HK1238147B - Oxytocin receptor antagonist therapy in the luteal phase for implantation and pregnancy in women undergoing assisted reproductive technologies - Google Patents

Description

Oxytocin receptor antagonist therapy during the luteal phase of implantation and pregnancy in women undergoing assisted reproductive technology

Citations of Related Applications

This application claims priority from European Application No. 14199709.8, filed on December 22, 2014, and U.S. Application No. 14/643307, filed on March 10, 2015, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the use of oxytocin receptor antagonists in females undergoing embryo transfer as part of assisted reproductive technology. In particular, methods are provided for increasing ongoing implantation rates, increasing ongoing pregnancy rates, increasing clinical pregnancy rates, and/or increasing live birth rates in female subjects undergoing embryo transfer. Specifically, the antagonist is released during the luteal phase when the endometrium is receptive to embryo implantation and/or when the embryo reaches the blastocyst stage.

Background Art

In vitro fertilization (IVF) is a method for establishing pregnancy in female subjects. The step generally involves ovarian stimulation using one or more hormones, mainly follicle stimulating hormone (FSH), and human chorionic gonadotropin (hCG) is usually given to trigger final follicular maturation. Oocyte retrieval usually occurs 2 days (about 36 hours) after hCG is given. The oocyte is then fertilized in vitro, cultured for several days, and transplanted to the uterus. The embryo can be placed in cryopreservation and transplanted (i.e., frozen embryo transfer) after several months or even several years.

Improving the implantation rate of transferred embryos is one of the major challenges in assisted reproductive technology (ART) treatments. For IVF/intracytoplasmic sperm injection (ICSI), only approximately one-third of transferred embryos implant in women undergoing controlled ovarian stimulation. Implantation and pregnancy rates are influenced by a variety of factors, including patient age and other characteristics, the magnitude of the response to ovarian stimulation, the quality of the embryos obtained, endometrial receptivity, and the actual transfer procedure.

Uterine contractions are considered a potentially important factor affecting implantation and pregnancy rates in IVF/ICSI cycles (Fanchin et al. 1998; Schoolcraft et al. 2001; Bulleti and de Ziegler 2005). High frequency of uterine contractions at the time of transfer appears to have a negative impact on outcomes, possibly by excluding embryos from the uterine cavity or by displacing them, thereby reducing implantation and pregnancy rates.

Observational data show that clinical pregnancy rates decrease with increasing contraction frequency at the time of cleavage-stage embryo transfer on hCG+4 day (i.e., four days after hCG administration, corresponding to day 2 after oocyte retrieval) (Fanchin et al. 1998). A prospective controlled study also observed that patients with a higher frequency of uterine contractions on the day of cleavage-stage embryo transfer (day 3 after oocyte retrieval) had a lower pregnancy rate than patients with a lower frequency of uterine contractions at the time of transfer (Zhu et al. 2014).

Uterine contractions during controlled ovarian stimulation cycles were compared to normal menstrual cycles (Ayoubi et al. 2003). The frequency of uterine contractions was found to be similar between the time of hCG administration in controlled ovarian stimulation cycles and the time of the luteinizing hormone (LH) surge in the natural cycle. During the luteal phase, the frequency of uterine contractions was higher on hCG+4 days (corresponding to day 2 after oocyte retrieval) in controlled ovarian stimulation cycles compared to LH+4 days (i.e., four days after the LH surge) in the natural cycle (Ayoubi et al. 2003). However, the frequency of uterine contractions on LH+6 days and hCG+6 days (corresponding to day 4 after oocyte retrieval) did not differ and was low in both cases, indicating that the level of uterine quiescence was the same at that time point in the controlled ovarian stimulation cycle and the natural cycle (Ayoubi et al. 2003). In another study, uterine contractions were assessed on the day of hCG administration, hCG+4 days (corresponding to day 2 after oocyte retrieval), and hCG+7 days (corresponding to day 5 after oocyte retrieval) in women undergoing a controlled ovarian stimulation cycle (Fanchin et al. 2001). The frequency of uterine contractions was highest on the day of hCG administration, slightly decreased during the early luteal phase as assessed on hCG+4 days, and almost reached a static state on hCG+7 days (corresponding to day 5 after oocyte retrieval). Another study reported a decrease in the number of juncture contractions in oocyte donors from day 2 to day 3 in the early luteal phase and to day 4 after oocyte retrieval (Lesny et al. 1999). Similarly, an assessment of uterine contractions in oocyte donors undergoing controlled ovarian stimulation and receiving luteal phase supplementation with exogenous progesterone showed that the frequency of uterine contractions decreased significantly from day 2 after oocyte retrieval to day 5 after oocyte retrieval (Blockeel et al. 2009).

The highest level of uterine contractions occurs at the end of controlled ovarian stimulation (the day of hCG administration) and is attributed to the high serum estradiol concentration and low serum progesterone concentration at this time point. The reduction in uterine contractions during the luteal phase is believed to be a result of exposure to endogenous progesterone caused by luteal function in response to hCG administration, as well as exposure to exogenous progesterone luteal supplementation used in IVF/ICSI cycles. Although progesterone supplementation is used for luteal phase support in IVF/ICSI patients and can reduce uterine contractions, there is still elevated uterine activity during the early luteal phase (days 2 or 3 after oocyte retrieval) when cleavage-stage embryo transfer is performed.

Because uterine contractions are elevated during the early luteal phase (days 2 or 3 after oocyte retrieval) when cleavage-stage embryo transfer is performed, studies evaluating the effects of different interventions to improve implantation on uterine contractions were performed during the early luteal phase (days 2 and 3 after oocyte retrieval; hCG+4). Compounds that reduce uterine contractility, such as atosiban (Kim et al 2008; Moraloglu et al 2010; Ng et al 2014), indomethacin (Bernabeu et al 2006), and piroxicam (Moon et al 2004), have been reported in randomized controlled trials (Moon et al 2004; Bernabeu et al 2006; Kim et al 2008; Ng et al 2014), quasi-randomized controlled trials (Moraloglu et al 2010), putative studies in fresh and frozen embryo exchange cycles (Chou et al 2011; Lan et al 2012), or case studies in fresh and frozen embryo exchange cycles (Pierzynski et al 2007; Liang et al 2009), all administered 2 or 3 days after oocyte retrieval, at the time of cleavage-stage embryo transfer.

A recent randomized controlled trial (Ng et al. 2014) compared the outcomes of IVF/ICSI patients treated with atosiban or placebo followed by cleavage-stage embryo transfer on day 2 or 3 after oocyte retrieval. This large study was designed to determine whether anecdotal evidence from previous smaller studies could be confirmed. This well-designed, large (N = 800), double-blind, randomized, controlled trial found no significant increase in implantation or live birth rates with atosiban compared with placebo, as shown by live birth rates of 39.8% versus 38.0%, respectively (Ng et al. 2014). Therefore, atosiban administered on day 2 or 3 after oocyte retrieval does not significantly increase implantation or live birth rates.

