal, vaginal, subcu- taneous, intramuscular and intravenous administration may all be suitable routes for dosing. 45 [0071] In some embodiments, the pharmaceutical composition can be delivered in a sustained or delayed release system. For example, the antagonist may be ad- wherein Q is (CH2)n-NH-A, n is 1-6 and A is H or -C(=NH) ministered using a transdermal patch or formulated in NH2 , lipophilic depots (e.g. fatty acids, waxes, oils). As used and wherein R is CH3 or C2H5; 50 herein, a sustained or delayed release system ensures and the moiety S is: that the antagonist is also present in the subject at a time point after administration, e.g., several hours or even sev- eral days after administration. Such sustained or delayed release systems allow the administration of the receptor 55 antagonists before the female is in a receptive endometri- um stage. The sustained or delayed release, however, wherein Mpa, lie, Asn and Abu have the following mean- ensures that a sufficient amount (or rather a therapeuti- ing: cally effective amount) of the antagonist is still present

8 15 EP 3 501 533 A1 16 when the female enters the receptive endometrium stage va H, Garcia-Velasco JA, Mrazek M, Chou P-Y, Wu and/or when the embryo has reached the blastocyst- M-H, Pan H-A, Hung K-H, Chang F-M. Use of an stage. oxytocin antagonist in in vitro fertilization-embryo [0072] In embodiments where the female is undergo- transfer for women with repeated implantation fail- ing transfer of a cleavage-stage embryo, the antagonist 5 ure: a retrospective study. Taiwan J Obstet Gynecol should bereleased when the cleavage-stage embryo has 2011; 40: 136-140. developed into a blastocyst-stage embryo and/or when Fanchin R, Righini C, Olivennes F, Taylor S, de Zie- the female has reached the receptive endometrium gler D, Frydman R. Uterine contractions at the time stage. If an immediate release formulation is used, then of embryo transfer alter pregnancy rates after in-vitro the antagonist should be administered several days after 10 fertilization. Hum Reprod 1998; 13: 1968-1974. embryo transfer, preferably two or three days after em- Fanchin R, Righini C, de Ziegler D, Olivennes F, bryo transfer as this will correspond to the time when the Ledée N, Frydman R. Effects of vaginal progester- cleavage-stage embryo has developed into a blastocyst- one administration on uterine contractility at the time stage embryo and/or when the female has reached the of embryo transfer. Fertil Steril 2001; 75: 1136-1140. receptive endometrium stage. If sustained or delayed re- 15 Fuchs A-R, Behrens O, Maschek H, Kupsch E, Ein- lease formulations are used, these may be administered spanier A. Oxytocin and vasopressin receptors in earlier, for example on the day of embryo transfer, pro- human and uterine myomas during menstrual cycle vided that the antagonist is released when the cleavage- and early pregnancy. Hum Reprod Update 1998; 4: stage embryo has developed into a blastocyst-stage em- 594-604. bryo and/or when the female has reached the receptive 20 Killick SR. Ultrasound and the receptivity of the my- endometrium stage. ometrium. RBM Online 2007; 15: 63-67. [0073] In embodiments where the female is undergo- Kim CH, Lee JW, Jeon IK, Park E, Lee YJ, Kim SH, ing transfer of a blastocyst-stage embryo, the antagonist Chae H, Kang BM, Lee HA. Administration of oxy- should be released on the same day as embryo transfer tocin antagonist improves the implantation rates in (within the same 24 hour period), as this will correspond 25 patients with repeated failure of IVF/ICSI treatment. to the time when the embryo has reached the blastocyst- Hum Reprod 2008; 23 (Suppl. 