Therefore, improving the implantation of transplanted embryos remains one of the major challenges in assisted reproductive technology (ART) treatment. The present disclosure aims to improve the implantation rate, thereby increasing pregnancy and live birth rates.

Summary of the Invention

One aspect of the present disclosure provides an oxytocin receptor antagonist for increasing the ongoing implantation rate, increasing the ongoing pregnancy rate, increasing the clinical pregnancy rate, and/or increasing the live birth rate in female subjects undergoing embryo transfer as part of an assisted reproductive technology, wherein the antagonist is provided to the female such that the antagonist effect is present when the female is in (or overlapping with) the receptive endometrium stage and/or when the effect of the antagonist coincides with the embryo reaching the blastocyst stage. Preferably, the antagonist is provided such that it is released at the receptive endometrium stage and/or when the embryo reaches the blastocyst stage. Preferably, the antagonist is administered when the female is in the receptive endometrium stage and/or when the embryo reaches the blastocyst stage. In certain embodiments, the antagonist is formulated for immediate release. In other embodiments, the antagonist is formulated as a sustained-release or delayed-release formulation, such as a depot, and is administered prior to the receptive endometrial stage and/or while the embryo is still in the cleavage stage, such that once the receptive endometrial stage is reached and/or the embryo reaches the blastocyst stage, the antagonist is released or sustained-released. Preferably, the antagonist is provided such that a therapeutically effective amount of the antagonist is present when the female is at (or overlapping with) the receptive endometrial stage and/or when the effect of the antagonist coincides with the embryo reaching the blastocyst stage.

In a preferred embodiment, the receptive endometrial stage corresponds to:

a) between LH+6 and LH+9, preferably between LH+6 and LH+8, and most preferably on LH+7, of a natural ovulation cycle;

b) on hCG+6 to hCG+9 days, preferably on hCG+6 to hCG+8 days, and most preferably on hCG+7 days, of the ovulation induction cycle;

c) between day 4 and day 7, preferably between day 4 and day 6, more preferably on day 5 or day 6, most preferably on day 5, of luteal phase support, wherein luteal phase support begins on the day after egg retrieval in an IVF cycle, preferably wherein the female undergoes ovarian stimulation; or

d) between day 4 and day 9 of luteal phase support, preferably between day 5 and day 7, more preferably on day 5 or day 6, preferably in preparation for frozen embryo transfer or third party IVF, and preferably wherein luteal support is initiated after at least 6 days of priming of the endometrium with exogenous estrogen.

Preferably, luteal phase support comprises supplemental progesterone, human chorionic gonadotropin, estradiol with progesterone, progestogen and/or a gonadotropin-releasing hormone (GnRH) agonist.

Therefore, the present disclosure provides oxytocin receptor antagonists, which can be used to prepare medicaments for increasing the sustained implantation rate, increasing the sustained pregnancy rate, increasing the clinical pregnancy rate, and/or increasing the live birth rate in female subjects undergoing embryo transplantation as part of assisted reproductive technology. The present disclosure also encompasses the use of oxytocin receptor antagonists for the preparation of medicaments for females undergoing transplantation of blastocyst stage embryos. Preferably, the drug is administered so that its action overlaps with the receptive endometrial stage and/or when the embryo reaches the blastocyst stage. Preferably, the antagonist in the drug is released in the female when the female is in the receptive endometrial stage and/or when the embryo reaches the blastocyst stage. Preferably, the antagonist is provided so that a therapeutically effective amount of the antagonist is present when the female is in (or overlaps with) the receptive endometrial stage and/or when the action of the antagonist coincides with the embryo reaching the blastocyst stage.

The present disclosure further includes a method for increasing the sustained implantation rate, the sustained pregnancy rate, the clinical pregnancy rate, and/or the live birth rate in a female subject undergoing embryo transplantation as a part of an assisted reproductive technology, including administering an oxytocin receptor antagonist to a female so that the effect of the antagonist overlaps with the receptive endometrial stage and/or when the embryo (e.g., the transplanted embryo) reaches the blastocyst stage. Preferably, a method for increasing the sustained implantation rate, the sustained pregnancy rate, the clinical pregnancy rate, and/or the live birth rate in a female is provided, the method comprising administering an oxytocin receptor antagonist to a female before and/or after embryo transplantation so that there is a therapeutically effective amount of the antagonist during at least a portion of the receptive endometrial stage of the female and/or the blastocyst stage of the embryo transplanted in the female, thereby increasing the sustained implantation rate, the sustained pregnancy rate, the clinical pregnancy rate, and/or the live birth rate relative to a control. In a preferred embodiment, the method further includes transplanting an embryo to the uterus, uterine cavity, or fallopian tube of a female, preferably wherein transplanting a blastocyst stage embryo.

The present disclosure further provides a method for implanting an embryo in a female subject, comprising transferring the embryo to the uterus, uterine cavity, or fallopian tube of the female, and administering an oxytocin receptor antagonist to the female such that the effect of the antagonist overlaps with the blastocyst stage of the embryo and/or the female is in a receptive endometrial stage. Preferably, the antagonist is provided such that a therapeutically effective amount of the antagonist is present when the female is in (or overlaps with) the receptive endometrial stage and/or when the effect of the antagonist is consistent with an embryo reaching the blastocyst stage.

Also provided are methods for implanting an embryo in a female subject, the method comprising transferring the embryo to the uterus, uterine cavity, or fallopian tube of the female and administering an oxytocin receptor antagonist to the female such that a therapeutically effective amount of the antagonist is present during at least a portion of the receptive endometrial stage of the female and/or during at least a portion of the blastocyst stage of the transferred embryo in the female.

In a preferred embodiment, the female undergoes transfer of a blastocyst stage embryo and the antagonist is administered to the female such that the antagonist is released to the female on the same day of embryo transfer. Preferably, the antagonist is administered between 2 hours before and 2 hours after embryo transfer (e.g., in an immediate release formulation), preferably, wherein the antagonist is administered twice, preferably wherein the first administration occurs about 45 minutes before embryo transfer and the second administration occurs about 60 minutes after the first administration. Preferably, the blastocyst stage embryo has 3, 4, 5, or 6 expansion and hatching states, more preferably wherein the blastocyst stage embryo is a day 5 embryo after insemination.

In a preferred embodiment, the female undergoes transfer of a cleavage stage embryo and the antagonist is administered to the female such that the antagonist is released two or three days after embryo transfer. Preferably, the cleavage stage embryo has at least six blastomeres and 20% or less fragmentation, preferably wherein the cleavage stage embryo is a day 2 or 3 embryo after fertilization.

In a preferred embodiment, the antagonist is a selective oxytocin receptor antagonist or a vasopressin/oxytocin receptor antagonist. More preferably, the antagonist is a selective oxytocin receptor antagonist.

Preferably, the antagonist is baluxiban. Preferably, baluxiban is administered subcutaneously. Preferably, 30-80 mg, more preferably 50 mg, of baluxiban is administered. In some embodiments, the female undergoes transfer of a blastocyst-stage embryo and baluxiban is administered to the female as a depot prior to the day of embryo transfer. Preferably, the female undergoes transfer of a blastocyst-stage embryo and baluxiban is administered to the female on the same day of embryo transfer. Preferably, 40 mg of baluxiban is administered subcutaneously approximately 15, 30, 45, 60, or 75 minutes (e.g., 45 minutes) prior to blastocyst-stage embryo transfer and 10 mg of baluxiban is administered subcutaneously approximately 15, 30, 45, 60, or 75 minutes (e.g., 60 minutes) after the first administration.

BRIEF DESCRIPTION OF THE DRAWINGS

1A and 1B depict examples of oxytocin receptor antagonists.

Figure 2 depicts the odds ratios for sustained engraftment rate by transplant date for the (BASIC) clinical trial.

FIG3 is a graph showing sustained engraftment rates by day of transplant for the (BASIC) clinical trial.

DETAILED DESCRIPTION

As used herein, "comprise," "comprises," and its conjunctions are used in their non-limiting sense to mean that the items included in the following terms are included, but items not specifically mentioned are not excluded. In addition, the verb "consisting of" can be replaced by "consisting essentially of," meaning that the compound or accompanying compounds as defined herein may include additional components other than those specifically identified, which additional components do not alter the unique characteristics of the invention.

The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. As an example, "an element" means one element or more than one element.

The word "about" or "approximately" when used in combination with a numerical value (about 10, about 10) preferably means that the value may be 1% greater or less than the given value 10.

When ranges are mentioned herein, for example, as a range of days, the ranges are inclusive of both endpoints. For example, LH+6 days to LH+9 days encompasses LH+6 days, LH+7 days, LH+8 days, and LH+9 days.

As used herein, the term "embryo" means a fertilized egg up to 8 weeks after fertilization."Embryo transfer" is a procedure in which one or more embryos are placed in the uterus, uterine cavity, or fallopian tube of a female.

As used herein, a female subject is a mammal, including humans; companion animals such as dogs and cats; livestock animals such as pigs, horses, donkeys, sheep, lambs, llamas; and rare and endangered species. Preferably, the subject is a human.

Assisted reproductive technology (ART) refers to methods that use artificial means to achieve pregnancy. Preferably, ART refers to methods in which an in vitro fertilized embryo is transferred to a female subject, for example using IVF/ICSI.

"Fresh embryo transfer" means embryo transfer without first freezing the embryo.

Generally speaking, a natural ovulatory cycle ranges from 21 to 35 days, with an average length of 28 days. The first part of the cycle is the follicular phase, during which the ovarian follicles mature. Ovulation is followed by the release of the mature egg into the fallopian tube. The luteal phase refers to the period of the ovulatory cycle that begins with the formation of the corpus luteum at LH+1 and ends one day before the first day of menstruation.

As used herein, "sustained implantation rate" refers to the number of fetuses that survive in the uterus 10-11 weeks after implantation divided by the number of embryos/blastocysts implanted. Preferably, administration of an oxytocin receptor antagonist as disclosed herein increases the sustained implantation rate by at least 5%, more preferably by at least 10%, and most preferably by at least 20%.

As used herein, "ongoing pregnancy rate" refers to the number of pregnancies with at least one viable fetus in the uterus 10-11 weeks after transfer divided by the number of embryos/blastocysts transferred. Preferably, administration of an oxytocin receptor antagonist disclosed herein increases the ongoing pregnancy rate by at least 5%, more preferably by at least 10%, and most preferably by at least 20%.

As used herein, "implantation rate" refers to the number of gestational sacs in the uterus with a fetal heart 5-6 weeks after transplantation divided by the number of embryos/blastocysts transferred. Preferably, administration of an oxytocin receptor antagonist disclosed herein increases the sustained implantation rate by at least 5%, more preferably by at least 10%, and most preferably by at least 20%.

As used herein, "clinical pregnancy rate" refers to the number of pregnancies with at least one intrauterine gestational sac containing a fetal heart divided by the number of embryos/blastocysts transferred 5-6 weeks after transfer. Preferably, administration of an oxytocin receptor antagonist disclosed herein increases the clinical pregnancy rate by at least 5%, more preferably by at least 10%, and most preferably by at least 20%.

"Live birth rate" refers to the number of live births per treated woman. Preferably, administration of an oxytocin receptor antagonist as disclosed herein increases the live birth rate by at least 5%, more preferably by at least 10% and most preferably by at least 20%.

As used herein, "therapeutically effective amount" refers to the amount that produces the desired effect for which it is administered. In certain embodiments, the term refers to an amount that is sufficient to increase the sustained implantation rate, increase the sustained pregnancy rate, increase the clinical pregnancy rate, and/or increase the live birth rate when administered to a female subject undergoing embryo transfer according to a therapeutic dosing regimen. Those of ordinary skill in the art will understand that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular individual. On the contrary, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a significant number of subjects when administered to a female subject in need of such treatment. Those of ordinary skill in the art will understand that, in certain embodiments, a therapeutically effective amount of a particular agent or therapy can be formulated and/or administered in a single dose. In certain embodiments, a therapeutically effective agent can be formulated and/or administered in multiple doses, for example, as part of a dosing regimen.

One aspect of the present disclosure provides an oxytocin receptor antagonist for increasing the sustained implantation rate, increasing the ongoing pregnancy rate, increasing the clinical pregnancy rate, and/or increasing the live birth rate in females undergoing embryo transfer relative to a control. Preferably, the sustained implantation rate is increased relative to a control.

Previous large studies in this area have reported that oxytocin receptor antagonists were administered in the early luteal phase (corresponding to day 2 or 3 after oocyte retrieval) when uterine contraction frequency is high. These compounds, which reduce uterine contractions, are expected to improve embryo implantation. However, when administered in the early luteal phase, oxytocin receptor antagonists did not show an improved effect on implantation (Ng et al. 2014). Therefore, in certain embodiments, the present invention excludes immediate-release or substantially immediate-release formulations of oxytocin receptor antagonists administered in the early luteal phase (i.e., before the receptive endometrial stage).

The present disclosure demonstrates the effectiveness of oxytocin receptor antagonists on embryo implantation when provided after the early luteal phase, or at the receptive endometrial stage and/or when the embryo reaches the blastocyst stage (see Example 1). Since uterine contraction frequency returns to or nearly returns to baseline during this phase, it is surprising and unexpected that oxytocin receptor antagonists have an effect on implantation rates.

Implantation is a critical process in which the embryo attaches to, attaches to, and invades the endometrium. The uterus is receptive to implanting embryos only during a limited period described as the "implantation window" or "receptive window" (Makrigiannakis and Minas 2006; Strowitzki et al., 2006). The implantation window is a period of several days during which the endometrium reaches a receptive stage that allows embryo attachment and invasion (Koot and Macklon 2013). This stage is referred to herein as the "receptive endometrial phase."