1): i124. stage and the female has reached the receptive en- Koot YEM, Macklon NS. Embryo implantation: biol- dometrium stage. Preferably, the antagonist is adminis- ogy, evaluation, and enhancement. Curr Opin Ob- tered between 2 hours prior to and 2 hours post embryo stet Gynecol 2013; 25: 274-279. transfer. More preferably, the antagonist is administered 30 Lan VT, Khang VN, Nhu GH, Thuong HM. Atosiban twice. In an exemplary embodiment using barusiban, the improves implantation and pregnancy rates in pa- first administration may take place around 45 minutes tients with repeated implantation failure. RBM Online prior to embryo transfer and the second administration 2012; 25: 254-260. around 60 minutes after the first administration. Lesny P, Killick SR, Tetlow RL, Robinson J, [0074] As is well-known to a skilled person, the timing 35 Maguiness SD. Embryo transfer - can we learn an- of administrationis dependent ofthe particular antagonist ything new from the observation of junctional zone used, in particular on the half-life of the antagonist. An- contractions? Hum Reprod 1998; 13: 1540-1546. tagonists with relatively short half-lives may need to be Liang YL, Kuo TC, Hung KH, Chen TH, Wu MH. Ox- administered multiple times in order to ensure that their ytocin antagonist for repeated implantation failure effects overlap the blastocyst-stage and/or the receptive 40 and delay of delivery. Taiwan J Obstet Gynecol endometrium stage. 2009; 48: 314-316. Makrigiannakis A, Minas V. Mechanisms of implan- References tation. RBM Online 2006; 14: 102-109. Moon HS, Park SH, Lee JO, Kim KS, Joo BS. Treat- [0075] 45 ment with piroxicam before embryo transfer increas- es the pregnancy rate after in vitro fertilization and Ayoubi J-M,Epiney M, Brioschi PA,Fanchin R, Char- embryo transfer. Fertil Steril 2004; 82: 816-820. donnens D, de Ziegler D. Comparison of changes in Moraloglu O, Tonguc E, Turgut V, Zeyrek T, Batioglu uterine contraction frequency after ovulation in the S. Treatment with oxytocin antagonists before em- menstrual cycle and in in vitro fertilization cycles. 50 bryo transfer may increase implantation rates after Fertil Steril 2003; 79: 1101-1105. IVF. RBM Online 2010; 21: 338-343. Bergh PA, Navot D. The impact of embryonic devel- Ng EH, Li RH, Chen L, Lan VT, Tuong HM, Quan S. opment and endometrial maturity on the timing of A randomized double blind comparison of atosiban implantation. Fertil Steril 1992; 58: 537-542. in patients undergoing IVF treatment. Hum Reprod Bernabeu R, Roca M, Torres A, Ten J. Indomethacin 55 2014; 29: 2687-2694. effecton implantationrates in oocyte recipients. Hum Papanikolaou EG, Kolibianakis EM, Tournaye H, Reprod 2006; 21: 364-369. Venetis CA, Fatemi H, Tarlatzis B and Devroey P. Blockeel C, Pierson R, Popovic-Todorovic B, Visno- Live birth rates after transfer of equal number of blas-

9 17 EP 3 501 533 A1 18

tocysts or cleavage-stage embryos in IVF. A system- the barusiban group or the placebo group, with stratifi- atic review and meta-analysis. Hum Reprod 2008; cation according to the day of transfer (day 3 post-re- 23: 91-99. trieval or day 5 post-retrieval) and the number of embry- Pierzynski P, Reinheimer TM, Kuczynski W. Oxytoc- os/blastocysts to be transferred (1 or 2). In total, 255 in antagonists may improve infertility treatment. Fer- 5 IVF/ICSI patients were randomised in the trial and con- til Steril 2007; 88: e19-e22. tributing with 440 embryos/blastocysts. Richter ON, Bartz C, Dowaji J, Kupka M, Reinsberg [0079] Investigational medicinal product, i.e. barusi- J, Ulrich U, Rath W. Contractile reactivity of human ban or placebo according to randomisation, was admin- myometrium in isolated non-pregnant uteri. Hum Re- istered as subcutaneous injections at two time points: 1st prod 2006; 21: 36-45. 