Successful implantation depends not only on a receptive endometrium, but also on a functional embryo and synchronized communication between the embryo and maternal tissues. Therefore, the embryo also needs to be at the right stage during the receptive window for implantation. Implantation occurs after the blastocyst hatches from the zona pellucida. Therefore, it is well known in the field of ART that if a blastocyst-stage embryo is transferred, the woman should ideally be at a receptive endometrial stage so that the endometrium and embryo implant synchronously. If a cleavage-stage embryo is transferred, the woman should be at a pre-receptive stage. The endometrium and embryo will develop further so that when the embryo reaches the blastocyst stage, the endometrium will reach a receptive stage.

Therefore, administering antagonists disclosed herein allows the effect of the antagonist to overlap with the receptive endometrial stage and/or the embryo reaching the blastocyst stage. Preferably, providing the antagonist allows the antagonist to be released or sustained-released in the receptive endometrial stage and/or the embryo reaching the blastocyst stage. As further disclosed herein, antagonists are typically formulated as immediate-release compositions such that they are administered in the receptive endometrial stage. However, the disclosure also includes antagonists formulated as controlled or delayed-release formulations, for example, as a warehouse such that they are administered during the pre-receptive stage, but released in the receptive stage. Preferably, the therapeutically effective amount of the antagonist is present during at least a portion of the receptive endometrial stage of the female.

Many cellular and morphological changes are associated with the transformation of the pre-receptive endometrium to the receptive endometrium. Biomarkers that can be used to assess whether the endometrium is in the receptive stage have also been identified. For example, the Endometrial Receptivity Array from Ignomix ^™ analyzes 238 expression genes to determine whether the endometrium is in the receptive stage (see WO2010010201 and WO2010010213). Preferably, the receptive endometrial stage is defined as having a normal receptivity profile based on the expression profile of one or more (e.g., all) of the 238 genes on the Endometrial Receptivity Array (ERA).

The receptive endometrial phase can also be characterized based on the phase of a normal ovulatory cycle. Ovulation occurs after a surge in luteinizing hormone (LH), which typically occurs around day 14 of a normal ovulatory cycle. The precise phase of the ovulatory cycle can be characterized based on the timing of the LH surge. The LH surge can be measured by sampling blood samples on various days of a woman's cycle. The day of the LH surge is considered LH day 0. LH+1 then typically corresponds to day 15 of the cycle, and LH+7 is typically day 21. At approximately LH+7 of the natural cycle, the endometrium becomes receptive to implantation and remains receptive, typically for approximately four days (Bergh and Navot 1992), although this duration varies from woman to woman. In a preferred embodiment, the receptive endometrial phase corresponds to between LH+6 and LH+9 of the natural ovulatory cycle, more preferably between LH+6 and LH+8. The receptive window typically lasts only 2-3 days per ovulatory cycle. However, as is well known in the art, the length of this window and when it occurs vary between women.

In women undergoing egg retrieval for fresh embryo transfer, the acceptance window can be characterized based on the number of days after egg retrieval, the number of days of luteal phase support after egg retrieval, and/or the number of days after hCG administration.

In a typical IVF procedure, ovarian stimulation is used to stimulate the ovaries to produce multiple eggs. Gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists can be given to prevent premature ovulation, while human menopausal gonadotropin (hMG), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and clomiphene citrate can be given to stimulate the production of multiple eggs. Typically, 8 to 14 days of stimulation are required before the ovarian follicles fully develop. Human chorionic gonadotropin (hCG) is then usually given to ensure the final stage of maturation and that the eggs are retrieved before ovulation, typically about 36 hours after hCG is given. The day hCG is given is defined as hCG+0 and egg retrieval is performed at hCG+2.

In a preferred embodiment, the receptive endometrial stage corresponds to between hCG+6 days and hCG+9 days (or, 4 to 7 days after oocyte retrieval), preferably between hCG+6 days and hCG+8 days.

Egg retrieval is a minor surgical procedure that can be performed using, for example, vaginal ultrasound. The eggs can be examined microscopically and diagnosed to observe their morphological characteristics. In vitro fertilization is then performed, for example, by culturing the oocytes with sperm or by intracytoplasmic sperm injection (ICSI), in which sperm is injected into the egg with a microinjection needle. "Fertilization" refers to the penetration of the sperm into the egg and the combination of their genetic material, resulting in the formation of a zygote.

After fertilization, the embryos are cultured in vitro. Methods for culturing and staging embryos are well known in the art and are described, for example, in US 20140134632, US 20140017717, US 20120252119, and US 20120252119, the entire contents of which are incorporated herein by reference. Suitable culture media for supporting cell development and growth in vitro known in the art include human tubal fluid (HTF) (Irvine Scientific), N-2-hydroxyethylpiperazine-N'-2-ethane (HEPES) medium (Irvine Scientific), IVF-50 (Scandanavian IVF Science), S2 (Scandanavian IVF Science), G1 and G2 (Scandanavian IVF Science), UnilVF, ISM-1, BlastAssist, UTM medium (sold as culture medium by Origio A/S), modified Whittens medium, Wittinghams T6 medium, Ham's F-10 medium, and Earle's solution. G1 and G2 medium are specifically formulated to meet the physiological requirements of cleavage-stage embryos and embryos in 8 cells that develop through the blastocyst stage. U.S. Patent No. 6,605,468 discloses a culture medium for the proliferation of early-stage embryos to the blastocyst stage.

Embryo can also be subjected to morphology, dynamics and/or genetics test.Preferably, by microscope visual observation embryo to determine whether there is abnormal physical or morphological characteristics (reference, for example WO2013078312).Preimplantation genetic diagnosis is usually carried out to screen for genetic diseases.For this method, one or two cells are removed from the embryo to test for genetic diseases.

Methods of embryo transfer are well known in the art. One or more embryos can be inserted into a catheter and inserted into the uterus, uterine cavity, or fallopian tube.

In some embodiments, the cleavage stage embryo is transplanted." cleavage stage " embryo range is by 2 cells to 16 cells and can be characterized (with reference to review Prados et al.Human Reproduction 2012 27:50-71) according to the shortage of for example fragment, division symmetry and multinucleation. Fragment is usually characterized by the percentage ratio of the embryonic volume replaced by fragment. Preferably, the cleavage stage embryo is characterized as having 4 blastomeres and having 6-8 blastomeres 3 days after fertilization in 2 days after fertilization. Preferably, the cleavage stage embryo has at least 6 blastomeres and 20% or fragmentation still less.

Preferably, cleavage stage embryos are transferred on day 2 or day 3. In fresh embryo transfer, where the female undergoes egg retrieval, cleavage stage embryos are transferred on day 2 or day 3, respectively, typically 2 or 3 days after egg retrieval. Ideally, the embryos reach the blastocyst stage and the receptive endometrial stage simultaneously a few days after transfer.