10 administration of 40 mg barusiban or placebo was 45 min Schoolcraft WB, Surrey ES, Gardner DK. Embryo prior to transfer and the 2nd administration of 10 mg ba- transfer: techniques and variables affecting success. rusiban or placebo was 60 min after the 1st administra- Fertil Steril 2001; 76: 863-870. tion. Simon C, Martin JC, Pellicer A. Paracrine regulators [0080] Transfer was performed on day 3 (cleavage- of implantation. Clin Obstet Gynaecol 2000; 14:15 stage embryos) or day 5 (blastocysts) after oocyte re- 815-826. trieval. On day 3, only embryos of good quality defined Shukovski L, Healy DL, Findlay JK. Circulating im- as ≥ 6 blastomeres and ≤ 20% fragmentation could be munoreactive oxytocin during the human menstrual transferred. On day 5, blastocysts with expansion and cycle comes from the pituitary and is estradiol de- hatching status 3, 4, 5 or 6 could be transferred. The pendent. J Clin Endocrinol Metab 1989; 68: 455-460. 20 actual number of transferred embryos/blastocysts for Strowitzki T, Germeyer A, Popovici R, Wolff M. The each individual patient depended on the availability of human endometrium as a fertility-determining factor. embryos/blastocysts of the required morphological qual- Hum Reprod Update 2006; 12: 617-630. ity, local regulations and clinical practice for the patient’s Zhu L, Che HS, Xiao L, Li YP. Uterine peristalsis age, but the maximum number was 2. before embryo transfer affects the chance of clinical 25 [0081] Key aspects related to the transfer procedure pregnancy in fresh and frozen-thawed embryo trans- had been standardised. A speculum was inserted into fer cycles. Hum Reprod 2014; 29: 1238-1243. the vagina and cleaning of the vagina and cervix was done according to local practices but with minimal ma- [0076] All patent and literature references cited in the nipulation and disturbance. Soft or ultrasoft catheters present specification are hereby incorporated by refer- 30 were used. The outer sheath of the catheter was inserted ence in their entirety. just protruding to the internal os (i.e. keeping the outer [0077] The invention is further explained in the follow- sheath in the cervical canal). The embryo(s)/blasto- ing examples. These examples do not limit the scope of cyst(s) were loaded into the inner sheath which was then the invention, but merely serve to clarify the invention. inserted through the outer sheath. Using abdominal ul- 35 trasound guidance, the embryo(s)/blastocyst(s) were EXAMPLES placed 1.5-2.0 cm from the fundus. The time from loading the inner catheter to placing the embryo(s)/blastocyst(s) Example 1: A randomised, placebo-controlled, double- should not have exceeded 1 min. After placement, the blind, parallel groups, multinational, multicentre trial as- inner and outer catheters were withdrawn and checked sessing the effect of barusiban administered subcutane- 40 for retained embryo(s)/blastocyst(s), mucus and blood. ously on the day of transfer on implantation and preg- After confirmation that there were no embryo(s)/blasto- nancy rates in IVF/ICSI patients cyst(s) left in the catheters, the speculum was subse- quently removed; this occurred approximately within 2 Methodology min after placement of the embryo(s)/blastocyst(s). Any 45 difficulties/eventualities occurring during the transfer pro- [0078] BASIC was a randomised, double-blind, place- cedure were recorded. bo-controlled, parallel groups, multinational, multicentre [0082] Patients received vaginal progesterone tablets trial. It was designed to evaluate the effects of barusiban, 100 mg twice daily from the day after oocyte retrieval and administered either on the day of cleavage-stage embryo until the day of the clinical pregnancy visit. On the day of transfer or on the day of blastocyst transfer, on ongoing 50 transfer, patients should insert the progesterone tablets implantation rate in IVF/ICSI patients. The patients un- at least 3 hours before transfer and at least 3 hours after derwent controlled ovarian stimulation in the long GnRH transfer. Ongoing implantation rate (primary endpoint) agonist or GnRH antagonist protocol, received hCG for was defined as the number of intrauterine viable fetuses triggering of final follicular maturation, had undergone 10-11 weeks after transfer divided by number of embry- oocyte retrieval, and had daily luteal phase support by 55 os/blastocysts transferred. supplementation with vaginal progesterone starting on [0083] With respect to statistical methodology, the pri- day 1 post-retrieval, and transfer on day 3 or 5 post-re- mary hypothesis was tested using a logistic regression trieval. Patients were randomised in a 1:1 ratio to either model with ongoing implantation (yes/no) as the outcome

10 19 EP 3 501 533 A1 20 and treatment and randomisation strata as factors. The [0087] The BASIC trial identified the time window for treatment effect is presented on the odds ratio scale, as a clinically relevant impact of barusiban, or generally for this represent the outcome of the logistic regression anal- oxytocin antagonists and mixed oxytocin/vasopressin ysis; an analysis that allows for inclusion of factors and antagonists, on implantation, which was not predicted in co-variates. Adjustment for potential imbalanced distri- 5 advance of the trial. An effect on implantation rate is seen bution between treatment groups of for example quality when the oxytocin antagonist is administered at the time of the transferred embryos/blastocysts was described in of implantation, on day 5 post-retrieval (or later), but not thestatistical analyses plannedfor the BASICtrial. It must in the early luteal phase on day 2-3 post-retrieval. be stressed that the odds ratio based on the logistic re- [0088] The lack of a consistent effect between day 3 gressionmodel provides the mostappropriate way of rep- 10 post-retrieval cleavage-stage embryo transfers and day resenting the data and the basis for evaluation of treat- 5 post-retrieval blastocyst transfers is of importance for ment effect. the hypotheses on the mechanisms related to uterine contractility and consequences for cycle outcome. For Results both days, thedose and method of administration of phar- 15 macological intervention as well as the transfer proce- [0084] The impact on treatment outcome of the differ- dure were the same. ent receptivity stages and implantation potential of cleav- [0089] Therefore, the mechanisms proposed such as age-stage embryos and blastocysts was apparent, as expulsion or dispersion of embryos/blastocysts due to illustrated by overall ongoing implantation rates of 19% uterine contractility in the early luteal phase or due to the for day 3 post-retrieval transfers and 38% for day 5 post- 20 embryo/blastocyst transfer procedure should no longer retrieval transfers. The same pattern was observed in be considered potential explanations for improvement of both the barusiban and placebo groups. implantation rates with utero-relaxant agents, including [0085] The observed overall (day 3 + day 5) ongoing oxytocin antagonists or mixed oxytocin/vasopressin an- implantation rate in the trial was 27.1% for barusiban and tagonists. In case these mechanisms were the most rel- 28.2%1, corresponding to an odds ratio 2 of 1.1 (95% con- 25 evant, the findings should have been the same for day 3 fidence interval 0.7- 1.8; p=0.6960), i.e. in favour of ba- and day 5 post-retrieval observations. rusiban but not significant. Therefore, the primary end- point for the overall trial population was not met, but as described below this was because the day of transfer Claims had an interaction. Transfer of cleavage-stage embryos 30 on day 3 post-retrieval yielded an odds ratio of 0.3 1. An oxytocin receptor antagonist for use in increasing (0.3-1.2; p=0.1509) (Figure 2). Analysis of the day 5 post- ongoing implantation rate, increasing ongoing preg- retrieval strata resulted in an odds ratio of 2.3 (1.1-4.7; nancy rate, increasing clinical pregnancy rate, p=0.0270) and thereby demonstrating a significant treat- and/or increasing live birth rate, in a female subject ment effect of barusiban on ongoing