Preferably, blastocyst stage embryos are transplanted. A "blastocyst stage" embryo has an inner cell mass, an outer cell layer called the trophectoderm, and a fluid-filled blastocyst cavity containing the inner cell mass derived from the entirety of the embryo. Embryos generally reach this stage 5 or 6 days after egg retrieval. Blastocyst stage embryos can be characterized based on their expansion and hatching status. Expansion involves increasing the volume of the cavity (i.e., the blastocyst cavity), and hatching refers to the prolapse or detachment of the blastocyst from its membrane (i.e., the zona pellucida). The expansion and hatching status are characterized as follows:

1. Early blastocysts and blastocysts are less than half the size of embryos

2. A blastocyst with a blastocoel whose volume is half or more than half the volume of the embryo

3. Blastocyst with blastocyst cavity, completely filled with embryo

4. The expanded blastocyst has a blastocoel volume larger than that of the early embryo, with a thin band

5. The hatching blastocyst with trophectoderm begins to break out of the zone

6. Hatching the blastocyst, wherein the blastocyst is completely separated from the band.

In a preferred embodiment, the blastocyst stage embryos used for transfer have 3, 4, 5 or 6 expansion and hatching stages.

In fresh embryo transfers where the female has undergone egg retrieval, blastocyst stage embryos are typically transferred to the female 5 or 6 days after egg retrieval, preferably 5 days after egg retrieval.

For women undergoing fresh embryo transfer after egg retrieval, the endometrium is typically pre-receptive and not conducive to implantation on day 2 or 3 after egg retrieval. In a preferred embodiment, the receptive endometrial stage corresponds to between day 4 and day 7 after egg retrieval, preferably between day 5 and day 6. If hCG is used to induce ovulation or trigger final maturation, days 4 to 7 after egg retrieval typically correspond to hCG+6 to hCG+9.

Ovarian stimulation using fertility drugs usually leads to luteal phase deficiency. Therefore, standard procedures for luteal phase support are usually used for women after egg retrieval. Luteal phase support refers to therapeutic intervention during the luteal phase, which is intended to supplement luteal function to improve embryo implantation and early pregnancy development. Luteal phase support is known in the art and usually includes supplementing progesterone, estradiol and progesterone, progestogen, hCG and/or GnRH agonists, or administering exogenous progesterone, estradiol and progesterone, progestogen, hCG and/or GnRH agonists. Progesterone is usually administered intramuscularly or vaginally, while hCG is usually administered intramuscularly or subcutaneously. Preferably, luteal phase support begins on the first day after egg retrieval, i.e., day 1 after egg retrieval.

Preferably, the receptive endometrial stage corresponds to between day 4 and day 7 of luteal phase support in women undergoing egg retrieval, preferably between day 4 and day 6. In a preferred embodiment, the female undergoes ovarian stimulation in preparation for egg retrieval.

The present disclosure includes transferring embryos to females within a few days of insemination (i.e., fresh embryo transfer), as well as using frozen embryos. Frozen embryo transfer (FET) is a known procedure that utilizes frozen embryos from a previous cycle of in vitro fertilization or ICSI. The frozen embryos are thawed and transferred to the uterine cavity via a catheter. The present disclosure also includes using frozen eggs for fertilization. In these embodiments, the eggs can be thawed, fertilized, cultured, and transplanted later as described herein.

Rapid freezing can be used for these purposes, for example, in conjunction with cryopreservatives. Traditional cryopreservatives include glycols such as ethylene glycol, propylene glycol, and glycerol; 2-methylpentanediol (MPD); dimethyl sulfoxide (DMSO), and sucrose. Alternatively, vitrification can also be used to freeze eggs or embryos.

FET, as well as "third-party IVF" (egg donation, embryo donation), can be performed during a natural ovulation cycle. The receptive window for these women can be determined based on the natural ovulation cycle described herein. In certain embodiments, ovulation is induced, for example, by administration of hCG. Preferably, in these women, the receptive window corresponds to between hCG+6 and hCG+9, preferably between hCG+6 and hCG+8.

In certain embodiments, women undergoing FET or third-party IVF also receive luteal support as described above. Preferably, in these women, the acceptance window corresponds to between days 4 and 9 of luteal phase support, preferably between days 5 and 7. Luteal phase support is typically used when FET or third-party IVF is performed during an "artificial cycle." In these cases, the endometrium is prepared by administering estrogen and/or progesterone using methods known in the art. Preferably, luteal phase support begins at least 6 days after the endometrium has been primed with exogenous estrogen to induce an artificial cycle.

In an exemplary embodiment of FET or third-party IVF, estrogen is provided orally or vaginally at a daily dose of 4-8 mg for about 10 days, at which time luteal phase support is initiated with vaginal progesterone administration and blastocyst transfer occurs 6 days after initiation of progesterone.

In one embodiment of the present disclosure, the effect of the oxytocin receptor antagonist overlaps with the blastocyst stage of the embryo. Preferably, the antagonist is released when the embryo reaches the blastocyst stage. Preferably, a blastocyst stage embryo is transferred to the female and the antagonist is administered on the same day as the embryo transfer. Preferably, a therapeutically effective amount of the antagonist is present during at least a portion of the blastocyst stage of the transferred embryo.

The present disclosure also encompasses the transfer of cleavage-stage embryos. In these embodiments, the antagonist can be provided as a sustained-release or controlled-release formulation for release several days after transfer. Alternatively, the antagonist can be administered as an immediate-release formulation several days after transfer. As shown in the Examples, administration of baluxiban (an oxytocin receptor antagonist) to blastocyst-stage embryos resulted in a significant increase in the sustained implantation rate from 27% to 45%.

Oxytocin receptor antagonists are known in the art, and any oxytocin receptor antagonist can be used in the methods of the present disclosure. Preferably, the oxytocin receptor antagonist is selected from a selective oxytocin antagonist and a mixed vasopressin/oxytocin receptor antagonist. The selective oxytocin antagonist has a Ki for the oxytocin receptor that is at least one order of magnitude higher than the Ki for the vasopressin receptor.

Receptor antagonists include compounds that reduce the expression and/or activity of the oxytocin receptor. A preferred vasopressin/oxytocin receptor antagonist is atosiban (1-(3-mercaptopropionic acid)-2-(O-ethyl-D-tyrosine)-4-L-threonine-8-L-ornithine-oxytocin).

Oxytocin receptor antagonists also include RNA interference molecules or RNA antisense molecules directed against the receptor.

Preferably, the antagonist binds to the receptor and prevents receptor activity. Such antagonists include, for example, antibodies that directly antagonize the receptor (e.g., "neutralizing antibodies") and small molecules. Preferably, the antagonist acts as a competitive antagonist and competes with oxytocin for binding to the oxytocin receptor. Preferably, the antagonist is a small molecule that binds to the receptor and antagonizes receptor activity. Preferably, the selective oxytocin antagonist is baluxiban.

Suitable oxytocin receptor antagonists are well known to the skilled artisan and can be readily identified based on known screening methods, for example, using receptor activation and/or receptor binding as readouts. Suitable antagonists include those disclosed in US Pat. No. 6,143,722, the entire contents of which are incorporated herein by reference. US Pat. No. 6,143,722 discloses heptapeptide analogs, or pharmaceutically acceptable salts thereof, having oxytocin antagonist activity and consisting of a hexapeptide portion S and a C-terminal β-amino alcohol residue Z bound to portion S via an amide bond, wherein the β-amino alcohol Z is:

wherein Q is (CH2)n-NH-A, n is 1-6, and A is H or -C(=NH)NH2,

and wherein R is CH3 or C2H5;

And part S is

Wherein, Mpa, Ile, Asn and Abu have the following meanings:

Mpa 3 mercaptopropionic acid residue

Ile isoleucine residue

Asn Asparagine residue

Abu α-aminobutyric acid residue;

and wherein X is a D-aromatic α-amino acid; and Y is an aliphatic α-amino acid. Preferred oxytocin antagonists are listed in FIG1 .

Further antagonists include OBE001/AS-602305 (particularly its oral formulation), TT-235 (Northwestern University), the selective oxytocin receptor antagonist Epelsiban ((3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimethylpyridin-3-yl)-2-(morpholin-4-yl)-2-oxyethyl]-6-[(1S)-1-methylpropyl]pyrazine-2,5-dione); Retosiban ((3R,6R)-6-[(2S)-butane-2- PF-3274167 (5-(3-(3-(2-chloro-4-fluorophenoxy)azetidin-1-yl)-5-(methoxymethyl)-4H-1,2,4-triazol-4-yl)-2-methoxypyridine); and L-368,899 hydrochloride (CAS 148927-60-0); L-371,257 (1-[1-[4-(1-acetylpiperidin-4-yl)oxy-2-methoxybenzoyl]piperidin-4-yl]-4H-3,1-benzoxazin-2-one). Additional oxytocin antagonists are also described in, for example, WO2004020414 and WO2005/028452, the entire contents of which are incorporated herein by reference.Preferably, the antagonist is selected from baluxiban, atosiban, OBE001/AS-602305, PF-3274167, apoxiban and ritoxaban.

Preferably, the oxytocin receptor antagonist is formulated in a pharmaceutical composition. The composition may also include pharmaceutically acceptable additives such as preservatives, diluents, dispersants, agents that promote mucosal absorption (e.g., disclosed by Merkus, F.W.H.M. et al., J. Controlled Release 24, 201-208, 1993, and which include surfactants, bile acid, fusidic acid, phospholipids, and cyclodextrins), buffers, and flavorings. Such compositions can be formulated as solids (e.g., tablets, capsules, or powders) or liquids (e.g., solutions or suspensions), including creams and emulsions herein, for oral or parenteral administration. Oral (including sublingual and buccal), nasal, pulmonary, transdermal, rectal, vaginal, subcutaneous, intramuscular, and intravenous administration can be suitable routes of administration.

In certain embodiments, the pharmaceutical composition can be delivered in a sustained release or delayed release system. For example, the antagonist can be administered using a transdermal patch or formulated in a lipophilic depot (e.g., fatty acid, wax, oil). As used herein, a sustained release or delayed release system ensures that the antagonist is also present in the subject at the time point after administration, such as a few hours or even days after administration. This sustained release or delayed release system allows the receptor antagonist to be administered before the female is in the receptive endometrial stage. However, sustained release or delayed release ensures that when the female enters the receptive endometrial stage and/or when the embryo reaches the blastocyst stage, there is still a sufficient amount (or therapeutically effective amount) of antagonist.

In the embodiment of female experience cleavage stage embryo transplantation, antagonist should be when this cleavage stage embryo has developed into blastocyst stage embryo and/or when female reaches the receptivity endometrium stage, discharge.If use immediate release preparation, then antagonist should be given in a few days after embryo transplantation, preferably two or three days after embryo transplantation, this should develop into blastocyst stage embryo and/or when female reaches the time of receptivity endometrium stage.If use sustained release or delayed release preparation, these can be early days giving, for example, on embryo transplantation day, condition is when cleavage stage embryo develops into blastocyst stage embryo and/or when female reaches the receptivity endometrium stage, antagonist discharges.

In embodiments where the female undergoes blastocyst stage embryo transfer, the antagonist should be released on the same day as the embryo transfer (e.g., within the same 24-hour period), corresponding to the time when the embryo reaches the blastocyst stage and the female has reached the receptive endometrial stage. Preferably, the antagonist is administered between 2 hours before and 2 hours after embryo transfer. More preferably, the antagonist is administered twice. In an exemplary embodiment using baluxiban, the first administration can occur approximately 45 minutes before embryo transfer and the second administration can occur approximately 60 minutes after the first administration.

As is well known to the skilled artisan, the timing of administration depends on the particular antagonist used, in particular the half-life of the antagonist. Antagonists with relatively short half-lives may need to be administered multiple times to ensure that their effects overlap with the blastocyst stage and/or receptive endometrial stage.

References

Ayoubi J-M, Epiney M, Brioschi PA, Fanchin R, Chardonnens D, de Ziegler D. Comparison of changes in uterine contraction frequency after ovulation in the menstrual cycle and in in vitro fertilization cycles. Fertil Steril 2003;79:1101-1105.

Bergh PA, Navot D. The impact of embryonic development and endometrialmaturity on the timing of implantation. Fertil Steril 1992;58:537-542.

Bernabeu R, Roca M, Torres A, Ten J. Indomethacin effect on implantationrates in oocyte recipients. Hum Reprod 2006;21:364-369.

Blockeel C, Pierson R, Popovic-Todorovic B, Visnova H, García-Velasco JA, Mrázek M, Chou P-Y, Wu M-H, Pan H-A, Hung K-H, Chang F-M. Use of an oxytocinantagonist in in vitro fertilization-embryo transfer for women with repeated implantation failure: a retrospective study. Taiwan J Obstet Gynecol 2011;40:136-140.

Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998;13:1968-1974.

Fanchin R, Righini C, de Ziegler D, Olivennes F, Ledée N, Frydman R. Effects of vaginal progesterone administration on uterine contractility at the time of embryo transter. Fertil Steril 2001;75:1136-1140.

Fuchs A-R, Behrens O, Maschek H, Kupsch E, Einspanier A. Oxytocin and vasopressin receptors in human and uterine myomas during menstrual cycle and early pregnancy. Hum Reprod Update 1998;4:594-604.

Killick SR.Ultrasound and the recepyivity of the myometrium.RBMOnline 2007;15:63-67.