implantation rate for 35 undergoing transfer of a blastocyst-stage embryo as blastocyst transfers (Figure 2). An odds ratio of 2.3 cor- part of an assisted reproductive technology, wherein responds to adjusted means of ongoing implantation said oxytocin receptor antagonist is administered to rates for blastocyst transfers of 45% for barusiban vs the female subject on the day of embryo transfer, 27% for placebo (relative Δ of 67%) (Figure 3). and wherein the oxytocin receptor antagonist is 1 Data presented in this document are for the per-protocol 40 OBE001. (PP) population. Similar results were observed for the intention-to-treat (ITT) population. For example, the on- 2. The oxytocin receptor antagonist for use according going implantation rate for the ITT population was 26.2% to claim 1, wherein said OBE001 is provided as an for barusiban and 27.9% for placebo. oral formulation. 2 The odds ratios are based on the logistic regression 45 model, for which the analyses are adjusted for site, pri- 3. The oxytocin receptor antagonist for use according mary reason for infertility and embryo/blastocyst quality. to claim 1 or 2, wherein the blastocyst-stage embryo [0086] The results from the BASIC trial indicated that has an expansion and hatching status of 3, 4, 5, or 6. interpretation of the effects of an oxytocin antagonist on implantation rate was affected by day of transfer; cleav- 50 4. The oxytocin receptor antagonist for use according age-stage embryo transfer (day 3 post-retrieval) or blas- to any one of claims 1-3, wherein the blastocyst- tocysttransfer (day 5post-retrieval). Noeffect on ongoing stage embryo is a day 5 post-fertilization embryo. implantation rate was established for barusiban when cleavage-stage embryo transfer was done on day 3 (pre- 5. The oxytocin receptor antagonist for use according receptive stage). However, a significant (p=0.0270) ef- 55 to any one of claims 1-4, wherein the embryo transfer fect of barusiban on improving ongoing implantation rate is a fresh embryo transfer. was observed when blastocyst transfer was done on day 5 (receptive stage). 6. The oxytocin receptor antagonist for use according

11 21 EP 3 501 533 A1 22 to any one of claims 1-5, wherein the antagonist is administered between 2 hours prior to and 2 hours post embryo transfer.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• WO 2010010201 A [0041] • US 6605468 B [0047] • WO 2010010213 A [0041] • WO 2013078312 A [0048] • US 20140134632 A [0047] • US 6143722 A [0068] • US 20140017717 A [0047] • WO 2004020414 A [0069] • US 20120252119 A [0047] • WO 2005028452 A [0069]

Non-patent literature cited in the description

• PRADOS et al. Human Reproduction, 2012, vol. 27, • KILLICK SR. Ultrasound and the receptivity of the 50-71 [0050] myometrium. RBM Online, 2007, vol. 15, 63-67 • CHEMICAL ABSTRACTS, 148927-60-0 [0069] [0075] • MERKUS, F. W. H. M. et al. J. Controlled Release, •KIMCH; LEE JW ; JEON IK ; PARK E ; LEE YJ ; 1993, vol. 24, 201-208 [0070] KIM SH ; CHAE H ; KANG BM ; LEE HA. Adminis- • AYOUBI J-M ; EPINEY M ; BRIOSCHI PA ; FAN- tration of oxytocin antagonist improves the implanta- CHIN R; CHARDONNENS D; DE ZIEGLER D. tion rates in patients with repeated failure of IVF/ICSI Comparison of changes in uterine contraction fre- treatment. Hum Reprod, 2008, vol. 23 (1), i124 [0075] quency after ovulation in the menstrual cycle and in • KOOT YEM ; MACKLON NS. Embryo implantation: in vitro fertilization cycles. Fertil Steril, 2003, vol. 79, biology,evaluation, and enhancement. CurrOpin Ob- 1101-1105 [0075] stet Gynecol, 2013, vol. 25, 274-279 [0075] • BERGH PA ; NAVOT D. The impact of embryonic •LANVT; KHANG VN ; NHU GH ; THUONG HM. development and endometrial maturity on the timing Atosiban improves implantation and pregnancy rates of implantation. Fertil Steril, 1992, vol. 58, 537-542 in patients with repeated implantation failure.RBM [0075] Online, 2012, vol. 25, 254-260 [0075] • BERNABEU R ; ROCA M ; TORRES A ; TEN J. In- • LESNY P ; KILLICK SR ; TETLOWRL ; ROBINSON domethacin effect on implantation rates in oocyte re- J; MAGUINESS SD. Embryo transfer - can we learn cipients. Hum Reprod, 2006, vol. 21, 364-369 [0075] anything new from the observation of junctional zone • BLOCKEEL C ; PIERSON R ; POPOVIC-TODOR- contractions?. Hum Reprod, 1998, vol. 13, OVIC B ; VISNOVA H ; GARCIA-VELASCO JA ; 1540-1546 [0075] MRAZEK M ; CHOU P-Y ; WU M-H ; PAN H-A ; • LIANG YL ; KUO TC ; HUNG KH ; CHEN TH ; WU HUNG K-H. Use of an oxytocin antagonist in in vitro MH. Oxytocin antagonist for repeated implantation fertilization-embryo transfer for women with repeated failure and delay of delivery. Taiwan J Obstet Gyne- implantation failure: a retrospective study. Taiwan J col, 2009, vol. 48, 314-316 [0075] Obstet Gynecol, 2011, vol. 40, 136-140 [0075] • MAKRIGIANNAKIS A ; MINAS V. Mechanisms of • FANCHIN R ; RIGHINI C ; OLIVENNES F ; TAYLOR implantation. RBM Online, 2006, vol. 14, 102-109 S; DE ZIEGLER D ; FRYDMAN R. Uterine contrac- [0075] tions at the time of embryo transfer alter pregnancy • MOON HS ; PARK SH ; LEE JO ; KIM KS ; JOO BS. rates after in-vitro fertilization. Hum Reprod, 1998, Treatment with piroxicam before embryo transfer in- vol. 13, 1968-1974 [0075] creases the pregnancy rate after in vitro fertilization • FANCHIN R ; RIGHINI C ; DE ZIEGLER D; OLI- and embryo transfer. Fertil Steril, 2004, vol. 82, VENNES F ; LEDÉE N ; FRYDMAN R. Effects of 816-820 [0075] vaginal progesterone administration on uterine con- • MORALOGLU O ; TONGUC E ; TURGUT V ; tractility at the time of embryo transfer.Fertil Steril, ZEYREK T ; BATIOGLU S. Treatment with oxytocin 2001, vol. 75, 1136-1140 [0075] antagonists before embryo transfer may increase im- • FUCHS A-R ; BEHRENS O ; MASCHEK H ; KUP- plantation rates after IVF. RBM Online, 2010, vol. 21, SCH E ; EINSPANIER A. Oxytocin and vasopressin 338-343 [0075] receptors in human and uterine myomas during men- strual cycle and early pregnancy. Hum Reprod Up- date, 1998, vol. 4, 594-604 [0075]

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•NGEH; LI RH ; CHEN L ; LAN VT ; TUONG HM ; •SIMONC; MARTIN JC ; PELLICER A. Paracrine QUAN S. A randomized double blind comparison of regulators of implantation.Clin Obstet Gynaecol, atosiban in patients undergoing IVF treatment. Hum 2000, vol. 14, 815-826 [0075] Reprod, 2014, vol. 29, 2687-2694 [0075] • SHUKOVSKI L ; HEALY DL ; FINDLAY JK. Circu- • PAPANIKOLAOU EG ; KOLIBIANAKIS EM ; lating immunoreactive oxytocin during the human TOURNAYE H ; VENETIS CA ; FATEMI H ; TAR- menstrual cycle comes from the pituitary and is es- LATZIS B ; DEVROEY P. Live birth rates after trans- tradioldependent. J Clin Endocrinol Metab, 1989, vol. fer of equal number of blastocysts or cleavage-stage 68, 455-460 [0075] embryos in IVF. A systematic review and meta-anal- • STROWITZKI T ; GERMEYER A ; POPOVICI R ; ysis. Hum Reprod, 2008, vol. 23, 91-99 [0075] WOLFF M. The human endometrium as a fertility-de- • PIERZYNSKI P ; REINHEIMER TM ; KUCZYNSKI termining factor. Hum Reprod Update, 2006, vol. 12, W. Oxytocin antagonists may improve infertility treat- 617-630 [0075] ment. Fertil Steril, 2007, vol. 88, e19-e22 [0075] •ZHUL; CHE HS ; XIAO L ; LI YP. Uterine peristalsis • RICHTER ON ; BARTZ C ; DOWAJI J ; KUPKA M ; before embryo transfer affects the chance of clinical REINSBERG J ; ULRICH U ; RATH W. Contractile pregnancy in fresh and frozen-thawed embryo trans- reactivity of human myometrium in isolated non-preg- fer cycles. Hum Reprod, 2014, vol. 29, 1238-1243 nant uteri. Hum Reprod, 2006, vol. 21, 36-45 [0075] [0075] • SCHOOLCRAFT WB ; SURREY ES ; GARDNER DK. Embryo transfer: techniques and variables af- fecting success. Fertil Steril, 2001, vol. 76, 863-870 [0075]

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