Kim CH, Lee JW, Jeon IK, Park E, Lee YJ, Kim SH, Chae H, Kang BM, LeeHA. Administration of oxytocin antagonist improves the implantation rates inpatients with repeated failure of IVF/ICSI treatment. Hum Reprod 2008;23(Suppl.1):i124.

Koot YEM, Macklon NS. Embryo implantation: biology, evaluation, andenhancement. Curr Opin Obstet Gynecol 2013;25:274-279.

Lan VT, Khang VN, Nhu GH, Thuong HM. Atosiban improves implantation andpregnancy rates in patients with repeated implantation failure. RBM Online2012;25:254-260.

Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer-can we learn anything new from the observation of junctional zonecontractions? Hum Reprod 1998;13:1540-1546.

Liang YL, Kuo TC, Hung KH, Chen TH, Wu MH. Oxytocin antagonist forrepeated implantation failure and delay of delivery. Taiwan J Obstet Gynecol2009;48:314-316.

Makrigiannakis A, Minas V. Mechanisms of implantation. RBM Online 2006;14:102-109.

Moon HS, Park SH, Lee JO, Kim KS, Joo BS. Treatment with piroxicam beforeembryo transfer increases the pregnancy rate after in vitro fertilization andembryo transfer. Fertil Steril 2004;82:816-820.

Moraloglu O, Tongue E, Turgut V, Zeyrek T, Batioglu S. Treatment with oxytocin antagonists before embryo transfer may increase implantation rates after IVF. RBM Online 2010;21:338-343.

Ng EH. Li RH, Chen L, Lan VT, Tuong HM, Quan S. A randomized double blind comparison of atosiban in patients undergoing IVF treatment. Hum Reprod 2014;29:2687-2694.

Papanikolaou EG, Kolibianakis EM, Tournaye H, Venetis CA, Fatemi H, Tarlatzis B and Devroey P. Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. Hum Reprod 2008;23:91-99.

Pierzynski P, Reinheimer TM, Kuczynski W. Oxytocin antagonists may improve infertility treatment. Fertil Steril 2007;88:e19-e22.

Richter ON, Bartz C, Dowaji J, Kupka M, Reinsberg J, Ulrich U, Rath W. Contractile reactivity of human myometrium in isolated non-pregnantuteri. Hum Reprod 2006;21:36-45.

Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001;76:863-870.

Simón C, Martin JC, Pellicer A. Paracrine regulators of implantation. Clin Obstet Gynaecol 2000;14:815-826.

Shukovski L, Healy DL, Findlay JK. Circulating immunoreactive oxytocinduring the human menstrual cycle comes from the pituitary and is estradioldependent. J Clin Endocrinol Metab1989;68:455-460.

Strowitzki T, Germeyer A, Popovici R, Wolff M. The human endometrium as afertility-determining factor. Hum Reprod Update 2006;12:617-630.

Zhu L, Che HS, Xiao L, Li YP. Uterine peristalsis before embryo transferaffects the chance of clinical pregnancy in fresh and frozen-thawed embryotransfer cycles. Hum Reprod 2014;29:1238-1243.

All patents and literature cited in this specification are incorporated herein by reference in their entirety.

The present invention is further described in detail with reference to the following examples, which are not intended to limit the scope of the present invention but are merely intended to illustrate the present invention.

Example

Example 1: A randomized, placebo-controlled, double-blind, parallel-group, multinational, multicenter trial evaluated the effects of subcutaneous administration of baluxiban on implantation and pregnancy rates on the day of transfer in IVF/ICSI patients.

method

BASIC is a randomized, double-blind, placebo-controlled, parallel group, multinational, multicenter trial. It is designed to evaluate the effect of baluxiban on the day of cleavage-stage embryo transfer or blastocyst transfer for the sustained implantation rate in IVF/ICSI patients. Patients undergoing controlled ovarian stimulation in a long GnRH agonist or GnRH antagonist regimen receive hCG to trigger final follicular maturation, undergo oocyte retrieval, and have daily luteal phase support supplemented by vaginal progesterone starting on day 1 after oocyte retrieval, and transplanted on day 3 or 5 after oocyte retrieval. Patients were randomly divided into the baluxiban group or the placebo group in a 1:1 ratio, stratified according to the day of transfer (day 3 or 5 after oocyte retrieval) and the number of embryos/blastocysts to be transferred (1 or 2). In total, 255 IVF/ICSI patients were randomly assigned to this trial and donated 440 embryos/blastocysts.

The study drug product, i.e., baluxiban or placebo according to randomization, was administered as a subcutaneous injection at two time points: a first dose of 40 mg of baluxiban or placebo 45 minutes before transplantation and a second dose of 10 mg of baluxiban or placebo 60 minutes after the first dose.

Transfers were performed on day 3 (cleavage-stage embryos) or day 5 (blastocysts) after oocyte retrieval. On day 3, only embryos of good quality, defined as ≥6 blastomeres and ≤20% fragments, were transferred. On day 5, blastocysts with expansion and hatching status 3, 4, 5, or 6 were transferred. The actual number of embryos/blastocysts transferred for each individual patient depended on the desired morphological quality for the patient's age, local conditioning, and the availability of embryos/blastocysts for the clinical protocol, but was a maximum of 2.

The key aspects of the transfer procedure have been standardized. A vaginal speculum is inserted into the vagina and the vagina and cervix are cleaned according to local practice but with minimal manipulation and interference. A soft or ultra-soft catheter is used. The outer sheath of the catheter is inserted just to the internal os of the cervix (i.e., keeping the outer sheath in the cervical canal). The embryo/blastocyst is loaded into the inner sheath, which is then inserted through the outer sheath. Using abdominal ultrasound guidance, the embryo/blastocyst is placed 1.5-2.0 cm from the fundus of the uterus. The time from loading the inner catheter to placing the embryo/blastocyst should not exceed 1 minute. After placement, the inner and outer catheters are withdrawn and confirmed for retained embryos/blastocysts, mucosa, and blood. After confirming that no embryo/blastocyst remains in the catheter, the vaginal speculum is then removed; this occurs within approximately 2 minutes after the embryo/blastocyst is placed. Any difficulties/incidents that occur during the transfer procedure are recorded.

Patients received vaginal progesterone tablets 100 mg twice daily starting on the day of egg retrieval until the day of the clinical pregnancy visit. On the day of transfer, patients should insert the progesterone tablets at least 3 hours before and at least 3 hours after transfer. The ongoing implantation rate (primary endpoint) was defined as the number of viable fetuses in the uterus 10-11 weeks after transfer divided by the number of embryos/blastocysts transferred.

Regarding statistical methods, the primary hypothesis was tested using a logistic regression model with sustained engraftment (yes/no) as the outcome and treatment and randomization stratum as factors. Treatment effects are presented on an odds ratio scale, representing the results of the logistic regression analysis; the analysis allowed for the inclusion of factors and covariates. It is important to emphasize that the odds ratios, based on the logistic regression model, provide the optimal method for performance data and the benchmark for evaluating treatment effects.

Several factors affect the probability of successful outcome of embryo transplantation, including the number of the embryo/blastocyst of transplantation day, and the quality of the embryo/blastocyst of transplantation. Randomization was used to ensure the first two of these factors of comparable groups through stratification. However, because embryonic development is continuous, stratification for the quality of the embryo/blastocyst of transplantation is not easy. Alternatively, the quality adjustment for the embryo/blastocyst was also included in the main analysis.

result

The effect of treatment on the different receptive stages and implantation potential of cleavage-stage embryos and blastocysts was evident, as demonstrated by the overall ongoing implantation rate of 19% on day 3 after oocyte retrieval and transfer and 38% on day 5 after oocyte retrieval and transfer. The same pattern was seen in both the baluxiban and placebo groups.

The overall (Day 3 + Day 5) ongoing implantation rates observed in the trial ^were 27.1% and 28.2% for baluxiban, corresponding to an odds ratio of ² of 1.1 (95% confidence interval 0.7-1.8; p=0.6960), i.e., favoring baluxiban but not significantly. Therefore, the primary endpoint was not met for the entire trial group, but as described below, this was due to a day-of-transfer interaction. Transfer of cleavage-stage embryos on Day 3 after oocyte retrieval resulted in an odds ratio of 0.3 (0.3-1.2; p=0.1509) (Figure 2). The analysis stratified by Day 5 after oocyte retrieval resulted in an odds ratio of 2.3 (1.1-4.7; p=0.0270), demonstrating a significant treatment effect of baluxiban on the ongoing implantation rate for blastocyst transfer (Figure 2). The odds ratio of 2.3 corresponds to an adjusted means for sustained implantation rate for blastocyst transfer of 45% for baluxiban versus 27% for placebo (relative delta of 67%) ( FIG3 ).

Results from the BASIC trial showed that the effect of oxytocin antagonists on implantation rates was influenced by the day of transfer, whether cleavage-stage embryo transfer (day 3 after oocyte retrieval) or blastocyst transfer (day 5 after oocyte retrieval). When cleavage-stage embryo transfer was performed on day 3 (pre-receptive stage), there was no effect of baluxiban on sustained implantation rates. However, when blastocyst transfer was performed on day 5 (receptive stage), a significant (p=0.0270) effect of baluxiban on improved sustained implantation rates was observed.

The BASIC trial identified a clinically relevant time window for the effects of baluxiban, or a general oxytocin antagonist, and a mixed oxytocin/vasopressin antagonist on implantation that could not be predicted before the trial. An effect on implantation rates was observed when the oxytocin antagonist was given at the time of implantation, on day 5 (or later) after oocyte retrieval, but not in the early luteal phase, on days 2-3 after oocyte retrieval.

The lack of consistent efficacy between cleavage-stage embryo transfer on day 3 post-ovulation and blastocyst transfer on day 5 post-ovulation is important for hypothesizing the mechanisms linking uterine contractions to subsequent cycle outcomes. The pharmacologically coordinated doses and methods of administration, as well as the transfer procedures, were identical for both days. Therefore, putative mechanisms such as uterine contractions due to early luteal phase or embryo/blastocyst expulsion or dispersion during the embryo/blastocyst transfer procedure should no longer be considered as potentially explaining the improved implantation rates with uterine relaxants, including oxytocin antagonists or mixed oxytocin/vasopressin antagonists. In cases where these mechanisms are most relevant, the observations on days 3 and 5 post-ovulation should be identical.

Claims (19)

1. Use of an oxytocin receptor antagonist in the preparation of a medicament for increasing sustained implantation rate, sustained pregnancy rate, clinical pregnancy rate, and/or live birth rate in female subjects undergoing embryo transfer at the blastocyst stage as part of assisted reproductive technology, wherein the antagonist is provided to the female such that the action of the antagonist overlaps with the receptive endometrial stage in the female, and the action of the antagonist is present when the embryo reaches the blastocyst stage, wherein the oxytocin receptor antagonist is selected from parfusiban and OBE001. 2. The use according to claim 1, wherein the receptive endometrial stage corresponds to: a) Between LH+6 and LH+9 in a natural ovulation cycle; b) Between hCG+6 and hCG+9 during the ovulation induction cycle; c) Support during the luteal phase from day 4 to day 7 after egg retrieval; d) Luteal support is provided between day 4 and day 9, wherein luteal support begins at least 6 days after the endometrium is induced by exogenous estrogen. 3. The use according to claim 2, wherein the luteal phase support comprises supplementation with human chorionic gonadotropin, progesterone, and/or a gonadotropin-releasing hormone agonist. 4. The use according to claim 2, wherein the luteal phase support comprises progesterone supplementation, or estradiol and progesterone. 5. The use according to any one of the preceding claims, wherein the antagonist is provided to the female such that the antagonist is released on the day of embryo transfer. 6. The use according to any one of claims 1-4, wherein the antagonist is administered to the female on the day of embryo transfer. 7. The use according to any one of claims 1-4, wherein the antagonist is administered between 2 hours before and 2 hours after embryo transfer. 8. The use according to any one of claims 1-4, wherein the antagonist is administered twice, and wherein the first administration occurs 45 minutes before embryo transfer and the second administration occurs 60 minutes after the first administration. 9. The use according to any one of claims 1-4, wherein the blastocyst stage embryo is an embryo on day 5 after fertilization. 10. Use of a sustained- or delayed-release formulation of an oxytocin receptor antagonist in the preparation of a medicament for increasing sustained implantation rate, sustained pregnancy rate, clinical pregnancy rate, and/or live birth rate in female subjects undergoing embryo transfer, wherein the antagonist is administered to the female such that the antagonist is effective when the embryo reaches the blastocyst stage, wherein the female undergoes transfer of a cleavage-stage embryo and the antagonist is administered to the female such that the antagonist is released two or three days after embryo transfer, wherein the oxytocin receptor antagonist is selected from baluciban and OBE001. 11. The use according to claim 10, wherein the cleavage-stage embryo has at least 6 blastomeres and 20% or less of fragments. 12. The use according to claim 10, wherein the cleavage stage embryo is an embryo on day 2 or day 3 after fertilization. 13. The use according to any one of claims 1-4 or claim 10, wherein the antagonist is baruciban. 14. The use according to claim 13, wherein parfusiban is provided subcutaneously. 15. The use according to claim 13, wherein the dosage of baluciban is between 30 and 80 mg. 16. The use according to claim 15, wherein 50 mg of baluciban is administered. 17. The use according to claim 13, wherein the female undergoes transfer of a blastocyst stage embryo and parfusiban is given to the female on the same day as the embryo transfer. 18. The use according to claim 17, wherein 40 mg of baluciban is administered subcutaneously 45 minutes before embryo transfer and 10 mg of baluciban is administered subcutaneously 60 minutes after the first administration. 19. The use according to any one of claims 1-4 or claim 10, wherein the antagonist is OBE001.